Patent Grant
11034963
The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled 20661007US371SEQLST.txt, created on May 7, 2019, which is 2,515,112 bytes in size. The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety.
The present invention relates to nucleic acid based signaling polynucleotides (SPNs) for detecting allergen proteins. Detection agents comprising SPNs of the present invention can be used as novel biosensor platforms.
Allergy is a serious medical condition affecting millions of people worldwide, with about 15 million people in the United States, including many children. During an allergic reaction, the immune system mistakenly targets an allergen as a threat and attacks it. The allergic reaction may affect the skin, the digestive system, the gastrointestinal tract, the respiratory system, the circulatory system and the cardiovascular system; in some allergic reactions, multiple organ systems are affected. Allergic reactions range from mild to severe or life-threatening. Severe symptoms may include difficulty in breathing, low blood pressure, chest pain, loss of consciousness, and anaphylaxis. People having allergies currently manage their allergies by avoiding any food that might contain that specific allergen. These restrictions have a major impact on the patients' quality of life and there remains no method for assessing the true allergen content of food. In the United States, food allergy symptoms send someone to the emergency room every three minutes.
Though antibody-based immunoassays are commonly used for food allergen detection, given the fact that antibodies to allergens are not easily to be obtained, there is an unmet demand of new platform technology to improve in vitro allergen detection in both clinical and non-clinical settings. Nucleic acids, for example, nucleic acid aptamers are promising alternatives or supplements to antibodies for this purpose. These small RNA/DNA molecules can form secondary and tertiary structures capable of specifically binding proteins or other targets. Aptamers can be developed against a seemingly unlimited range of targets such as small inorganic ions, drugs, organic peptides, proteins and even complex cells. Aptamers are thermally stable, so they can be stored and transported easily. These properties make aptamers good agents for analyte detection in a sample, protein/nucleic acid purification and other aspects of biological researches.
Methods using aptamers specific allergen proteins have been reported for detection of several common allergens, for example, gluten (PCT Publication PCT/ES2013/000133 to Amaya-Gonzalez, et al.), and toxic ß-conglutin (Lup an 1) (Nadal, et al., PLoS ONE, 2012, 7(4): e35253; Nadal, et al., Anal. Bioanal. Chem. 2013, 405: 9343-9349; and Mairal, et al., Biosensors and Bioelectronics, 2014, 54: 207-210.) The present inventors have recognized that allergen detection in various matrices of food products can be conveniently performed using aptamer-based detector sequences such as signaling polynucleotides (SPNs), which are particularly well suited for use in a simple and portable sensor that can be used repetitively with high sensitivity and reproducibility at ambient temperature to ensure food safety. Recent patent applications by the inventors disclose allergen specific aptamers, SPNs and their applications in allergen detection, including PCT patent application publication NO.: WO2015/066027; PCT Patent Application Serial NO.: PCT/US 16/29356; and U.S. Patent Application Ser. No. 62/308,377; the contents of each of which are incorporated by reference in their entirety.
In addition to sensitive ligands (e.g., nucleic acid aptamers) that bind analytes of interest (e.g., an allergen protein), materials and surfaces that enhance the analytic performance of the ligands and biosensors are also important. Recently A wide variety of materials including magnetic particles, gold or latex particles and electrode surfaces have been used in many settings as strategies for signaling amplification. The properties of magnetic particles, both nano- and micro-size dimensions, have proved to be promising substrates to be coupled with detection ligands for the design of cost-effective biosensing platforms.
The present application discloses magnetic particle conjugates comprising SPNs and/or SPN complexes that are suitable for allergen detection with high sensitivity and specificity. Such SPN-magnetic particle conjugates may be used as detection agents for allergen detection in a variety of analyte detection assays, kits, devices and systems.
The present invention provides signaling polynucleotides (SPNs), SPN-complement complexes, magnetic particle conjugates, DNA printed glass slides and detection agents for allergen detection, and biosensors, assays and method for detecting allergen proteins in food samples using the compositions and agents discussed in the present disclosure.
In one embodiment, SPNs derived from nucleic acid aptamers having nucleic acid sequences that bind allergens with high specificity and affinity are provided. In some aspects, the signaling polynucleotides comprise nucleic acid sequences of SEQ ID NOs.: 1-696, which bind specifically to eight common food allergens. In some aspects, the SPN comprises a nucleic acid sequence that specifically binds to cashew selected from SEQ ID NOs.: 1-12. In some aspects, the SPN may comprise a nucleic acid sequence that specifically binds to peanut selected from SEQ ID NOs.: 13-24, 96 and 97-496. In some aspects, the SPN may comprise a nucleic acid sequence that specifically binds to tree nut selected from SEQ ID NOs.: 497-696. In some aspects, the SPN may comprise a nucleic acid sequence that specifically binds to milk selected from SEQ ID NOs.: 25-34. In some aspects, the SPN may comprise a nucleic acid sequence that specifically binds to fish allergen selected from SEQ ID NOs.: 35-46. In some aspects, the SPN may comprise a nucleic acid sequence that specifically binds to egg selected from SEQ ID NOs.: 47-58 and 93-94. In some aspects, the SPN may comprise a nucleic acid sequence that specifically binds to gluten selected from SEQ ID NOs.: 59-70 and 91-92. In some aspects, the SPN may comprise a nucleic acid sequence that specifically binds to soy allergen selected from SEQ ID NOs.: 71-80. In other aspects, the SPN may comprise a nucleic acid sequence that specifically binds to crustacean selected from SEQ ID NOs.: 81-90.
In one embodiment, SPN-complement complexes are provided in accordance with the present invention. The SPN-complement complex comprises a SPN and a short nucleic acid sequence complementary to a portion of the sequence of the SPN, wherein the SPN and its corresponding complementary sequence are hybridized to form the complex. In one example, the complement is complementary to either the 5′ end or 3′ end sequence of the SPN. In some aspects, the complementary sequence contains about 5-20 nucleotide residues. In one preferred example, the complementary sequence contains 5-10 nucleotide residues.
In one embodiment, magnetic particle conjugates are provided in accordance with the present invention. The magnetic particle conjugates comprise SPN-complement complexes covalently immobilized on the surface of the particles. The magnetic particles may be micro-magnetic particles (MMPs) or nano-magnetic particles (NMPs). In some aspects, the 3′ end of the SPN is linked to magnetic particles, for example, through a 3′ amine or 3′ thiol group, or the addition of a poly-A sequence at the end, or a biotin modification. In other aspects, the 5′ end of the SPN is linked to magnetic particles, for example, through a 5′ amine or 5′ thiol group, or a biotin modification. Accordingly, the complementary sequence contains nucleotide sequences complementary to the free end of the SPN. In further other aspects, the SPN is linked to magnetic particles through its complementary sequence which is covalently immobilized on the surface of magnetic particles. In some examples, the complementary sequence may be attached to the surface of magnetic beads through amine, or thiol, or biotin-streptavidin linkage. In some aspects, magnetic particles may be further coated with PEG polymers or ethanolamine-PEG linkers.
In another embodiment. SPNs and SPN-complement complexes may be attached to the surface of a solid support; such solid support may be a glass slide, a silicon chip or wafer or a microwell plate. The DNA-printed surface may be used as a biosensing platform in a variety of settings for allergen detection. For example, a DNA printed glass slide may be inserted into a detection sensor for detection of a target allergen. In some aspects, one end of the SPN is attached to the two-dimensional surface of the solid support. The complement and allergen protein compete each other for binding to the attached SPN. In other aspects, the short complement is attached to the two-dimensional surface of the solid support. The SPN is hybridized with the complement to form complexes.
The covalent conjugation of nucleic acid molecules of the present invention (e.g., aptamer. SPN and SPN complement) to a surface, either a three-dimensional surface of magnetic particle or a two-dimensional surface of a glass slide can be through a thiol-maleimide mediated covalent chemical reaction, or an amine-mediated reaction, or a biotin-streptavidin linkage. In one example, the surface of a solid substrate (e.g. magnetic particles and glass slides) may be further coated with PEG polymers or ethanolamine-PEG linkers.
In some aspects, the nucleic acid sequence may be directly synthesized in situ on the solid substrate. In one embodiment, the short complementary oligonucleotide is directly synthesized on the solid substrate at a low density. A stable non-cleavable linker and optionally a spacer group may be used for in situ synthetic reaction.
In one embodiment, detection agents comprising SPNs, SPN-complement complexes, magnetic particle conjugates and/or DNA printed solid glasses are provided in accordance with the present invention. The detection agents may be used in any biosensor platform for capturing and detecting allergen proteins in food samples. In some aspects, the agents may be labeled with a fluorescent marker. The fluorescent marker may be conjugated to the agent in a variety of configurations. In one example, one end of the complementary sequence may be labeled with a fluorophore. In this configuration, a target allergen protein when binding to the SPN, will detach the complementary sequence hybridized with the SPN, resulting in “fluorescent signal of”. In another embodiment, one end of the complementary sequence is labeled with a quencher and the free end of the SPN is labeled with a fluorophore. Therefore, the fluorescent signal is quenched in the SPN-complement complex. The binding of a target allergen will detach the complementary sequence hybridized with the SPN, resulting in the quenched fluorescent signal back on. In further another example, one end of the complementary sequence and the free end of the SPN are labeled with different fluorophores. The binding of a target allergen will detach the complementary sequence hybridized with the SPN, resulting in a change in fluorescent signal. In each signal configuration, changes in fluorescent signals can be used to determine the presence and/or absence of the target allergen in food samples.
In accordance with the present invention, kits, biosensors and devices dependent on the present compositions and agents are also provided.
In other embodiments, the present invention provides assays and methods for detecting the presence, absence and/or quantity of an allergen of interest in a sample.
The details of one or more embodiments of the invention are set forth in the accompanying description below. Although any materials and methods similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred materials and methods are now described. Other features, objects and advantages of the invention will be apparent from the description. In the description, the singular forms also include the plural unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In the case of conflict, the present description will control.
The present invention provides compositions, conjugates and detection agents for allergen detection. The functional/core component of a detection agent is a nucleic acid aptamer that specifically binds an allergen protein. In accordance with the present invention, an allergen specific aptamer may be modified to form a signal polynucleotide (SPN) (described in detail herein below). The SPN may be used as a free detection agent, or conjugated to a particle or a solid surface as a complex detection agent. In some aspects, the SPNs alone work as both binding ligands and signaling molecules or labels. In other aspects, complexes composed of SPNs and their complementary sequences work as both binding ligands and signaling molecules or labels. Aptamers, SPNs and/or SPN-complement complexes can be conjugated to any particles such as metal nanoparticles, redox compounds and magnetic particles, magnetic microparticles, and other solid surfaces such as glass slides and silicon chips, forming biosensor platforms.
Aptamers comprising nucleic acid sequences that bind common food allergens are isolated through SELEX selections. Aptamers are then modified to generate signal polynucleotides (SPNs) which work as capturing and detecting ligands for allergen proteins. In particular, the SPN may comprise a nucleic acid sequence selected from SEQ ID NOs.: 1-696.
In some aspects, the SPN comprises a nucleic acid sequence that specifically binds to cashew selected from SEQ ID NOs.: 1-12; the cashew specific SPN has a unique sequence of SEQ ID. NOs.: 1-6. In some aspects, the SPN may comprise a nucleic acid sequence that specifically binds to peanut selected from SEQ ID NOs.: 13-24, and 96-696; the peanut specific SPN has a unique sequence of SEQ ID NOs.: 13-18 and 97-296. In some aspects, the SPN may comprise a nucleic acid sequence that specifically binds to tree nut selected from SEQ ID NOs.: 497-696; the tree nut specific SPN has a unique sequence of SEQ ID NOs.: 497-596. In some aspects, the SPN may comprise a nucleic acid sequence that specifically binds to milk selected from SEQ ID NOs.: 25-34; the milk specific SPN has a unique sequence of SEQ ID NOs.: 25-29. In some aspects, the SPN may comprise a nucleic acid sequence that specifically binds to fish allergen selected from SEQ ID NOs.: 35-46; the fish specific SPN has a unique sequence of SEQ ID NOs.: 35-40. In some aspects, the SPN may comprise a nucleic acid sequence that specifically binds to egg selected from SEQ ID NOs.: 47-58 and 93-94; the egg specific SPN has a unique sequence of SEQ ID NOs.: 47-52. In some aspects, the SPN may comprise a nucleic acid sequence that specifically binds to gluten selected from SEQ ID NOs.: 59-70 and 91-92, the gluten specific SPN has a unique sequence of SEQ ID NOs.: 59-64. In some aspects, the SPN may comprise a nucleic acid sequence that specifically binds to soy allergen selected from SEQ ID NOs.: 71-80; the soy specific SPN has a unique sequence of SEQ ID NOs.: 71-75. In other aspects, the SPN may comprise a nucleic acid sequence that specifically binds to crustacean selected from SEQ ID NOs.: 81-90; the crustacean specific SPN has a unique sequence of SEQ ID NOs.: 81-85.
In some embodiments, a SPN and a short nucleic acid sequence which is complementary to a portion of the sequence of the SPN (e.g., the sequence at either the 5′ end or 3′ end of the SPN) may be hybridized to form a complex (referred to “SPN-complement complex”). The SPN-complement complexes may be attached to the surface of magnetic particles or other solid surfaces through either the SPN or the complementary sequence.
In some embodiments, SPNs of the present invention are conjugated to the surface of magnetic particles or a solid substrate (e.g., a glass slide) at one end, e.g. the 5′ end or 3′end (e.g., as shown in
In this context, the opposite end of the SPN which is free from the attached magnetic particles may be hybridized with the complementary sequence, forming a SPN-complement complex. The short complementary sequences may be labeled with a fluorescent dye, such as Alex 647, Cy5, Cy3-FITC and Texas red. The binding of an allergen to the SPN will detach the complementary sequence from the complex, resulting in a signaling change (as shown in
In some embodiments, the complementary sequences of the SPNs may be conjugated to the surface of magnetic particles or other solid substrates (e.g., printed glass surface) through covalent bindings as discussed herein. In this context, the printed glass surface having such complementary sequences may be exposed to the SPNs and allergen proteins. In this context, either an attachment assay or detachment assay may be developed for detection of allergen proteins (
In other embodiments, nucleic acid molecules of the present invention may be attached to solid surfaces by in situ oligonucleotide synthesis.
Aptamers, Signaling Polynucleotides (SPNs) and SPN-Complement Complex
Aptamers (sometimes also called chemical antibodies) are single-stranded oligonucleotides (RNA or single stranded DNA) that form stable but unique three-dimensional confirmations capable of binding with high affinity and specificity to a variety of molecular targets. Aptamers bind to protein targets in much the same manner as antibodies and modulate protein function. Thus, aptamers are also referred to as “chemical antibodies”. Aptamers have advantages over antibodies in that they are poorly immunogenic, stable, and often bind to a target molecule more strongly than do antibodies. Generally aptamers can be synthesized easily and in large quantities by in vitro transcription, PCR, or chemical synthesis (Annu. Rev. Med. 2005, 56, 555-583; Nat. Rev. Drug Discov. 2006, 5, 123-132), and target-specific aptamers can be selected from random-sequence, single-stranded nucleic acid libraries by an in vitro selection and amplification procedure known as SELEX (systematic evolution of ligands by exponential enrichment). The selected aptamers are small single-stranded nucleic acids that fold into a well-defined three-dimensional structure. They show a high affinity and specificity for their target molecules and inhibit their biological functions. Furthermore, aptamers have important properties that simplify its industrialization. For example, aptamers are thermally stable, so they can be stored and transported easily. Aptamers can be produced or modified in large scale, with minimal batch-to-batch variation, given the well-established chemical synthesis and modification technologies.
Aptamers are useful and cost-effective tools for biochemical analyses. Also, they can be developed quickly against a seemingly unlimited range of targets. To date, specific aptamers against diverse targets have been successfully developed, including small inorganic irons, organic peptides, drugs, proteins, lipids and even complex cells. One of the more recent reviews of aptamer-based analysis in context of food safety control indicated that the selection of aptamers for this group of ingredients is emerging (Amaya-Gonzalez et al., Sensors 2013, 13: 16292-16311, the contents of which are incorporated herein by reference in its entirety).
1. Selection of Aptamers Specific to a Target
Aptamers can be artificially generated by a method called systematic evolution of ligands by exponential enrichment (SELEX) (Tuerk and Gold, Science, 1990, 249, 505-510). More recently, a new improved separation technology for aptamer selection was introduced, capillary electrophoresis (CE)-SELEX (Mosing and Bowser, Methods Mol Biol., 2009, 535: 33-43).
Aptamers that bind to virtually any particular target can be selected by using an iterative process called SELEX™ (Systemic Evolution of Ligands by Exponential Enrichment). The process is described in, for example U.S. Pat. Nos. 5,270,163 and 5,475,096. The SELEX™ process is based on the unique insight that nucleic acids have sufficient capacity for forming a variety of two- and three-dimensional structures and sufficient chemical versatility available within their monomers to act as ligands (i.e., form specific binding pairs) with virtually any chemical compound, whether monomeric or polymeric.
The SELEX™ process relies, as a starting point, upon a large library or pool of single stranded oligonucleotides comprising randomized sequences. The oligonucleotides can be modified or unmodified DNA, RNA, or DNA/RNA hybrids. In some examples, the pool comprises 100% random or partially random oligonucleotides. In other examples, the pool comprises random or partially random oligonucleotides containing at least one fixed sequence and/or conserved sequence incorporated within randomized sequence. In other examples, the pool comprises random or partially random oligonucleotides containing at least one fixed sequence and/or conserved sequence at its 5′ and/or 3′ end which may comprise a sequence shared by all the molecules of the oligonucleotide pool. Fixed sequences are sequences common to oligonucleotides in the pool which are incorporated for a preselected purpose such as, CpG motifs, hybridization sites for PCR primers, promoter sequences for RNA polymerases (e.g., T3, T4, T7, and SP6), restriction sites, or homopolymeric sequences, such as poly A or poly T tracts, catalytic cores, sites for selective binding to affinity columns, and other sequences to facilitate cloning and/or sequencing of an oligonucleotide of interest. Conserved sequences are sequences, other than the previously described fixed sequences, shared by a number of aptamers that bind to the same target.
The oligonucleotides of the pool preferably include a randomized sequence portion as well as fixed sequences necessary for efficient amplification. Typically, the oligonucleotides of the starting pool contain fixed 5′ and 3′ terminal sequences which flank an internal region of 30-50 random nucleotides. The randomized nucleotides can be produced in a number of ways including chemical synthesis and size selection from randomly cleaved cellular nucleic acids. Sequence variation in the test nucleic acids can also be introduced or increased by mutagenesis before or during the selection/amplification iterations.
The random sequence portion of the oligonucleotide can be of any length and can comprise ribonucleotides and/or deoxyribonucleotides and can include modified or non-natural nucleotides or nucleotide analogs (see for example U.S. Pat. Nos. 5,958,691 and 5,660,985). Random oligonucleotides can be synthesized from phosphodiester-linked nucleotides using solid phase oligonucleotide synthesis techniques well known in the art. Random oligonucleotides can also be synthesized using solution phase methods such as triester synthesis methods. Typical syntheses carried out on automated DNA synthesis equipment yield 1014-1016 individual molecules, a number sufficient for most SELEX™ experiments.
The starting library of oligonucleotides may be generated by automated chemical synthesis on a DNA synthesizer. Partially random sequences can be created by adding the four nucleotides in different molar ratios at each addition step.
The library of oligonucleotides for aptamer selection may be either RNA or DNA. A RNA library of oligonucleotides is typically generated by transcribing a DNA library of oligonucleotides in vitro using T7 RNA polymerase or modified T7 RNA polymerases and purified. The RNA or DNA library is then mixed with the target under conditions favorable for binding and subjected to step-wise iterations of binding, partitioning and amplification, using the same general selection scheme, to achieve the desired criterion of binding affinity and selectivity as defined in the present application.
More specifically, starting with a mixture containing the starting pool of nucleic acids, the SELEX™ method includes steps of: (a) contacting the mixture with the target under conditions favorable for binding; (b) partitioning unbound nucleic acids from those nucleic acids which have bound specifically to target molecules; (c) dissociating the nucleic acid-target complexes; (d) amplifying the nucleic acids dissociated from the nucleic acid-target complexes to yield a ligand-enriched mixture of nucleic acids; and (e) reiterating the steps of binding, partitioning, dissociating and amplifying through as many cycles as desired to yield highly specific, high affinity nucleic acid ligands to the target molecule. Cycles of selection and amplification are repeated until a desired goal is achieved. Generally this is until no significant improvement in binding strength is achieved on repetition of the cycle. Typically, nucleic acid aptamer molecules are selected in a 5 to 20 cycle procedure.
A variety of nucleic acid primary, secondary and tertiary structures are known to exist. The structures or motifs that have been shown most commonly to be involved in non-Watson-Crick type interactions are referred to as hairpin loops, symmetric and asymmetric bulges, pseudoknots and myriad combinations of the same. The core SELEX™ method has been modified to achieve a number of specific objectives, such as selection of aptamers with particular secondary structures. Examples of SELEX processes can be found in U.S. Pat. Nos. 5,270,163 and 5,475,096. For example, U.S. Pat. No. 5,707,796 describes the use of SELEX™ in conjunction with gel electrophoresis to select nucleic acid molecules with specific structural characteristics, such as bent DNA. U.S. Pat. No. 5,763,177 describes SELEX™ based methods for selecting nucleic acid ligands containing photo reactive groups capable of binding and/or photo-crosslinking to and/or photo-inactivating a target molecule U.S. Pat. Nos. 5,567,588 and 5,861,254 describe SELEX™ based methods which achieve highly efficient partitioning between oligonucleotides having high and low affinity for a target molecule. U.S. Pat. No. 5,496,938 describes methods for obtaining improved nucleic acid ligands after the SELEX™ process has been performed. U.S. Pat. No. 5,705,337 describes methods for covalently linking a ligand to its target, the contents of each of which are incorporated herein by reference in their entirety.
Counter-SELEX™ is a method for improving the specificity of nucleic acid ligands to a target molecule by eliminating nucleic acid ligand sequences with cross-reactivity to one or more non-target molecules. Counter-SELEX™ is comprised of the steps of: (a) preparing a candidate mixture of nucleic acids; (b) contacting the candidate mixture with the target, wherein nucleic acids having an increased affinity to the target relative to the candidate mixture may be partitioned from the remainder of the candidate mixture; (c) partitioning the increased affinity nucleic acids from the remainder of the candidate mixture; (d) dissociating the increased affinity nucleic acids from the target; (e) contacting the increased affinity nucleic acids with one or more non-target molecules such that nucleic acid ligands with specific affinity for the non-target molecule(s) are removed; and (f) amplifying the nucleic acids with specific affinity only to the target molecule to yield a mixture of nucleic acids enriched for nucleic acid sequences with a relatively higher affinity and specificity for binding to the target molecule. As described above for SELEX™, cycles of selection and amplification are repeated as necessary until a desired goal is achieved.
The binding affinity describes the measure of the strength of the binding or affinity of molecules to each other. Binding affinity of the aptamer herein with respect to targets and other molecules is defined in terms of Kd. The dissociation constant can be determined by methods known in the art. It has been observed, however, that for some small oligonucleotides, direct determination of Kd is difficult, and can lead to misleadingly high results. Under these circumstances, a competitive binding assay for the target molecule or other candidate substance can be conducted with respect to substances known to bind the target or candidate. The value of the concentration at which 50% inhibition occurs (K,) is, under ideal conditions, equivalent to Kd.
In accordance with the present invention, a SELEX approach was used to select core binding aptamers that bind 8 major food allergens (i.e. cashew, egg, milk, peanuts, gluten, fish, crustacean and soy). Several aptamers with sequences that can specifically recognize a target allergen were selected and the nucleic acid sequences of selected aptamers were further modified to generate signaling polynucleotides. The aptamers with high selectivity, specificity and stability are selected and further labeled as detection agents. The sequences of the selected aptamers for the 8 major allergens are listed in Table 1. For example, an aptamer having a full sequence (SEQ ID NO.: 7) is one of the SPNs that bind cashew. The full sequence includes the primers used for the screen and the core binding sequence of the aptamer (SEQ ID NO.: 1), the full sequence will be further modified to generate signaling polynucleotides (SPNs) specific to cashew, as discussed herein below.
2. Aptamer Modifications
In accordance with the present invention, oligonucleotides and aptamers may be further modified to improve their stability. The present invention also includes analogs as described herein and/or additional modifications designed to improve one or more characteristics of aptamers such as protection from nuclease digestion. Oligonucleotide modifications contemplated in the present invention include, but are not limited to, those which provide other chemical groups that incorporate additional charge, polarizability, hydrophobicity, hydrogen bonding, electrostatic interaction, and fluxionality to the nucleic acid ligand bases or to the nucleic acid ligand as a whole.
Modifications to generate oligonucleotides which are resistant to nucleases can also include one or more substitute internucleotide linkages, altered sugars, altered bases, or combinations thereof. Such modifications include 2′-position sugar modifications, 5-position pyrimidine modifications, 8-position purine modifications, modifications at exocyclic amines, substitution of 4-thiouridine, substitution of 5-bromo or 5-iodo-uracil, backbone modifications, phosphorothioate or alkyl phosphate modifications, methylations, and unusual base-pairing combinations such as the isobases isocytidine and isoguanosine, 3′ and 5′ modifications such as capping; conjugation to a high molecular weight, non-immunogenic compound; conjugation to a lipophilic compound; and phosphate backbone modification.
Nucleic acid aptamers may be ribonucleic acid, deoxyribonucleic acid, or mixed ribonucleic acid and deoxyribonucleic acid. Aptamers may be single stranded ribonucleic acid, deoxyribonucleic acid or mixed ribonucleic acid and deoxyribonucleic acid.
Nucleic acid aptamers comprise a series of linked nucleosides or nucleotides. The term “nucleic acid,” in its broadest sense, includes any compound and/or substance that comprise a polymer of nucleotides. These polymers are often referred to as polynucleotides. Exemplary nucleic acid molecules or polynucleotides of the invention include, but are not limited to, either D- or L-nucleic acids, ribonucleic acids (RNAs), deoxyribonucleic acids (DNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs, including LNA having a β-D-ribo configuration, α-LNA having an α-L-ribo configuration (a diastereomer of LNA), 2′-amino-LNA having a 2′-amino functionalization, and 2′-amino-α-LNA having a 2′-amino functionalization) or hybrids thereof.
The skilled artisan will recognize that the term “RNA molecule” or “ribonucleic acid molecule” encompasses not only RNA molecules as expressed or found in nature, but also analogs and derivatives of RNA comprising one or more ribonucleotide/ribonucleoside analogs or derivatives as described herein or as known in the art. Strictly speaking, a “ribonucleoside” includes a nucleoside base and a ribose sugar, and a “ribonucleotide” is a ribonucleoside with one, two or three phosphate moieties. However, the terms “ribonucleoside” and “ribonucleotide” can be considered to be equivalent as used herein. The RNA can be modified in the nucleobase structure, the ribofuranosyl ring or in the ribose-phosphate backbone.
In some embodiments, the aptamer comprises at least one chemical modification. In some embodiments, the chemical modification is selected from a chemical substitution of the nucleic acid at a sugar position, a chemical substitution at a phosphate position and a chemical substitution at a base position. In other embodiments, the chemical modification is selected from incorporation of a modified nucleotide; 3′ capping; conjugation to a high molecular weight, non-immunogenic compound; conjugation to a lipophilic compound; and incorporation of phosphorothioate into the phosphate backbone. In a preferred embodiment, the high molecular weight, non-immunogenic compound is polyalkylene glycol, and more preferably is polyethylene glycol (PEG). The process of covalent conjugation of PEG to another molecule, normally a drug or therapeutic protein is known as PEGylation. PEGylation is routinely achieved by incubation of a reactive derivative of PEG with the target molecule. The covalent attachment of PEG to a drug or therapeutic protein can mask the agent from the host's immune system, thereby providing reduced immunogenicity and antigenicity, and increase the hydrodynamic size (size in solution) of the agent which prolongs its circulatory time by reducing renal clearance. PEGylation can also provide water solubility to hydrophobic drugs and proteins.
In another preferred embodiment, the 3′ cap is an inverted deoxythymidine cap.
In some embodiments, nucleic acid aptamers are provided in which the P(O)O group is replaced by P(O)S (“thioate”), P(S)S (“dithioate”). P(O)NR2 (“amidate”), P(O)R, P(O)OR′, CO or CH2 (“formacetal”) or 3′-amine (—NH—CH2-CH2-), wherein each R or R′ is independently H or substituted or unsubstituted alkyl. Linkage groups can be attached to adjacent nucleotide through a —O—, —N—, or —S— linkage. Not all linkages in the nucleic acid aptamers are required to be identical.
As non-limiting examples, a nucleic acid aptamer can include D-ribose or L-ribose nucleic acid residues and can also include at least one modified ribonucleoside including but not limited to a 2′-O-methyl modified nucleoside, a nucleoside comprising a 5′ phosphorothioate group, a terminal nucleoside linked to a cholesteryl derivative or dodecanoic acid bisdecylamide group, a locked nucleoside, an abasic nucleoside, an inverted deoxynucleoside or inverted ribonucleoside, a 2′-deoxy-2′-fluoro-modified nucleoside, a 2′-amino-modified nucleoside, a 2′-alkyl-modified nucleoside, a morpholino nucleoside, a phosphoramidate or a non-natural base comprising nucleoside, or any combination thereof. Alternatively, a nucleic acid aptamer can comprise at least two modified ribonucleosides, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20 or more modified ribonucleosides, up to the entire length of the molecule. The modifications need not be the same for each of such a plurality of modified deoxy- or ribonucleosides in a nucleic acid molecule.
Aptamer may comprise modified nucleobase (often referred to in the art simply as “base”) for increasing the affinity and specificity for their target protein. As used herein, “unmodified” or “natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). For example, the modified base may be a pyrimidine modified by a hydrophobic group, such as benzyl group, a naphthyl group, or a pyrrolebenzyl group, at its 5-position. Modified nucleoside may be exemplified as 5-(N-benzylcarboxyamide)-2′-deoxyuridine (called BzdU), 5-(N-naphthylcarboxyamide)-2′-deoxyuridine (called NapdU), 5-(N-4-pyrrolebenzyl carboxyamide)-2′-deoxyuridine (called 4-PBdU), 5-(N-benzylcarboxyamide)-2′-deoxycytidine (called BzdC), 5-(N-naphthylcarboxyamide)-2′-deoxycytidine (called NapdC), 5-(N-4-pyrrolebenzylcarboxyamide)-2′-deoxycytidine (called 4-PBdC), 5-(N-benzylcarboxyamide)-2′-uridine (called BzU), 5-(N-naphthylcarboxyamide)-2′-uridine (called NapU), 5-(N-4-pyrrolebenzylcarboxyamide)-2′-uridine (called 4-PBU), 5-(N-benzylcarboxyamide)-2′-cytidine (called BzC), 5-(N-naphthylcarboxyamide)-2′-cytidine (called NapC), 5-(N-4-pyrrolebenzyl carboxyamide)-2′-cytidine (called 4-PBC), and the like, but not be limited thereto. Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P. ed. Wiley-VCH, 2008; those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. L, ed. John Wiley & Sons, 1990, those disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y S., Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B., Ed., CRC Press, 1993.
In accordance with the present invention, a suitable nucleotide length for an aptamer ranges from about 15 to about 100 nucleotides (nt), and in various other preferred embodiments, 15-30 nt, 20-25 nt, 30-100 nt, 30-60 nt, 25-70 nt, 25-60 nt, 40-60 nt, 25-40 nt, 30-40 nt, any of 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 nt or 40-70 nt in length. In some embodiments, an aptamer may be 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70 nt in length. In other embodiments, an aptamer may 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100 nt in length. However, the sequence can be designed with sufficient flexibility such that it can accommodate interactions of aptamers with two targets at the distances described herein.
In some embodiments, the nucleic acid aptamer comprises one or more regions of double-stranded character. Such double stranded regions may arise from internal self-complementarity or complementarity with a second or further aptamers or oligonucleotide molecule. In some embodiments the double stranded region may be from 4-12, 4-10, 4-8 base pairs in length. In some embodiments the double stranded region may be 5, 6, 7, 8, 9, 10, 11 or 12 base pairs. In some embodiments the double stranded region may form a stem region. Such extended stem regions having double stranded character can serve to stabilize the nucleic acid aptamer. As used herein, the term “double stranded character” means that over any length of two nucleic acid molecules, their sequences form base pairings (standard or nonstandard) of more than 50 percent of the length.
Aptamers may be further modified to provide protection from nuclease and other enzymatic activities. The aptamer sequence can be modified by any suitable methods known in the art. For example, phosphorothioate can be incorporated into the backbone, and 5′-modified pyrimidine can be included in 5′ end of ssDNA for DNA aptamers. For RNA aptamers, modified nucleotides such as substitutions of the 2′-OH groups of the ribose backbone, e.g., with 2′-deoxy-NTP or 2′-fluoro-NTP, can be incorporated into the RNA molecule using T7 RNA polymerase mutants. The resistance of these modified aptamers to nuclease can be tested by incubating them with either purified nucleases or nuclease from mouse serum, and the integrity of aptamers can be analyzed by gel electrophoresis.
In some embodiments, such modified nucleic acid aptamers may be synthesized entirely of modified nucleotides, or with a subset of modified nucleotides. The modifications can be the same or different. All nucleotides may be modified, and all may contain the same modification. All nucleotides may be modified, but contain different modifications, e.g., all nucleotides containing the same base may have one type of modification, while nucleotides containing other bases may have different types of modifications. For example, all purine nucleotides may have one type of modification (or are unmodified), while all pyrimidine nucleotides have another, different type of modification (or are unmodified). In this way, oligonucleotides, or libraries of oligonucleotides are generated using any combination of modifications as disclosed herein.
Aptamers may be either monovalent or multivalent. Aptamers may be monomeric, dimeric, trimeric, tetrameric or higher multimeric. Individual aptamer monomers may be linked to form multimeric aptamer fusion molecules. As a non-limiting example, a linking oligonucleotide (i.e., linker) may be designed to contain sequences complementary to both 5′-arm and 3′-arm regions of random aptamers to form dimeric aptamers. For trimeric or tetrameric aptamers, a small trimeric or tetrameric (i.e., a Holiday junction-like) DNA nanostructure will be engineered to include sequences complementary to the 3′-arm region of the random aptamers, therefore creating multimeric aptamer fusion through hybridization. In addition, 3 to 5 or 5 to 10 dT rich nucleotides can be engineered into the linker polynucleotides as a single stranded region between the aptamer-binding motifs, which offers flexibility and freedom of multiple aptamers to coordinate and synergize multivalent interactions with cellular ligands or receptors. Alternatively, multimeric aptamers can also be formed by mixing biotinylated aptamers with streptavidin.
As used herein, the term “multimeric aptamer” or “multivalent aptamer” refers to an aptamer that comprises multiple monomeric units, wherein each of the monomeric units can be an aptamer on its own. Multivalent aptamers have multivalent binding characteristics. A multimeric aptamer can be a homomultimer or a heteromultimer. The term “homomultimer” refers to a multimeric aptamer that comprises multiple binding units of the same kind, i.e., each unit binds to the same binding site of the same target molecule. The term “heteromultimer” refers to a multimeric aptamer that comprises multiple binding units of different kinds, i.e., each binding unit binds to a different binding site of the same target molecule, or each binding unit binds to a binding site on different target molecule. Thus, a heteromultimer can refer to a multimeric aptamer that binds to one target molecule at different binding sties or a multimeric aptamer that binds to different target molecules. A heteromultimer that binds to different target molecules can also be referred to as a multi-specific multimer.
According to certain embodiments of the present invention, variants and derivatives of aptamers are provided. The term “derivative” is used synonymously with the term “variant” and refers to a molecule that has been modified or changed in any way relative to a reference or starting aptamer. The nucleic acid sequence of aptamer variants may possess substitutions, deletions, and/or insertions at certain positions within the nucleotide sequence, as compared to a reference or starting sequence. Ordinarily, variants will possess at least about 50% identity (homology) to a reference sequence, and preferably, they will be at least about 80%, more preferably at least about 90% identical (homologous) to a reference sequence.
In some embodiments, variant mimics of aptamers of the present invention are provided. As used herein, the term “variant mimic” is one which contains one or more nucleic acids which would mimic an activated sequence. The nucleic acid sequences of variant mimics may comprise naturally occurring nucleic acids, or alternatively, non-naturally occurring nucleic acids.
3. Signaling Polynucleotides (SPNs)
Aptamers selected through the process mentioned above herein may be used as signaling polynucleotides (SPNs) for detection of target allergens. Signaling polynucleotides based on aptamer core/binding sequences are advantageous with respect to the objective of development of simple, yet effective detection assays for biomolecule sensors. In accordance with the present invention, a signaling polynucleotide may be developed from the selected aptamers which specifically bind a target allergen molecule. The polynucleotide sequences are detectable when bound at high affinity and specificity to molecular targets.
In some embodiments, signaling polynucleotides (SPNs) of the present invention comprise the core binding sequences which determine the specificity and affinity of SPNs to a target allergen molecule. The full sequence of a selected aptamer can be shortened by deleting the primers used for aptamer selection without impacting the binding sequence to a target allergen. Additional nucleotides may also be added at the 5′terminus and/or the 3′ terminus, without impacting the binding (core) sequence of each aptamer. 3D structures of such SPNs are predicted using standard structure prediction software. The resulting polynucleotide may form a stable 3D structure. In other aspects, nucleotides added at the termini may increase the stability of the polynucleotide and facilitate magnetic particle conjugation, and/or other modifications. For example, a short polyA sequence may be added to the terminus of a SPN to increase the distance between the SPN and magnetic particles (or other solid surface). The length and sequence of additional nucleotides may vary in the context of the core binding sequence of a signaling polynucleotide. SPNs generated from aptamers against common allergens are listed in Tables 1-4.
In some aspects, the SPN comprises a nucleic acid sequence that specifically binds to cashew selected from SEQ ID NOs.: 1-12; the cashew specific SPN has a unique sequence of SEQ ID. NOs.: 1-6. In some aspects, the SPN may comprise a nucleic acid sequence that specifically binds to peanut selected from SEQ ID NOs.: 13-24, and 97-496; the peanut specific SPN has a unique sequence of SEQ ID NOs.: 13-18 and 97-296. In some aspects, the SPN may comprise a nucleic acid sequence that specifically binds to tree nut selected from SEQ ID NOs.: 497-696; the tree nut specific SPN has a unique sequence of SEQ ID NOs.: 497-596. In some aspects, the SPN may comprise a nucleic acid sequence that specifically binds to milk selected from SEQ ID NOs.: 25-34; the milk specific SPN has a unique sequence of SEQ ID NOs.: 25-29. In some aspects, the SPN may comprise a nucleic acid sequence that specifically binds to fish allergen selected from SEQ ID NOs.: 35-46; the fish specific SPN has a unique sequence of SEQ ID NOs.: 35-40. In some aspects, the SPN may comprise a nucleic acid sequence that specifically binds to egg selected from SEQ ID NOs.: 47-58 and 93-94; the egg specific SPN has a unique sequence of SEQ ID NOs.: 47-52. In some aspects, the SPN may comprise a nucleic acid sequence that specifically binds to gluten selected from SEQ ID NOs.: 59-70 and 91-92; the gluten specific SPN has a unique sequence of SEQ ID NOs.: 59-64. In some aspects, the SPN may comprise a nucleic acid sequence that specifically binds to soy allergen selected from SEQ ID NOs.: 71-80; the soy specific SPN has a unique sequence of SEQ ID NOs.: 71-75. In other aspects, the SPN may comprise a nucleic acid sequence that specifically binds to crustacean selected from SEQ ID NOs.: 81-90; the crustacean specific SPN has a unique sequence of SEQ ID NOs.: 81-85.
In some embodiments, signaling polynucleotides of the present invention may be generated by modifying the original allergen binding aptamers disclosed in the literature. The parent sequence of each aptamer against a specific allergen is modified to comprise the shortest sequence without changing the binding specificity and affinity of the aptamer. Some exemplary signaling polynucleotides modified from known parent sequences are listed in Table 2.
In some embodiments, aptamer sequences that specific bind to peanut allergen and tree nut allergen were selected through SELEX and top 100 sequences with high relative abundance were selected from each SELEX selection. Their unique sequences and full sequences including the primer sequences are listed in Table 3 (peanut) and Table 4 (tree nut). Complementary sequences specific to an aptamer may be synthesized and screened for the best binding specificity to its corresponding SPN and to the solid support.
4. SPN-Complement Complexes
In some embodiments, SPN-complement complexes are provided. A nucleic acid sequence that is complementary to a portion of the sequence of a signaling polynucleotide (SPN) (e.g., either the 5′end or 3′end sequence of the SPN) is hybridized with the corresponding SPN sequence, forming a SPN-complement complex. In some aspects, the complementary sequence may contain about 5 to 20 nucleotide residues, or about 5 to 10 nucleotide residues, or about 10 to 15 nucleotide residues, or about 10-20 nucleotide residues. In particular, it may contain 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotide residues. In one embodiment, the complementary sequence contains 5 nucleotide residues. In another embodiment, the complementary sequence contains 10 nucleotide residues. In some aspects, the complementary sequence may be at least 100% complementary to the SPN sequence, or at least 99% complementary to the SPN sequence, or at least 95% complementary to the SPN sequence, or at least 90% complementary to the SPN sequence, or least 80% complementary to the SPN sequence. In another embodiment, the complementary sequence may have additional polyA nucleotides. The short complementary sequence can easily detach from the corresponding SPN, in particular when a target allergen protein competes and binds to the SPN, enabling a highly sensitive detection assay.
In some aspects, the complementary sequence is labeled with a detection signal moiety e.g., a fluorophore, at either the 5′end or 3′end of the sequence. As non-limiting examples, the fluorophore may be selected from Alex Fluor® @ fluorophores (such as Alex 514, Alex 532, Alex 546, Alex 555, Alex 568, Alex 594, Alex 610, Alex 633, Alex 635, Alex 647, Alex 660, Alex 680, Alexa 700, Alex 750, Alex 800, Alex 610-R-phycoerythrin (R-PE), Alex 647-R-phycoerythrin (R-PE), Alex 680-R-phycoerythrin (R-PE), and Alex 680-Allophycocyanin (APC)), Allophycocyanin (APC) and its derivatives, Cy fluorophores (e.g., Cy3.5, Cy3-FITC, CY5, CY 5.5, CY7, CY7-APC, CY5.5-APC), Qdots, TRITC, R-PE, Tamara, Rhodamine Red-X, Rox, TruRed, SYPRO red, BODIPY TR, Propidium iodide and Texas red. In some examples, the fluorophore is Alex 647, Cy5, Cy3-FITC or Texas red.
Detection Agents
Aptamers, signaling polynucleotides (SPNs) and SPN-complement complexes can be used as detection agents in a variety of allergen detection assays, biosensors, detection systems and devices as disclosed in the prior art, either as free agents or conjugates to other support substances. For example, aptamers, SPNs and SPN-complement complexes of the present invention may be used as surface bound affinity molecules that bind the surfaces of solid substrates. The solid surface may be a three-dimensional surface such as micro-spheres (e.g., magnetic beads/particles), or a two-dimensional surface such as the glass or silicon surface.
In one embodiment, aptamers, SPNs and SPN-complement complexes of the present invention are immobilized on the surface of magnetic particles to form functionalized magnetic particles which can capture a target analyte (e.g., allergen) in a fluid sample; the particles will be suitable for magnetic manipulations in a detection assay and method.
In some aspects, aptamers, SPNs and SPN-complement complexes of the present invention may be covalently immobilized on the surface of magnetic particles. In some aspects, SPNs may be covalently immobilized on the surface of magnetic particles through an amine (—NH2) group, or a thiol group (—SH) at one end the SPN sequence. In other aspects, complementary sequence of SPNs may be covalently immobilized on the surface of magnetic particles, from which the SPNs are attached to the particles. Concentrations of SPNs, complementary sequences and magnetic particles are optimized for the most effective binding to each other. In some aspects, SPNs and their complementary sequences may be at a ratio of 1:5, or at a ratio of 1:4, or at a ratio of 1:3, or at a ratio of 1:2.
In another embodiment, aptamers, SPNs and SPN-complement complexes of the present invention may be attached to a two-dimensional solid surface; said two dimensional solid surface may be a glass surface or the surface of a silicon chip. Such surfaces printed/coated with aptamers, SPNs, complements and/or SPN-complement complexes of the present invention may be used as biosensing platforms for a variety of assays and applications.
In some embodiments, detection agents of the present invention may be labeled with a fluorescent marker which generates detectable fluorescent signals. Either the SPN or its complementary sequence may be labeled with a fluorophore. In some aspects, the fluorophore is added to one end of the complement sequence, either the 5′end or 3′end, as illustrated in
1. Magnetic Particles/Beads
Magnetic particles have several advantages that make them attractive substrates for use as signal transducers, including their biological inertness, physical stability, and the absence of competing magnetic signals in biological materials (Gijs et al., Chem Rev. 2010; Vol 110(3), 1518-1563).
Magnetic particles may be any particle materials that can be separated by magnetic forces. Magnetic particles for bioresearch may consist of one or more magnetic cores with a coating matrix of polymers, silica or hydroxylapatite with terminal functionalized groups. The magnetic core generally consists either of magnetite (Fe3O4) or maghemite (γ-Fe2O3) with superparamagnetic or ferromagnetic properties. For example, magnetic cores can be made with magnetic ferrites, such as cobalt ferrite or manganese ferrite. Such magnetic micro- or nanospheres can be separated easily and quickly by magnetic forces and can be used together with bioaffine ligands, e.g. antibodies or aptamers with a high affinity to the target.
In one example, magnetic particles or beads may be synthesized by dispersing ferrite crystals in a suspension of styrene, divinylbenzene monomers and polymerizing the cocktails into microparticles. In another example, magnetic particles/beads may be synthesized by coating one or more layers of magnetite onto a polystyrene core. Multiple layers of magnetite may increase the speed by which the particle/bead responds to a magnetic field. The synthesized microparticles are encapsulated with inert polymers such as polystyrene to prevent the iron from interfering with any subsequent biochemical reactions. Polystyrene magnetic beads can be used for with both carboxylic acid and amine surface chemistries.
Additional polymers that may be used to prepare magnetic particles/beads include, but are not limited to, alginate, dextran, polyacrylamide, polycaprolactone, polyethylenimine (PEI), polyisopropene, poly(2-cinnamoylethyl methacrylate), poly(acetoacetoxyethyl methacrylate), poly(dimethylsiloxane), poly(ethylene glycol)-poly(aspartic acid) (PEG-PAsp), poly(ethylene oxide), poly(glutamic acid), poly(glycidyl methacrylate), poly(lactide), poly(lactide-co-glycolide), poly(1-lactic acid), poly(l-lactide-co-glycolide), poly(methyl methacrylate-divinylbenzene), poly(N-isopropyl acrylamide) (PNIPA), poly(N-vinylcaprolactam), poly(styrene-acetoacetoxyethylmethacrylate) (PSAAEM), poly(styrene-butyl acrylate-methacrylic acid), poly(styrene-co-acrylamide), poly(styrene-co-acrylic acid), poly(styrene-co-butyl acrylate), poly(styrene-divinylbenzene-glycidyl methacrylate), poly(styrene-methacrylic acidacrylamide), poly(tert-butyl acrylate), poly(vinyl alcohol) (PVA), and any combinations thereof.
Magnetic particles may also be coated with streptavidin, maleimide, amino groups or carboxyl groups to optimize aptamer affinity. DNA specificity and non-specific adsorption of DNAs on their surfaces.
In addition to chemical modifications on the surface, magnetic particles may be in a size with maximal efficiency and affinity to the conjugated nucleic acids. As used herein, the particle size (or particle diameter) is given as a hydrodynamic diameter, which includes the core diameter and two times the diameter of the cover matrix. The size of the particle determines the surface area available for the attachment of functional groups. Increasing the size reduces the surface-to-volume ratio therefore resulting in a decrease of the available surface area per volume for modification, but speeding up its response in a magnetic field which makes it easier to be manipulated. In some examples, magnetic particles may be in a wide range of average particle sizes, from 100 nm to 5.0 μm in the diameter, having optimized parameters such as sedimentation rate, available binding sites, and magnetic volume. In some aspects, the average magnetic particle size may be from 100 nm to 800 nm, or from 200 nm to 500 nm, or from 0.1 μm to 3.0 μm, the size may be 100 nm, 125 nm, 150 nm, 200 nm, 250 nm, 300 nm, 350 nm, 400 nm, 450 nm, 500 nm, 550 nm, 600 nm, 700 nm, 800 nm, 900 nm, 0.2 μm, 0.3 μm, 0.4 μm, 0.5 μm, 0.6 μm, 0.7 μm, 0.8 μm, 0.9 μm, 1.0 μm, 1.5 μm, 2.0 μm, 2.5 μm, 3.0 μm, 3.5 μm, 4.0 μm, 4.5 μm, or 5.0 μm.
In some cases, magnetic particles may have an irregular or rough surface. As curvature can introduce additional surface area, irregular or rough surface increases the overall surface area available for the attachment of nucleic acid molecules.
As non-limiting examples, magnetic particles may be fluidMAG particles (which is hydrophilic). SiMAG particles (which are magnetic silica beads with superparamagnetic or ferromagnetic properties and possess either a highly porous or a non-porous silica surface), mHPA-particles (which are non-spherical with hydroxylapatite coated ferromagnetic particles with a diameter of 2 μm, consisting of calcium phosphate), ZeoliteMAG (which are magnetic zeolite particles, which consist of a superparamagnetic iron oxide core and a high-porous aluminosilicate matrix), beadMAG-particles (which are magnetic particles with a diameter of 1 μm, covered with a hydrophilic matrix of crosslinked starch with terminal cation-exchange phosphate groups), and magTosyl-magnetic beads or other appropriately derived magnetic beads for nucleic acid conjugations. In some embodiments, polystyrene magnetic particles may be used. The magnetic particles may also be sepharose magnetic microbeads and agarose magnetic microbeads.
In some embodiments, magnetic particles, in the absence of a magnetic field, may exhibit no net magnetization, but within a magnetic field, the magnetic moments of the bead align with the field, making the beads magnetic.
2. Other Solid Substrates
Aptamers, SPNs and/or SPN-complement complexes may be attached to any two-dimensional solid surfaces such as microtiter plates, silicon chips and printed glass surfaces (e.g. epoxy saline-derived glass surfaces). Other examples of suitable solid substrates (also called solid supports) may include, but are not limited to, those made of silica or silica-based materials, functionalized glass, modified silicon, inorganic glasses, plastics, resins, polysaccharides, carbon, metals, nylon, natural fibers such as silk, wool and cotton, and polymers. Solid substrates may have any useful form including thin films or membranes, beads, microwell plates, dishes, slides, fibers, woven fibers, shaped polymers, particles, chips, wafers and microparticles. Solid substrates may be porous or non-porous.
In some embodiments, the two-dimensional surface may be a surface of a glass or silicon slide. Glass is a readily available and inexpensive support medium that has low intrinsic fluorescence. The surface of the glass or silicon slide may be modified or unmodified, although most attachment protocols involve chemically modifying the glass surface to facilitate attachment of the oligonucleotides. Glass has a relatively homogeneous chemical surface whose properties have been well studied and is amenable to chemical modification using very versatile and well developed silanization chemistry. In certain embodiments, the surface of the glass or silicon slide is unmodified. For example, silianized oligonucleotides can be covalently linked to an unmodified glass surface (Kumar et al., Nucleic Acids Res. 2000 Jul. 15; 28(14): e71).
In certain embodiments, the surface of glass or silicon is chemically modified. Glass surface can be treated with an amino silane to have a uniform layer of primary amines or epoxides. In one example, oligonucleotides modified with an NH2 group can be immobilized onto epoxy silane-derivatized or isothiocyanate coated glass slides. In another example, succinylated oligonucleotides can be coupled to aminophenyl- or aminopropyl-derivitized glass slides by peptide bonds. In yet another example, disulfide-modified oligonucleotides can be immobilized onto a mercaptosilanized glass support by a thiol/disulfide exchange reactions or through chemical cross linkers.
In some embodiments, graphene oxide (GO) may be used as signal transducers in replacement of magnetic particles. Graphene oxide (GO) is a single-atom-thick two-dimensional carbon nanomaterial widely used in biosensor applications due to its unique optical electronic, thermal, and long-lasting biocompatibility properties (also known as graphene oxide nanosheet and graphene nanosheet). Most importantly. GO has superior fluorescence quenching capacity and unique adsorption characteristics for biomolecules. The quenching capacity is due to self-assembly of graphene oxide through specific π-π interactions.
GO is graphite oxidized to intersperse the carbon layers with oxygen molecules, and then reduced to separate the carbon layers completely into individual or few layer graphene. GO can be synthesized by the oxidative treatment of graphite by one of the principle methods developed by Brodie, Hummers or Staudenmeir in the art. A number of modified methods are also available (Paulchamy, et al., J Nanomed Nanotechnol, 2015, 6:1. doi: 10.4172/2157-7439.1000253; Jasim, et al., 2D Mater. 2016, 3, 014006. doi: 10.1088/2053-1583/3/1/014006; and Shahriary and Athawale, Int. J. Renew. Energy Environ. Eng, 2014, Vol. 02 (01): 58-63; the contents of each of which are incorporated herein by reference in their entirety.)
In some embodiments, SPNs and/or SPN-complement complexes may be attached onto the GO surface through physical adsorption. In this aspect, SPNs or SPN-complement complexes may be labeled with Qdots. When Qdots-aptamer conjugates are attached onto GO, the fluorescence signal is quenched due to non-radioactive electronic excitation energy transfer between the fluorophore and GO. In the presence of a target molecule, the interaction between the target molecule and the aptamer is stronger than that between the aptamer and GO, resulting in the release of the aptamer and therefore recovery of the fluorescence signal.
3. Conjugation Chemistry and Methods
Aptamers, SPNs and SPN complementary sequences can be conjugated to magnetic particles by any method known in the art which are used to conjugate nucleic acid molecules to solid surfaces. The methods may be irreversible immobilization methods or reversible immobilization methods. As non-limiting examples, methods may include biotin-streptavidin system, and EDC mediated carboxyl-to-amine crosslinking (e.g., covalent binding of amino-modified aptamer (SPNs) to carboxyl-functionalized magnetic particles), Aldehyde-activated sugars; and cross linkers used to modify nucleic acids and solid surfaces for conjugations (Scientific, Thermo (2012), Crosslinking Technical Handbook, Thermo Fisher Scientific Inc., Waltham, USA).
In some embodiments, the short complementary sequences may be bound to a solid surface. In other embodiments, the SPN may be bound to a solid surface.
In some embodiments, nucleic acid molecules (e.g. aptamers, SPNs, and SPN complement) of the present invention are conjugated to the surface of magnetic particles at one end, e.g. the 5′ end or 3′end (e.g., as shown in
In some aspects, an amino group may be attached to the 5′ or 3′ end of the nucleic acid sequence. Various amino modifiers such as β-cyanoethyl phosphoramidites can be added to the 5′ end of a nucleic acid molecule. 5′ amino modifiers can be simple amino groups with a six or twelve carbon spacers, a Uni-Link amino modifier, or amino modified thymidine or cytosine. Amino modifiers that can be added to the 3′ end of a nucleic acid molecule (e.g., SPN), may be CPGs. In one example, a primary amine (—NH2) modifier may be placed to the 5′end, or 3′end of a nucleic acid molecule (e.g., SPN), or internally using an amino-C or amino-T modified base. The amino-modified nucleic acid molecules may be attached to magnetic particles using an acylating reagent, including but not limiting to Carbodiimide (EDC), Isothiocyanate, Sulfonyl chloride, and Succinimidyl esters (NHS-ester). In one aspect, amine-modified nucleic acids can then be reacted with carboxylate-modified microparticles with carbodiimide mediated acylation in a one-step coupling, which is fast and inexpensive. In addition, Acylation of a carboxyl group generates a stable carbonyl amide. One example of carbodiimide may be water soluble EDAC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride). Alternatively, amine-modified nucleic acids may be attached to magnetic particles with Isothiocyanates, which form thioureas linkage upon reaction with amines. The thioureas linkage is relatively stable.
In other aspects, a thiol group (e.g., thiol meliamide) is attached to the 5′ or 3′ end of a nucleic acid molecule (e.g., a SPN). The thiol (—SH) modifier enables covalent attachment of a nucleic acid molecule to a variety of substrates including magnetic particles, via disulfide bond (—S—S—) or maleimide linkages. The incorporation of a thiol group at the 5′ end of a nucleic acid molecule may be achieved with S-trityl-6-mercaptohexyl derivatives. The 3′-thiol modifier C3 S—S CPG may be used to introduce a thiol group to the 3′-end of a nucleic acid molecule.
In one particular example, a short polyA sequence (e.g., 5 nt) may be added at the end of the SPN or the complementary sequence. The polyA tail then is modified with a thiol group. The reaction between thiol-DNA and carboxylated magnetic beads is mediated by maleimide which is attached to Poly(ethylene glycol) (PEG) polymer coated solid substrates. It is believed that the distance created by the addition of polyA residues and PEG, increases the binding between the bead and DNA and the stability of DNA on the surface of magnetic particles. At the same time, PEG linkers may reduce non-specific binding.
In some examples, cross linkers may be used to attach thiol, or amine-modified nucleic acids to the surface of magnetic beads. As used herein, the term “cross-linker” means a functional chemical group that covalently binds two distinct chemical groups and generates a physical space between the two entities which provides greater accessibility and or freedom to each of the linked biomolecules. A number of cross-linkers may be used to develop covalent attachment of thiol or amine modified nucleic acids to solid surfaces, such as succinimidyl 4-[maleimidophenyl]butyrate (SMPB).
Magnetic particles may also be coated with streptavidin, maleimide, other amino groups or carboxyl groups to optimize aptamer affinity, DNA specificity and non-specific adsorption of DNAs on their surfaces.
In one example, the coupling of nucleic acids and surfaces may be through the binding of biotin-labeled nucleic acids to streptavidin-coated beads. The biotin-streptavidin linkage can tether nucleic acid molecules to the solid surface (e.g. magnetic particles). In another example, nucleic acid molecules and/or solid substrates may be coupled by ethanolamine and heterobifunctional poly (ethylene glycol) (PEG) linkers, which allow the covalent binding of nucleic acid molecules to the solid substrates (e.g. magnetic particles and glass slides). Accordingly, magnetic beads (or other solid substrates such as glass slides) are coated with PEG polymers. PEG polymers coated magnetic particles are further modified to add an active agent such as a carboxyl group, an amine group, a maleimide and a neutravidin. The PEG linkers will change the surface force, nucleic acid stability and non-specific binding. A detailed protocol is described by Janissen et al (Nucleic acid Research, 2014, 42(18): e137; the contents of which are incorporated herein by reference in its entirety).
In addition to chemical modifications on the surface, magnetic beads may be in a size with maximal efficiency and affinity to the linked nucleic acids. Magnetic particles or beads may be synthesized by dispersing ferrite crystals in a suspension of styrene, divinylbenzene monomers and polymerizing the cocktails into microparticles. (Polystyrene magnetic beads can be used for with both carboxylic acid and amine surface chemistries.)
In some aspects, acid treated magnetic particles containing hydroxyl (OH) groups on the surface can be used to conjugate ligands including aptamers as disclosed in U.S. Patent application publication No.: US2014/0206822, the contents of which are incorporated herein by reference in its entirety.
In further another example, molecular spacers may be used to mediate the coupling between aptamers and magnetic particles. The method can avoid interaction between the solid surface and the aptamer conformation.
Aptamers, SPNs and SPN-complement complexes may also be conjugated to a glass surface or other two-dimensional surfaces through chemical modifications known in the art. In some aspects, the solid surface treatment may include, but is not limited to epoxy silane coated glass, isothiocyanate coated glass, aminophenyl or aminopropyl-derivatized glass, mercaptosilanized glass support, aldehyde or epoxide treatment. Amine (NH2), succinylation, disulfide and hydrazide (e.g., I-Linker™) may be used to modify oligonucleotide to facilitate the attachment of DNA to different surfaces.
A two-dimensional surface may be prepared by treating the glass surface with an amino silane which will result in a uniform layer of primary amine or epoxides. Parameters that affect the binding of oligonucleotides to the surface may be optimized to achieve the greatest specificity and DNA density. A high surface coverage of the oligonucleotide may generate a higher signaling due to higher hybridization between SPNs and their complementary sequences. The fluorescence background, chemical stability, complexity, amenability to chemical modification, surface area, loading capacity, and the degree of non-specific binding of detect molecule are adjusted for choosing appropriate surface support and conjugating chemistry.
For example, to increase the loading capacity of oligonucleotides on the planar surface structures of glasses, acrylamide gels can be applied to glasses to construct a—three-dimensional surface which will increase the surface area for DNA attachment.
4. In Situ Oligonucleotide Synthesis on Solid Support
In addition to attaching a synthesized aptamer, SPN or a SPN-complementary sequence to a solid support, nucleic acid molecules (e.g. aptamers, SPNs, and SPN complementary sequence) of the present invention can also be synthesized in situ on a solid support such as on magnetic particles, glass slides, wafers, microwell plates and silicon chips. Compared to the conventional method which requires the oligonucleotides to be pre-synthesized with specific end modifications to fit the surface attachment chemistry, this method can streamline the process of synthetic preparation by eliminating these steps.
The nucleic acid molecule can be synthesized by (a) covalently attaching one or more phosphoramidite linkers to the functional groups on the solid substrate to produce a derivatized support. (b) reacting the derivatized support with a nucleoside phosphoramidite corresponding to the first nucleotide of desired nucleotide sequence, and (c) adding nucleoside phosphoramidite stepwise until the entire oligonucleotide is assembled. Nucleoside phosphoramidites are preferred because naturally occurring nucleotides and their phosphodiester analogs are insufficiently reactive for an expeditious synthesis of oligonucleotides in high yields. Non-nucleoside phosphoramidites may be used to produce modified oligonucleotides.
External forces may be used to immobilize the solid substance during synthesis. For example, nucleic acid molecules may be synthesized on paramagnetic beads using the methods described in U.S. patent application Ser. No. 14/280,609 and Jensen et al., J Biotechnol, 2013, Sep. 20; 167(4); the contents of which are hereby incorporated by reference. The approach uses an external magnet to hold the paramagnetic beads in place during synthesis.
The nucleic acid synthesis can be performed in either the 5′ to 3′ direction or the 3′ to 5′ direction by suitable choice of nucleoside phosphoramidite reagents. For example, 3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidites can be used for synthesis in the 3′ to 5′ direction. Alternatively, 5′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidites (Glen Research) can be used for synthesis in the 5′ to 3′ direction.
In certain embodiments, the substrate functional group used for attachment of the linker is a hydroxyl group or an amino group. For example, a hydroxylated substrate, such as hydroxylated polystyrene may be used for attachment of the linker. Phosphoramidite linkers are used to reduce steric hindrance associated with neighboring base-base interactions. In certain embodiments, the phosphoramidite linker is between 30 and 60 atoms in length. Exemplary phosphoramidite linkers include those disclosed in U.S. patent application Ser. No. 14/280,609; the contents of which are incorporated herein in its entirety.
5. Optimizing Oligonucleotide Synthesis on a Solid Support
In some embodiments, the short oligonucleotide sequences directly synthesized on a solid surface may be further optimized to fit the purpose of detection agents. In accordance with the present invention, a short sequence that is directly synthesized on a solid support (e.g., the surface of magnetic beads) will form a complex with the aptamer. The short oligonucleotide is complementary to one end of the aptamer sequence. The aptamer-complement complexes linked on the solid support are then used as detection agents. To fulfill this purpose, short complement sequences attached on a solid surface may meet the following features: (1) the oligonucleotides cannot be cleaved off from the surface following synthesis; (2) the oligonucleotides are being held away from the surface to allow hybridization with the complementary sequence on the aptamer to form aptamer-complement complexes; and (3) only low to moderate density of the complementary oligonucleotides are synthesized on the solid surface.
As described above, a standard 3′ to 5′ phosphoramidite chemical reaction may be used to for oligonucleotide synthesis. Automated synthesizers may be used to synthesize the short oligonucleotides using magnetic beads as supports. In one example, highly cross-linked polystyrene (PS) beads with proper particle sizes and good moisture exclusion properties may be used for efficient oligonucleotide synthesis. The polystyrene polymer may further bind together with magnetic particles (e.g., Fe3O4 particles) to form the solid support for direct oligonucleotide synthesis.
To stabilize synthesized oligonucleotide sequences on beads, non-cleavable stable linkers may be used for oligonucleotide synthesis. A non-cleavable linker is a chemical moiety immobilized on a solid support and not substantially cleaved under synthesis conditions (e.g., deprotection conditions). The linker may be covalently bound to a solid support (e.g., functionalized glass and magnetic beads).
Exemplary stable linkers may include, but are not limited to, amino linkers such as siloxane linkers, linkers containing alkyl groups such as —(OCH2CH2)n— (n=1-20), —(CH2)m—C(O)NH—(CH2)n— and —(OCH2CH2)m—C(O)NH—(OCH2CH2H2)n— (m and n can be the same or different and m and n are from about 1 to about 20), linkers containing amide groups like —R1—C(O)NH—R2—, monomethoxytrityl (MMT)-protected amino linker, monomethoxytrityl (MMT)-protected amino linker, trifluoroacetyl (TFA)-protected pentyl (C5) amino linker, and trifluoroacetyl (TFA)-protected hexyl (C6) amino linker. In some embodiments, the linker may be a complex linker comprising more than one functional group. Exemplary complex linkers may include those discussed in U.S. Pat. Nos. 7,553,958; 8,053,187; and 9,303,055; the contents of each of which are incorporated herein by reference in their entirety. Other non-cleavable linkers to solid supports may also include these disclosed in U.S. Pat. Nos. 8,569,515; and 8,853,132, the contents of each of which are incorporated herein by reference in their entirety.
In other embodiments, a spacer may be used to hold synthesized oligonucleotides away from the beads, therefore allow the short oligonucleotides to bind effectively to its complement on the end of the aptamer. As used herein, the term “spacer” refers to a chemical group connected to a linker that is used to extend the length of the linker moiety and as a site for initiating synthesis of a polymer chain. Examples of spacer include, but are not limited to, ethyleneglycol polymer, alkyl, oligonucleotides, peptides, and peptditomimetics. In other aspects, the spacer may be an anchor group that is attached to one end of the linker. The anchor group may be served as the site of oligonucleotide synthesis. At the end of oligonucleotide synthesis, a 5′DMT (4.4′-dimethoxytrityl) protecting group may be added to the last nucleoside, the role of which is to prevent interaction with the solid supports and between oligonucleotide polymers.
In some embodiments, low to moderate density of short oligonucleotides may be synthesized on the beads. The controlled loading allows for independent short oligonucleotides that have space to interact with the complementary sequence on the aptamer. The density of aptamer-complement complexes on the solid support may further be optimized to provide adequate signals when the conjugates are used in a detection assay.
In some embodiments, solid supports (e.g., magnetic beads and functionalized glasses) with synthesized oligonucleotides may be separated from the reaction suspension. For example, Magnet force may be used to pull down the beads from the reaction suspension. Separated beads are then read for fluorescent signals.
Target Allergens
Compositions, SPNs, SPN-complement complexes, magnetic particle conjugates and detection agents of the present invention may work as ligands for any target analyte. As stated below, the target analyte may be an allergen protein or variants thereof. In some embodiments, compositions, SPNs. SPN-complement complexes, magnetic particle conjugates and detection agents of the present invention may be designed to bind or associate with allergen proteins or other biomolecules which themselves associated with the allergen.
As used herein, the term “allergen” refers to a substance that can cause allergic reaction. An allergen is then a type of antigen that triggers an abnormally vigorous immune response in body.
Allergens include those from food products, the environment such as pollen, or animals such as a domestic pet dander. Food allergens include, but are not limited to proteins in legumes such as peanuts, peas, lentils and beans, tree nuts, wheat, milk, fish, egg white and sea food. Other allergens may be from the environment such as pollens, other animals (e.g., pet), pathogens and medicines. A comprehensive list of allergenic proteins from various sources is discussed below.
In some embodiments, allergens are food allergens. Examples of allergenic proteins associated with food include, but are not limited to, Brine shrimp (Art fr 5), Crab (Cha f 1), North Sea Shrimp (Cra c 1. Cra c 2, Cra c 4, Cra c 5, Cra c 6, Cra c 8), American lobster (Hom a 1, Hom a 3, Hom a 6), white shrimp (Lit v 1, Lit v 2, Lit v 3, Lit v4), giant freshwater prawn (Mac r 1), shrimp (Met e 1, Pen a 1, Pen i 1), northern shrimp (Pan b 1), spiny lobster (Pan s 1), black tiger shrimp (Pen m 1, Pen m 2, Pen m 3, Pen m 4, Pen m 6), narrow-clawed crayfish (Pon i 4, Pon i 7), blue swimmer crab (Por p 1), domestic cattle (Bos d 4, Bos d 5, Bos d 6, Bos d 7, Bos d 8, Bos d 9, Bos d 10, Bos d 11, Bos d 12), Atlantic herring (Clu h 1), common carp (Cyp c 1), Baltic cod (Gad c 1). Atlantic cod (Gad m 1, Gad m 2, Gad m 3), cod (Gad c 1), chicken (Gal d 1, Gal d 2, Gal d 3. Gal d 4, Gal d 5), Barramunda (Lat c 1), Lepidorhombus whiffiagonis (Lep w 1), chum salmon (One k 5), Atlantic salmon (Sal s 1, Sal s 2, Sal s 3) rainbow trout (One m 1), Mozambique tilapia (Ore m 4), edible frog (Ran e 1, Ran e 2), pacific pilchard (Sar sa 1), ocean perch (Seb m 1), yellowfin tuna (Thu a 1, Thu a 2, Thu a 3), swordfish (Xip g 1), abalone (Hal m 1), brown garden snail (Hel as 1), Squid (Tod p 1), pineapple (Ana c 1, Ana c 2), asparagus (Aspa o 1), barley (Hor v 12, Hor v 15, Hor v 16, Hor v 17, Hor v 20, Hor v 21), banana (Mus a 1, Mus a 2, Mus a 3, Mus a 4, Mus a 5), banana (Musxp1), rice (Ory s 12), rye (Sec c 20), wheat (Tri a 12, Tri a 14, Tri a 18, Tri a 19, Tri a 25, Tri a 26, Tri a 36, Tri a 37), maize (corn) (Zea m 14, Zea m 25), kiwi fruit (Act c1, Act c 2, Act c 5, Act c 8, Act c 10, Act d 1, Act d 2, Act d 3, Act d 4, Act d 5, Act d 6, Act d 7, Act d 8, Act d 9, Act d 10, Act d 1), cashew (Ana o 1, Ana o 2. Ana o 3), celery (Api g 1, Api g 2, Api g 3, Api g 4, Api g 5, Api g 6), peanut (Ara h 1, Ara h 2, Ara h 3, Ara h 4, Ara h 5, Ara h 6, Ara h 7, Ara h 8, Ara h 9, Ara h 10, Ara h 11, Ara h 12, Ara h 13), brazil nut (Ber e 1, Ber e 2), oriental mustard (Bra j 1), rapeseed (Bra n 1), cabbage (Bra o 3), turnip (Bra r 1, Bra r 2), bell pepper (Cap a 1w, Cap a 2), pecan (Car i 1, Car i 4), chestnut (Cas s 1, Cas s 5, Cas s 8, Cas s 9), lemon (Cit I 3), tangerine (Cit r 3), sweet orange (Cit s 1, Cit s 2, Cit s 3), Hazel (Cor a 1, Cor a 2, Cor a 8, Cor a 9, Cor a 11, Cor a 12, Cor a 13, Cor a 14), muskmelon (Cuc m 1, Cuc m 2, Cuc m 3), carrot (Dau c 1, Dau c 4, Dau c 5), common buckwheat (Fag e 2, Fag e 3), tartarian buckwheat (Fag t 2), strawberry (Fra a 1, Fra a 3, Fra a 4), soybean (Gly m 1, Gly m 2, Gly m 3, Gly m 4, Gly m 5, Gly m 6, Gly m 7, Gly m 8), sunflower (Hel a1, Hel a 2, Hel a 3), black walnut (Jug n 1, Jug n 2), English walnut (Jug r 1, Jug r 2, Jug r 3, Jug r 4), Cultivated lettuce (Lac s 1), Lentil (Len c 1, Len c 2, Len c 3), litchi (Lit c 1), narrow-leaved blue lupin (Lup an 1), apple (Mal d 1, Mal d 2, Mal d 3, Mal d 4), Cassava (Man e 5), mulberry (Mor n 3), avocado (Pers a 1), green bean (Pha v 3), pistachio (Pis v 1, Pis v 2, Pis v 3, Pis v 4, Pis v 5), pea (Pis s 1, Pis s 2), apricot (Pru ar 1, Pru ar 3), sweet cherry (Pru av 1, Pru av 2, Pru av 3, Pru av 4), European plum (Pru d 3), almond (Pru du 3, Pru du 4, Pru du 5, Pru du 6), peach (Pru p 1, Pru p 2, Pru p 3, Pru p 4, Pru p 7), pomegranate (Pun g 1), pear (Pyr c 1, Pyr c 3, Pyr c 4, Pyr c 5), castor bean (Ric c 1), red raspberry (Rub i 1, Rub i 3), Sesame (Ses i 1, Ses i 2, Ses i 3, Ses i 4, Ses i 5, Ses i 6, Ses i 7), yellow mustard (Sin a 1, Sin a 2, Sin a 3, Sin a 4), tomato (Sola I 1, Sola I 2, Sola I 3, Sola I 4), potato (Sola t 1, Sola t 2, Sola t 3, Sola t 4), Mung bean (Vig r 1, Vig r 2, Vig r 3, Vig r 4, Vig r 5, Vig r 6), grape (Vit v 1), Chinese date (Ziz m 1), Anacardium occidentale (Ana o 1.0101, Ana o 1.0102), Apium graveolens (Api g 1.0101, Api g 1.0201), Daucus carota (Dau c1.0101, Dau c1.0102, Dau c1.0103, Dau c1.0104, Dau c1.0105, Dau c1.0201), Citrus sinensis (Cit s3.0101, Cit s3.0102), Glycine max (Gly m1.0101, Gly m1.0102, Gly m3.0101, Gly m3.0102), Lens culinaris (Len c1.0101, Len c1.0102, Len c1.0103), Pisum sativum (Pis s1.0101, Pis s1.0102), Lycopersicon sativum (Lye e2.0101, Lye e2.0102), Fragaria ananassa (Fra a3.0101, Fra a3.0102, Fra a3.0201, Fr a3.0202, Fra a3.0203, Fra a3.0204, Fra a3.0301), Malus domestica (Mal d1.0101, Mal d1.0102, Mal d1.0103, Mal d1.0104, Mal d1.0105, Mal d1.0106, Mal d1.0107, Mal d1.0108, Mal d1.0109, Mal d1.0201, Mal d1.0202, Mal d1.0203, Mal d1.0204, Mal d1.0205, Mal d1.0206, Mal d1.0207, Mal d1.0208, Mal d1.0301, Mal d1.0302, Mal d1.0303, Mal d1.0304, Mal d1.0401, Mal d1.0402, Mal d1.0403, Mal d3.0101w, Mal d3.0102w, Mal d3.0201w, Mal d3.0202w, Mal d3.0203w, Mal d4.0101, Mal d4.0102, Mal d4.0201, Mal d4.0202, Mal d4.0301, Mal d4.0302), Prunus avium (Pru av1.0101, Pru av1.0201, Pru av 1.0202, Pru av1.0203), and Prunus persica (Pru p4.0101, Pru p4.0201); and any variants thereof. The names of allergens associated with food are systematically named and listed according to IUIS Allergen Nomenclature Sub-Committee (see, International Union of Immunological Societies Allergen Nomenclature Sub-Committee, List of isoallergens and variants.)
In addition to food allergens, SPNs, magnetic particle conjugates and compositions of the present invention may detect airborne particulates/allergens and other environmental allergens. Samples that contain allergens may be obtained from plants (e.g. weeds, grasses, trees, pollens), animals (e.g., allergens found in the dander, urine, saliva, blood or other bodily fluid of mammals such as cat, dog, cow, pig, sheep, horse, rabbit, rat, guinea pig, mouse and gerbil), fungi/mold, insects (e.g., stinging insects such as bee, wasp, and hornet and chirnomidae (non-biting midges), as well as other insects such as the housefly, fruit fly, sheep blow fly, screw worm fly, grain weevil, silkworm, honeybee, non-biting midge larvae, bee moth larvae, mealworm, cockroach and larvae of Tenibrio molitor beetle; spiders and mites such as the house dust mite), rubbers (e.g. latex), metals, chemicals (e.g. drugs, protein detergent additives) and autoallergens and human autoallergens (e.g. Hom s 1, Hom s 2, Hom s 3, Hom s 4, Hom s 5) (see, Allergen Nomenclature: International Union of Immunological Societies Allergen Nomenclature Sub-Committee, List of allergens and Allergen Nomenclature: International Union of Immunological Societies Allergen Nomenclature Sub-Committee, List of isoallergens and variants).
Examples of allergenic proteins from plants that can be detected using the compositions of the present invention include, but are not limited to, ash (Fra e 1), Japanese cypress (Cha o1, Cha o 2), sugi (Cry j 1, Cry j 2), cypress (Cup a 1), common cypress (Cup s 1, Cup s 3), mountain cedar (Jun a 1, Jun a 2, Jun a 3, Jun s 1), prickly juniper (Jun o 4), eastern red cedar (Jun v 1, Jun v 3), sweet vernal grass (Ant o 1), saffron crocus (Cro s 1, Cro s 2), Bermuda grass (Cyn d 1, Cyn d 7, Cyn d 12, Cyn d 15, Cyn d 22w, Cyn d 23, Cyn d 24), orchard grass (Dac g 1, Dac g 2, Dac g 3, Dac g 4, Dac g 5), meadow fescue (Fes p 4), velvet grass (Hol I 1, Hol I 5), barley (Hor v 1, Hor v 5), rye grass (Lol p 1, Lol p 2, Lol p 3, Lol p 4, Lol p 11), bahia grass (Pas n 1), canary grass (Pha a 1, Pha a 5), timothy (Phl p 1, Phi p 2, Phi p 4, Phi p 5, Phi p 6, Phl p 7, Phl p 11, Phl p 12, Phi p 13), date palm (Pho d 2), Kentucky blue grass (Poa p 1, Poa p 5), rye (Sec c 1, Sec c 5, Sec c 38), Johnson grass (Sor h 1), wheat (Tri a 15, Tri a 21, Tri a 27, Tri a 28, Tri a29, Tri a30, Tri a 31, Tri a32, Tri a 33, Tri a 34, Tri a 35, Tri a 39), maize (Zea m 1, Zea m 12), alder (Aln g 1, Aln g 4), redroot pigweed (Ama r 2), short ragweed (Amb a 1, Amb a 2, Amb a 3, Amb a 4, Amb a 5, Amb a 6, Amb a 7, Amb a 8, Amb a 9, Amb a 10, Amb a 11), western ragweed (Amb p 5), giant ragweed (Amb t 5), mugwort (Art v 1, Art v 2, Art v 3, Art v 4, Art v 5, Art v 6), sugar beet (Beta v 1, beta v 2), European white birch (Bet v 1, Bet v 2, Bet v 3, Bet v 4, Bet v 6, Bet v 7), turnip (Bra r 5), hornbeam (Car b 1), chestnut (Cas s 1), rosy periwinkle (Cat r 1), lamb's-quarters, pigweed (Che a 1, Che a 2, Che a 3), Arabian coffee (Cof a 1, Cof a 2, Cof a 3), Hazel (Cor a 6, Cor a 10), Hazel nut (Cor a1.04, Cor a2, Cor a8), European beech (Fag s 1), ash (Fra e 1), sunflower (Hel a 1, Hel a 2), para rubber tree (Hev b 1, Hev b 2, Hev b 3, Hev b 4, Hev b 5, Hev b 6, Hev b 7, Hev b 8, Hev b 9, Hev b 10, Hev b 11, Hev b 12, Hev b 13, Hev b 14). Japanese hop (Hum j 1), privet (Lig v 1), Mercurialis annua (Mer a 1), olive (Ole e 1, Ole e 2, Ole e 3, Ole e 4, Ole e 5, Ole e 6, Ole e 7, Ole e 8, Ole e 9, Ole e 10, Ole e 11), European hophornbeam (Ost c 1), Parietaria judaica (Par j 1, Par j 2, Par j 3, Par j 4), Parietaria officinalis (Par o 1), Plantago lanceolata (Pal I 1), London plane tree (Pla a 1, Pla a 2, Pla a 3), Platanus orientalis (Pla or 1, Pla or 2, Pla or 3), white oak (Que a 1), Russian thistle (Sal k 1, Sal k 2, Sal k 3, Sal k 4, Sal k 5), tomato (Sola I 5), Lilac (Syr v 1, Syr v 5), Russian-thistle (Sal k 1), English plantain (Pla 11), Ambrosia artemisiifolia (Amb a8.0101, Amb a8.0102, Amb a9.0101, Amb a9.0102), Plantago lanceolata (Pla 11.0101, Pla 11.0102, Pla 11.0103), Parietaria judaica (Par j 3.0102), Cynodon dactylon (Cyn d1.0101, Cyn d1.0102, Cyn d1.0103, Cyn d1.0104, Cyn d1.0105, Cyn d1.0106, Cyn d1.0107, Cyn d1.0201, Cyn d1.0202, Cyn d1.0203, Cyn d1.0204), Holcus lanatus (Hol 11.0101, Hol 11.0102), Lolium perenne (Phl p1.0101, Phl p1.0102, Phl p4.0101, Phl p4.0201, Phl p5.0101, Phl p5.0102, Phl p5.0103, Phi p5.0104, Phl p5.0105, Phl p5.0106, Phl p5.0107, Phl p5.0108, Phl p5.0201, Phl p5.0202), Secale cereale (Sec c20.0101. Sec c20.0201), Betula Verrucosa (Bet v1.0101, Bet v1.0102, Bet v 1.0103, Bet v 1.0201, Bet v 1.0301, Bet v1.0401, Bet v 1.0402, Bet v 1.0501, Bet v 1.0601, Bet v 1.0602, Bet v1.0701, Bet v1.0801, Bet v1.0901, Bet v1.1001, Bet v1.1101, Bet v1.1201, Bet v 1.1301, Bet v1.1401, Bet v1.1402, Bet v1.1501, Bet v1.1502, Bet v1.1601, Bet v1.1701, Bet v 1.1801, Bet v1.1901, Bet v1.2001, Bet v1.2101, Bet v1.2201, Bet v1.2301, Bet v1.2401, Bet v 1.2501, Bet v1.2601, Bet v1.2701, Bet v1.2801, Bet v1.2901, Bet v1.3001, Bet v1.3101, Bet v 6.0101, Bet v6.0102), Carpinus betulus (Car b1.0101, Car b1.0102, Car b1.0103, Car b1.0104, Car b1.0105, Car b1.0106, Car b1.0106, Car b1.0106, Car b1.0106, Car b1.0107, Car b1.0107, Car b1.0108, Car b1.0201, Car b1.0301, Car b1.0302), Corylus avellana (Cor a1.0101, Cor a1.0102, Cor a1.0103, Cor a1.0104, Cor a1.0201, Cor a1.0301. Cor a1.0401, Cor a1.0402, Cor a1.0403, Cor a1.0404), Ligustrum vulgare (Syr v1.0101, Syr v1.0102, Syr v1.0103), Cryptomeria japonica (Cry j2.0101, Cry j2.0102), and Cupressus sempervirens (Cup s1.0101, Cup s1.0102, Cup s1.0103, Cup s1.0104, Cup s1.0105), and any variants thereof.
Lupin is an herbaceous plant of the leguminous family belonging to the genus Lupinus. In Europe, lupin flour and seeds are widely used in bread, cookies, pastry, pasta, sauces, as well as in beverages as a substitute for milk or soy, and in gluten-free foods. The International Union of Immunological Societies (IUIS) allergen nomenclature subcommittee recently designated β-conglutin as the Lup an 1 allergen. (Nadal, et al., (2012) PLoS one, 7(4): e35253), and more recently, a high-affinity 11-mer DNA aptamer against Lup an 1 (β-conglutin) was reported (Nadal, et al., (2013), Anal. Bioanal. Chem. 405:9343-9349).
Examples of allergenic proteins from mites that can be detected using the compositions of the present invention include, but are not limited to, mite (Blo t 1, Blo t 3. Blo t 4, Blo t 5, Blo t 6, Blo t 10, Blo t 11, Blo t 12, Blo t 13, Blo t 19, Blot t 21); American house dust mite (Der f 1, Der f 2, Der f 3, Der f 7, Der f 10, Der f 11, Der f 13, Der f 14, Der f 15, Der f 16, Der f 17, Der f 18, Der f 22, Der f 24); Dermatophagoides microceras (house dust mite) (Der m 1); European house dust mite (Der p 1, Der p 2, Der p 3, Der p 4, Der p 5, Der p 6, Der p 7, Der p 8, Der p 9, Der p 10, Der p 11, Der p 14, Der p 15, Der p 20, Der p 21, Der p 23); Euroglyphus maynei (House dust mite) (Eur m 2, Eur m 2, Eur m 3, Eur m 4, Eur m 14); storage mite (Aca s 13, Gly d 2, Lep d 2, Lep d 5, Lep d 7, Lep d 10, Lep d 13, Tyr p 2, Tyr p 3, Tyr p 10, Tyr p 13. Tyr p 24), Dermatophagoides farinae (Der f1.0101, Der f1.0102, Der f1.0103, Der f1.0104, Der f1.0105, Der f2.0101, Der f2.0102, Der f2.0103, Der f2.0104, Der f2.0105, Der f2.0106, Der f2.0107, Der f2.0108, Der f2.0109, Der f2.0110, Der f2.0111, Der f2.0112, Der f2.0113, Der f2.0114, Der f2.0115, Der f2.0116, Der f2.0117), Dermatophagoides pteronyssinus (Der p 1.0101, Der p 1.0102, Der p 1.0103, Der p 1.0104, Der p1.0105, Der p1.0106, Der p1.0107, Der p1.0108, Der p1.0109, Der p1.0110, Der p1.0111. Der p1.0112, Der p1.0113, Der p1.0114, Der p1.0115, Der p1.0116, Der p1.0117, Der p1.0118, Der p1.0119, Der p1.0120, Der p1.0121, Der p1.0122, Der p1.0123, Der p2.0101, Der p2.0102, Der p2.0103, Der p2.0104, Der p2.0105, Der p2.0106, Der p2.0107, Der p2.0108, Der p2.0109, Der p2.0110, Der p2.0111, Der p2.0112, Der p2.0113), Euroglyphus maynei (Eur m2.0101, Eur m2.0102), Lepidoglyphus destructor (Lep d2.0101, Lep d2.0101, Lep d2.0101, Lep d2.0102, Lep d2.0201, Lep d2.020) and Glycphagus domesticus (Gly d2.0101, Gly d2.0201); and any variants thereof.
Examples of allergenic proteins from animals that can be detected using the compositions of the present invention include, but are not limited to, domestic cattle (Bos d 2, Bos d 3, Bos d 4, Bos d 5, Bos d 6, Bos d 7, Bos d 8), dog (Can f 1, Can f 2, Can f3, Can f 4, Can f 5, Can f 6), domestic horse (Equ c 1, Equ c 2, Equ c 3, Equ c 4, Equ c 5), cat (Fel d 1, Fel d 2, Fel d 3, Fel d 4, Fel d 5w, Fel d 6w, Fel d 7, Fel d 8), mouse (Mus m 1), guinea pig (Cav p 1, Cav p 2, Cav p 3, Cav p 4, Cav p 6), rabbit (Ory c 1, Ory c 3, Ory c 4) rat (Rat n 1), Bos domesticus (Bos d 2.0101, Bos d 2.0102, Bos d 2.0103) and Equus caballus (Equ c2.0101, Equ c 2.0102); and any variants thereof
Examples of allergenic proteins from insects that can be detected using the compositions of the present invention include, but are not limited to, yellow fever mosquito (Aed a 1, Aed a 2, Aed a 3). Eastern hive bee (Api c 1), giant honeybee (Api d 1), honey bee (Api m 1, Api m 2, Api m 3, Api m 4, Api m 5, Api m 6, Api m 7, Api m 8, Api m 9, Api m 10, Api m 11, Api m 12), pigeon tick (Arg r 1), German cockroach (Bla g 1, Bla g 2, Bla g 3, Bla g 4, Bla g 5, Bla g 6, Bla g 7, Bla g 8, Bla g 11), bumble bee (Bom p 1, Bom p 4, Bom t 1, Bom t 4), silk moth (Bomb m 1), midge (Chi k 10, Chi t 1, Chi t 1.01, Chi t 2, Chit 2. 0101, Chi t 2. 0102, Chit 3, Chit 4, Chit 5, Chit 6, Chi t 6.01, Chit 7, Chi t 8, Chit 9), cat flea (Cte f 1, Cte f 2, Cte f 3), yellow hornet (Dol a 5), white face hornet (Dol m 1, Dol m 2, Dol m 5), biting midge (For t 1, For t 2), Savannah Tsetse fly (Glo m 5), Asian ladybeetle (Har a 1, Har a 2), silverfish (Lep s 1), booklouse (Lip b 1), Australian jumper ant (Myr p 1, Myr p 2, Myr p 3), American cockroach (Per a 1, Per a 3, Per a 6, Per a 7, Per a 9, Per a 10), Indian meal moth (Plo i 1, Plo i 2), wasp (Pol a 1, Pol a 2, Pol a 5, Pole 1, Pol e 4, Pol e 5, Pol f 5, Pol g 1, Pol g 5, Pol m 5, Poly p 1, Poly s 5, Ves vi 5), Mediterranean paper wasp (Pol d 1, Pol d 4, Pol d 5), tropical fire ant (Sol g 2, Sol g 3, Sol g 4), Solenopsis invicta (red imported fire ant) (Sol I 1, Sol I 2, Sol I 3, Sol I 4), black fire ant (Sol r 2, Sol r 3), Brazilian fire ant (Sol s 2, Sol s 3), horsefly (Tab y 1, Tab y 2, Tab y 5), pine processionary moth (Tha p 1, Tha p 2), California kissing bug (Tria p 1), European hornet (Vesp c 1, Vesp c 5), Vespa magnifica (hornet) (Vesp ma 2, Vesp ma 5), Vespa mandarinia (Giant asian hornet) (Vesp m1, Vesp m 5), yellow jacket (Ves f 5, Ves g 5, Ves m 1, Ves m 2, Ves m 5), Vespula germanica (yellow jacket) (Ves p 5), Vespula squamosa (Yellow jacket) (Ves s 1, Ve s s5), Vespula vulgaris (Yellow jacket) (Ves v 1, Ves v 2, Ves v 3, Ves v 4, Ves v 5, Ves v 6), Blattella germanica (Bla g 1.0101, Bla g 1.0102, Bla g 1.0103, Bla g 1.02, Bla g 6.0101, Bla g 6.0201, Bla g 6.0301), Periplaneta Americana (Per a1.0101, Per a1.0102, Per a1.0103, Per a1.0104, Per a1.02, Per a3.01, Per a3.0201, Per a3.0202, Per a3.0203, Per a7.0101, Per a7.0102), Vespa crabo (Ves pc 5.0101, Ves pc 5.0101), Vespa mandarina (Vesp m 1.01, Vesp m 1.02); and any variants thereof.
Examples of allergenic proteins from fungi/mold that can be detected using the signaling polynucleotides and assays of the present invention include, but are not limited to, Alternaria alternata (Alternaria rot fungus) (Alt a 1, Alt a 3, Alt a 4, Alt a 5, Alt a 6, Alt a 7, Alt a 8, Alt a 10, Alt a 12, Alt a 13), Aspergillus flavus (fungus) (Asp fl 13), Aspergillus fumigatus (fungus) (Asp f 1, Asp f 2, Asp f 3, Asp f 4, Asp f 5, Asp f 6, Asp f 7, Asp f 8, Asp f9, Asp f 10, Asp f 11, Asp f 12, Asp f 13, Asp f 15, Asp f 16, Asp f 17, Asp f 18, Asp f22, Asp f 23, Asp f 27, Asp f 28, Asp f 29, Asp f 34), Aspergillus niger (Asp n 14, Asp n 18, Asp n 25), Aspergillus oryzae (Asp o 13, Asp o 21), Aspergillus versicolor (Asp v 13), Candida albicans (Yeast) (Cand a 1, Cand a 3), Candida boidinii (Yeast) (Cand b 2), Cladosporium cladosporioides (Cla c 9, Cla c 14), Cladosporium herbarum (Cla h 2, Cla h 5, Cla h 6, Cla h 7, Cla h 8, Cla h 9, Cla h 10, Cla h 12), Curvularia lunata (Synonym: Cochliobolus lunatus) (Cur I 1, Cur I 2, Cur I 3, Cur I 4), Epicoccum purpurascens (Soil fungus) (Epi p 1), Fusarium culmorum (N.A.) (Fus c 1, Fus c 2). Fusarium proliferatum (Fus p 4), Penicillium brevicompactum (Pen b 13, Pen b 26), Penicillium chrysogenum (Pen ch 13, Pen ch 18, Pen ch 20, Pen ch 31, Pen ch 33, Pen ch 35), Penicillium citrinum (Pen c 3. Pen c 13, Pen c 19, Pen c 22, Pen c 24, Pen c 30. Pen c 32), Penicillium crustosum (Pen cr 26), Penicillium oxalicum (Pen o 18), Stachybotrys chartarum (Sta c 3), Trichophyton rubrum (Tri r 2, Tri r 4), Trichophyton tonsurans (Tri t 1, Tri t 4), Psilocybe cubensis (Psi c 1, Psi c 2), Shaggy cap (Cop c 1, Cop c 2, Cop c 3, Cop c 5, Cop c 7), Rhodotorula mucilaginosa (Rho m 1, Rho m 2), Malassezia furfur (Malaf2, Malaf3, Malaf4), Malassezia sympodialis (Malas1, Malas5, Malas6, Malas7, Malas8, Malas9, Malas10, Malas11, Malas12, Malas13) and Alternaria alternate (Alt a1.0101, Alt a1.0102); and any variants thereof.
Examples of additional allergens include, but are not limited to, Nematode (Ani s 1, Ani s 2, Ani s 3, Ani s 4), worm (Asc s 1), soft coral (Den n 1), rubber (Latex) (Hev b 1, Hev b 2, Hev b 3, Hev b 5, Hev b 6, Hev b 7, Hev b 8, Hev b 9, Hev b 10, Hev b 11, Hev b 12, Hev b 13), obeche (Trip s 1) and Heveabrasiliensis (Hev b6.01, Hev b6.0201, Hev b6.0202, Hev b6.03, Hev b8.0101, Hev b8.0102, Hev b8.0201, Hev b8.0202, Hev b8.0203, Hev b8.0204, Hev b10.0101, Hev b10.0102, Hev b10.0103, Hev b11.0101, Hev b11.0102); and any variants thereof.
Table 5 provides a list of non-limiting examples of allergenic proteins from various species. In the table, in addition to the species and allergen name, provided are the biochemical name of the allergenic protein and the unique identification numbers from GenBank or UniProt databases. In some embodiments, SPNs, magnetic particle conjugates and compositions of the present invention may be used to detect any of the allergenic proteins listed in Table 5, or any variant, fragment or antigenic portion thereof.
Acarus siro (Storage
Aca s 13
Aedes aegypti (Yellow
Aed a 1
Aedes aegypti (Yellow
Aed a 2
Aedes aegypti (Yellow
Aed a 3
Aedes aegypti (Yellow
Aed a 4
Aedes aegypti (Yellow
Aed a 5
Aedes aegypti (Yellow
Aed a 6
Aedes aegypti (Yellow
Aed a 7
Aedes aegypti (Yellow
Aed a 8
Aedes aegypti (Yellow
Aed a
Aedes aegypti (Yellow
Aed a
Aedes aegypti (Yellow
Aed a 11
Apis cerana (Asiatic
Api c 1
Apis dorsata (Giant
Api d 1
Apis mellifera (Honey
Api m 1
Apis mellifera (Honey
Api m 2
Apis mellifera (Honey
Api m 3
Apis mellifera (Honey
Api m 4
Apis mellifera (Honey
Api m 5
Apis mellifera (Honey
Api m 6
Apis mellifera (Honey
Api m 7
Apis mellifera (Honey
Api m 8
Apis mellifera (Honey
Api m 9
Apis mellifera (Honey
Api m 10
Apis mellifera (Honey
Api m
Apis mellifera (Honey
Api m
Apis mellifera (Honey
Api m 12
Archaeopotamobius
Arc s 8
sibiriensis (Crustacean
Argas reflexus (Pigeon
Arg r 1
Artemia franciscana
Art fr 5
Blattella germanica
Bla g
Blattella germanica
Bla g
Blattella germanica
Bla g 2
Blattella germanica
Bla g 3
Blattella germanica
Bla g 4
Blattella germanica
Bla g 5
Blattella germanica
Bla g
Blattella germanica
Bla g
Blattella germanica
Bla g
Blattella germanica
Bla g 7
Blattella germanica
Bla g 8
Blattella germanica
Bla g 9
Blattella germanica
Bla g 11
Blomia tropicalis
Blo t
Blomia tropicalis
Blo t
Blomia tropicalis
Blo t
Blomia tropicalis
Blo t
Blomia tropicalis
Blo t
Blomia tropicalis
Blo t 3
Blomia tropicalis
Blo t 4
Blomia tropicalis
Blo t 5
Blomia tropicalis
Blo t 6
Blomia tropicalis
Blo t 7
Blomia tropicalis
Blo t 8
Blomia tropicalis
Blo t 10
Blomia tropicalis
Blo t 11
Blomia tropicalis
Blo t 12
Blomia tropicalis
Blo t 13
Blomia tropicalis
Blo t 19
Blomia tropicalis
Blo t 21
Bombus
Bom p 1
pennsylvanicus
Bombus
Bom p 4
pennsylvanicus
Bombus terrestris
Bom t 1
Bombus terrestris
Bom t 4
Bombyx mori (Silk
Bomb m
Charybdis feriatus
Cha f 1
Chironomus kiiensis
Chi k 10
Chironomus thummi
Chi t
thummi (Midge)
Chironomus thummi
Chi t
thummi (Midge)
Chironomus thummi
Chi t
thummi (Midge)
Chironomus thummi
Chi t
thummi (Midge)
Chironomus thummi
Chi t
thummi (Midge)
Chironomus thummi
Chi t
thummi (Midge)
Chironomus thummi
Chi t
thummi (Midge)
Chironomus thummi
Chi t
thummi (Midge)
Chironomus thummi
Chi t
thummi (Midge)
Chironomus thummi
Chi t
thummi (Midge)
Chironomus thummi
Chi t
thummi (Midge)
Chironomus thummi
Chi t
thummi (Midge)
Chironomus thummi
Chi t
thummi (Midge)
Chironomus thummi
Chi t
thummi (Midge)
Chironomus thummi
Chi t 4
thummi (Midge)
Chironomus thummi
Chi t 9
thummi (Midge)
Chortoglyphus arcuatus
Cho a 10
Crangon crangon
Cra c 1
Crangon crangon
Cra c 2
Crangon crangon
Cra c 4
Crangon crangon
Cra c 5
Crangon crangon
Cra c 6
Crangon crangon
Cra c 8
Ctenocephalides felis
Cte f 1
Ctenocephalides felis
Cte f 2
Ctenocephalides felis
Cte f 3
Dermatophagoides
Der f
farinae (American
Dermatophagoides
Der f
farinae (American
Dermatophagoides
Der f
farinae (American
Dermatophagoides
Der f
farinae (American
Dermatophagoides
Der f
farinae (American
Dermatophagoides
Der f
farinae (American
Dermatophagoides
Der f
farinae (American
Dermatophagoides
Der f
farinae (American
Dermatophagoides
Der f
farinae (American
Dermatophagoides
Der f
farinae (American
Dermatophagoides
Der f
farinae (American
Dermatophagoides
Der f
farinae (American
Dermatophagoides
Der f
farinae (American
Dermatophagoides
Der f
farinae (American
Dermatophagoides
Der f
farinae (American
Dermatophagoides
Der f
farinae (American
Dermatophagoides
Der f 3
farinae (American
Dermatophagoides
Der f 4
farinae (American
Dermatophagoides
Der f 6
farinae (American
Dermatophagoides
Der f 7
farinae (American
Dermatophagoides
Der f 8
farinae (American
Dermatophagoides
Der f 10
farinae (American
Dermatophagoides
Der f 11
farinae (American
Dermatophagoides
Der f 13
farinae (American
Dermatophagoides
Der f 14
farinae (American
Dermatophagoides
Der f 15
farinae (American
Dermatophagoides
Der f 16
farinae (American
Dermatophagoides
Der f 17
farinae (American
Dermatophagoides
Der f 18
farinae (American
Dermatophagoides
Der f
farinae (American
Dermatophagoides
Der f
farinae (American
Dermatophagoides
Der f 21
farinae (American
Dermatophagoides
Der f 22
farinae (American
Dermatophagoides
Der f 24
farinae (American
Dermatophagoides
Der f
farinae (American
Dermatophagoides
Der f
farinae (American
Dermatophagoides
Der f 26
farinae (American
Dermatophagoides
Der f 27
farinae (American
Dermatophagoides
Der f
farinae (American
Dermatophagoides
Der f
farinae (American
Dermatophagoides
Der f 29
farinae (American
Dermatophagoides
Der f 30
farinae (American
Dermatophagoides
Der f 31
farinae (American
Dermatophagoides
Der f 32
farinae (American
Dermatophagoides
Der f 33
farinae (American
Dermatophagoides
Der f 34
farinae (American
Dermatophagoides
Der f 35
farinae (American
Dermatophagoides
Der m 1
microceras (House dust
Dermatophagoides
Der p
pteronyssinus
Dermatophagoides
Der p
pteronyssinus
Dermatophagoides
Der p
pteronyssinus
Dermatophagoides
Der p
pteronyssinus
Dermatophagoides
Der p
pteronyssinus
Dermatophagoides
Der p
pteronyssinus
Dermatophagoides
Der p
pteronyssinus
Dermatophagoides
Der p
pteronyssinus
Dermatophagoides
Der p
pteronyssinus
Dermatophagoides
Der p
pteronyssinus
Dermatophagoides
Der p
pteronyssinus
Dermatophagoides
Der p
pteronyssinus
Dermatophagoides
Der p
pteronyssinus
Dermatophagoides
Der p
pteronyssinus
Dermatophagoides
Der p
pteronyssinus
Dermatophagoides
Der p
pteronyssinus
Dermatophagoides
Der p
pteronyssinus
Dermatophagoides
Der p
pteronyssinus
Dermatophagoides
Der p
pteronyssinus
Dermatophagoides
Der p
pteronyssinus
Dermatophagoides
Der p
pteronyssinus
Dermatophagoides
Der p
pteronyssinus
Dermatophagoides
Der p
pteronyssinus
Dermatophagoides
Der p
pteronyssinus
Dermatophagoides
Der p
pteronyssinus
Dermatophagoides
Der p
pteronyssinus
Dermatophagoides
Der p
pteronyssinus
Dermatophagoides
Der p
pteronyssinus
Dermatophagoides
Der p
pteronyssinus
Dermatophagoides
Der p
pteronyssinus
Dermatophagoides
Der p
pteronyssinus
Dermatophagoides
Der p
pteronyssinus
Dermatophagoides
Der p
pteronyssinus
Dermatophagoides
Der p
pteronyssinus
Dermatophagoides
Der p
pteronyssinus
Dermatophagoides
Der p 3
pteronyssinus
Dermatophagoides
Der p 4
pteronyssinus
Dermatophagoides
Der p
pteronyssinus
Dermatophagoides
Der p
pteronyssinus
Dermatophagoides
Der p 6
pteronyssinus
Dermatophagoides
Der p 7
pteronyssinus
Dermatophagoides
Der p 8
pteronyssinus
Dermatophagoides
Der p
pteronyssinus
Dermatophagoides
Der p
pteronyssinus
Dermatophagoides
Der p 10
pteronyssinus
Dermatophagoides
Der p 11
pteronyssinus
Dermatophagoides
Der p 13
pteronyssinus
Dermatophagoides
Der p 14
pteronyssinus
Dermatophagoides
Der p
pteronyssinus
Dermatophagoides
Der p
pteronyssinus
Dermatophagoides
Der p 18
pteronyssinus
Dermatophagoides
Der p 20
pteronyssinus
Dermatophagoides
Der p 21
pteronyssinus
Dermatophagoides
Der p 23
pteronyssinus
Dermatophagoides
Der p 24
pteronyssinus
Dolichovespula
Dol a 5
arenaria (Yellow
Dolichovespula
Dol m
maculata (White face
Dolichovespula
Dol m
maculata (White face
Dolichovespula
Dol m 2
maculata (White face
Dolichovespula
Dol m
maculata (White face
Dolichovespula
Dol m
maculata (White face
Eriocheir sinensis
Eri s 2
Euroglyphus maynei
Eur m
Euroglyphus maynei
Eur m
Euroglyphus maynei
Eur m
Euroglyphus maynei
Eur m
Euroglyphus maynei
Eur m 3
Euroglyphus maynei
Eur m 4
Euroglyphus maynei
Eur m 14
Forcipomyia taiwana
For t 1
Forcipomyia taiwana
For t 2
Glossina morsitans
Glo m 5
Glycyphagus
Gly d
domesticus (Storage
Glycyphagus
Gly d
domesticus (Storage
Harmonia axyridis
Har a 1
Harmonia axyridis
Har a 2
Homarus americanus
Hom a
Homarus americanus
Hom a
Homarus americanus
Hom a 3
Homarus americanus
Hom a 6
Lepidoglyphus
Lep d
destructor (Storage
Lepidoglyphus
Lep d
destructor (Storage
Lepidoglyphus
Lep d
destructor (Storage
Lepidoglyphus
Lep d
destructor (Storage
Lepidoglyphus
Lep d
destructor (Storage
Lepidoglyphus
Lep d
destructor (Storage
Lepidoglyphus
Lep d
destructor (Storage
Lepidoglyphus
Lep d 7
destructor (Storage
Lepidoglyphus
Lep d 10
destructor (Storage
Lepidoglyphus
Lep d 13
destructor (Storage
Lepisma saccharina
Lep s 1
Liposcelis
Lip b 1
bostrychophila
Litopenaeus vannamei
Lit v 1
Litopenaeus vannamei
Lit v 2
Litopenaeus vannamei
Lit v 3
Litopenaeus vannamei
Lit v 4
Macrobrachium
Mac r 1
rosenbergii (giant
Melicertus latisulcatus
Mel l 1
Metapenaeus ensis
Met e 1
Myrmecia pilosula
Myr p 1
Myrmecia pilosula
Myr p 2
Myrmecia pilosula
Myr p 3
Pachycondyla chinensis
Pac c 3
Pandalus borealis
Pan b 1
Panulirus stimpsoni
Pan s 1
Penaeus aztecus
Pen a 1
Penaeus indicus
Pen i 1
Penaeus monodon
Pen m 1
Penaeus monodon
Pen m 2
Penaeus monodon
Pen m 3
Penaeus monodon
Pen m 4
Penaeus monodon
Pen m 6
Periplaneta americana
Per a
Periplaneta americana
Per a
Periplaneta americana
Per a
Periplaneta americana
Per a
Periplaneta americana
Per a
Periplaneta americana
Per a 2
Periplaneta americana
Per a
Periplaneta americana
Per a
Periplaneta americana
Per a
Periplaneta americana
Per a
Periplaneta americana
Per a 6
Periplaneta americana
Per a 7
Periplaneta americana
Per a 9
Periplaneta americana
Per a 10
Periplaneta americana
Per a 11
Periplaneta americana
Per a 12
Plodia interpunctella
Plo i 1
Plodia interpunctella
Plo i 2
Polistes annularis
Pol a 1
Polistes annularis
Pol a 2
Polistes annularis
Pol a 5
Polistes dominulus
Pol d
Polistes dominulus
Pol d
Polistes dominulus
Pol d
Polistes dominulus
Pol d
Polistes dominulus
Pol d 4
Polistes dominulus
Pol d 5
Polistes exclamans
Pol e 1
Polistes exclamans
Pol e 4
Polistes exclamans
Pol e 5
Polistes fuscatus
Pol f 5
Polistes gallicus (Wasp)
Pol g 1
Polistes gallicus (Wasp)
Pol g 5
Polistes metricus
Pol m 5
Polybia paulista (Wasp)
Poly p 1
Polybia paulista (Wasp)
Poly p 2
Polybia paulista (Wasp)
Poly p
Polybia paulista (Wasp)
Poly p
Polybia scutellaris
Poly s 5
tastacus leptodactylus
Pon l 4
tastacus leptodactylus
Pon l 7
Portunus pelagicus
Por p 1
Solenopsis geminata
Sol g 2
Solenopsis geminata
Sol g 3
Solenopsis geminata
Sol g 4
Solenopsis invicta (Red
Sol i 1
Solenopsis invicta (Red
Sol i 2
Solenopsis invicta (Red
Sol i 3
Solenopsis invicta (Red
Sol i 4
Solenopsis richteri
Sol r 2
Solenopsis richteri
Sol r 3
Solenopsis saevissima
Sol s 2
Solenopsis saevissima
Sol s 3
Tabanus yao (Horsefly)
Tab y 1
Tabanus yao (Horsefly)
Tab y 2
Tabanus yao (Horsefly)
Tab y 5
Thaumetopoea
Tha p 1
pityocampa (Pine
Thaumetopoea
Tha p 2
pityocampa (Pine
Triatoma protracta
Tria p 1
Tyrophagus
Tyr p 2
putrescentiae (Storage
Tyrophagus
Tyr p 3
putrescentiae (Storage
Tyrophagus
Tyr p 10
putrescentiae (Storage
Tyrophagus
Tyr p 13
putrescentiae (Storage
Tyrophagus
Tyr p 28
putrescentiae (Storage
Tyrophagus
Tyr p 34
putrescentiae (Storage
Tyrophagus
Tyr p 35
putrescentiae (Storage
Tyrophagus
Tyr p 36
putrescentiae (Storage
Vespa crabro
Vesp c 1
Vespa crabro
Vesp c
Vespa crabro
Vesp c
Vespa magnifica
Vesp ma
Vespa magnifica
Vesp ma
Vespa mandarinia
Vesp m 1
Vespa mandarinia
Vesp m 5
Vespula flavopilosa
Ves f 5
Vespula germanica
Ves g 5
Vespula maculifrons
Ves m 1
Vespula maculifrons
Ves m 2
Vespula maculifrons
Ves m 5
Vespula pensylvanica
Ves p 5
Vespula squamosa
Ves s 1
Vespula squamosa
Ves s 5
Vespula vidua (Wasp)
Ves vi 5
Vespula vulgaris
Ves v 1
Vespula vulgaris
Ves v
Vespula vulgaris
Ves v
Vespula vulgaris
Ves v 3
Vespula vulgaris
Ves v 5
Vespula vulgaris
Ves v 6
Bos domesticus Bos
Bos d
taurus (domestic cattle)
Bos domesticus Bos
Bos d
taurus (domestic cattle)
Bos domesticus Bos
Bos d 3
taurus (domestic cattle)
Bos domesticus Bos
Bos d 4
taurus (domestic cattle)
Bos domesticus Bos
Bos d 5
taurus (domestic cattle)
Bos domesticus Bos
Bos d 6
taurus (domestic cattle)
Bos domesticus Bos
Bos d 7
taurus (domestic cattle)
Bos domesticus Bos
Bos d 8
taurus (domestic cattle)
Bos domesticus Bos
Bos d 9
taurus (domestic cattle)
Bos domesticus Bos
Bos d 10
taurus (domestic cattle)
Bos domesticus Bos
Bos d 11
taurus (domestic cattle)
Bos domesticus Bos
Bos d 12
taurus (domestic cattle)
Canis familiaris (dog)
Can f 1
Canis familiaris (dog)
Can f 2
Canis familiaris (dog)
Can f 3
Canis familiaris (dog)
Can f 4
Canis familiaris (dog)
Can f 5
Canis familiaris (dog)
Can f 6
Canis familiaris (dog)
Can f 7
Cavia porcellus (guinea
Cav p 1
Cavia porcellus (guinea
Cav p 2
Cavia porcellus (guinea
Cav p 3
Cavia porcellus (guinea
Cav p 4
Cavia porcellus (guinea
Cav p 6
Clupea harengus
Clu h
Clupea harengus
Clu h
Clupea harengus
Clu h
Cyprinus carpio
Cyp c
Cyprinus carpio
Cyp c
Equus caballus
Equ c 1
Equus caballus
Equ c
Equus caballus
Equ c
Equus caballus
Equ c 3
Equus caballus
Equ c 4
Felis domesticus (cat)
Fel d 1
Felis domesticus (cat)
Fel d 1
Felis domesticus (cat)
Fel d 2
Felis domesticus (cat)
Fel d 3
Felis domesticus (cat)
Fel d 4
Felis domesticus (cat)
Fel d 5w
Felis domesticus (cat)
Fel d 6w
Felis domesticus (cat)
Fel d 7
Felis domesticus (cat)
Fel d 8
Gadus callarias (Baltic
Gad c 1
Gadus morhua (Atlantic
Gad m
Gadus morhua (Atlantic
Gad m
Gadus morhua (Atlantic
Gad m
Gadus morhua (Atlantic
Gad m
Gadus morhua (Atlantic
Gad m 2
Gadus morhua (Atlantic
Gad m 3
Gallus domesticus
Gal d 1
Gallus domesticus
Gal d 2
Gallus domesticus
Gal d 3
Gallus domesticus
Gal d 4
Gallus domesticus
Gal d 5
Gallus domesticus
Gal d 6
Gallus domesticus
Gal d 7
Gallus domesticus
Gal d 8
Gallus domesticus
Gal d 9
Gallus domesticus
Gal d 10
Homo sapiens (human
Hom s 1
Homo sapiens (human
Hom s 2
Homo sapiens (human
Hom s 3
Homo sapiens (human
Hom s 4
Homo sapiens (human
Hom s 5
Lates calcarifer
Lat c
Lates calcarifer
Lat c
Lepidorhombus
Lep w 1
whiffiagonis (Megrim,
Mesocricetus auratus
Mes a 1
Mus musculus (mouse)
Mus m
Mus musculus (mouse)
Mus m
Oncorhynchus keta
Onc k 5
Oncorhynchus mykiss
Onc m
Oncorhynchus mykiss
Onc m
Oreochromis
Ore m 4
mossambicus
Oryctolagus cuniculus
Ory c 1
Oryctolagus cuniculus
Ory c
Oryctolagus cuniculus
Ory c
Oryctolagus cuniculus
Ory c 4
Phodopus sungorus
Phod s 1
Rana esculenta (edible
Ran e 1
Rana esculenta (edible
Ran e 2
Rastrelliger kanagurta
Ras k 1
Rattus norvegicus (Rat)
Rat n 1
Salmo salar (Atlantic
Sal s 1
Salmo salar (Atlantic
Sal s 2
Salmo salar (Atlantic
Sal s 3
Sardinops sagax
Sar sa 1
Sebastes marinus
Seb m
Sebastes marinus
Seb m
Sus scrofa (Domestic
Sus s 1
Thunnus albacares
Thu a 1
Thunnus albacares
Thu a 2
Thunnus albacares
Thu a 3
Xiphias gladius
Xip g 1
Dendronephthya sp.
Den n 1
Crassostrea
Cra g
gigas (Pacific Ovster)
Crassostrea
Cra g
gigas (Pacific Oyster)
Haliotis laevigata ×
Hal l 1
Haliotis rubra (Jade
Haliotis midae
Hal m 1
Helix aspersa (Brown
Hel as 1
odes pacificus
Tod p 1
Anisakis simplex
Ani s 1
Anisakis simplex
Ani s 2
Anisakis simplex
Ani s 3
Anisakis simplex
Ani s 4
Anisakis simplex
Ani s 5
Anisakis simplex
Ani s 6
Anisakis simplex
Ani s 7
Anisakis simplex
Ani s 8
Anisakis simplex
Ani s 9
Anisakis simplex
Ani s 10
Anisakis simplex
Ani s 11
Anisakis simplex
Ani s 12
Anisakis simplex
Ani s 13
Anisakis simplex
Ani s 14
Ascaris lumbricoides
Asc l 3
Ascaris lumbricoides
Asc l 13
Ascaris suum (Pig
Asc s 1
Ascaris suum (Pig
Asc s 13
Alternaria alternata
Alt a
Alternaria alternata
Alt a
Alternaria alternata
Alt a 3
Alternaria alternata
Alt a 4
Alternaria alternata
Alt a 5
Alternaria alternata
Alt a 6
Alternaria alternata
Alt a 7
Alternaria alternata
Alt a 8
Alternaria alternata
Alt a 10
Alternaria alternata
Alt a 12
Alternaria alternata
Alt a 13
Alternaria alternata
Alt a 14
Alternaria alternata
Alt a 15
Aspergillus flavus
Asp fl 13
Aspergillus fumigatus
Asp f 1
Aspergillus fumigatus
Asp f 2
Aspergillus fumigatus
Asp f 3
Aspergillus fumigatus
Asp f 4
Aspergillus fumigatus
Asp f 5
Aspergillus fumigatus
Asp f 6
Aspergillus fumigatus
Asp f 7
Aspergillus fumigatus
Asp f 8
Aspergillus fumigatus
Asp f 9
Aspergillus fumigatus
Asp f 10
Aspergillus fumigatus
Asp f 11
Aspergillus fumigatus
Asp f 12
Aspergillus fumigatus
Asp f 13
Aspergillus fumigatus
Asp f 15
Aspergillus fumigatus
Asp f 16
Aspergillus fumigatus
Asp f 17
Aspergillus fumigatus
Asp f 18
Aspergillus fumigatus
Asp f 22
Aspergillus fumigatus
Asp f 23
Aspergillus fumigatus
Asp f 27
Aspergillus fumigatus
Asp f 28
Aspergillus fumigatus
Asp f 29
Aspergillus fumigatus
Asp f 34
Aspergillus niger
Asp n 14
Aspergillus niger
Asp n 18
Aspergillus niger
Asp n 25
Aspergillus oryzae
Asp o 13
Aspergillus oryzae
Asp o 21
Aspergillus versicolor
Asp v 13
Candida albicans
Cand a 1
Candida albicans
Cand a 3
Candida boidinii
Cand b 2
Cladosporium
Cla c 9
cladosporioides
Cladosporium
Cla c 14
cladosporioides
Cladosporium
Cla h 2
herbarum (Fungus of
Cladosporium
Cla h 5
herbarum (Fungus of
Cladosporium
Cla h 6
herbarum (Fungus of
Cladosporium
Cla h 7
herbarum (Fungus of
Cladosporium
Cla h 8
herbarum (Fungus of
Cladosporium
Cla h 9
herbarum (Fungus of
Cladosporium
Cla h 10
herbarum (Fungus of
Cladosporium
Cla h 12
herbarum (Fungus of
Curvularia lunata
Cur l 1
Cochliobolus lunatus)
Curvularia lunata
Cur l 2
Cochliobolus lunatus)
Curvularia lunata
Cur l 3
Cochliobolus lunatus)
Curvularia lunata
Cur l 4
Cochliobolus lunatus)
Epicoccum
Epi p 1
purpurascens (Soil
Fusarium culmorum
Fus c 1
Fusarium culmorum
Fus c 2
Fusarium proliferatum
Fus p 4
Fusarium proliferatum
Fus p 9
Penicillium
Pen b 13
brevicompactum
Penicillium
Pen b 26
brevicompactum
Penicillium
Pen ch
chrysogenum
Penicillium
Pen ch
chrysogenum
Penicillium
Pen ch
chrysogenum
Penicillium
Pen ch
chrysogenum
Penicillium
Pen ch
chrysogenum
Penicillium
Pen ch
chrysogenum
lium citrinum
Pen c 3
lium citrinum
Pen c 13
lium citrinum
Pen c 19
lium citrinum
Pen c 22
lium citrinum
Pen c 24
lium citrinum
Pen c 30
lium citrinum
Pen c 32
Penicillium crustosum
Pen cr 26
Penicillium oxalicum
Pen o 18
Stachybotrys chartarum
Sta c 3
Trichophyton rubrum
Tri r 2
Trichophyton rubrum
Tri r 4
Trichophyton tonsurans
Tri t 1
Trichophyton tonsurans
Tri t 4
Coprinus comatus
Cop c 1
Coprinus comatus
Cop c 2
Coprinus comatus
Cop c 3
Coprinus comatus
Cop c 5
Coprinus comatus
Cop c 7
Malassezia furfur
Mala f 2
Malassezia furfur
Mala f 3
Malassezia furfur
Mala f 4
Malassezia sympodialis
Mala s 1
Malassezia sympodialis
Mala s 5
Malassezia sympodialis
Mala s 6
Malassezia sympodialis
Mala s 7
Malassezia sympodialis
Mala s 8
Malassezia sympodialis
Mala s 9
Malassezia sympodialis
Mala s
Malassezia sympodialis
Mala s
Malassezia sympodialis
Mala s
Malassezia sympodialis
Mala s
Psilocybe cubensis
Psi c 1
Psilocybe cubensis
Psi c 2
Rhodotorula
Rho m 1
mucilaginosa (Yeast)
Rhodotorula
Rho m 2
mucilaginosa (Yeast)
Schizophyllum
Sch c 1
commune
Rhizopus oryzae (Bread
Rhi o 1
Rhizopus oryzae (Bread
Rhi o 2
Ananas comosus
Ana c 1
Ananas comosus
Ana c 2
Anthoxanthum
Ant o 1
odoratum (Sweet vernal
Asparagus officinalis
Aspa o 1
Cocos nucifera
Coc n 1
Crocus sativus (Saffron
Cro s 1
Crocus sativus (Saffron
Cro s 2
Cynodon dactylon
Cyn d
Cynodon dactylon
Cyn d
Cynodon dactylon
Cyn d
Cynodon dactylon
Cyn d
Cynodon dactylon
Cyn d
Cynodon dactylon
Cyn d 7
Cynodon dactylon
Cyn d 12
Cynodon dactylon
Cyn d 15
Cynodon dactylon
Cyn d
Cynodon dactylon
Cyn d 23
Cynodon dactylon
Cyn d 24
Dactylis glomerata
Dac g 1
Dactylis glomerata
Dac g 2
Dactylis glomerata
Dac g 3
Dactylis glomerata
Dac g 4
Dactylis glomerata
Dac g 5
Festuca pratensis
Fes p 4
Holcus lanatus (Velvet
Hol l 1
Holcus lanatus (Velvet
Hol l
Holcus lanatus (Velvet
Hol l
Hordeum vulgare
Hor v 5
Hordeum vulgare
Hor v 12
Hordeum vulgare
Hor v 15
Hordeum vulgare
Hor v 16
Hordeum vulgare
Hor v 17
Hordeum vulgare
Hor v 20
Lolium perenne (Rye
Lol p
Lolium perenne (Rye
Lol p
Lolium perenne (Rye
Lol p 2
Lolium perenne (Rye
Lol p 3
Lolium perenne (Rye
Lol p 4
Lolium perenne (Rye
Lol p 5
Lolium perenne (Rye
Lol p 11
Musa acuminata
Mus a 1
Musa acuminata
Mus a 2
Musa acuminata
Mus a 3
Musa acuminata
Mus a 4
Musa acuminata
Mus a 5
Musa acuminata
Mus a 6
Oryza sativa (Rice)
Ory s 1
Oryza sativa (Rice)
Ory s 12
Paspalum notatum
Pas n 1
Phalaris aquatica
Pha a 1
Phalaris aquatica
Pha a 5
Phleum pratense
Phl p
Phleum pratense
Phl p
Phleum pratense
Phl p 2
Phleum pratense
Phl p
Phleum pratense
Phl p
Phleum pratense
Phl p
Phleum pratense
Phl p
Phleum pratense
Phl p
Phleum pratense
Phl p
Phleum pratense
Phl p
Phleum pratense
Phl p
Phleum pratense
Phl p
Phleum pratense
Phl p
Phleum pratense
Phl p
Phleum pratense
Phl p
Phleum pratense
Phl p
Phleum pratense
Phl p
Phleum pratense
Phl p
Phleum pratense
Phl p
Phleum pratense
Phl p
Phleum pratense
Phl p 7
Phleum pratense
Phl p 11
Phleum pratense
Phl p
Phleum pratense
Phl p
Phleum pratense
Phl p
Phleum pratense
Phl p 13
Phoenix dactylifera
Pho d 2
Poa pratensis
Poa p 1
Poa pratensis
Poa p 5
Secale cereale (Rye)
Sec c 5
Secale cereale (Rye)
Sec c
Secale cereale (Rye)
Sec c
Secale cereale (Rye)
Sec c 38
Sorghum halepense
Sor h
Sorghum halepense
Sor h
Sorghum halepense
Sor h
Sorghum halepense
Sor h
Sorghum halepense
Sor h
Sorghum halepense
Sor h
Triticum aestivum
Tri a
Triticum aestivum
Tri a
Triticum aestivum
Tri a
Triticum aestivum
Tri a
Triticum aestivum
Tri a 14
Triticum aestivum
Tri a 15
Triticum aestivum
Tri a 18
Triticum aestivum
Tri a 19
Triticum aestivum
Tri a 20
Triticum aestivum
Tri a 21
Triticum aestivum
Tri a 25
Triticum aestivum
Tri a
Triticum aestivum
Tri a
Triticum aestivum
Tri a 27
Triticum aestivum
Tri a 28
Triticum aestivum
Tri a
Triticum aestivum
Tri a
Triticum aestivum
Tri a 30
Triticum aestivum
Tri a 31
Triticum aestivum
Tri a 32
Triticum aestivum
Tri a 33
Triticum aestivum
Tri a 34
Triticum aestivum
Tri a 35
Triticum aestivum
Tri a 36
Triticum aestivum
Tri a 37
Triticum aestivum
Tri a 39
Triticum aestivum
Tri a 40
Triticum aestivum
Tri a 41
Triticum aestivum
Tri a 42
Triticum aestivum
Tri a 43
Triticum aestivum
Tri a 44
Triticum aestivum
Tri a 45
Zea mays (Maize)
Zea m 1
Zea mays (Maize)
Zea m 8
Zea mays (Maize)
Zea m
Zea mays (Maize)
Zea m
Zea mays (Maize)
Zea m
Zea mays (Maize)
Zea m
Zea mays (Maize)
Zea m
Zea mays (Maize)
Zea m
Zea mays (Maize)
Zea m
Zea mays (Maize)
Zea m 25
Acacia farnesiana
Aca f 1
Acacia farnesiana
Aca f 2
Actinidia chinensis
Act c
Actinidia chinensis
Act c
Actinidia chinensis
Act c 8
Actinidia chinensis
Act c 10
Actinidia deliciosa
Act d 1
Actinidia deliciosa
Act d 2
Actinidia deliciosa
Act d 3
Actinidia deliciosa
Act d 4
Actinidia deliciosa
Act d 5
Actinidia deliciosa
Act d 6
Actinidia deliciosa
Act d 7
Actinidia deliciosa
Act d 8
Actinidia deliciosa
Act d 9
Actinidia deliciosa
Act d
Actinidia deliciosa
Act d
Actinidia deliciosa
Act d 11
Actinidia deliciosa
Act d
Actinidia deliciosa
Act d
Actinidia deliciosa
Act d 13
Alnus glutinosa (Alder)
Aln g 1
Alnus glutinosa (Alder)
Aln g 4
Amaranthus retroflexus
Ama r 1
Amaranthus retroflexus
Ama r 2
Ambrosia artemisiifolia
Amb a
Ambrosia artemisiifolia
Amb a
Ambrosia artemisiifolia
Amb a
Ambrosia artemisiifolia
Amb a
Ambrosia artemisiifolia
Amb a
Ambrosia artemisiifolia
Amb a
Ambrosia artemisiifolia
Amb a
Ambrosia artemisiifolia
Amb a
Ambrosia artemisiifolia
Amb a
Ambrosia artemisiifolia
Amb a
Ambrosia artemisiifolia
Amb a
Ambrosia artemisiifolia
Amb a
Ambrosia artemisiifolia
Amb a 3
Ambrosia artemisiifolia
Amb a 4
Ambrosia artemisiifolia
Amb a 5
Ambrosia artemisiifolia
Amb a 6
Ambrosia artemisiifolia
Amb a 7
Ambrosia artemisiifolia
Amb a
Ambrosia artemisiifolia
Amb a
Ambrosia artemisiifolia
Amb a
Ambrosia artemisiifolia
Amb a
Ambrosia artemisiifolia
Amb a
Ambrosia artemisiifolia
Amb a
Ambrosia artemisiifolia
Amb a
Ambrosia artemisiifolia
Amb a
Ambrosia psilostachya
Amb p
Ambrosia psilostachya
Amb p
Ambrosia trifida (Giant
Amb t 5
Anacardium
Ana 0
occidentale (Cashew)
Anacardium
Ana o
occidentale (Cashew)
Anacardium
Ana o 2
occidentale (Cashew)
Anacardium
Ana o 3
occidentale (Cashew)
Apium graveolens
Api g
Apium graveolens
Api g
Apium graveolens
Api g 2
Apium graveolens
Api g 3
Apium graveolens
Api g 4
Apium graveolens
Api g 5
Apium graveolens
Api g 6
Arachis hypogaea
Ara h 1
Arachis hypogaea
Ara h 2
Arachis hypogaea
Ara h
Arachis hypogaea
Ara h
Arachis hypogaea
Ara h 5
Arachis hypogaea
Ara h 6
Arachis hypogaea
Ara h
Arachis hypogaea
Ara h
Arachis hypogaea
Ara h
Arachis hypogaea
Ara h
Arachis hypogaea
Ara h
Arachis hypogaea
Ara h
Arachis hypogaea
Ara h
Arachis hypogaea
Ara h
Arachis hypogaea
Ara h
Arachis hypogaea
Ara h
Arachis hypogaea
Ara h 12
Arachis hypogaea
Ara h 13
Arachis hypogaea
Ara h
Arachis hypogaea
Ara h
Arachis hypogaea
Ara h
Arachis hypogaea
Ara h 15
Arachis hypogaea
Ara h 16
Arachis hypogaea
Ara h 17
Artemisia annua (Sweet
Art an 7
Artemisia vulgaris
Art v 1
Artemisia vulgaris
Art v 2
Artemisia vulgaris
Art v
Artemisia vulgaris
Art v
Artemisia vulgaris
Art v
Artemisia vulgaris
Art v
Artemisia vulgaris
Art v
Artemisia vulgaris
Art v
Artemisia vulgaris
Art v 5
Artemisia vulgaris
Art v 6
Bertholletia excelsa
Ber e 1
Bertholletia excelsa
Ber e 2
Beta vulgaris (Sugar
Beta v 1
Beta vulgaris (Sugar
Beta v 2
Betula verrucosa
Bet v
Betula verrucosa
Bet v
Betula verrucosa
Bet v
Betula verrucosa
Bet v
Betula verrucosa
Bet v
Betula verrucosa
Bet v
Betula verrucosa
Bet v
Betula verrucosa
Bet v
Betula verrucosa
Bet v
Betula verrucosa
Bet v
Betula verrucosa
Bet v
Betula verrucosa
Bet v
Betula verrucosa
Bet v
Betula verrucosa
Bet v
Betula verrucosa
Bet v
Betula verrucosa
Bet v
Betula verrucosa
Bet v
Betula verrucosa
Bet v
Betula verrucosa
Bet v
Betula verrucosa
Bet v
Betula verrucosa
Bet v
Betula verrucosa
Bet v
Betula verrucosa
Bet v
Betula verrucosa
Bet v
Betula verrucosa
Bet v
Betula verrucosa
Bet v
Betula verrucosa
Bet v
Betula verrucosa
Bet v 2
Betula verrucosa
Bet v 3
Betula verrucosa
Bet v 4
Betula verrucosa
Bet v
Betula verrucosa
Bet v
Betula verrucosa
Bet v 7
Brassica juncea (Indian
Bra j 1
Brassica napus
Bra n 1
Brassica oleracea
Bra o 3
Brassica rapa (Field
Bra r 1
Brassica rapa (Field
Bra r 2
Brassica rapa (Field
Bra r 5
Cannabis sativa (Indian
Can s 3
Capsicum annuum
Cap a 1
Capsicum annuum
Cap a 2
Carpinus betulus
Car b
Carpinus betulus
Car b
Carya illinoinensis
Car i 1
Carya illinoinensis
Car i 2
Carya illinoinensis
Car i 4
Castanea sativa
Cas s 1
Castanea sativa
Cas s 5
Castanea sativa
Cas s 8
Castanea sativa
Cas s 9
Catharanthus roseus
Cat r 1
Chenopodium album
Che a 1
Chenopodium album
Che a 2
Chenopodium album
Che a 3
Citrullus lanatus
Citr l 2
Citrus limon (Lemon)
Cit l 3
Citrus reticulata
Cit r 3
Citrus sinensis (Sweet
Cit s 1
Citrus sinensis (Sweet
Cit s 2
Citrus sinensis (Sweet
Cit s 3
Citrus sinensis (Sweet
Cit s 7
Coffea arabica (Arabian
Cof a 1
Coffea arabica (Arabian
Cof a 2
Coffea arabica (Arabian
Cof a 3
Corylus avellana
Cor a
Corylus avellana
Cor a
Corylus avellana
Cor a
Corylus avellana
Cor a
Corylus avellana
Cor a
Corylus avellana
Cor a
Corylus avellana
Cor a
Corylus avellana
Cor a
Corylus avellana
Cor a
Corylus avellana
Cor a 6
Corylus avellana
Cor a 8
Corylus avellana
Cor a 9
Corylus avellana
Cor a 10
Corylus avellana
Cor a 11
Corylus avellana
Cor a 12
Corylus avellana
Cor a 13
Corylus avellana
Cor a 14
Cucumis melo
Cuc m 1
Cucumis melo
Cuc m 2
Cucumis melo
Cuc m 3
Daucus carota (Carrot)
Dau c
Daucus carota (Carrot)
Dau c
Daucus carota (Carrot)
Dau c
Daucus carota (Carrot)
Dau c 4
Daucus carota (Carrot)
Dau c 5
Fagopyrum esculentum
Fag e 2
Fagopyrum esculentum
Fag e 3
Fagopyrum esculentum
Fag e
Fagopyrum esculentum
Fag e
Fagopyrum esculentum
Fag e 5
Fagopyrum tataricum
Fag t 2
Fagus sylvatica
Fag s 1
Fragaria ananassa
Fra a 1
Fragaria ananassa
Fra a
Fragaria ananassa
Fra a
Fragaria ananassa
Fra a
Fragaria ananassa
Fra a
Fragaria ananassa
Fra a 4
Fraxinus excelsior
Fra e
Fraxinus excelsior
Fra e
Fraxinus excelsior
Fra e
Glycine max (Soybean)
Gly m 1
Glycine max (Soybean)
Gly m 2
Glycine max (Soybean)
Gly m
Glycine max (Soybean)
Gly m
Glycine max (Soybean)
Gly m 4
Glycine max (Soybean)
Gly m
Glycine max (Soybean)
Gly m
Glycine max (Soybean)
Gly m
Glycine max (Soybean)
Gly m
Glycine max (Soybean)
Gly m
Glycine max (Soybean)
Gly m
Glycine max (Soybean)
Gly m
Glycine max (Soybean)
Gly m
Glycine max (Soybean)
Gly m 7
Glycine max (Soybean)
Gly m 8
Helianthus annuus
Hel a 1
Helianthus annuus
Hel a 2
Helianthus annuus
Hel a 3
Hevea brasiliensis (Para
Hev b 1
Hevea brasiliensis (Para
Hev b 2
Hevea brasiliensis (Para
Hev b 3
Hevea brasiliensis (Para
Hev b 4
Hevea brasiliensis (Para
Hev b 5
Hevea brasiliensis (Para
Hev b 6
Hevea brasiliensis (Para
Hev b 7
Hevea brasiliensis (Para
Hev b
Hevea brasiliensis (Para
Hev b
Hevea brasiliensis (Para
Hev b
Hevea brasiliensis (Para
Hev b
Hevea brasiliensis (Para
Hev b
Hevea brasiliensis (Para
Hev b
Hevea brasiliensis (Para
Hev b 9
Hevea brasiliensis (Para
Hev b
Hevea brasiliensis (Para
Hev b
Hevea brasiliensis (Para
Hev b
Hevea brasiliensis (Para
Hev b
Hevea brasiliensis (Para
Hev b
Hevea brasiliensis (Para
Hev b 12
Hevea brasiliensis (Para
Hev b 13
Hevea brasiliensis (Para
Hev b 14
Hevea brasiliensis (Para
Hev b 15
Humulus japonicus
Hum j 1
Juglans nigra (Black
Jug n 1
Juglans nigra (Black
Jug n 2
Juglans nigra (Black
Jug n 4
Juglans regia (English
Jug r 1
Juglans regia (English
Jug r 2
Juglans regia (English
Jug r 3
Juglans regia (English
Jug r 4
Juglans regia (English
Jug r 5
Juglans regia (English
Jug r 6
Kochia scoparia
Koc s 1
Kochia scoparia
Koc s 2
Lactuca sativa
Lac s 1
Lens culinaris (Lentil)
Len c
Lens culinaris (Lentil)
Len c
Lens culinaris (Lentil)
Len c 2
Lens culinaris (Lentil)
Len c 3
Ligustrum vulgare
Lig v 1
Litchi chinensis
Lit c 1
Lupinus (albus )
Lup a 5
Lupinus angustifolius
Lup an 1
Malus domestica
Mal d
Malus domestica
Mal d
Malus domestica
Mal d
Malus domestica
Mal d
Malus domestica
Mal d
Malus domestica
Mal d
Malus domestica
Mal d
Malus domestica
Mal d
Malus domestica
Mal d
Malus domestica
Mal d
Malus domestica
Mal d
Malus domestica
Mal d
Malus domestica
Mal d
Malus domestica
Mal d
Malus domestica
Mal d
Malus domestica
Mal d
Malus domestica
Mal d
Malus domestica
Mal d
Malus domestica
Mal d
Malus domestica
Mal d
Malus domestica
Mal d
Malus domestica
Mal d
Malus domestica
Mal d
Malus domestica
Mal d 2
Malus domestica
Mal d
Malus domestica
Mal d
Malus domestica
Mal d
Malus domestica
Mal d
Malus domestica
Mal d
Malus domestica
Mal d
Malus domestica
Mal d
Malus domestica
Mal d
Malus domestica
Mal d
Malus domestica
Mal d
Malus domestica
Mal d
Manihot esculenta
Man e 5
Mercurialis annua
Mer a 1
Morus nigra (Mulberry)
Mor n 3
Olea europaea (Olive)
Ole e 1
Olea europaea (Olive)
Ole e 2
Olea europaea (Olive)
Ole e 3
Olea europaea (Olive)
Ole e 4
Olea europaea (Olive)
Ole e 5
Olea europaea (Olive)
Ole e 6
Olea europaea (Olive)
Ole e 7
Olea europaea (Olive)
Ole e 8
Olea europaea (Olive)
Ole e 9
Olea europaea (Olive)
Ole e 10
Olea europaea (Olive)
Ole e 11
Ostrya carpinifolia
Ost c 1
Parietaria judaica
Par j
Parietaria judaica
Par j
Parietaria judaica
Par j
Parietaria judaica
Par j
Parietaria judaica
Par j
Parietaria judaica
Par j
Parietaria judaica
Par j
Parietaria judaica
Par j
Parietaria judaica
Par j
Parietaria judaica
Par j 4
ietaria officinalis
Par o 1
Parthenium
Par h 1
hysterophorus
Persea americana
Pers a 1
Phaseolus vulgaris
Pha v
Phaseolus vulgaris
Pha v
Pistacia vera (Pistachio)
Pis v 1
Pistacia vera (Pistachio)
Pis v
Pistacia vera (Pistachio)
Pis v
Pistacia vera (Pistachio)
Pis v 3
Pistacia vera (Pistachio)
Pis v 4
Pistacia vera (Pistachio)
Pis v 5
Pisum sativum (Pea)
Pis s
Pisum sativum (Pea)
Pis s
Pisum sativum (Pea)
Pis s 2
Pisum sativum (Pea)
Pis s 3
Plantago lanceolata
Pla l
Plantago lanceolata
Pla l
Plantago lanceolata
Pla l
Plantago lanceolata
Pla l 2
Platanus acerifolia
Pla a 1
Platanus acerifolia
Pla a 2
Platanus acerifolia
Pla a 3
Platanus orientalis
Pla or 1
Platanus orientalis
Pla or 2
Platanus orientalis
Pla or 3
Prosopis juliflora
Pro j 1
Prosopis juliflora
Pro j 2
Prunus armeniaca
Pru ar 1
Prunus armeniaca
Pru ar 3
Prunus avium (Sweet
Pru av
Prunus avium (Sweet
Pru av
Prunus avium (Sweet
Pru av
Prunus avium (Sweet
Pru av
Prunus avium (Sweet
Pru av 2
Prunus avium (Sweet
Pru av 3
Prunus avium (Sweet
Pru av 4
Prunus domestica
Pru d 3
Prunus dulcis (Almond)
Pru du 3
Prunus dulcis (Almond)
Pru du 4
Prunus dulcis (Almond)
Pru du 5
Prunus dulcis (Almond)
Pru du
Prunus dulcis (Almond)
Pru du
Prunus mume (Japanese
Pru m 7
Prunus persica (Peach)
Pru p 1
Prunus persica (Peach)
Pru p
Prunus persica (Peach)
Pru p
Prunus persica (Peach)
Pru p
Prunus persica (Peach)
Pru p
Prunus persica (Peach)
Pru p
Prunus persica (Peach)
Pru p 4
Prunus persica (Peach)
Pru p 7
Punica granatum
Pun g
Punica granatum
Pun g
Punica granatum
Pun g
Pyrus communis (Pear)
Pyr c 1
Pyrus communis (Pear)
Pyr c 3
Pyrus communis (Pear)
Pyr c 4
Pyrus communis (Pear)
Pyr c 5
Quercus alba (White
Que a
Quercus alba (White
Que a
Quercus alba (White
Que a
Ricinus communis
Ric c 1
Rubus idaeus (Red
Rub i 1
Rubus idaeus (Red
Rub i 3
Salsola kali (Russian
Sal k
Salsola kali (Russian
Sal k
Salsola kali (Russian
Sal k
Salsola kali (Russian
Sal k
Salsola kali (Russian
Sal k 2
Salsola kali (Russian
Sal k 3
Salsola kali (Russian
Sal k
Salsola kali (Russian
Sal k
Salsola kali (Russian
Sal k 5
Sesamum indicum
Ses i 1
Sesamum indicum
Ses i 2
Sesamum indicum
Ses i 3
Sesamum indicum
Ses i 4
Sesamum indicum
Ses i 5
Sesamum indicum
Ses i 6
Sesamum indicum
Ses i 7
Sinapis alba (Yellow
Sin a 1
Sinapis alba (Yellow
Sin a 2
Sinapis alba (Yellow
Sin a 3
Sinapis alba (Yellow
Sin a 4
Solanum lycopersicum
Sola l 1
esculentum) (Tomato)
Solanum lycopersicum
Sola l
esculentum) (Tomato)
Solanum lycopersicum
Sola l
esculentum) (Tomato)
Solanum lycopersicum
Sola l 3
esculentum) (Tomato)
Solanum lycopersicum
Sola l
esculentum) (Tomato)
Solanum lycopersicum
Sola l
esculentum) (Tomato)
Solanum lycopersicum
Sola l 5
esculentum) (Tomato)
Solanum lycopersicum
Sola l 6
esculentum) (Tomato)
Solanum lycopersicum
Sola l 7
esculentum) (Tomato)
Solanum tuberosum
Sola t 1
Solanum tuberosum
Sola t 2
Solanum tuberosum
Sola t
Solanum tuberosum
Sola t
Solanum tuberosum
Sola t 4
Syringa vulgaris (Lilac)
Syr v
Syringa vulgaris (Lilac)
Syr v
Syringa vulgaris (Lilac)
Syr v 3
Triplochiton
Trip s 1
scleroxylon (Obeche)
Vigna radiata (Mung
Vig r 1
Vigna radiata (Mung
Vig r
Vigna radiata (Mung
Vig r
Vigna radiata (Mung
Vig r 4
Vigna radiata (Mung
Vig r 6
Vitis vinifera (Grape)
Vit v 1
Ziziphus mauritiana
Ziz m 1
Chamaecyparis obtusa
Cha o 1
Chamaecyparis obtusa
Cha o 2
Chamaecyparis obtusa
Cha o 3
Cryptomeria japonica
Cry j
Cryptomeria japonica
Cry j
Cryptomeria japonica
Cry j 2
Cupressus arizonica
Cup a 1
Cupressus
Cup s
sempervirens (Common
Cupressus
Cup s
sempervirens (Common
Cupressus
Cup s
sempervirens (Common
Cupressus
Cup s
sempervirens (Common
Cupressus
Cup s
sempervirens (Common
Cupressus
Cup s 2
sempervirens (Common
Cupressus
Cup s
sempervirens (Common
Cupressus
Cup s
sempervirens (Common
Juniperus ashei
Jun a
Juniperus ashei
Jun a
Juniperus ashei
Jun a 2
Juniperus ashei
Jun a 3
Juniperus oxycedrus
Jun o 4
Juniperus sabinoides
Jun s 1
Juniperus virginiana
Jun v
Juniperus virginiana
Jun v
Juniperus virginiana
Jun v 3
Pinus koraiensis
Pin k 2
Pinus pinea (Stone
Pin p 1
In some embodiments, SPNs, magnetic particle conjugates and compositions of the present invention may detect a specific epitope of a target allergen. As used herein, an “epitope” is the part of the allergen that is recognized by the immune system (e.g., antibodies. B cells, or T cells). Generally, the whole allergen is not involved in the immune response, but rather, the epitopes of allergens, which are recognized by a T-cell receptor or an IgE-antibody, contribute to allergic reactions. SPNs, magnetic particle conjugates and compositions of the present invention may bind to an epitope of a target allergen in a similar manner as antibodies or T-cell receptors.
Epitopes of an allergen protein may be identified using methods and techniques known in the art. For example, IgE-binding epitopes may be determined by methods such as X-ray co-crystallography of allergens and immunocomplexes, array-based oligo-peptide scanning, mutagenesis mapping, or nuclear magnetic resonance. For example, T-cell epitopes may be identified using short peptide fragments that overlap the entire amino acid sequence of the target allergen and allergen-specific T-cell lines derived from peripheral blood mononuclear cells. Peptides that induce T-cell proliferation contain T-cell epitopes. Additional techniques that may be used to determine epitopes on an allergen protein include crosslinking coupled mass spectrometry, hydrogen-deuterium exchange, and computer-based in silico analysis. Many epitopes of common food allergens have been identified, such as those listed in Tables 1-5 of Matsuo H et al., Allergol Int. 2015 October; 64(4):332-43.
In some embodiments, SPNs, magnetic particle conjugates and compositions of the present invention may be used in a hospital for clinical food allergy or allergy test and to identify food/allergen(s) to which a patient is allergic. In addition, SPNs, magnetic particle conjugates and compositions of the present invention may be used as a carry-on tester for people who have food/environmental allergy, for example at home to test commercial food, or at restaurant to check dishes they ordered. The food sample could be fresh food, frozen food, cooled food or processed food containing animal derived meat and/or vegetables.
In some embodiments, SPNs, magnetic particle conjugates and compositions of the present invention may detect other target molecules, including but not limited to, pathogens from a pathogenic microorganism in a sample, such as bacteria, yeasts, fungi, spores, viruses or prions; disease proteins (e.g., biomarkers for diseases diagnosis and prognosis); pesticides and fertilizers remained in the environment; and toxins. In other embodiments, SPNs and compositions of the present invention may bind to non-protein targets such as minerals and small molecules (e.g., antibiotics), drugs and inorganic ion.
Compositions, SPNs, SPN-complement complexes, magnetic particle conjugates, DNA-printed solid substrates and detection agents of the present invention may be combined with other ingredients or reagents or prepared as components of kits or other retail products for commercial sale or distribution.
The kit will contain compositions of the present invention, along with instructions regarding administration and/or use of the kit. The kit may also contain one or more of the following: a syringe, a bag or bottle.
Formulations and/or compositions of the present invention can be packaged for use in a variety of pharmaceutically or diagnostically acceptable containers using any acceptable container closure, as the formulations are compatible with PVC-containing and PVC-free containers and container closures. Examples of acceptable containers include, but are not limited to, ampules and pre-filled syringes, cartridges and the like.
Alternatively, the formulation may contain SPNs, SPN-complement complexes, magnetic particle conjugates in one compartment of an admix bag and an acceptable solvent in a separate compartment of the admix bag such that the two compartments may be mixed together prior to use. Acceptable containers are well known in the art and commercially available.
In some embodiments, biosensors comprising compositions, SPN-complement complexes, magnetic particle conjugates, DNA-printed solid substrates and detection agents of the present invention are provided. The biosensor may be an on-chip magnetic particle biosensor. For example, a plurality of magnetic particle sensors are embedded in the integrated circuit; the magnetic particle sensors are capable of detecting magnetic particles specifically bound to the one or more sensor areas on the surface of the integrated circuit. The affinity molecules on the surface of the sensor area may be SPNs, or SPN-complement complexes of the present invention.
In some embodiments, a graphene-oxide (GO) based biosensor is provided, wherein the sensing agents are SPNs conjugated to the graphene-oxide surface.
In some aspects, it may be a “signal on” biosensor. The SPN and its complementary sequence may be labeled with a fluorophore and a quencher at one end of the nucleic acid sequence, respectively. The signal produced by the sensor is dependent on the structural changes in the SPNs following the binding of target allergens. The binding of the target allergen to the SPN disrupts the SPN-complement complex and replaces the complementary sequence in the complex, resulting in the quenched fluorescent signal back on (
In some aspects, it may be a “signal off” biosensor, in which the complementary sequence is labeled with a fluorophore at one end of the sequence. The binding of the target allergen to the SPN detaches the complementary sequence in the complex, causing the fluorescent signal off (
In other aspects, it may be a “dual signal” biosensor, in which the SPN and the complementary sequence are labeled with different fluorophores. The competition between the target allergen and the complement will change the fluorescent signals (
In accordance with the present invention, aptamers, SPNs and SPN-complement complexes, magnetic particle conjugates, DNA-printed solid substrates, detection agents and compositions of the present invention may be used to, in a broad concept, detect any molecules in a sample in a large variety of applications, such as food safety, diagnostic and prognostic tests in civilian and battlefield settings, environmental monitoring/control, and military use for detection of biological weapons. Various methods and assays may be used in combination with aptamers, SPNs, SPN-complement complexes, magnetic particle conjugates, DNA-printed solid substrates, detection agents and compositions of the present invention, the choice may depend on the application field.
Particularly the present invention provides methods of determining the absence, presence and/or quantity of one or more target allergens in a sample using detection agents comprising SPN-magnetic particle conjugates and printed solid substrates. In some embodiments, the detection assays and methods can be used in a hospital for clinical food allergy or allergy test and to identify food/allergen(s) to which a patient is allergic. Such assays and methods may be used to monitor allergen contamination in food industry. Additionally, they may also be used at home or in a restaurant by a person who has allergy to test the allergen content before he/she consumes the food.
Examples of foods are eggs, milk, meat, fishes, crustacea and mollusks, cereals, legumes and nuts, fruits, vegetables, beer yeast, and gelatin; more particularly, egg white and egg yolk of the eggs, milk and cheese of the milk, pork, beef, chicken and mutton of the meat, mackerel, horse mackerel, sardine, tuna, salmon, codfish, flatfish and salmon caviar of the fishes, crab, shrimp, blue mussel, squid, octopus, lobster and abalone of the crustacea and mollusks, wheat, rice, buckwheat, rye, barley, oat, corn, millet, foxtail millet and barnyardgrass of the cereals, soybean, peanut, cacao, pea, kidney bean, hazelnut, Brazil nut, almond, coconut and walnut of the legumes and nuts, apple, banana, orange, peach, kiwi, strawberry, melon, avocado, grapefruit, mango, pear, sesame and mustard of the fruits, tomato, carrot, potato, spinach, onion, garlic, bamboo shoot, pumpkin, sweet potato, celery, parsley, yam and Matsutake mushroom of the vegetables, the foods containing them, and the ingredients thereof (e.g., ovoalbumin, ovomucoid, lysozyme, casein, beta-lactoglobulin, alpha-lactoalbumin, gluten, and alpha-amylase inhibitor).
The foods could be fresh foods, frozen foods, cooled foods or processed foods containing animal derived meat and/or vegetables. These foods may be processed by heating, freezing, drying, salting, fermentation, enzymatic processing, etc.
In some embodiments, more than one agents may be used, depending on the nature of the food matrixes. Some food contains several allergenic proteins, e.g., at least eight peanut proteins, such as Ara h1 and Ara h2, can potentially cause an immunological response. In such case, more than one SPNs against more than one allergenic protein may be used in a mixed cocktail for detecting the absence or presence of peanut. In other aspects, some food matrixes such as fish, shellfish and mollusks, contain only one major allergenic protein. One or more SPNs that specifically bind to this major allergen protein may be used for allergen detection.
In some embodiments, allergen detection assays and methods of the present invention can detect a lower concentration of allergen in a food sample. The sensitivity of nucleic acid aptamers makes it possible to detect the presence of an allergen as low as 0.0001 ppm. In some aspects, the concentration or mass of allergen that can be detected may range from 0.001 ppm to 5 ppm, or from 0.001 ppm to 0.1 ppm, or from 0.1 ppm to 3 ppm, or from 1 ppm to 5 ppm, or from 5 ppm to 10 ppm, or from 10 ppm to 50 ppm, or from 10 ppm to 20 ppm, or from 10 ppm to 100 ppm. In some aspects, the concentration or mass of allergen in a food sample that can be detected may be 0.001 ppm, 0.002 ppm, 0.003 ppm, 0.004 ppm, 0.005 ppm, 0.006 ppm, 0.007 ppm, 0.008 ppm, 0.009 ppm, 0.01 ppm, 0.02 ppm, 0.03 ppm, 0.04 ppm, 0.05 ppm, 0.06 ppm, 0.07 ppm, 0.08 ppm, 0.09 ppm, 0.1 ppm, 0.2 ppm, 0.3 ppm, 0.4 ppm, 0.5 ppm, 0.6 ppm, 0.7 ppm, 0.8 ppm, 0.9 ppm, 1.0 ppm, 1.5 ppm, 2 ppm, 2.5 ppm, 3 ppm, 3.5 ppm, 4 ppm, 4.5 ppm, 5 ppm or 10 ppm. In some embodiments, the concentration or mass of allergen in a food sample that can be detected may be 5 ppm, 1 ppm, 15 ppm, 20 ppm, 25 ppm, 30 ppm, 35 ppm, 40 ppm, 45 ppm, 50 ppm, 55 ppm, 60 ppm, 65 ppm, 70 ppm, 75 ppm, 80 ppm, 85 ppm, 90 ppm, 95 ppm or 100 ppm. In one embodiment, the concentration or mass of allergen in a food sample that can be detected may be at low as 50 ppm. In another embodiment, the concentration or mass of allergen in a food sample that can be detected may be at low as 12.5 ppm
In accordance with the present aptamer derived SPN based detection assays, the sensitivity can be achieved regardless of food matrices. The diverse food matrices with different salt, fat and sugar content, different color, texture, and pH can be detected at the present sensitivity.
In some embodiments, methods of the present invention for detecting the absence, presence and/or quantity of a target allergen in a test sample comprise (a). obtaining and processing a test sample which is suspected to contain an allergen of interest; (b). contacting the processed test sample with detection agents comprising SPNs conjugated to solid substrates, wherein the SPNs are single-stranded nucleic acid molecules having 20 to 200 nucleotides capable of specifically binding to the target allergen; (c) washing the mixture of the detection agents and the test sample formed in step (b); (d). detecting the allergen-SPN complexes formed during the assay; and (e). processing and analyzing the detection signals to determine whether the target allergen is present in the food sample and/or the quantity of the target allergen in the sample.
In some aspects, the detection agents further comprise a nucleic acid molecule complementary to a region of the SPN sequence which binds to the SPN only when the SPN is not bound to its target allergen protein; the SPN and the complementary sequence forms the SPN-complement complex, in which the SPN and the complementary sequence work as binding ligand (to the target allergen) and detection molecule or label, respectively. In some examples, the SPN is covalently immobilized on the surface of the solid substrate through its 5′end or 3′end. In other examples, the complementary sequence is covalently immobilized on the surface of the solid substrate.
In some examples, the solid substrate is magnetic particles. In accordance, the magnetic particles may be held using a magnetic field during the wash step. Optionally, the washed reaction mixture may be shaken, prior to detecting the allergen-SPN-magnetic particle complexes formed during the assay.
Assays and methods of the present invention may have various forms depending on biosensors, detection kits and detection devices and systems used to implement the assays. SPN/complement-magnetic particle conjugates may be used in any steps of the assays, such as capture agents to capture a target analyte (e.g., a target allergen or a specific epitope of the target allergen) in a sample; or detector agents for signaling detection; or competitive binding agents, or affinity agents to selected a target analyte (e.g., an allergen) bound magnetic particles.
In some embodiments, SPNs comprise the nucleic acid sequences of SEQ ID NOs.: 1-696 (See Tables 1-4). The complementary sequences may contain about 5-20 nucleotide residues. The SPN and its complementary sequence may be at a ratio of 1:5, or at a ratio of 1:4, or at a ratio of 1:3, or at a ratio of 1:2.
In some embodiments, an attachment assay may be developed using the compositions and agents of the present invention (as shown in
In other embodiments, a detachment assay may be developed using the compositions and agents of the present invention (as shown in
In some embodiments, SPNs with different sequences but having a high affinity and specificity to the same target allergen may be used in combination. In other aspects, SPNs of the present invention may be used in combination with antibodies that bind the same target allergen in a detection assay.
In some embodiments, the detection assay of the present invention can be optimized depending on the testing condition such as food matrix. The reaction buffer used may be decreased in volume. In some aspects. The reaction volume can be limited to less than 5 mL, for example, less than 4 mL, or less than 3 mL, or less than 2 mL, or less than 1 mL. In one example, the volume may be 1.5 mL, 2 mL, 2.5 mL, 3 mL, 3.5 mL, 4.5 mL, and 5.0 mL.
In some embodiments, the sample size can be reduced, limiting the requirement of a large size sample. The assay sensitivity can be achieved with a small portion of test sample, for example, less than 5 mg, or less than 4.5 mg, or less than 4 mg, or less than 3.5 mg, or less than 3 mg, or less than 2.5 mg, or less than 2 mg, or less than 1.5 mg.
In some embodiments, the detection assay of the present invention can be completed in a short period of time, for example less than 5 minutes. In some aspects, the detection reaction can be completed from 10 seconds to 5 minutes, or from 10 seconds to 60 seconds, or from 1 minute to 2 minutes. In some examples, the detection assay can be completed in about 10 seconds, about 20 seconds, about 30 seconds, about 35 seconds, about 40 seconds, about 45 seconds, about 50 seconds, about 1 minute, about 1.5 minutes, about 2 minutes, about 2.5 minutes, about 3 minutes, about 4.5 minutes, or about 5 minutes.
In some embodiments, the detection assays of the present invention can be optimized to achieve the greatest signal intensity. The step optimization may include, but are not limited to, changing the concentration of SPN molecules, controlling the sample and SPN incubation conditions and time, controlling the flow rate and maximizing the exposure of solid surface to SPNs.
In some embodiments, the detection assay of the present invention further comprising an internal control signal. The built-in control signal may be measured using pre-incubated free SPNs and/or free food samples without SPN binding. These control signals will reduce the background signal and non-specific reading during the detection reaction.
In some embodiments, the detection signals are processed and displayed by a reader device. The reader device may be a computer, an iPad and/or a cellphone, or other processors that can execute one or more methods for detecting the absence, presence and/or quantity of the target allergen.
In accordance with the present invention, the detection agents and other reaction agents of the detection assay are stable and can be stored for a long-period of time. In general, short nucleic acid molecules are stable at routine storage condition. In some aspects, the SPNs, SPN-magnetic bead conjugates and nucleic acid coated solid surfaces are stable and can maintain their activity for at least one month, or at least two months, or at least three months, or at least four months, or at least five months, or at least six months. They may be stable from about one month to about one year. The reaction agents such as the extraction buffer can be stable at various temperature for at least nine months, or at least ten months, or at least one year.
At various places in the present specification, substituents of compounds of the present disclosure are disclosed in groups or in ranges. It is specifically intended that the present disclosure include each and every individual sub-combination of the members of such groups and ranges. The following is a non-limiting list of term definitions.
Activity: As used herein, the term “activity” refers to the condition in which things are happening or being done. Compositions of the invention may have activity and this activity may involve the binding to a target molecule.
Allergen: as used herein, the term “allergen” means a compound, substance or composition that causes, elicits or triggers and immune reaction in a subject. As such, allergens are typically referred to as antigens. An allergen is typically a protein or a polypeptide.
Allergen detection agent: As used herein, the term “an allergen detection agent” refers to Any agent which is capable of, or does, interact with and/or bind to one or more allergens in a way that allows detection of such allergen in a sample is referred to herein as an “allergen detection agent” or “detection agent”.
Analyte: As used herein, an “analyte” is a target of interest that can specifically interact with (bind to) an aptamer and be detected and/or measured. In the context of the present invention, an analyte may be an allergen.
Aptamer: as used herein, the term“aptamer” refers to single stranded nucleic acid. In general, aptamers refer to either an oligonucleotide of a single defined sequence or a mixture of said oligonucleotides, wherein the mixture retains the properties of binding specifically to a target allergen. A RNA aptamer is an aptamer comprising ribonucleoside units. RNA aptamer also meant to encompass RNA analogs as defined herein. A DNA aptamer an aptamer comprising deoxy-ribonucleoside units. DNA aptamer also meant to encompass DNA analogs as defined herein.
Binding affinity: as used herein, the term “binding affinity” is intended to refer to the tendency of an aptamer to bind or not bind a target and describes the measure of the strength of the binding or affinity of the aptamer to bind the target.
Complementary: As used herein, the term “complementary” or “complement” refer to the natural binding of polynucleotides by base pairing such as A-T(U) and C-G pairs. Two single-stranded molecules may be partially complementary such that only some of the nucleic acids bind, or it may be “complete,” such that total complementarity exists between the single stranded molecules. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of the hybridization between the nucleic acid strands.
Detection: As used herein, the term “detection” means an extraction of a particular target protein from a mixture of many non-target proteins, indicating the absence, presence, and/or amount of a target protein from a mixture of many non-target proteins.
Hybridization: As used herein, the term “hybridization” refers to the process by which a polynucleotide strand anneals with a complementary strand through base pairing under defined hybridization conditions. Specific hybridization is an indication that two nucleic acid sequences share a high degree of identity such as a SPN of the present invention and a short complementary sequence of the SPN. Specific hybridization complexes form under permissive annealing conditions and remain hybridized after the “washing” step(s). The washing step(s) is particularly important in determining the stringency of the hybridization process, with more stringent conditions allowing less non-specific binding. i.e., binding between pairs of nucleic acid strands that are not perfectly matched.
Magnetic particles: As used herein, the term “magnetic particles” refer to ( ). Magnetic particles may include magnetic microbeads and/or nanoparticles.
Oligonucleotide: as used herein, the term “oligonucleotide” is generic to polydeoxyribonucleotides (containing 2′-deoxy-D-ribose or modified forms thereof), i.e. DNA, to polyribonucleotides (containing D ribose or modified forms thereof), i.e. RNA, and to any other type of polynucleotide which is an N-glycoside or C-glycoside of a purine or pyrimidine base, or modified purine or pyrimidine base or abasic nucleotides. In the context of the present invention, the “oligonucleotide” includes not only those with conventional bases, sugar residues and internucleotide linkages, but also those that contain modifications of any or all of these three moieties.
As used herein, the terms “nucleic acid” “polynucleotide” and “oligonucleotide” are used interchangeable herein and refer to a deoxyribonucleotide or ribonucleotide polymer in either single- or double-stranded form, and unless otherwise limited, encompasses known analogs of natural nucleotides that hybridize to nucleic acids in a manner similar to naturally-occurring nucleotides. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, and peptide-nucleic acids (PNAs).
Sample: As used herein, the term “sample” refers to any composition that might contain a target of interest to be analyzed including, but not limited to, biological samples obtained from subjects (including humans and animals as detailed below), samples obtained from the environment for example soil samples, water samples, agriculture samples (including plant and crop samples), or food samples. Food samples may be obtained from fresh food, processed/cooked food or frozen food.
Sensitivity: As used herein, the term “sensitivity” means the ability of a detection molecule to bind to a target molecule.
Specifically binds: as used herein, the term “specifically binds” means that an aptamer reacts or associates more frequently, more rapidly, with greater duration and with greater affinity with a particular target molecule, than it does with alternative target molecules. For example, an aptamer that specifically binds to a target allergen binds that allergen or a structural part or fragment thereof with greater affinity, avidity, more readily, and/or with greater duration than it binds to unrelated allergen protein and/or parts or fragments thereof. It is also understood by reading this definition that, for example, an aptamer that specifically binds to a first target may or may not specifically bind to a second target. As such. “specific binding” does not necessarily require exclusive binding or non-detectable binding of another molecule, this is encompassed by the term “selective binding”. The specificity of binding is defined in terms of the comparative dissociation constants (Kd) of the aptamer for target as compared to the dissociation constant with respect to the aptamer and other materials in the environment or unrelated molecules in general. Typically, the Kd for the aptamer with respect to the target will be 2-fold, 5-fold, or 10-fold less than the Kd with respect to the target and the unrelated molecule or accompanying molecule in the environment. Even more preferably, the Kd will be 50-fold, 100-fold or 200-fold less.
Target: as used herein, the term “target” and “target molecule” refers to a molecule which may be found in a tested sample and which is capable of binding to a detection molecule such as an aptamer or an antibody.
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments in accordance with the invention described herein. The scope of the present invention is not intended to be limited to the above Description, but rather is as set forth in the appended claims.
In the claims, articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention includes embodiments in which more than one, or the entire group members are present in, employed in, or otherwise relevant to a given product or process.
It is also noted that the term “comprising” is intended to be open and permits but does not require the inclusion of additional elements or steps. When the term “comprising” is used herein, the term “consisting of” is thus also encompassed and disclosed.
Where ranges are given, endpoints are included. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or subrange within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.
In addition, it is to be understood that any particular embodiment of the present invention that falls within the prior art may be explicitly excluded from any one or more of the claims. Since such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiment of the compositions of the invention (e.g., any antibiotic, therapeutic or active ingredient; any method of production, any method of use; etc.) can be excluded from any one or more claims, for any reason, whether or not related to the existence of prior art.
It is to be understood that the words which have been used are words of description rather than limitation, and that changes may be made within the purview of the appended claims without departing from the true scope and spirit of the invention in its broader aspects.
While the present invention has been described at some length and with some particularity with respect to the several described embodiments, it is not intended that it should be limited to any such particulars or embodiments or any particular embodiment, but it is to be construed with references to the appended claims so as to provide the broadest possible interpretation of such claims in view of the prior art and, therefore, to effectively encompass the intended scope of the invention.
An aptamer against the Ara h 1 allergen protein which is described by Tran et al. in Selection of aptamers against Ara h 1 protein for FO-SPR biosensing of peanut allergens in food matrices. Biosensors and Bioelectronics, 2013, 43, 245-251 (incorporated herein by reference in its entirety), was used to design a signaling polynucleotide and conjugated to magnetic beads. The sequence of this aptamer is shown below (SEQ ID NO: 96).
5′TCGCACATTCCGCTTCTACCGGGGGTCGAGCTGAGTGGATGCGAA TCTGTGGGTGGGCCGTAAGTCCGTGTGTGCGAA3′ (SEQ ID NO.: 96)
A short nucleic acid sequence containing 5 nucleotide residues was designed to be complementary to the 3′-end residues of the T-modified aptamer of SEQ ID NO: 96. Alternatively, the sequence may be complementary to the 5′-end residues of the T-modified aptamer of SEQ ID NO: 96. One end of the complementary sequence was labeled with a fluorophore, Texas Red.
Three reactions were used to conjugate the aptamer to magnetic beads. The sequence was modified with a thiol (—SH) modifier at either the 5′end or 3′end (
Alternatively, the aptamer was modified with a primary amine group at the 5′end or 3′end. The amine modified aptamer was attached to magnetic beads via carbodiimide chemical reaction. Magnetic carboxylated speedbeads were used (GE Healthcare Life Science, Cat. Log No.: XX). The Texas Red labeled complementary sequence was annealed to the aptamer at the 3′end, forming a magnetic bead conjugated signaling polynucleotide (SPN) (shown in
Amine Reaction
Amine-modified DNA molecules at either 5′ end of 3′ end were coupled to magnetic carboxylated speedbeads (GE Healthcare, USA) through carbodiimide chemical reaction. Carboxyl modified beads were first incubated with EDC/NHS (N-hydroxysuccinimide) prior to the addition of amine-modified DNA molecules. After EDC/NHS reaction, the carboxyl groups (—COOH) were activated for direct reaction with primary amines via amide bond formation. Amine-modified DNA molecules were mixed with the beads and the coupling reaction of beads and DNA occurred in water. After the reaction, a TRIS buffer was used to block/occupy the unreacted sites.
Thiol Reaction
Thiol-modified DNA molecules at either 5′ end of 3′ end were coupled to magnetic carboxylated speedbeads (GE Healthcare. USA) through amino-polyethylene-glycol maleimide linkage (2HN-PEG-Maleimide). The carboxyl groups (—COOH) on the surface of beads were activated first using water soluble EDC (1-ethyl-3-(˜3-dimethylaminopropyl) carbodiimide hydrochloride) and then by 2-HN-methoxypolyethylene glycol (PEG). The beads were incubated with EDC and PEGs in MES (4-morpholinoethanesulfonic acid) reaction buffer for 2 hours (30 min×4 sonicator bath). Activated beads were then mixed with thiol-modified DNA molecules and incubated with DTT (Dithiothreitol) in water.
The study tested an experimental procedure for obtaining fluorescence readings and thereby determining the effectiveness of signaling polynucleotides in identifying and quantifying a molecular target such as an allergen protein in a food sample.
Peanut was diluted to an appropriate concentration in double-distilled water, ranging from 0 ppm to 500 ppm. SPN-magnetic bead conjugates as described in Example 1 were diluted in solution/buffer either HEPES/Tween, PBS/BSA or T-buffer (Tris pH8 100 mM NaCl, 50 mM KCl, 10 mM MgCl 0.5% Sodium Deoxcholate 0.1% Gelatin 1% Triton 0.1% SDS) at various concentrations. Peanut samples and SPN-magnetic bead conjugates were mixed and incubated for a period of time. The fluorescent signals before and after the reaction were detected and measured. In one study in which magnetic beads and SPN-complement were configured as illustrated in
Food samples spiked with peanut including Poptart, Graham crackers, Oreo, Blue Frosting were tested for signal decrease using 5′thiol modified SPN conjugated magnetic beads. As illustrated in
Comparison of signal detection using magnetic beads conjugated with 5′thiol modified SPNs and magnetic beads conjugated with polyA3′thiol modified SPNs suggest that 5′thiol modification displays more significant decrease than polyA3′thiol modification (See Table 6). About 40% of signal decrease was detected with magnetic beads conjugated with 5′thiol modified SPNs, while only about 20% of signal decrease was detected with magnetic beads conjugated with polyA3′thiol modified SPNs. The fluorescent measure also indicates that thiol modification displays more significant decrease than amine modification (data not shown).
As described in Example 1, amine-modified SPNs were annealed with the 5-nt complementary sequences (at a ratio of 1:5). The SPN-complement complex was conjugated to magnetic beads. The complementary sequence was labeled with Cy5 or Alex647, respectively. The SPN-magnetic bead conjugates were incubated with different concentrations of peanut flour (from 0 ppm to 500 ppm) at 37° C. Fluorescent signals were recorded after the incubation. As shown in Table 7, both dyes display significant signal decreases as peanut concentration increases, indicating more allergen proteins bind the SPN and detach the complementary sequences from the SPNs.
As described in Example 1, thiol-modified oligonucleotides were annealed with the 5-nt complementary sequences (at a ratio of 1:5). The SPN-complement complexes were conjugated to magnetic beads. The complementary sequence was labeled with Cy5 or Alex647, respectively. The SPN-magnetic bead conjugates were incubated with different concentrations of peanut flour (from 0 ppm to 500 ppm) at 37° C. Fluorescent signals were recorded after the incubation. As shown in Table 8, both dyes display significant signal decreases as peanut concentration increases, indicating more allergen proteins bind the SPN and detach the complementary sequences from the SPNs. As shown in Table 8, when the SPN is at high concentration, 1000 nM, Cy5 labeled signal decreases as the peanut concentration increases.
Various parameters may affect the stability of nucleic acids immobilized on magnetic beads and their interaction with target allergens in a test sample. Tables 9 and 10 illustrate the fluorescent reading in various conditions tested. The data suggests that doubling Maleimide concentration enables signal decrease using a higher concentration of SPN. However the decrease doesn't retain more than 50% (Table 9). Shaking the SPN solution prior to the signal reading to re-suspend magnetic beads is critical for accurate signal measurement. It can cause about 65% decrease in signal (Table 10). The data also suggests that the step of washing beads after reaction, extraction buffer and annealing conditions affect the signal detection (Table 10).
Effect of Wash Components on Signal Detection
Reaction mixtures comprising polyA-thiol modified SPNs and peanuts were washed before signal detection using PBS buffer containing BSA (Bovine serum albumin). As compared with the signal after washing with PBS only, noticeable signal decrease is detected as peanut concentration is increased, indicating that BSA can facilitate removal of the complementary sequences after allergen binding (Table 11).
Washing Buffer Containing Detergent
SPN-complement complexes with Texas Red, complementary sequences labeled with Texas red, and fluorophore Texas Red alone were washed with PBS buffer containing Tween-20 at various concentrations. The fluorescent signals (RFU) after washing were detected and compared.
As shown in Examples 3 and 4, the high ratio 1:5 of aptamer oligonucleotides and complementary sequences gives a high fluorescent signal. A ratio 1:2 of aptamer oligonucleotides and complementary sequences was tested for signal detection.
Magnetic beads conjugated with complexes of amine-SPN and its complementary sequences labeled with Cy5 in which the amine-SPN molecules and CY-5-labeled complementary sequences is at a ratio of 1:2, were incubated with peanut flour at concentrations from 0 to 500 ppm. Signals were detected and as shown in Table 14, signal decreases as peanut concentration increases.
To test if the same amount of beads are analyzed during target detection, the absorbance signals were analyzed in parallel with the fluorescent signal. Magnetic beads conjugated with SPN-complement complexes of amine-SPN and its complementary sequence (5nt in length) labeled with Cy5 in which the amine-SPN molecules and CY-5-labeled complementary sequences is at a ratio of 1:2, were incubated with peanut flour at concentrations from 0 to 500 ppm. Absorbance by beads was measured. The absorbance data indicates a change in absorbance when magnetic beaded conjugated to SPNs are exposed to higher concentrations of peanut flour (See Table 15).
Similarly, magnetic beads conjugated with SPN-complement complexes of thiol-SPN and its complementary sequence (10 nt in length) labeled with Texas red in which the amine-SPN molecules and Texas red-labeled complementary sequences is at a ratio of 1:2, were incubated with peanut flour at concentrations from 0 to 500 ppm. Absorbance by beads was measured. As shown in Table 16, the absorption data indicates no changes in absorbance with beads conjugated to thiol-SPNs.
Optimize the Printing Protocol
3 complementary sequences ranging in length between 10-15 nucleotides with a 3′amine modification are used for the test. The DNA sequences are attached and printed on the surface of the glass slides using a variety of DNA loading densities and slide coatings.
Screen for Complementary Sequences for a Selected Aptamer
A microarray-based screen is used to select complementary DNA sequences that only bind the aptamer when it is not bound to the allergen protein. An aptamer (SEQ ID NO. 353 and SEQ ID NO. 307) specific to peanut allergen protein AraH1 and an aptamer (SEQ ID NO. 564) specific to nut were selected. About 40 short sequences that are complementary to different portions of each aptamer are designed based on the aptamer sequence (See Table 17). These sequences range in length between 10-20 nucleotides. The short sequences (also referred to as anchor or linker sequences) are printed on the surface of the glass slide using the printing protocol optimized previously.
Each of 16-array slides is coated with 3 copies of the 50 sequences, giving a total of 150 sequences per array (see chip layout in Table 18) using the printing protocol optimized previously. The binding of the short sequences to its target aptamer is tested using either an attachment or detachment protocol detailed below. The aptamer is labeled on the 5′ end with a fluorescent dye Cy5 which is used to quantify the aptamer bound to the plate. The allergenic protein is prepared into a serial dilution of five different concentrations (e.g., 0 ppm, 1 ppm, 10 ppm, 100 ppm, and 1000 ppm). The output of the experiment is relative Cy5 reads from the protein curve as well as the negative controls. Table 17 shows an example of the screen chip layout. The screen consists of three 16-array slides. Each array is designed to have 3 copies of the 50 sequences (150 samples total). For each experimental slide, a 5-point protein curve (×3) and a buffer control are included (a full 16 array slide).
Attachment Assay
For the attachment protocol, a fixed concentration of aptamer is mixed with increasing concentrations of the allergen protein for 1 min to allow the aptamer to bind to the allergen protein. The protein-aptamer mixture is then added to the wells of the plate and incubated for 1 min at room temperature, washed, and measured for fluorescence. The controls for the experiment include determining fluorescence of the microarray wells with either only the aptamer or protein not previously incubated with the aptamer, as well as fluorescence with only the short sequences present. Aptamer that is protein-free attaches to the short sequences immobilized on the plate and contributes to the fluorescence reading. Protein-bound aptamer does not attach to the plate and is washed away. If the short immobilized DNA sequence only binds the protein-free aptamer, decreasing fluorescence signal is observed with an increasing concentration of the protein, as the higher the protein concentration the less protein-free aptamer is available to bind to the short sequence on the microarray. If the short sequence cannot differentiate between protein-bound and unbound aptamer, a similar fluorescence intensity is observed regardless of the concentration of the protein.
Detachment Assay
For the detachment protocol, the aptamer is added to the wells of the plate and incubated for 1 min at room temperature, washed, and measured for fluorescence. Protein is added to the plate at five different concentrations, incubated for 1 min, washed, and measured for fluorescence again. The controls of the experiment are the same as described for the attachment protocol. If the short sequence only binds the protein-free aptamer, a drop in fluorescence signal is observed when protein is added. The magnitude of the drop increases with an increasing concentration of the protein, as the higher the protein concentration the more aptamers detach from the short immobilized sequence. If the short sequence cannot differentiate between protein-bound and unbound aptamer, a similar fluorescence intensity is observed regardless of the concentration of the protein.
The top 3 short sequences are selected based on the screen results and validated using a second microarray. Each one of the 3 sequences is printed in 50 spots per array, giving a total of 150 sequences. The binding to the short sequences is tested using either an attachment or detachment protocol as described above. The validation assay incorporates control proteins and control test matrices (see chip layout in Table 18). Relative Cy5 reads from different protein concentrations as well as the negative controls are compared. The best candidate meets the following criteria: (a) specific binding to the corresponding aptamer, (b) easy detachment from the bound aptamer in the presence of the allergenic protein, and (c) minimal background/non-specific binding. The selected complementary sequence is used to form SPN-complement complexes.
Table 19 shows the chip layout for the validation assay. A set of 12, 16-array slides are used for the attachment assay and another 12 are used for the detachment assay. For each slide, a 5-point curve (×3) and a control are included (a full 16 array slide). Each spot of the 16 array slides contains 50 copies of the 3 candidates selected from the screen (150 samples total).
As discussed in Example 1, 5′ Thiol modified short complementary sequences were conjugated to magnetic beads and aptamer specific to peanut allergen (SEQ ID NO. 96: 5′ (Texas Red) TCGCACATTCCGCTTCTACCGGGGGGTCGAGCTGAGTGGATGCGAATCTGTGGG TGGGCCGTAAGTCCGTGTGTGCGAA3′) was hybrided with the short linker nucleic acid molecules. The aptamer was labeled with Texas red at the 5′ end. The short linker sequence is AAAAATCAAGTGGTC with 5′ Thiol modification (SEQ ID NO. 778)
The SPN-magnetic bead conjugates were used as detection agents to validate the detection sensitivity in detection assays. Peanut was diluted in either buffer or cookie at 0 ppm, 5 ppm, 50 ppm and 500 ppm, respectively. The prepared peanut samples were reacted with SPN-magnetic bead conjugates (loaded in 96-well microplate) and signals were read using lab-grade LED based reader after incubation and wash. Under bench-top reaction conditions, peanut present either in buffer or in cookie can be detected at a low ppm using SPN-magnetic bead conjugates. The recorded signal indicates that the fluorescent signal is correlatively reduced when the concentration of peanut increases. At the concentration of 50 ppm (peanut commodity), a significant fluorescent signal reduction is recorded in buffer (
In order to further confirm the sensitivity of the SPN-magnetic bead conjugates based assay, over 20 different food matrices were spiked with peanut and tested for signal detection and its sensitivity. The 50 ppm sensitivity was confirmed on representative foods from each category as shown in Table 20.
The detection sensitivity of SPN-magnetic bead conjugates was further confirmed using different fluorescent signal readers. The signal reading was recorded and analyzed using a lab-grade LED based reader as well as a designed optical sensor. When the detection assay was carried out using Data shown in Table 21 indicate that the detection sensitivity is not affected when using different signal readers. The designed optical sensor is comparable to the laboratory-standard microplate reader (e.g.,
Chips were coated with short complementary DNA sequences that bind the aptamer only when it is not bound to the allergen protein. These short nucleic acid anchor sequences specific to the aptamers (SEQ ID NO. 307, SEQ ID NO. 404 and SEQ ID NO. 304) which bind to peanut allergen protein were used to coat the glass. These anchor sequences are about 10 nt in length. The short anchor sequences are further modified to contain 5 (polyA) at one end to pull the sequence away from the surface. A 6 carbon or 12 Carbon linker is further added to the sequences to pull them further away from the surface. An amine attachment is added to print the sequences to the surface of the chip. The sequences and modification of the anchor/linker DNA molecules are listed in Table 22. Prepared peanut containing samples were incubated with a fixed concentration of SPNs specific to the peanut allergen and filtered to remove unbound food particles. The aptamers were labeled with Cy5 at the 5′ end. The mixture was incubated with the chip coated with short linker/anchor sequences. After incubation and wash, the fluorescent reading was recorded using a lab grade LED based reader, as well as the designed optical sensor. The anchor sequences that generate the most specific and sensitive signals are selected for further validation.
Peanut detection using nucleic acid coated chips was evaluated in different detection assays and with different food matrices. The sensitivity of the detection assay based on nucleic acid coated chips was validated and confirmed. In this study, the anchor/linker sequence (5′ 12C-Amine) AAAAATTCGCACACA (SEQ ID NO. 789) was printed on the surface of the chip. As shown in
Detection assays with nucleic acid coated chip were tested both on a designed reaction system (e.g., the rig), and the benchtop reaction and LED read. More than 20 food samples were tested and the detection sensitivity of the SPN in solid surface based assay is repeatable using different solid surfaces (see, e.g.,
Detection assays using SPN-magnetic bead conjugates and nucleic acid coated chips were validated in various conditions using a variety of different food samples. The detection sensitivity is confirmed in different conditions. For assays using SPN-magnetic bead conjugates, food samples without or with 50 ppm peanut were prepared using GentleMACS homogenizer and centrifuged. The processed samples were then incubated with SPN-magnetic bead conjugates and fluorescent signals were recorded using lab-grade LED based reader. The 50 ppm sensitivity was confirmed in 20 food samples tested (
Detection assays were also performed using nucleic acid coated chips as described in Example 10. Food samples without or with 50 ppm peanut were prepared using GentleMACS homogenizer and centrifuged, or alternatively prepared using rotor-dependent homogenizer and filtered using 0.1 mm PES filter. The processed samples were then incubated with a fixed concentration of aptamer specific to peanut allergen. The mixed samples were added to the nucleic acid coated chips. After incubation and wash, the fluorescent signals were recorded using designed optical sensors. The 50 ppm sensitivity was achieved in different food samples (
The sensitivity of SPN-magnetic beads and nucleic acid coated chips was also tested in different reaction settings such as standard bench-top reaction chamber and a designed reaction chamber (e.g., the rig). The rig is a benchtop fixture that includes a motor, syringe pump and valving the enables the homogenization of the food sample, filtering through a filter bed and running the sample over the reaction chamber in which the DNA coated chip is located. The rig enables controlled washing. The detailed conditions are listed in Table 23. In all conditions tested, the 50 ppm sensitivity can be achieved.
Limited Reaction Buffer
The detection assay run on nucleic acid coated solid surface (e.g., chip) was optimized in several aspects and its sensitivity was validated in various reaction conditions. The effect of buffer volume on the detection sensitivity was tested in one study. In the nucleic acid chip assay, when the reaction volume is decreased by 50% (2.5 ml) and only half of the sample size (0.25 g per sample) was used, the detection result indicates that limited buffer volume and reduced sample size has minimal effect on the detection sensitivity of SPNs and solid surface assays (
Internal Control
To reduce background recording and signals from non-specific bindings, control signals to subtract these non-specific readings are important to optimize the assay. An internal control signal was recorded to correct signal measurement. The two control panels as shown in
Concentration of MgCl2
Fluorescent signal is likely be affected by many factors such as salts and buffer concentration, etc. The concentration of MgCl2 in reaction buffer is to optimize the detection assay to achieve high-sensitive and intense signal. 0 mM to 120 mM MgCl2 was tested and MgCl2 was added either to extraction buffer or after food sample extraction and filtration. The results suggest that addition of MgCl2 to extraction buffer completely eliminates sensitivity. The MgCl2 concentration needs to be tightly controlled between 25 mM and 50 mM. Lower or higher concentration of MgCl2 affects both sensitivity and signal intensity (
Optimization of Detection Assay on the Rig
In addition to internal control, various parameters that affect the efficiency of detection assay were optimized including the steps of pre-blocking the DNA coated chips, decreasing reaction time and washing. It is found that DNA-chip, the solid surface can be effectively blocked by BSA, which reduce non-specific signal. During the process of assaying, the reaction time can be decreased from 6 minutes to about 64 seconds. Optimized steps include removing air bubbles for both the reaction and wash, decreasing incubation time of food sample and SPNs, transitioning to a slow flow of incubation, increasing volume of reaction to flow over the chip and increasing flow rate of solution to accommodate the volume in 30 seconds. One example of the optimized reaction step and time is shown in Table 24.
In this protocol of detection reaction within 64 seconds, the assay sensitivity is retained (
Filtration
Different filters were tested and compared for efficiency in removing food particles and its effect on signal read. Three different filters: fish/netting, cotton/glass, and cotton/netting were tested and compared in 6 different food matrices spiked with peanut (Table 25). Dependent of food matrices, a different combination of filters may be used to increase allergen extraction from the sample; and to deplete other components such as fat and other particles.
Components used for the present detection assay need to be stable at ambient temperature. Examples of agents and other parts employed to run an allergen assay, include extraction buffer, wash buffer, Flagged-SPN and DNA chips. The stability test indicates that fluorescent labeled SPN (e.g., Texas-Red labeled SPN) is stable and its activity remains at least for one month at various temperatures. (Table 26) as measured with fluorescent polarization (FP). After 9 month storage, significant fluorescent signal can still be detected (
This application is a 35 U.S.C. § 371 U.S. National Stage Entry of International Application No. PCT/US2017/060487 filed Nov. 8, 2017 which claims priority to U.S. Provisional Application Ser. No. 62/418,984, filed on Nov. 8, 2016; U.S. Provisional Application Ser. No. 62/435,106, filed on Dec. 16, 2016; and U.S. Provisional Application Ser. No. 62/512,299, filed on May 30, 2017; the contents of each of which are incorporated herein by reference in their entirety.
| Filing Document | Filing Date | Country | Kind |
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| PCT/US2017/060487 | 11/8/2017 | WO | 00 |
| Publishing Document | Publishing Date | Country | Kind |
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| WO2018/089391 | 5/17/2018 | WO | A |
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| 2010121223 | Oct 2010 | WO |
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| 2013116527 | Aug 2013 | WO |
| WO-2015066027 | May 2015 | WO |
| 2018089391 | May 2018 | WO |
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| Number | Date | Country | |
|---|---|---|---|
| 20190292544 A1 | Sep 2019 | US |
| Number | Date | Country | |
|---|---|---|---|
| 62512299 | May 2017 | US | |
| 62435106 | Dec 2016 | US | |
| 62418984 | Nov 2016 | US |
