Patent Application
20110195862
The present disclosure relates to devices methods and systems for target detection. In particular, the present disclosure relates to devices methods and systems for detection of targets on a polymer array.
The application of molecular techniques has rapidly advanced the detection and identification of targets in sample where targets of various chemical natures are included. Several techniques are available that allow detection of target molecules such as polymers, and in particular biopolymers, for various purposes, including, for example, identification of microorganisms and microbial systems.
However, reproducibility and/or quantification of targets can still be challenging in particular when detection is performed using a polymer array. Chemical similarities between the target molecules can interfere with the ability to accurately detect multiple targets. In certain cases ability to predict the extent of hybridization and sensitivity of some related reporting techniques can make detection of specific molecules and related quantitation quite challenging.
Provided herein are devices, methods and systems configured for target detection through Secondary Ion Mass Spectrometry (SIMS), which, in several embodiments, allow quantitative and/or sensitive detection of targets bound to a polymer array. In particular, in several embodiments, devices methods and systems herein described allow quantitative and/or sensitive detection of target polymers presenting SIMS detectable labels following binding of the target polymers with the polymer array.
According to a first aspect a method for quantitative detection of a target is described. The method comprises, labeling a target with a SIMS detectable label, which can in particular be formed by stable isotope probes, to provide a SIMS labeled target, the SIMS labeled target capable of binding a polymer of a polymer array herein described. The method further comprises contacting the SIMS labeled target with the polymer array for a time and under conditions that allow binding of the SIMS labeled target molecule to the polymer array. The method also comprises performing SIMS detection of the polymer array following the contacting to detect the SIMS labeled target bound to the polymer array. For the polymer array, the platform comprises a substrate coated with an electrically conductive layer and the polymer is attached to the platform through a functional linker molecule attached to the electrically conductive layer.
According to a second aspect a method to detect a target in a sample is described: The method comprises exposing the sample to a label detectable by Secondary Ion Mass Spectrometry (SIMS label) for a time and under conditions that allow binding of the SIMS label with the target. The method further comprises contacting a polymer array with the sample to allow binding of the labeled target to the polymer array. The method also comprises performing Secondary Ion Mass Spectrometry on the polymer array following the contacting in order to detect the SIMS labeled target. In the polymer array, the platform comprises a substrate coated with an electrically conductive layer and the polymer is attached to the platform through a functional linker molecule attached to the electrically conductive layer.
According to a third aspect, a system for detection of a target is described, that comprises a polymer array herein described, and a SIMS detectable label. In some embodiments, the system can further include SIMS detecting elements, such as suitable pieces of equipment to perform detection of a target comprising the SIMS detectable label.
According to a fourth aspect, a functionalized platform is described, that comprises a substrate having an electrically conductive surface, the electrically conductive surface attaching a functionalized linker molecule comprising an organosilane presenting an organosilane functional group. The functionalized platform is also configured to be associated, during operation, with a polymer array, through attachment of the polymers of the polymer array with the functionalized linker molecule, and the polymer array is configured for SIMS detection of a target attached to a polymer on the polymer array, through a SIMS detectable label attached to the target.
According to a fifth aspect, a polymer array is described that is configured to allow SIMS detection of a target attached to the polymer through a SIMS detectable label attached to the target. The polymer array comprises a polymer attached to a functionalized platform described herein wherein the polymer is attached to the functionalized linker molecules of the platform.
According to a sixth aspect, a bio-chip is described that comprises a polymer array herein described.
The platforms, arrays, methods and systems described allow in several embodiments quantitative detection of targets such as a polymers and in particular biopolymer comprising nucleic acids, polypeptides and additional polymers identifiable by a skilled person.
The platforms, arrays, methods and systems described herein can be used in connection with applications wherein quantitative detection sorting and/or analysis of targets of interest and in particular nucleic acid molecules through an array is desired, including but not limited to medical application, biological analysis and diagnostics including but not limited to clinical applications.
The details of one or more embodiments of the disclosure are set forth in the accompanying drawings and the description below. Other features, objects, and advantages will be apparent from the description and drawings, and from the claims.
The accompanying drawings, which are incorporated into and constitute a part of this specification, illustrate one or more embodiments of the present disclosure and, together with the detailed description and the examples, serve to explain the principles and implementations of the disclosure.
Devices, arrays methods and systems described herein are also indicated as “Chip-SIP” that in several embodiments allow detection of a target on a polymer array through Secondary Ion Mass Spectrometry.
The term “detect” or “detection” as used herein indicates the determination of the existence, presence or fact of a target or signal in a limited portion of space, including but not limited to a sample, a reaction mixture, a molecular complex and a substrate including a platform and an array. A detection is “quantitative” when it refers, relates to, or involves the measurement of quantity or amount of the target or signal (also referred as quantitation), which includes but is not limited to any analysis designed to determine the amounts or proportions of the target or signal. A detection is “qualitative” when it refers, relates to, or involves identification of a quality or kind of the target or signal in terms of relative abundance to another target or signal, which is not quantified. In several embodiments, the Chip-SIP devices, methods and systems allows quantitative detection of single or multiple targets.
The term “target” as used herein indicates an analyte of interest. The term “analyte” refers to a substance, compound or component whose presence or absence in a sample has to be detected. Analytes include but are not limited to biomolecules and in particular biomarkers. The term “biomolecule” as used herein indicates a substance compound or component associated with a biological environment, especially the nucleic acids DNA and RNA. The term “biomarker” indicates a biomolecule that is associated with a specific state of a biological environment including but not limited to a phase of cellular cycle, health and disease state. The presence, absence, reduction, upregulation of the biomarker is associated with and is indicative of a particular state. Biomolecules that are detectable through Chip-SIP include in particular biopolymers, which in certain embodiments can also be used as biomarkers.
According to various embodiments the Chip-SIP herein described, detection of a target can be performed through Secondary Ion Mass Spectrometry analysis of a polymer array presenting a target, typically formed by one or more biopolymers.
The term “polymer array” as used herein indicates a regular and imposing grouping or arrangement of polymer molecules immobilized on an appropriate or compatible substrate in an ordered manner, herein also indicated as probe polymers. More particularly, the term polymer array indicates an ordered grouping of probe polymers arranged so to allow, under appropriate conditions, specific binding of a target to at least one of the polymer composing the polymer array and subsequent detection of the target bound to the polymer.
In Chip-SIP detection, polymer arrays are attached on a functionalized platform through linkage with functional linker molecules attached on an electrically conductive layer and presenting functional groups for binding with probe polymers.
The term “platform” as used herein indicates a physical and usually flat structure suitable for carrying a polymer array. A platform typically comprises a substrate functionalized to be capable of reacting with a polymer of the polymer array and the polymer array.
The term “substrate” as used herein indicates a base material on which processing can be conducted to modify the chemical nature of at least one surface of the base material. Exemplary chemical modifications include functionalization and/or depositing on the modified surface a layer of a second material chemically different from the base material. Exemplary substrates in the sense of the present disclosure include but are not limited to glass, such as silica-based glass, plastics, such as cyclo-olefin copolymer, carbonates and the like, and silicon materials, such as the ones used in the electronic industry. The substrate can be two dimensional such as a typical glass microscope slide of standard dimension, i.e. 25 mm×75 mm.
In platform described herein a substrate is coated with a functionalized electrically conductive layer that can be formed by a metal oxide layer. The term “layer” as used herein indicates a single thickness of material covering a surface. Accordingly, a metal oxide layer is a thickness of a metal oxide compound covering a substrate surface of the substrate of the platform or a portion thereof.
The term “metal oxide” as used herein indicates a compound including at least one oxygen atom bound to a metal atom. Exemplary metal oxides include in particular amphoteric metal oxide such as aluminum oxide and other metal oxides wherein the metal element is in a +3 oxidation state, tin oxide other metal oxides wherein the metal element is in a +4 oxidation state or mixture thereof. In an embodiment, the metal oxide comprises Indium Tin Oxide, a solid solution of indium (III) oxide (In2O3) and tin oxide (SnO2), typically 90% In2O3, 10% SnO2 by weight, which is a particularly suitable electrically conductive material.
In platform herein described, a metal oxide thickness can be applied to the substrate by deposition of the metal oxide performed by techniques identifiable by a skilled person. In particular, in several embodiments herein disclosed, the surface of a substrate is coated by the metal oxide, wherein the term “coat” and “coating” indicates a covering of the metal oxide applied to the surface using techniques known in the art. Exemplary techniques suitable to apply a coating to a substrate include chemical vapor deposition, conversion coating, plating and other techniques identifiable by a skilled person. In case of ITO thin films of indium tin oxide coating procedures can be performed by electron beam evaporation, physical vapor deposition, or a range of sputter deposition techniques. Concentration of charge carriers during deposition is selected in view of the desired electrical conductivity since a high concentration will increase the material's conductivity, but decrease its transparency.
In platforms and the microarray herein described, the metal oxide is functionalized to allow attachment of a polymer array. The terms “functionalize” and “functionalization” as used herein, indicates the appropriate chemical modifications of a molecular structure (including a substrate or a compound) resulting in attachment of a functional group to the molecular structure. The term “functional group” as used herein indicates specific groups of atoms within a molecular structure that are responsible for the characteristic chemical reactions of that structure. Exemplary functional groups include, hydrocarbons, groups containing halogen, groups containing oxygen, groups containing nitrogen and groups containing phosphorus and sulfur all identifiable by a skilled person. The term “attach” or “attached” as used herein, refers to connecting or uniting by a bond, link, force or tie in order to keep two or more components together, which encompasses either direct or indirect attachment such that for example where a first compound is directly bound to a second compound or material, and the embodiments wherein one or more intermediate compounds, and in particular molecules, are disposed between the first compound and the second compound or material.
In platforms herein described the electrically conductive layer is functionalized to attach an alkyl phosphonate compound that presents an alkyl phosphonate functional group and/or with organosilanes that presents an organosilane functional group. The term “present” as used herein with reference to a compound or functional group indicates attachment performed to maintain the chemical reactivity of the compound or functional group as attached. Accordingly, a functional group presented on a surface is able to perform under the appropriate conditions the one or more chemical reactions that chemically characterize the functional group.
In particular, in some embodiments, the metal oxide layer is treated with a solution of a functionalized alkyl phosphonate compound. In those embodiments, the phosphonates form an ordered monolayer on the metal oxide surface and are covalently linked to the metal oxide via formation of stable metal-phosphodiester bonds as has been well-established in published scientific literature. In some embodiments, the metal oxide is functionalized with an organosilane, e.g. triethoxyaminoproply silane or other organosilane identifiable by a skilled person. The alkylphosphonate functional group and/or organosilane functional groups are used to attach probe polymers of a polymer array.
The term “polymer” as used herein indicates a large molecule (macromolecule) composed of repeating structural units typically connected by covalent chemical bonds. Polymers constitute a large class of natural and synthetic materials with a variety of properties and purposes and include bio-polymers which are the typical polymer component of polymer arrays as identified herewith. Biopolymers comprise polysaccharides polymers made up of many monosaccharides joined together by glycosidic bonds, polynucleotide and polypeptides that are originally produced by living organisms including viruses.
The term “polynucleotide” as used herein indicates an organic polymer composed of two or more monomers including nucleotides, nucleosides or analogs thereof. The term “nucleotide” refers to any of several compounds that consist of a ribose or deoxyribose sugar joined to a purine or pyrimidine base and to a phosphate group and that is the basic structural unit of nucleic acids. The term “nucleoside” refers to a compound (such as guanosine or adenosine) that consists of a purine or pyrimidine base combined with deoxyribose or ribose and is found especially in nucleic acids. The term “nucleotide analog” or “nucleoside analog” refers respectively to a nucleotide or nucleoside in which one or more individual atoms have been replaced with a different atom or a with a different functional group. Accordingly, the term polynucleotide includes nucleic acids of any length including DNA, RNA, DNA or RNA analogs and fragments thereof. A polynucleotide of three or more nucleotides is also called a nucleotidic oligomers or oligonucleotide. Exemplary polynucleotides composing arrays herein disclosed are DNA molecules, and in particular DNA oligomers, peptide nucleic acids (PNAs), locked nucleic acid polymers (LNAs) and the like.
The term “peptide nucleic acid” indicates an artificially synthesized polymer similar to DNA or RNA and is used in biological research and medical treatments. PNA is not known to occur naturally. In particular, while DNA and RNA have a deoxyribose and ribose sugar backbone, respectively, whereas PNA's backbone is composed of repeating N-(2-aminoethyl)-glycine units linked by peptide bonds. The various purine and pyrimidine bases are linked to the backbone by methylene carbonyl bonds. PNAs are depicted like peptides, with the N-terminus at the first (left) position and the C-terminus at the right.
The term “locked nucleic acid”, often referred to as inaccessible RNA, indicates a modified RNA nucleotide. The ribose moiety of an LNA nucleotide is modified with an extra bridge connecting the 2′ and 4′ carbons. The bridge “locks” the ribose in the 3′-endo structural conformation, which is often found in the A-form of DNA or RNA. LNA nucleotides can be mixed with DNA or RNA bases in the oligonucleotide whenever desired. Such oligomers are commercially available. The locked ribose conformation enhances base stacking and backbone pre-organization. This significantly increases the thermal stability (melting temperature) of oligonucleotides. LNA nucleotides are used to increase the sensitivity and specificity of expression in DNA microarrays, FISH probes, real-time PCR probes and other molecular biology techniques based on oligonucleotides. For the in situ detection of miRNA the use of LNA is currently (2005) the only efficient method. A triplet of LNA nucleotides surrounding a single-base mismatch site maximizes LNA probe specificity unless the probe contains the guanine base of G-T mismatch.
The term “polypeptide” as used herein indicates an organic polymer composed of two or more amino acid monomers and/or analogs thereof. The term “polypeptide” includes amino acid polymers of any length including full length proteins and peptides, as well as analogs and fragments thereof. A polypeptide of three or more amino acids is also called a protein oligomer or oligopeptide. As used herein the term “amino acid”, “amino acidic monomer”, or “amino acid residue” refers to any of the twenty naturally occurring amino acids including synthetic amino acids with unnatural side chains and including both D and L optical isomers. The term “amino acid analog” refers to an amino acid in which one or more individual atoms have been replaced, either with a different atom, isotope, or with a different functional group but is otherwise identical to its natural amino acid analog.
The term “protein” as used herein indicates a polypeptide with a particular secondary and tertiary structure that can participate in, but not limited to, interactions with other biomolecules including other proteins, DNA, RNA, lipids, metabolites, hormones, chemokines, and small molecules. Exemplary proteins composing arrays herein described are antibodies.
The term “antibody” as used herein refers to a protein that is produced by activated B cells after stimulation by an antigen and binds specifically to the antigen promoting an immune response in biological systems and that typically consists of four subunits including two heavy chains and two light chains. The term antibody includes natural and synthetic antibodies, including but not limited to monoclonal antibodies, polyclonal antibodies or fragments thereof. Exemplary antibodies include IgA, IgD, IgG1, IgG2, IgG3, IgM and the like. Exemplary fragments include Fab Fv, Fab′ F(ab′)2 and the like. A monoclonal antibody is an antibody that specifically binds to and is thereby defined as complementary to a single particular spatial and polar organization of another biomolecule which is termed an “epitope”. A polyclonal antibody refers to a mixture of monoclonal antibodies with each monoclonal antibody binding to a different antigenic epitope. Antibodies can be prepared by techniques that are well known in the art, such as immunization of a host and collection of sera (polyclonal) or by preparing continuous hybridoma cell lines and collecting the secreted protein (monoclonal).
In polymer array herein described, any of the above polymers can be synthesized or added and in particular spotted on a coated substrate according to techniques identifiable by a skilled person.
Applicants have surprisingly found that probe polymer arrays on functionalized platforms herein described allow detection of properly labeled target performed through SIMS.
The term “SIMS” or “Secondary Ion Mass Spectrometry” as used herein indicates a technique typically used in materials science and surface science to analyze the composition of solid surfaces and thin films by sputtering the surface of the specimen with a focused primary ion beam and collecting and analyzing ejected secondary ions. These secondary ions are typically measured with a mass spectrometer or other SIMS detecting elements to determine the elemental, isotopic, or molecular composition of the surface. In several applications, SIMS is one of the most sensitive surface analysis techniques able to detect elements present in the parts per billion range. Exemplary procedures and detecting elements suitable for SIMS analysis are described for example in the enclosed references (see e.g. ref (1) and ref (15)). A skilled person will be able to identify additional instruments and procedures that are suitable for the implementation of Chip-SIP herein described upon reading of the present disclosure.
A detection performed through SIMS, or “SIMS detection” is performed through measurement of a SIMS detectable signal typically issued by a SIMS label on a surface following sputtering of the surface with a focused primary ion beam. The terms “label” and “labeled molecule” as used herein refer to any elemental label capable of detection, which in general comprise radioactive isotopes, stable isotopes, halogenated oligonucleotide probes, metal ions, nanoparticles, and the like. As a consequence the wording “labeling signal” indicates in general the signal emitted from the label that allows detection of the label.
A “SIMS label” as used herein indicates a label capable of issuing a SIMS detectable signal on a surface following sputtering of the surface with a focused primary ion beam. A “SIMS detectable signal” indicates a signal that is detectable through the use of SIMS detecting elements, (e.g. a sector, quadrupole, and time-of-flight mass analyzer) A SIMS detectable signal is typically in the form of characteristic secondary ions detectable through any appropriate SIMS detecting element as will be understood by a skilled person. Exemplary SIMS labels comprise stable isotopes wherein the term “stable isotope” refers to a non-radioactive isotopic form of an element, which can include, but is not limited to, 13C or 15N, 18O, 19E, 127I, 79Br, or 197Au.
Metal oxide layers and in particular layers formed by comprising Indium Tin Oxide (ITO), are particularly suitable for SIMS analysis because of their conductive properties and stability under reduced pressure. Presentation of probe polymers on such an electrically conducting layer, made possible by use of functionalized linker molecules such as phosphohonate or organosilane, in combination with use of SIMS detectable label has enabled a detection of single and in particular multiple targets that in some embodiments, is significantly more sensitive of corresponding approach of the art. Additional details concerning procedures specific embodiments of platform presenting alkyl phosphonate functional groups are described in US Pat. Application US 2009-0203549 and International Application WO 2009/100201, each of which is herein incorporated in its entirety.
In several embodiments of the Chip-SIP devices, methods and systems, detection of a SIMS signal issued by a SIMS labeled target bound on a polymer array herein described allows quantitative and/or qualitative detection of target.
In particular in some embodiments a quantitative detection of a target can be performed by labeling the target with a SIMS detectable label to provide a SIMS labeled target, the SIMS labeled target capable of binding a polymer of a polymer array herein described.
Suitable labeling procedures depend on the target and desired detection. For example nucleic acids can be labeled by incorporating 13C and/or 15N in the nucleic acids during synthesis which can be performed within a cell, or in vitro e.g. in a cell free system. Additional labeling can be performed by attaching gold nanoparticles or halogen atoms (F, I, Br) to DNA or RNA. In an embodiment, the labeling can be performed by exposing a sample to a SIMS-label to allow binding of the SIMS label with a target whose quantity or presence in the sample one wants to detect. The term “exposing” or “expose” or “to expose” as used herein refers to a contacting of the sample performed to allow the introduction of SIMS-label (e.g. stable isotopes) to a sample, to allow attachment of the SIMS-label in the target if present in the sample. By way of example, in embodiments where detection of nucleic acids in microbes is desired, a bacterial population can be grown on a substrate enriched with a SIMS label formed by stable isotopes. By way of example, bacteria can be grown in a liquid media substance containing stable isotopes wherein the bacteria feed off the stable isotope-containing liquid media. Additional methods for enabling the attachment of a SIMS-label onto a target in a sample are identifiable by a skilled person depending on the specific target and label selected for the detection.
In an embodiment, the SIMS labeled target resulting from a labeling procedure is then contacted with a polymer array for a time and under conditions to allow binding of the SIMS labeled target molecule to the polymer array.
In some embodiments, the contacting is performed by isolating the labeled target from a sample comprising the target (e.g. by extraction of nucleic acids from an organism) and then contacting the isolated target with a polymer array herein described.
In particular, in embodiments, where the target are also biopolymers the contacting can be performed by hybridization of the probe polymers with the target polymer. The term “hybridize” or “hybridization” or “hybridized” as used herein refers to a process by which single strands of nucleic acid sequences form double-helical segments via hydrogen bonding between complementary nucleotides covalently bonded to a functionalized platform. Other forms of specific binding between probe polymers and target herein described will be identifiable by a skilled person. Additional forms of contacting include protein-protein interactions, antigen-antibody interaction, nucleic acid protein interaction and additional interactions identifiable by a skilled person upon reading of the present disclosure.
In some embodiments, the contacting is performed with a polymer array that comprises an arrayed series of thousands of microscopic spots of the polymer of interest, called features, each containing a small amount, (e.g. picomoles) of a specific probe polymer and in particular a probe biopolymer, (for example a DNA polymer having a specific sequence). Exemplary biopolymers include, a short section of a gene or other DNA element that are used as stationary probes capable of binding to added sample molecule (target) under conditions or varying binding stringency. In an embodiment, arrays can include but are not limited to: features ranging in size from 25 square microns (μ2) to 250 square microns (μ2) that are made by mechanically (robotically) or manually spotting a defined volume of polymer on the substrate surface. In an embodiment microarrays can include but are not limited to features ranging in size from 5 square microns (μ2) to 250 square microns (μ2) that are prepared by de novo synthesis of a plurality of defined biopolymer material, e.g. DNA probes; using established solid phase synthetic chemistry. In some embodiments, probe polymers are used the comprise oligonucleotides between 25 and 50 base pairs, although one skilled in the art would recognize that oligonucleotides that are much shorter than 25 base pairs, or significantly longer than 50 base pairs could be used. Probe arrangement suitable for SIMS includes any organized arrangement where probe spots are a consistent distance apart, ideally laid out in a precise grid pattern.
Following contacting, SIMS detection of the polymer array is performed to qualitative and/or quantitatively detect the SIMS labeled target bound to the polymer array. Detecting can be performed with SIMS detecting elements which comprise many SIMS instruments having a resolution of about 10 microns or less, (e.g. a ToF-SIMS). In principle, any SIMS instrument can be used to detect the presence of stable isotopes as described above provided it can rater over a sample feature between 13-15 micron.
In embodiments, wherein an oligonucleotide array is being used to detect or sort a population of nucleic acids, the Chip-SIP approach will allow one to measure the relative amount of hybridization of the target and the surface probe. In particular, in some of those embodiments, Chip-SIP allows relatively rapid, high sensitivity measurements of complex populations of target such as RNA fragments with rapid throughput and high resolution. As demonstrated by Applicants (see Example 5 and in
In some embodiments, Chip-SIP also allows multiple labels to be used simultaneously. SIMS detection with Chip-SIP further allows for quantification of label incorporation. In some of those embodiments, Chip-SIP can be used for multiplex detection and can be used in applications such as molecular biology and in medicine to analyze/detect molecular recognition, e.g. hybridization between complementary strands of DNA and other chemical and biological properties associated with molecular recognition between biopolymers of interest.
In an embodiment, the Chip-SIP method combines polymer microarray methodology with nano-scale secondary ion mass spectrometry (NanoSIMS) analysis. In particular, Chip-SIP can be accomplished by SIMS-labeling targets, such as microbial nucleic acids (e.g. by exposing organisms and/or microbial communities to isotopically enriched substrates), contacting the SIMS labeled target with a polymer array configured for SIMS detection (e.g. hybridizing the SIMS labeled microbial nucleic acid to an engineered high-density oligonucleotide microarray as described herein), and then analyzing the polymer array binding the SIMS labeled target through NanoSIMS.
NanoSIMS is an imaging secondary ion mass spectrometer with the unprecedented combination of high spatial resolution (50 nm), high sensitivity (1 of every 20 C/N atoms) and high mass specificity (2, 3). For example, when an ITO microarray is hybridized to isotopically labeled RNA fragments, the added oligonucleotides can be quantified with NanoSIMS imaging; the conductive ITO layer uniquely facilitates generation of secondary ions for measurement and quantification. If the population of DNA oligonucleotides are assembled as a microarray on the ITO surface, a test population of complimentary nucleic acid polymers, e.g. DNA, RNA or analogs thereof (PNAs and the like) containing a stable isotope can be hybridized, and the extent of hybridization can be measured and quantified directly by NanoSIMS. Some of the current methods require substantial (15-50 atom %) enrichment of the stable-isotope, whereas Chip-SIP is able to detect small isotopic enrichments (<1 atom %). This provides for the ability to measure the level of isotopic enrichment of DNA/RNA hybridization to pre-synthesized DNA array probes, which can be used for various purposes including linking the identity of microbes to their functional roles.
In some embodiments, CHIP-SIP can be used to: connect identity to physiological function of microorganisms in most environmental or medical settings (i.e. soils. sediments, lake water marine water, insect gut, human tissue) and/or to quantify hybridization or molecular recognition events of nucleic acids on microarray surfaces. Functional roles of microorganisms include, but are not limited to, microbial biofilms pathogenic to human tissues, microbial communities involved in bioremediation, microorganisms controlling the fate of greenhouse gases, microbial communities present in a wide variety of engineered bioreactors, biodegradation of pollutants, and additional functional roles identifiable by a skilled person
In some embodiments, Chip-SIP is accomplished by isotopically-labeling microbial nucleic acids by exposing organisms and/or microbial communities to isotopically enriched substrates. The nucleic acids are then hybridized to the engineered high-density oligonucleotide microarray as described herein, and then analyzed by NanoSIMS.
In several embodiments, Chip-SIP can be used to decipher of wide variety of microbial systems having unique functional roles: microbial biofilms pathogenic to human tissues, microbial communities involved in bioremediation, microorganisms controlling the fate of greenhouse gases, microbial communities present in a wide variety of engineered bioreactors, biodegradation of pollutants, etc.
Microbial systems refer to systems formed by microorganisms. The term “microorganism” as used herein refers to prokaryotic and eukaryotic cells, which grow as single cells, or when growing in association with other cells, do not form organs. Microorganisms include, but are not limited to, bacteria, yeast, molds, protozoa, plankton and fungi. Exemplary microbial system that can be investigated with Chip SIP comprise Pseudomonas stutzeri, Vibria cholera, Bacillus cereus, Francisellia tularensis, and the cellulose-degrading and N-fixing microorganisms found in the guts of the passalid beetle Odontotaenius disjunctus In an embodiment, Chip-SIP analysis can be performed on microorganisms that are collected from a marine and/or estuarine environment.
In particular in some embodiments, nucleic acid stable isotope probing techniques (4, 5) can be used to directly connect specific substrate utilization to microbial identity. In an exemplary approach natural microbial communities are incubated in the presence of substrates enriched in rare isotopes (e.g., 13C or 15N). The organisms, including their nucleic acids, incorporate the substrate and become isotopically enriched over time. DNA- and RNA-Stable isotope probing technique exposure requires high substrate concentrations in order to meet the sensitivity threshold of density gradient separation (in many systems >20% 13C DNA) (6) and can be extremely difficult to perform with 15N substrates (>40% 15N DNA required) (7). Traditional SIP further requires long exposure times (risking community cross-feeding), low-throughput (1-2 weeks lab processing time per sample batch), and incomplete quantification. Related culture-independent approaches can link microbial identity to function and can also have ideal qualities such as high sensitivity or in situ resolution (e.g. 13C-PLFA (8); EL FISH (9), FISH MAR (10), isotope arrays (11)). In contrast, the multiple stable isotope (e.g. 15N and 13C) incorporation made possible with the Chip-SIP method combines high throughput, sensitivity, taxonomic resolution, and quantitative estimation.
Molecular approaches for detection of microbes typically target conserved biomarkers present in all organisms of interest, such as the small subunit ribosomal RNA molecule (16S rRNA for prokaryotes and 18S rRNA for eukaryotes). Detection and monitoring of bacteria and archaea routinely rely upon classifying heterogeneous 16S rRNA molecules, either as RNA or as gene fragments amplified by universal PCR.
In an embodiment described herein, cellular RNA is used as the nucleic acid to identify organisms because one skilled in the art would recognize that the higher synthesis rates of cellular RNA allows rapid response to environmental stimuli.
An embodiment described herein, rRNA is used as the nucleic acid to identify organisms. One skilled in the art would recognize that the use of rRNA facilitates the identification of organisms with higher ribosome content, which is the active fraction of a microbial community. One skilled in the art would recognize, however, that any type of natural or synthesized nucleic acid can be used with the methods and system described herein.
As described herein, following extraction from a sample population of interest, isotopically hybridized nucleic acids can be hybridized to a functional platform using probes complementary to active community microorganism. Hybridization allows the identification of each probe having a target match, as evidenced by a fluorescent signal.
In an embodiment, 16S rRNA microarrays can be used to analyze the prokaryotic composition of complex environmental samples, such as those obtained from bioaerosols (12), soils ((13) and water(14). Such microarrays take advantage of the potential for array technology to identify individual components and assess multiple samples simultaneously. The 16S rRNA PhyloChip consists of almost 9,000 sets of 25-mer oligonucleotide probes, and is exemplary of a type of 16S rRNA microarray that can be used as a functionalized platform. Each set is specific for one 16S-rRNA gene of a particular species or group of related species. Each probe set is composed of at least 11 individual perfect-match probes and their corresponding single mismatch probes, which contain one centrally located sequence mismatch. The mismatch probe allows for the assessment and control of non-specific hybridization. For data analysis using the 16S rRNA PhyloChip, a summary statistic that describes the quantity of sequence-specific hybridization to each probe set can be calculated from the ratio of perfect-match to mismatch probe fluorescence for each probe and the consistency in fluorescence across all the probes within a given probe set.
In an embodiment, 18S DNA microarrays can be used to analyze the eukaryotic composition of complex environmental samples.
In an embodiment, hybridized mixtures of 13C-RNA are combined with mixtures of RNA from multiple organisms. Such an approach can provide both a qualitative and quantitative measure (e.g. a spot can be identified as either enriched or not, and the degree of enrichment can be known by the heavy/light isotope ratio of the spot). Additionally, different organisms can be labeled to differing degrees, creating a standard curve of 13C-RNA samples, with which it can be determined the sensitivity limits and ability to generate quantitative information based upon the degree of isotope incorporation and thus intensity of 13C in individual spots. Hybridized RNA containing stable isotopes can then be quantified trough SIM detection for example using NanoSIMS as herein described.
As disclosed herein, the functionalized platform, probe polymers, polymer arrays and SIMS-label, can be provided as a part of systems to detect targets according to any of the methods described herein. The systems can be provided in the form of kits of parts.
In a kit of parts, the functionalized platform, probe polymers, polymer arrays and SIMS-label and other reagents to perform the methods can be comprised in the kit independently. One or more probe polymers and SIMS-labels can be included in one or more compositions alone or in mixtures identifiable by a skilled person. Each of the one or more of probe polymers or SIMS labels or other reagents can be in a composition together with a suitable vehicle.
Additional reagents can include molecules suitable to enhance or favor the contacting according to any embodiments herein described and/or molecules, standards and/or equipment to allow detection of pressure temperature and possibly other suitable conditions.
In particular, the components of the kit can be provided, with suitable instructions and other necessary reagents, in order to perform the methods here described. The kit will normally contain the compositions in separate containers. Instructions, for example written or audio instructions, on paper or electronic support such as tapes or CD-ROMs, for carrying out the assay, will usually be included in the kit. The kit can also contain, depending on the particular method used, other packaged reagents and materials (i.e. wash buffers and the like).
Further advantages and characteristics of the present disclosure will become more apparent hereinafter from the following detailed disclosure by way of illustration only with reference to an experimental section.
The platforms, arrays, methods and systems herein disclosed are further illustrated in the following examples, which are provided by way of illustration and are not intended to be limiting.
In particular, in the following examples platforms, array and related methods and systems are described that use ITO coated glass slide attaching nucleic acid probes through organosilanes or alkyl phosphonate and directed to detection of target biopolymers such as DNA or RNA. A skilled person will be able to adapt the exemplary materials, structures and procedure to additional supports, conductive material probes functionalized linker probes and targets in accordance with the present disclosure.
A custom conductive surface for the microarrays is used to eliminate charging during SIMS analysis. Glass slides coated with indium-tin oxide (ITO; Sigma) are treated with an amino- or hydroxy-alkyl phosphonate to provide a starting matrix for DNA synthesis (
Custom-designed microarrays (feature size=15 μm) are synthesized using a photolabile deprotection strategy (15) on the LLNL Maskless Array Synthesizer (MAS)(Roche Nimblegen, Madison, Wis.). Reagents for synthesis (Roche Nimblegen) are delivered via an automated DNA synthesizer (Expedite, PerSeptive Biosystems). For quality control (to determine that DNA synthesis was successful), each slide contains a set of DNA probes to Arabidopsis calmodulin protein kinase 6 (CPK6); the latter is detected using complimentary oligonucleotides labeled with Cy3 (Integrated DNA Technologies).
If synthesis is successful, hybridization with Cy3 or Cy5-labeled complimentary targets reveals a series of ordered fiducial marks (probe spots with the complementary sequence synthesized throughout the array area). Probes targeting microbial taxa are arranged in a densely packed formation to decrease the total area analyzed by imaging secondary ion mass spectrometry the NanoSIMS. Hybridized arrays are later analyzed using a Cameca NanoSIMS 50 which provides the critical capacity to detect isotopic enrichment in the captured ribosomal RNA fragments.
RNA from pelleted cells (for pure culture laboratory strains) and filters (for aquatic field samples) are extracted with the Qiagen RNEasy kit according to manufacturer's instructions, with slight modifications for field samples.
This protocol was used for pure cultures of P. stutzeri, V cholera and B. cereus, and it has also worked for the complex communities found in seawater and insect hindguts.
Filters are incubated in 200 μL TE buffer with 5 mg mL−1 lysozyme and vortexed for 10 min at RT. RLT buffer (800 μL, Qiagen) is then added, vortexed, centrifuged, and the supernatant transferred to a new tube. Ethanol (560 μl) is added, mixed gently, and the sample is applied to the kit-provided mini-column.
The remaining manufacturer's protocol is subsequently followed. At this point, RNA samples are split: one fraction saved for fluorescent labeling (see below), the other saved unlabeled for NanoSIMS analysis. This procedure is used because the labeling protocol introduces background carbon (mostly 12C) that dilutes the 13C signal (data not shown). Alexafluor 546 labeling is done with the Ulysis kit (Invitrogen) for 10 min at 90° C. (2 μL RNA, 10 μL labeling buffer, 2 μL Alexafluor reagent), followed by fragmentation. All RNA (fluorescently labeled or not) is fragmented using 5× fragmentation buffer (Affymetrix) for 10 min at 90° C. before hybridization. Labeled RNA is purified using a SPIN-OUT™mini-column (Millipore), and RNA is concentrated by ethanol precipitation to a final concentration of 500 ng μL−1.
For array hybridization, RNA samples in 1× Hybridization buffer (Nimblegen) are placed on Nimblegen X4 mixer slides and incubated inside a Maui hybridization system (BIOMICRO® Systems) for 18 hrs at 42° C. and subsequently washed according to manufacturer's instructions (Nimblegen). Arrays with fluorescently labeled RNA are imaged with a Genepix 4000B fluorescence scanner at pmt=650 units. Arrays with RNA that is not fluorescently labeled are marked with a diamond pen and also imaged with the fluorescence scanner to subsequently navigate to the analysis spots in the NanoSIMS.
These spots are observable in the fluorescence image because fiducial probe spots are synthesized around the outline of the area to be analyzed by NanoSIMS. Prior to NanoSIMS analysis, samples are not metal coated to avoid further dilution of the RNA's isotope ratio or loss of material Finally, slides are trimmed and mounted in custom-built stainless steel holders.
Secondary ion mass spectrometry analysis of microarrays hybridized with 13C and/or 15N rRNA is performed with a Cameca NanoSIMS 50 (Cameca, Gennevilliers, France).
A Cs+ primary ion beam is used to enhance the generation of negative secondary ions. Carbon and nitrogen isotopic ratios are determined by electrostatic peak switching on electron multipliers in pulse counting mode, alternately measuring 12C14N− and 12C15N− simultaneously for the 15N/14N ratio, and then measuring 12C14N− and 13C14N− and simultaneously for the 13C/12C ratio. Peak switching strategy is used because the secondary ion count rate for the CN− species in these samples is 5-10 times higher than any of the other carbon species (e.g., C−, CH−, C2−), and therefore higher precision is achieved even though total analytical time is split between the two CN− species at mass 27.
If only one isotopic ratio was needed, peak switching was not performed. Mass resolution is set to ˜10,000 mass resolving power to minimize the contribution of isobaric interferences to the species of interest (e.g., 11B16O− contribution to 13C14N−<1/100; 13C2− contribution to 12C14N−<1/1000). Analyses are performed in imaging mode to generate digital ion images of the sample for each ion species. Analytical conditions are optimized for speed of analysis, ability to spatially resolve adjacent hybridization locations, and analytical stability. The primary beam current is set to 5 to 7 pA Cs+, which yields spatial resolution of 200-400 nm and a maximum count rate on the detectors of ˜300,000 cps 12C14N. Analysis area is 50×50 μm2 with a pixel density of 256×256 with 0.5 or 1 ms/pixel dwell time. For peak switching, one scan of the analysis area is made per species set, resulting in two scans per analytical cycle. With these conditions, reproducible secondary ion ratios can be measured for a maximum of 4 cycles through the two sets of measurements before the sample is largely consumed.
Data are collected for 2 to 4 cycles. Based on total counts for analyzed cycles, precision of 2-3% for 13C14N and 1-4% for 15N12C can be achieved depending on the enrichment and hybridization intensity. A single microarray analysis of approximately 2500 probes, with an area of 0.75 mm2 and the acquisition of 300 images, was carried out using the Cameca software automated chain analysis in 16 hours. Ion images are stitched together and processed to generate isotopic ratios with custom software (L'IMAGE, L. Nittler, Carnegie Institution of Washington). Ion counts are corrected for detector dead time on a pixel by pixel basis.
Hybridization locations are selected by hand or with the auto-ROI function, and isotopic ratios are calculated for the selected regions over all cycles to produce the location isotopic ratios. Isotopic ratios are converted to delta values using δ=[(Rmeas/Rstandard) 1]×1000, where R the measured ratio and Rstandard is the standard ratio (0.00367 for 15N/14N and 0.011237 for Data are corrected for natural abundance ratios measured in unhybridized locations of the sample.
For each taxon identified by a microarray probe spot, isotopic enrichment of individual probe spots is plotted against fluorescence and the linear regression slope is calculated with the y-intercept constrained to natural isotope abundances (zero permil for 15N data and −20 permil for 13C data).
This calculated slope (permil/fluorescence), referred to as hybridization-corrected enrichment (HCE), is a metric that can be used to compare the relative incorporation of a given substrate by different taxa. It should be noted that due to the different natural concentrations of 13C and 15N, and more importantly, different background contributions from the microarray, HCEs for 15N substrates and 13C substrates are not comparable.
Initial tests spotted slides with synthetic DNA oligonucleotides representing/covering the genome of a strain of Francisellia tularensis, creating a DNA array. Microscopic examination of the autofluorescence of the arrays provides initial visual assessment of spotting efficiency and sample-substrate interaction.
The features on an alkyl phosphonate spotted array are approximately 150 um in diameter (
The results of the test illustrated in
A further series of experiments showed that an ITO coated array slide can be successfully analyzed by NanoSIMS. 5 μm region of ITO coated microarray was sputtered for 20 minutes. The ion plot of carbon (12C), oxygen (16O) and silicon (28Si) generated during NanoSIMS analysis of an ITO-coated microarray is illustrated in
As shown in the illustration of
A skilled person will understand that the exemplary results shown in
The suitability and stability of ITO coated arrays for SIMS analysis are demonstrated with a further series of experiments resulting in NanoSIMS analysis images following sputtering of a 5 μm region for 20 minutes.
The results illustrated in
In further proof of concept experiments, after extracting RNA from microbial cultures of Pseudomonas stuzeri exposed to 13C glucose, NanoSIMS was used to detect isotopic enrichment in P. stuzeri rRNA hybridized to oligonucleotide probe spots on a microarray.
The results of hybridization of extracted RNA from a single bacterial species (Pseudomonas stutzeri) grown on 13C-glucose as the sole carbon source are illustrated in
By imaging multiple probe spots simultaneously with the NanoSIMS, isotopically enriched nucleic acids were identified against the large background of non-enriched genes in a mixed microbial community RNA sample (
NanoSIMS measurements demonstrate detection of 13C in successfully hybridized probe spots (
Results of RNA hybridization to ITO microarray and then detection via NanoSIMS illustrated in
To demonstrate that the approach works with mixtures of nucleic acids, enriched to differing degrees, isolates of two bacterial strains (Vibrio cholerae and Bacillus cereus) were grown on multiple different enrichment levels of 13C glucose. Fluorescence and NanoSIMS analysis of the mixed 13C and 15N V. cholerae and B. cereus RNA on ITO arrays hybridized with differential isotopic enrichment shows clear separation of the two different RNA types (
This exemplary series of experiments demonstrates that the Chip-SIP method works with mixtures of RNA (from different taxa) and also with mixtures of both low and high isotope enrichment.
Additional experiments with simple two-member communities including Pseudomonas stutzeri grown on 25% 15N ammonium and Bacillus cereus grown on natural abundance ammonium demonstrate that unenriched RNA is not detected via false positive measurements. From each culture, RNA was extracted, mixed in equal concentrations, and hybridized to an ITO-coated array. Array fluorescence (
The results illustrated in
Additional experiments with simple two-member communities including Pseudomonas stutzeri grown on 100% 13C glucose and Vibrio cholera grown on 20% 13C glucose demonstrate that two different types of RNA, enriched to different levels and mixed, can be statistically separated with the Chip-SIP method (
Additionally, the experiments of
The Chip-SIP method of analyzing isotopic or elementally labeled RNA fragments on a high density ITO microarray, can be particularly useful when applied to naturally occurring environmental microbes in which a 16S rRNA and 18S rRNA microarray for common marine microbial taxa (bacteria, archaea, and protists) has been designed to target specific phylotypes (approximately at the species/genus level). In such cases, the technique allows simultaneous identification of a taxa's identity and its physiology.
Currently little is known about organic carbon incorporation patterns in marine and estuarine environments, partly because the dominant organisms are uncultured and cannot be directly interrogated in the laboratory. Applicants used the Chip-SIP method to test whether different taxa incorporate amino acids, fatty acids, and starch for their carbon growth requirements.
A Target taxa selection was performed by PhyloChip analysis and de novo probe design. RNA extracts from SF Bay SIP experiment samples were treated with DNAse I and reverse-transcribed to produce cDNA using the Genechip Expression 3′ amplification one-cycle cDNA synthesis kit (Affymetrix). The cDNA was PCR amplified with bacterial and archaeal primers, fragmented, fluorescently labeled, and hybridized to the G2 PhyloChip (6). Taxa (16S operating taxonomic units, OTU) considered to be present in the samples were identified based on 90% of the probes for that taxon being responsive, defined as the signal of the perfect match probe >1.3 times the signal from the mismatch probe. From approximately 1500 positively identified taxa, we chose a subset of 100 taxa commonly found in marine environments to target with chip-SIP. We also did not target OTUs previously identified from soil, sewage, and bioreactors as our goal was to characterize the activity of marine microorganisms. Using the Greengenes database (18) implemented in ARB (19), Applicants designed 25 probes (25 by long), to create a ‘probe set’ for each taxon (see SEQ ID NO: 1 to SEQ ID NO: 2805 of the annexed Sequence Listing incorporated herein by reference in its entirety), as well as general probes for the three domains of life. Probes for single laboratory strains (Pseudomonas stutzeri, Bacillus cereus, and Vibrio cholerae) were also designed with ARB (SEQ ID NO: 1 to SEQ ID NO: 2805 of the annexed Sequence Listing incorporated herein by reference in its entirety). Sequences of the probes are also reported in the following table
The Chip-SIP method was applied to San Francisco Bay water collected at the Berkeley Calif. pier, incubated in the presence of 200 uM 15N ammonium for 24 hours and sampled over this time. An array designed to target marine microorganisms was designed using ARB software; where each row on the array represents a series of probes designed to hybridize to a different taxon (microbial species).
A collected environmental water sample was analyzed by Chip-SIP. In particular San Francisco Bay water was collected at the Berkeley pier, and incubated with 200 uM 15N ammonium for 24 hours. An array designed to target marine microorganisms was designed using built with ARB software.
To construct the network diagram of
Agreia sp. PHSC20C1
Algoriphagus sp. PR1
Aurantimonas sp. SI85-9A1
Bacillus sp. B14905
Bacillus sp. NRRL B-14911
Bacillus sp. SG-1
Beggiatoa sp. PS
Bermanella marisrubri
Blastopirellula marina DSM 3645
Caminibacter mediatlanticus TB-2
Candidatus Blochmannia
pennsylvanicus BPEN
Candidatus Pelagibacter ubique
Carnobacterium sp. AT7
Congregibacter litoralis KT71
Croceibacter atlanticus HTCC2559
Cyanothece sp. CCY 0110
Dokdonia donghaensis MED134
Erythrobacter litoralis HTCC2594
Erythrobacter sp. NAP1
Erythrobacter sp. SD-21
Finegoldia magna ATCC 29328
Flavobacteria bacterium BAL38
Flavobacteria bacterium BBFL7
Flavobacteriales bacterium ALC-1
Flavobacteriales bacterium
Fulvimarina pelagi HTCC2506
Hoeflea phototrophica DFL-43
Hydrogenivirga sp. 128-5-R1-1
Idiomarina baltica OS145
Janibacter sp. HTCC2649
Kordia algicida OT-1
Labrenzia aggregata IAM 12614
Leeuwenhoekiella blandensis
Lentisphaera araneosa HTCC2155
Limnobacter sp. MED105
Loktanella vestfoldensis SKA53
Lyngbya sp. PCC 8106
Marinobacter algicola DG893
Marinobacter sp. ELB17
Marinomonas sp. MED121
Mariprofundus ferrooxydans PV-1
Methylophilales bacterium
Microscilla marina ATCC 23134
Moritella sp. PE36
Neptuniibacter caesariensis
Nisaea sp. BAL199
Nitrobacter sp. Nb-311A
Nitrococcus mobilis Nb-231
Nodularia spumigena CCY9414
Oceanibulbus indolifex HEL-45
Oceanicaulis alexandrii HTCC2633
Oceanicola batsensis HTCC2597
Oceanicola granulosus HTCC2516
Parvularcula bermudensis
Pedobacter sp. BAL39
Pelotomaculum thermopropionicum
Phaeobacter gallaeciensis 2.10
Phaeobacter gallaeciensis BS107
Photobacterium angustum S14
Photobacterium profundum 3TCK
Photobacterium sp. SKA34
Planctomyces maris DSM 8797
Plesiocystis pacifica SIR-1
Polaribacter irgensii 23-P
Polaribacter sp. MED152
Prochlorococcus marinus AS9601
Prochlorococcus marinus MIT 9211
Prochlorococcus marinus MIT 9301
Prochlorococcus marinus MIT 9303
Prochlorococcus marinus MIT 9515
Prochlorococcus marinus NATL1A
Pseudoalteromonas sp. TW-7
Pseudoalteromonas tunicata D2
Psychroflexus torquis ATCC
Psychromonas sp. CNPT3
Reinekea sp. MED297
Rhodobacterales bacterium
Rhodobacterales bacterium
Rhodobacterales sp. HTCC2255
Roseobacter litoralis Och 149
Roseobacter sp. AzwK-3b
Roseobacter sp. CCS2
Roseobacter sp. MED193
Roseobacter sp. SK209-2-6
Roseovarius nubinhibens ISM
Roseovarius sp. 217
Roseovarius sp. HTCC2601
Roseovarius sp. TM1035
Sagittula stellata E-37
Shewanella benthica KT99
Sphingomonas sp. SKA58
Sulfitobacter sp. EE-36
Sulfitobacter sp. NAS-14.1
Synechococcus sp. BL107
Synechococcus sp. RS9916
Synechococcus sp. RS9917
Synechococcus sp. WH 5701
Synechococcus sp. WH 7805
Ulvibacter sp. SCB49
Vibrio alginolyticus 12G01
Vibrio campbellii AND4
Vibrio harveyi HY01
Vibrio shilonii AK1
Vibrio sp. MED222
Vibrio splendidus 12B01
Vibrionales bacterium SWAT-3
For phylogenetic relationships the global 16S rRNA phylogeny in the Greengenes database (18) was opened in ARB (19) and all taxa except the targets of the array analysis were removed with the taxon pruning function.
As evident in the hybridization patterns measured (
In a similar experiment, where isotopically labeled nucleic acids, amino acids, and fatty acids were added as microbial substrates, Chip-SIP was able to identify substrate specialist and generalist taxa (
The results illustrated in
A functionalized microarray was manufactured comprising a defined plurality of single-strand DNA molecules that have been chemically synthesized on the surface of a standard glass microscope slide. Importantly, the latter has been coated with a conductive layer consisting of inorganic indium-tin-oxide (16) between 300 and 1500 angstroms in thickness—such glass microscope slides are commercially available from Sigma Chemical Company, St. Louis, Mo. The ITO surface is treated with a linker molecule to provide a starting point for DNA synthesis.
Such linker molecules contain a chemical group that reacts specifically with the ITO surface, e.g. silanes, phosphonates and the like; as well as a chemical group that provides a starting point for DNA synthesis, e.g. hydroxyl (—OH), amino (—NH2) and the like. These functionalized glass microscope slides are placed in a Maskless Array Synthesizer (MAS) unit; the MAS is programmed to synthesize a plurality of unique single-strand DNA molecules each within a feature size between 13-15 micron2. Subsequent hybridization with complementary single strand oligonucleotides containing stable isotopes, e.g. C13 and N15, results in double-stranded molecular assemblies labeled with stable isotopes. The latter can be detected and quantified by secondary mass spectrometry or SIMS as described herein.
In summary, in several embodiments, polymer arrays are described that are suitable to perform quantitative and qualitative detection as well as sorting of a target molecules and related devices methods and systems.
The examples set forth above are provided to give those of ordinary skill in the art a complete disclosure and description of how to make and use the embodiments of the compositions, systems and methods of the disclosure, and are not intended to limit the scope of what the inventors regard as their disclosure. All patents and publications mentioned in the specification are indicative of the levels of skill of those skilled in the art to which the disclosure pertains.
The entire disclosure of each document cited (including patents, patent applications, journal articles, abstracts, laboratory manuals, books, or other disclosures) in the Background, Summary, Detailed Description, and Examples is hereby incorporated herein by reference. All references cited in this disclosure are incorporated by reference to the same extent as if each reference had been incorporated by reference in its entirety individually.
Further, the sequence listing annexed herewith in computer readable form forms integral part of this description and is incorporated herein by reference in its entirety.
It is to be understood that the disclosures are not limited to particular compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise. The term “plurality” includes two or more referents unless the content 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 the disclosure pertains.
When a Markush group or other grouping is used herein, all individual members of the group and all combinations and possible subcombinations of the group are intended to be individually included in the disclosure. Every combination of components or materials described or exemplified herein can be used to practice the disclosure, unless otherwise stated. One of ordinary skill in the art will appreciate that methods, device elements, and materials other than those specifically exemplified can be employed in the practice of the disclosure without resort to undue experimentation. All art-known functional equivalents, of any such methods, device elements, and materials are intended to be included in this disclosure. Whenever a range is given in the specification, for example, a temperature range, a frequency range, a time range, or a composition range, all intermediate ranges and all subranges, as well as, all individual values included in the ranges given are intended to be included in the disclosure. Any one or more individual members of a range or group disclosed herein can be excluded from a claim of this disclosure. The disclosure illustratively described herein suitably can be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein.
Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the products, methods and system of the present disclosure, exemplary appropriate materials and methods are described herein.
A number of embodiments of the disclosure have been described. The specific embodiments provided herein are examples of useful embodiments of the disclosure and it will be apparent to one skilled in the art that the disclosure can be carried out using a large number of variations of the functionalized platforms, arrays, compositions, methods steps, and systems set forth in the present description. As will be obvious to one of skill in the art, methods and devices useful for the present methods can include a large number of optional composition and processing elements and steps.
It will be understood that various modifications may be made without departing from the spirit and scope of the present disclosure. Accordingly, other embodiments are within the scope of the following claims.
This application claims priority to U.S. Provisional Application No. 61/302,535 entitled “Using Phylogenetic Probes For Quantification Of Stable Isotope Labeling And Microbial Community Analysis” filed on Feb. 8, 2010, docket IB-2774P1 and with U.S. Provisional Application No. 61/302,827 entitled “Chip-SIP: Quantification of Nucleic Acid Stable Isotope Labeling with Biopolymer Microarrays and Secondary Ionization Mass Spectrometry (SIMS)” filed on Feb. 9, 2010, with docket number IL-12105, each of which is incorporated herein by reference in its entirety. The present application may also be related to U.S. application Ser. No. 12/366,476 entitled “ ”Functionalized platform for arrays configured for optical detection of targets and related arrays, methods and systems” filed on Feb. 5, 2009 with docket IL-11703, and to U.S. application Ser. No. ______, entitled “Using Phylogenetic Probes For Quantification Of Stable Isotope Labeling And Microbial Community Analysis” filed on Feb. 8, 2011 with docket IB-2774, each of which is herein also incorporated by reference in its entirety.
The United States Government has rights in this invention pursuant to Contract No. DE-AC52-07NA27344 between the United States Department of Energy and Lawrence Livermore National Security, LLC for the operation of Lawrence Livermore National Laboratory
| Number | Date | Country | |
|---|---|---|---|
| 61302535 | Feb 2010 | US | |
| 61302827 | Feb 2010 | US |
