Patent Application
20150031571
1. Field of the Invention
The present invention is directed to compositions of matter for use in devices for the chemiluminescent-based detection of analytes.
2. Description of Related Art
Chemiluminescence is a process in which visible light is emitted as a result of chemical reactions. It has been widely utilized for forensic investigations by spraying luminol (5-amino-2,3-dihydro-1,4-phthalazine-dione) to identify dried bloodstains. Recently, luminol was attached to gold nanoparticles using 3-mercaptopropionic acid and then functionalized with antibodies (luminol-AuNP-Ab) and stored in solution at 4° C. for use in a sandwich immunoassay for the detection of carcinoembryonic antigen in serum involving antibody immobilized magnetic beads (MBs-Ab) as described in Yang et al., Luminol/antibody labeled gold nanoparticles for chemiluminescence immunoassay of carcinoembryonic antigen, Anal Chim Acta 666 (1-2) 91-96 (2010). Such an assay requires the luminol-AuNP-Ab solution to be refrigerated, requires expensive equipment to perform, and requires incubation of times of several hours to permit the immunocomplex to form. The poor stability, short shelf life, and lack of specificity to particular targets (microbes or pathogens) of antibodies limit this method for broad applications. Thus, there remains a need for improved chemiluminescent-based detection systems.
The present invention is directed to novel chemiluminescent nanoparticles and their use in chemiluminescent detection systems. In one aspect, the detection system for detecting an analyte in a sample comprises a light-shielding container having a fiberoptic cable for transmitting light generated within the light-shielding container to a photodetector; a plurality of functionalized nanoparticles deposited in solid form on or within a support, such that the support is located within the light-shielding container; wherein the functionalized nanoparticles comprise nanoparticles covalently attached to one or more chemiluminescent moieties; and a reagent system which causes the chemiluminescent moieties to produce light in the presence of the reagent system and the analyte in the sample.
In another aspect, the detection system is designed to detecting a bacterium or virus in a sample and comprises a light-shielding container having a fiberoptic cable for transmitting light generated within the light-shielding container to a photodetector; a support located within the light-shielding container, the support having a sample application region, a test region, and a control region; a plurality of first functionalized nanoparticles deposited in solid form on or within the sample application region of the support, wherein the first functionalized nanoparticles comprise nanoparticles covalently attached to a chemiluminescent moiety and a first oligonucleotide probe capable of selectively hybridizing to the bacterium or virus nucleic acids; a plurality of second particles functionalized with a second oligonucleotide probe capable of selectively hybridizing to the bacterium or virus nucleic acids, the second particles immobilized on or within the test region of the support; a plurality of third particles functionalized with a third oligonucleotide probe capable of selectively hybridizing to the first oligonucleotide probe, the third particles immobilized on or within the control region of the support; and a reagent system which causes the chemiluminescent moiety to produce light in the presence of the reagent system and the first functionalized nanoparticles.
Additional aspects of the invention, together with the advantages and novel features appurtenant thereto, will be set forth in part in the description which follows, and in part will become apparent to those skilled in the art upon examination of the following, or may be learned from the practice of the invention. The objects and advantages of the invention may be realized and attained by means of the instrumentalities and combinations particularly pointed out in the appended claims.
The present invention is directed to chemiluminescent compositions of matter comprising nanoparticles 10 covalently attached via one or more linkers 20 to one or more chemiluminescent moieties 30 that are optionally further functionalized with one or more probe moieties 40. The chemiluminescent compositions of matter of the present invention are generally shown in
The nanoparticles of the present invention are preferably less than about 1 micron in size, for example, about 1000, 900, 800, 700, 600, 500, 400, 300, 200, or 100 nm. In some embodiments, the average or mean diameter of the nanoparticles is between about 2 to about 100 nm, and most preferably between about 2 to 50 nm. In some embodiments, the average or mean diameter of the nanoparticles is about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 nm (or some range therebetween).
The nanoparticles may have various morphologies or structures. Non-limiting examples of suitable regular shapes of the metal nanoparticles include spheres, oblate spheres, prolate spheroids, ellipsoids, rods, cylinders, cones, disks, cubes, and rectangles. Preferably, the nanoparticles are generally spherical in shape.
The nanoparticles may be comprised of various materials used in conventional diagnostic assays. Non-limiting examples include Al2O3, TiO2, ZrO2, Y2O3, SiO2, ferric oxide, ferrous oxide, a rare earth metal oxide, a transitional metal oxide, mixtures thereof, and alloys thereof. Additional non-limiting examples of metals include aluminum, gold, silver, stainless steel, iron, titanium, cobalt, nickel, and alloys thereof. In certain embodiments, nanoparticle may be comprised of biodegradable polymers, non-biodegradable water-soluble polymers, non-biodegradable non-water soluble polymers, and biopolymers. Non-limiting examples include such materials as poly(styrene), poly(urethane), poly(lactic acid), poly(glycolic acid), poly(ester), poly(alpha-hydroxy acid), poly(epsilon-caprolactone), poly(dioxanone), poly(orthoester), poly(ether-ester), poly(lactone), poly(carbonate), poly(phosphazene), poly(phosphonate), poly(ether), poly(anhydride), mixtures thereof and copolymers thereof. In one aspect, the nanoparticles are preferably comprised of metals, and most preferably, the nanoparticles used in the compositions of the present invention are comprised of gold. It is believed that the chemiluminescent signal is enhanced due to (a) charge transfer at the gold nanoparticles, and (b) aggregation of the gold nanoparticles.
The linker is preferably a linear molecule with functional groups at the two ends, one of which can form covalent bond with the nanoparticles and the other one is preferably a carboxylic group. The example linker to be used on gold nanoparticles is preferably derived from carboxylic acid having a terminal thiol group. Exemplary carboxylic acids include the C5 to C20 carboxylic acids (e.g., C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, or C20), such as mercaptoundecanoic acid (“MUA”). A carboxy activating agent is used for the coupling of primary amines in the chemiluminescent material to yield amide bonds. Preferably, diimides and amine-reactive N-hydro-succinimide esters, such as 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (“EDC”) and N-hydrosuccinimide (“NHS”), are used for this coupling step. The linker may be used to attach the chemiluminescent moiety to the nanoparticle and/or to attach the probe moiety to the nanoparticle.
Suitable chemiluminescent agents include amine-reactive luminol derivatives, microperoxidasies, acridinium esters, peroxidases, and derivatives thereof. The chemiluminescent moiety is preferably a diacylhydrazides. Exemplary chemiluminescent moieties include, but are not limited to, luminol, N-(4-aminobutyl)-N-ethylisoluminol, 4-aminophthalhydrazide monohydrate, bis(2-carbopentyloxy-3,5,6-trichlorophenyl) oxalate, 9,10-bis(phenylethynyl)anthracene, 5,12-bis(phenylethynyl)naphthacene, 2-chloro-9,10-bis(phenylethynyl)anthracene, 1,8-dichloro-9,10-bis(phenylethynyl)anthracene, Lucifer Yellow CH dipotassium salt, Lucifer yellow VS dilithium salt, 85% (dye content), 2,4,5-Triphenylimidazole, 9,10-Diphenylanthracene, Rubrene, or Tetrakis(dimethylamino)ethylene.
The chemiluminescent nanoparticles may also optionally include a probe moiety having a recognition portion that can recognize and bind to the target of interest (analyte). In the present invention, the nanoparticles are preferably functionalized with an oligonucleotide probe that is complementary to and capable of selectively hybridizing to a target polynucleotide. The term “hybridization” as used herein refers to the process in which two single-stranded polynucleotides bind non-covalently to form a stable double-stranded polynucleotide; triple-stranded hybridization is also theoretically possible.
The oligonucleotide probe is preferably capable of selectively hybridizing to a segment of the nucleic acid contents (the analyte) in a target bacterium or virus. The target virus may be single or double stranded or DNA-based or RNA-based. In one aspect, the target virus is selected from the group consisting of Parvoviridae, Papovaviridae, Adenoviridae, Hepadnaviridae, Herpesviridae, Iridoviridae, and Poxyiridae. For example, it is intended that the present invention encompass methods for the detection of any DNA-containing virus, including, but not limited to Hepatitis B, parvoviruses, dependoviruses, papillomaviruses, polyomaviruses, mastadenoviruses, aviadenoviruses, hepadnaviruses, simplexviruses (such as herpes simplex virus 1 and 2), varicelloviruses, cytomegaloviruses, muromegaloviruses, lymphocryptoviruses, thetalymphocryptoviruses, rhadinoviruses, iridoviruses, ranaviruses, pisciniviruses, orthopoxviruses, parapoxviruses, avipoxviruses, capripoxviruses, leporipoxviruses, suipoxviruses, yatapoxviruses, and mulluscipoxvirus). Thus, it is not intended that the present invention be limited to any DNA virus family. In further embodiments, the target virus is selected from the group consisting of Picornaviridae, Caliciviridae, Reoviridae, Togaviridae, Flaviviridae, Orthomyxoviridae, Paramyxoviridae, Arenaviridae, Rhabdoviridae, Coronaviridae, Bunyaviridae, and Retroviridae. For example, it is intended that the present invention encompass methods for the detection of RNA-containing virus, including, but not limited to enteroviruses (e.g., polioviruses, Coxsackieviruses, echoviruses, enteroviruses, hepatitis A virus, encephalomyocarditis virus, mengovirus, rhinoviruses, and aphthoviruses), caliciviruses, reoviruses, orbiviruses, rotaviruses, Birnaviruses, alphaviruses, rubiviruses, pestiviruses, flaviviruses (e.g., hepatitis C virus, yellow fever viruses, dengue, Japanese, Murray Valley, and St. Louis encephalitis viruses, West Nile fever virus, Kyanasur Forest disease virus, Omsk hemorrhagic fever virus, European and Far Eastern tick-borne encephalitis viruses, and louping ill virus), influenza viruses (e.g., types A, B, and C), paramyxoviruses, morbilliviruses, pneumoviruses, veisculoviruses, lyssaviruses, filoviruses, coronaviruses, bunyaviruses, phleboviruses, nairoviruses, uukuviruses, hantaviruses, sarcoma and leukemia viruses, oncoviruses, HTLV, spumaviruses, lentiviruses, arenaviruses, and human immunodeficiency virus (HIV). Thus, it is not intended that the present invention be limited to any RNA virus family.
The target bacterium may be any suitable bacterial species (spp.), for example, Bacillus spp. (e.g., Bacillus anthracis), Bordetella spp. (e.g., Bordetella pertussis), Borrelia spp. (e.g., Borrelia burgdorferi), Brucella spp. (e.g., Brucella abortus, Brucella canis, Brucella melitensis, Brucella suis), Campylobacter spp. (e.g., Campylobacter jejuni), Chlamydia spp. (e.g., Chlamydia pneumoniae, Chlamydia psittaci, Chlamydia trachomatis), Clostridium spp. (e.g., Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani), Corynebacterium spp. (e.g., Corynebacterium diptheriae), Enterococcus spp. (e.g., Enterococcus faecalis, Enterococcus faecum), Escherichia spp. (e.g., Escherichia coli), Francisella spp. (e.g., Francisella tularensis), Haemophilus spp. (e.g., Haemophilus influenza), Helicobacter spp. (e.g., Helicobacter pylori), Legionella spp. (e.g., Legionella pneumophila), Leptospira spp. (e.g., Leptospira interrogans), Listeria spp. (e.g., Listeria monocytogenes), Mycobacterium spp. (e.g., Mycobacterium leprae, Mycobacterium tuberculosis), Mycoplasma spp. (e.g., Mycoplasma pneumoniae), Neisseria spp. (e.g., Neisseria gonorrhea, Neisseria meningitidis), Pseudomonas spp. (e.g., Pseudomonas aeruginosa), Rickettsia spp. (e.g., Rickettsia rickettsii), Salmonella spp. (e.g., Salmonella enterica, Salmonella typhi, Salmonella typhinurium), Shigella spp. (e.g., Shigella sonnei), Staphylococcus spp. (e.g., Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, coagulase negative staphylococcus (e.g., U.S. Pat. No. 7,473,762)), Streptococcus spp. (e.g., Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyrogenes), Treponema spp. (e.g., Treponema pallidum), Vibrio spp. (e.g., Vibrio cholerae), and Yersinia spp. (e.g., Yersinia pestis). Other bacterial species not listed above can also be detected as would be understood by one of skill in the art.
The oligonucleotide probe is typically comprised of 10 to 100 deoxyribonucleotides or ribonucleotides, preferably about 20 to 50 nucleotides (e.g., 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, or 50 nt in length), and most preferably about 20 to 30 nucleotides.
In a preferred aspect, the oligonucleotide probe is capable of selectively hybridizing the RNAs of the Hepatitis C virus (“HCV”) or the reverse-transcripted DNAs. HCV is a member of the Flaviviridae family. More specifically, HCV has about 9.5 kb sized (+)-RNA (single stranded positive-sense RNA) genome inside its membrane. The RNA genome consists of an untranslational region (“UTR”) at 5′ and 3′ ends and a long open reading frame (“ORF”). This ORF is expressed as a polyprotein including 3,010 to 3,040 amino acids by host cell enzymes and divided into 3 structural proteins and 6 nonstructural proteins by the host cell and its own protease. Also, there is a uniformly conserved region in the 5′ and 3′ end of the genome, respectively. This region is believed to play an important role for protein formation and RNA replication of the virus. The long ORF is expressed as a polyprotein, and through co-translational or post-translational processing, it is processed into structural proteins, i.e., core antigen protein (core) and surface antigen protein (E1, E2), and nonstructural proteins, NS2 (protease), NS3 (serine protease, helicase), NS4A (serine protease cofactor), NS4B (protease cofactor, involved in resistance), NS5A, and NS5B (RNA dependent RNA polymerase, RdRp), each contributing to replication of virus. The structural proteins are divided into core, E1 and E2 by signal peptidase of the host cell. Meanwhile, the nonstructural proteins are processed by serine protease (“NS3”) and cofactor (“NS2,” “NS4A,” and “NS4B”) of the virus. The core antigen protein together with surface antigen protein of the structural protein compose a capsid of the virus, and the nonstructural proteins like NS3 and NS5B play an important part of the RNA replication of the virus (see Bartenschager, Molecular targets in inhibition of hepatitis C virus replication, Antivir. Chem. Chemother. 8 281-301 (1997)).
Similar to other Flaviviruses, the 5′ and 3′ ends of the virus RNA has a uniformly conserved untranslational region. Generally, this region is known to play a very important role in replication of the virus. The 5′ end has 5′-UTR composed of 341 nucleotides, and this part has the structure of 4 stem and loop (I, II, III, and IV). Actually, this part functions as an internal ribosome entry site (“IRES”) necessary for translation processing to express protein. Particularly, the stem III, which has the biggest and most stable structure and has a conserved sequence, has been reported to play the most essential part for ribosome binding. In addition, it is known that proteins of the virus are expressed by initiating translation processing from AUG that exists in the single RNA of the stem IV (see Stanley et al., Internal ribosome entry sites within the RNA genomes of hepatitis C virus and other Flaviviruses, seminars in Virology 8 274-288 (1997)). Moreover, the 3′ end has 3′-UTR composed of 318 nucleotides. This part is known to play a very important role in initiation step of binding of NS5B, an essential enzyme of RNA replication. The 3′-UTR, according to the sequence and tertiary structure, is composed of three different parts: -X-tail-5′ starting from the 5′ end to 98th nucleotide (98 nt), -poly(U)- having UTP consecutively, and the rest of 3′-UTR-. More specifically, X-tail-5′ part consists of 98 nucleotides having a very conserved sequence, and has three stem and loop structures, thereby forming a very stable tertiary structure. Probably, this is why X-tail-5′ part is considered very essential of NS5B binding. Also, it is reported that -poly(U)- part induces a pyrimidine track, thereby facilitating RNA polymerase effect. Lastly, the rest of the 3′-UTR has the tertiary structure of loop and plays an important role in NS5B binding. However, its structure is somewhat unstable. Overall, the 3′ end region of HCV RNA is known to have an essential structure in NS5B binding when the RNA replication starts (see Yamada et al., Genetic organization and diversity of the hepatitis C virus genome, Virology 223 255-281 (1996)). Oligonucleotide probes complementary to these regions are most preferred. Most preferred oligonucleotide probes are complementary to the X-tail and are set forth below:
It will be appreciated that the sequence of the oligonucleotide probe will be a function of the target virus or bacterium. Four exemplary sequences for the influenza hemagglutinin (HA) virus are as follows:
Likewise, three exemplary sequences for influenza neuraminidase (NA) are as follows:
Exemplary sequences for Chlamydia trachomatis DNA sequences are as follows:
The probes may be of any length that would selectively hybridize to the target bacterium or virus, and for example may be, for example, about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 50, 75, 100, 150, 200, 300, 400, or about 500 nucleotides in length. Probes may also include additional sequence at their 5′ and/or 3′ ends so that they extent beyond the target sequence with which they hybridize. Variant nucleotide sequences may also be used, such as those having about 75%, 80%, 85%, desirably about 90% to 95% or more, and more suitably about 98% or more sequence identity to that particular nucleotide sequence as determined by sequence alignment programs known in the art. The term “sequence identity” means that two polynucleotide sequences are identical (i.e., on a nucleotide-by-nucleotide basis) over the window of comparison. The term percentage of sequence identity means that two polynucleotide sequences are identical (i.e., on a nucleotide-by-nucleotide basis) over the window of comparison. The term “percentage of sequence identity” is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, U, or I) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. Thus, in one aspect, the oligonucleotide probe comprises a nucleotide sequence that shares at least 75, 80, 85, 90, 95, 98, or 100% sequence identity with the sequence of any one of SEQ ID NO: 1 to 9.
The term “selectively hybridize” means to detectably and specifically bind. The probes selectively hybridize to nucleic acid strands under hybridization conditions that minimize appreciable amounts of detectable binding to nonspecific nucleic acids. High stringency conditions can be used to achieve selective hybridization conditions as known in the art and discussed herein.
In a preferred embodiment, the oligonucleotide probe is attached to the nanoparticle via a linker. The linker may be the same or different from the linker used to attach the chemiluminescent moiety to the nanoparticle. The linker for gold nanoparticles is preferably derived from carboxylic acid having a terminal thiol group. Exemplary carboxylic acids include the C5 to C18 carboxylic acids, such as MUA. A carboxy activating agent is for the coupling of primary amines in the chemiluminescent material to yield amide bonds. Preferably, diimides and amine-reactive N-hydro-succinimide esters, such as EDC and NHS are used for this coupling step. The linker may be used to attach the chemiluminescent agent to the nanoparticle and/or to attach the probe moiety to the nanoparticle.
In another aspect, the nanoparticles may be functionalized with an oligonucleotide probe as is generally described in Mirkin et al., U.S. Application No. 2009/0325812, which is incorporated by reference in its entirety. For instance, oligonucleotides functionalized with alkanethiols at their 3′-termini or 5′-termini readily attach to gold nanoparticles. See Whitesides, Proceedings of the Robert A. Welch Foundation 39th Conference On Chemical Research Nanophase Chemistry, Houston, Tex. pages 109-121 (1995). See also, Mucic et al., Synthesis and characterization of DNA with ferrocenyl groups attached to their 5′-termini: electrochemical characterization of a redox-active nucleotide monolayer, Chem. Commun. 555-557 (1996) (describes a method of attaching 3′ thiol DNA to flat gold surfaces; this method can be used to attach oligonucleotides to nanoparticles). The alkanethiol method can also be used to attach oligonucleotides to other metal, semiconductor, and magnetic colloids and to the other nanoparticles listed above. Other functional groups for attaching oligonucleotides to solid surfaces include phosphorothioate groups (see, e.g., U.S. Pat. No. 5,472,881 for the binding of oligonucleotide-phosphorothioates to gold surfaces), substituted alkylsiloxanes (see, e.g., Burwell, Chemical Technology, 4, 370-377 (1974) and Matteucci et al., Synthesis of deoxyoligonucleotides on a polymer support, J. Am. Chem. Soc. 103 3185-3191 (1981) for binding of oligonucleotides to silica and glass surfaces, and Grabar et al., Preparation and characterization of Au colloid monolayers, Anal. Chem. 67 735-743 (1995) for binding of aminoalkylsiloxanes and for similar binding of mercaptoaklylsiloxanes). Oligonucleotides terminated with a 5′ thionucleoside or a 3′ thionucleoside may also be used for attaching oligonucleotides to solid surfaces. The following references describe other methods which may be employed to attached oligonucleotides to nanoparticles: Nuzzo et al., J. Am. Chem. Soc. 109, 2358 (1987) (disulfides on gold); Allara and Nuzzo, Langmuir 1, 45 (1985) (carboxylic acids on aluminum); Allara and Tompkins, J. Colloid Interface Sci. 49 410-421 (1974) (carboxylic acids on copper); Iler, The Chemistry Of Silica, Chapter 6 (Wiley 1979) (carboxylic acids on silica); Timmons and Zisman, J. Phys. Chem. 69 984-990 (1965) (carboxylic acids on platinum); Soriaga and Hubbard, J. Am. Chem. Soc. 104, 3937 (1982) (aromatic ring compounds on platinum); Hubbard, Acc. Chem. Res. 13, 177 (1980) (sulfolanes, sulfoxides and other functionalized solvents on platinum); Hickman et al., J. Am. Chem. Soc. 111, 7271 (1989) (isonitriles on platinum); Maoz and Sagiv, Langmuir 3, 1045 (1987) (silanes on silica); Maoz and Sagiv, Langmuir 3, 1034 (1987) (silanes on silica); Wasserman et al., Langmuir 5, 1074 (1989) (silanes on silica); Eltekova and Eltekova, Langmuir 3, 951 (1987) (aromatic carboxylic acids, aldehydes, alcohols and methoxy groups on titanium dioxide and silica); Lee et al., J. Phys. Chem. 92, 2597 (1988) (rigid phosphates on metals).
The analyte of interest (typically the nucleic acids of a target bacterium or virus) is contained in the sample to be tested. The term “sample” as used herein refers to any sample that could contain an analyte for detection. The sample may be of entirely natural origin, of entirely non-natural origin (such as of synthetic origin), or a combination of natural and non-natural origins. A sample may include whole cells (such as prokaryotic cells, bacterial cells, eukaryotic cells, plant cells, fungal cells, or cells from multi-cellular organisms including invertebrates, vertebrates, mammals, and humans), tissues, organs, lysates, or biological fluids (such as, but not limited to, blood, serum, plasma, urine, semen, and cerebrospinal fluid). Thus, a sample includes but is not limited to, a cell, a tissue (e.g., a biopsy), the lysates, a biological fluid (e.g., blood, plasma, serum, cerebrospinal fluid, amniotic fluid, synovial fluid, urine, lymph, saliva, anal and vaginal secretions, perspiration, semen, lacrimal secretions of virtually any organism, with mammalian samples being preferred and human samples being particularly preferred). A sample may be an extract made from biological materials, such as from prokaryotes, bacteria, eukaryotes, plants, fungi, multi-cellular organisms or animals, invertebrates, vertebrates, mammals, non-human mammals, and humans. A sample may be an extract made from whole organisms or portions of organisms, cells, organs, tissues, fluids, whole cultures, or portions of cultures, or environmental samples or portions thereof. In addition to the target analyte, in some embodiments the sample may comprise any number of other substances or compounds, as known in the art. In some embodiments, sample refers to the original sample modified prior to analysis by any steps or actions required. Such preparative steps may include washing, fixing, staining, diluting, concentrating, decontaminating, lysis, or other actions to facilitate analysis. A sample may need minimal preparation (for example, collection into a suitable container) for use in a method of the present invention, or more extensive preparation (such as, but not limited to removal, inactivation, or blocking of undesirable material or contaminants, filtration, size selection, affinity purification, cell lysis or tissue digestion, concentration, or dilution).
As discussed below, the nanoparticles serve as carriers with a relatively large surface area to ensure the functionalization of a relatively large quantity of chemiluminescent molecules (and optionally oligonucleotide probes).
In one aspect, the present invention enables the application of nanoparticle-functionalized chemiluminescence for detection of analytes contained in solution. However, the functionalized chemiluminescent nanoparticles are preferably deposited in dry form on a support and may be stored at ambient temperatures. As used herein, “support” is interchangeable with terms such as “solid support,” “solid carrier,” “solid phase”, “surface,” “membrane” or “resin.” All supports comprise at least one surface. Surfaces can be planar, substantially planar, or non-planar.
A support can be comprised of organic polymers such as polystyrene, polyethylene, polypropylene, polyfluoroethylene, polyethyleneoxy, polyacrylamide, polisiloxanes, as well as co-polymers and grafts of any of the foregoing. Some other exemplary support materials include, but are not limited to, latex, polystyrene, polytetrafluoroethylene (“PTFE”), polyvinylidene difluoride (“PVDF”), nylon, polyacrylamide, or poly(styrenedivinylbenzene), or polydimethylsiloxane (“PDMS”). A support can also be inorganic, such as glass, silica, or controlled-pore-glass (“CPG”). The configuration of a support can be in the form of a bead, a sphere, a particle, a granule, a gel, or a membrane. Some non-limiting examples of suitable supports include, but are not limited to, microparticles, nanoparticles, chromatography supports, membranes, or microwell surfaces. Supports can be porous or non-porous, and can have swelling or non-swelling characteristics. Supports can be rigid or can be pliable. A support can be configured in the form of a well, depression, or other container, vessel, feature, or location. A plurality of supports can be configured in an array at various locations, addressable for robotic delivery of reagents, or by detection methods and/or instruments.
Thus, in an exemplary aspect, the present invention is directed to a device comprising a light-shielding container having a fiberoptic cable for transmitting light generated within said light-shielding container to a photon detector. A plurality of functionalized nanoparticles are deposited or captured on or within the support in solid form and placed inside the container. Typically, the functionalized nanoparticles are deposited in a carrier solution, and then the carrier solution is allowed to evaporate leaving the nanoparticles in solid form on or within the support. The area on the support that the nanoparticles are deposited may be the loading region on a lateral flow device (test strip or thin film chromatography).
In one aspect, the chemiluminescent compositions of matter of the present invention are used in a multiwell format. An exemplary device is illustrated in panel a of
More specifically, in panel a of
The nanoparticles of the present invention are also well suited for use in a test strip format based on specific affinity binding between the probe (e.g., oligonucleotide probe) and the analyte (the nucleic acid contents of the target bacterium or virus of interest). An exemplary device is illustrated in panel b of
Preferred materials for the test strip pad include thin layer of silica gel, aluminum oxide, or cellulose on glass, plastic, or aluminum foil, etc. Further, similar to the common diagnostic test strips (also called rapid lateral flow test strips), and preferred for water sample, the device may comprise a single strip or a stack of several porous films including paper, nitrocellulose membranes, woven meshes, cellulose filters, thin mats of pre-spun fibers of cellulose, glass, or plastic (such as polyester, polypropylene, or polyethylene).
To immobilize particles on the test strip pads, commercially available membrane sheets (in normal paper size) can be used. Ink printing or a drawing pin can be used to deposit the latex microparticles (or other microparticles such as silica, alumina, iron oxides, etc.) on the testing and control lines. After the solvent is evaporated, the microparticles are left on the membrane surface. The latex beads are covalently attached with the testing probes and control probes, respectively.
In use, the sample is placed before the application pad region 260. As the sample 205 flows through the test pad (typically via capillary action), the bacterium or viral analyte (e.g., the nucleic acids of a Hepatitis C virus) selectively hybridizes to the oligonucleotide probe of the luminol-functionalized nanoparticles 10. The mobile bacterium or virus/Probe-CL-NP hybrid is then captured in the test region 270 by the second hybridization reaction between the nucleic acids of bacterium or virus analyte and the immobilized particles functionalized with the second oligonucleotide probe 275. The immobilized particles functionalized with the third oligonucleotide probe 285 capture the remaining functionalized nanoparticles (Probe-CL-NP) as they flow through the control pad region 280.
When the reagent system 265 (e.g., NaOH and hydrogen peroxide and iron) is added to the test pad region 270 and confined within a blackened elastomer tubing wrapping around the bundle of fiberoptics 220 and reagent-delivery microtubing by pressing the assembly against the test strip, the chemiluminescent moiety of the functionalized nanoparticles 10 will produce light. A substantial portion of the light is collected and transmitted via the fiberoptic cable 220 to the photo detector 230. The assembly is then raised, moved on top of the control pad region 280, and pressed down for similar reagent injection and chemiluminescence reading. If the nucleic acids of the target bacterium or virus are present in the sample, chemiluminescence will be observed in the test pad region 270 because the bacterium or virus/Probe-CL-NP hybrid will be captured in the test pad region 270. In the absence of the nucleic acids of the target bacterium or virus, no chemiluminescence is observed in the test pad region 270. The observation of chemiluminescence in the control pad region 280, however, illustrates that the test pad is working properly since excess Probe-CL-NP will be captured in the control pad region 280.
It will be appreciated that the test strip of the present invention may take a shape of a rectangle, circle, oval, triangle, and other various shapes, provided that there should be at least one direction along which a test solution moves by capillarity. In case of an oval or circular shape, in which the test solution is initially applied to the center thereof, there are different flow directions. However, what is taken into consideration is that the test solution should move in at least one direction toward a predetermined position containing the immobilized second probe. The thickness of the test strip according to the present invention is usually 0.1 to 2 mm, more usually 0.15 to 1 mm, preferably 0.2 to 0.7 mm, though it is not important. In general, a minimum thickness is determined depending on the strength of the strip material, the sorption capability for providing the capillary lateral flow, and needs for producing a readily detectable signal while a maximum thickness is determined depending on handling ease and cost of reagents. In order to maintain reagents and provide a sample of a defined size, the strip is constructed to have a relatively narrow width, usually less than 20 mm, preferably less than 10 mm. In general, the width of the strip should be at least about 1.0 mm, typically in a range of about 2 mm to 12 mm, preferably in a range of about 4 mm to 8 mm.
The test strip may also include a backing (not shown). The backing is typically made of water-insoluble, non-porous, and rigid material and has a length and width equal to the pads situated thereon, along which the sample develops, but may have a dimension being less or greater than the pad. In preparation of the backing, various natural and synthetic organic and inorganic materials can be used, provided that the backing prepared from the material should not hinder capillary actions of the absorption material, nor non-specifically bind to an analyte, nor interfere with the reaction of the analyte with a detector. Representative examples of polymers usable in the present invention include, but are not limited to, polyethylene, polyester, polypropylene, poly(4-methylbutene), polystyrene, polymethacrylate, poly(ethylene terephthalate), nylon, poly(vinyl butyrate), glass, ceramic, metal, and the like. On the backing, a variety of pads are adhered by means of adhesives. Proper selection of adhesives may improve the performance of the strip and lengthen the shelf life of the strip. According to the present invention, pressure-sensitive adhesives (“PSA”) may be representatively used in the lateral flow assay strip. Typically, the adhesion of different pads of the lateral flow assay strip is accomplished as the adhesive penetrates into pores of the pads, thereby binding pads together with the backing.
The application pad region 260 basically acts to receive the fluid sample containing an analyte. It includes the unimmobilized chemiluminescent-probe-labeled nanoparticles 10 for selectively hybridizing to the bacterium or virus of interest in the sample 205. The material in the application pad region 260 preferably had a rapid filtering speed and a good ability to hold particles. As such, synthetic material such as polyester and glass fiber filter can be used. Other materials include paper, cotton, polyester, glass, nylon, mixed cellulose esters, spun polyethylene, polysulfones, and the like. Preferably, nitrocellulose, nylon, or mixed cellulose esters are used for the analyte detection membrane strip 12. Methods for depositing the functionalized nanoparticles onto the application pad region 260 include an impregnation process in which a pad such as glass fiber is immersed in a solution of the functionalized nanoparticles reagent particularly formulated, followed by drying. The functionalized pads on the control region 250 and test pad regions 270 may be deposited using inkjet printing methods.
Now, the present invention will be described in detail using embodiments shown in the following examples. However, the examples are for illustration of the present invention and do not limit the scope of the present invention thereto.
In the following examples, citrate protected GNPs (8.0-12.0 nm in diameter), and mercaptoundecanoic acid were purchased from Sigma Aldrich. Luminol (“LUM”), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride, N-hydrosuccinimide, Tween 20, potassium ferricyanide (K3Fe(CN)6), phosphate buffer saline (“PBS”), sodium hydroxide, and hydrogen peroxide (H2O2) were obtained from Fisher Scientific. Polydimethylsiloxane (“PDMS”) was ordered from Dow Corning. All chemicals used in this study were analytical grade. Deionized (“DI”) water with a resistivity of 18.2 MΩ-cm from a portable filtration system (Easy Pure II, Milipore) was used in all the experiments.
For the blood sample preparation, whole sheep blood was obtained from HemoStat Laboratories (Dixon, Calif.). The concentration of the sheep red blood cells in the stock blood solution was measured as about 4.6×109 cells/ml using Petroff Hausser counting chamber under an upright optical microscope (AxioSkop II, Carl Zeiss). The received blood sample was stored at about 4° C. Before chemiluminescent experiments, the sample was inspected under an optical microscope, to make sure that the cells were intact. In the experiments using lysed cells, 100 μL blood samples were frozen at −20° C. and thawed on ice before use. This resulted in complete cell lysis.
UV-visible absorption spectra were recorded using Beckman DU640 spectrophotometer in a 360 μL microcuvette with an optical path length of 10.0 mm. Infrared spectroscopy (“IR”) was performed on a Nicolet 380 FT-IR spectrophotometer with neat solid samples in transmission mode. Transmission electron microscopy (“TEM”) measurements were carried out using FEI Tecnai F20 XT field emission system.
Chemiluminescent experiments were carried out using a IVIS Lumina II system (Caliper Life Sciences, CA), which utilizes a highly sensitive, −90° C. cooled, and back illuminated CCD camera as the detector. A layer of PDMS of about 1.5 mm in thickness was laid on a glass microscope slide (3″×1″×1 mm) in which an array of oval shaped holes (3 mm×4 mm) was punched through to form chemiluminescent reaction wells of about 12 μl in volume. The GNP-MUA-LUM solution was dropped in the well and dried before chemiluminescent measurements. Each well contained a known number of luminol modified GNPs. Typical chemiluminescent experiments involved mixing 4 μl of 0.033 M NaOH, 4 μl of 0.47 M H2O2, and 4 μl of 1 mM Fe(CN)63− solution or, in some experiments, blood samples (at varied concentrations) in different PDMS wells. The slide was then quickly placed in the light tight black box of the IVIS Lumina II system. A bright field reference photograph was first recorded using the CCD camera (this process takes about 3 seconds), and then the chemiluminescent signal (Photon flux) was recorded in the kinetic mode (i.e., flux of photons vs. time) with an exposure time of 10 seconds to the CCD camera. The chemiluminescent signal is represented in a pseudocolor image by overlaying the bright-field and chemiluminescent images. The elapse between consecutive chemiluminescent snap shots in the kinetic mode is approximately 13 seconds (i.e., 3 seconds for reference photograph, and 10 seconds to collect chemiluminescent signal). Normally, 10 such chemiluminescent snapshot images were taken and the integrated photon flux over the designated PDMS well was plotted vs. time.
In this example, GNPs were functionalized with a chemiluminescent material according to the scheme outline in
In sum, the two-step strategy to functionalize luminol on GNPs and the scheme of using such functionalized GNPs for detecting Fe3+ containing analytes are illustrated in
The UV-visible absorption spectra in
TEM images in
After functionalizing GNPs with chemiluminescent luminol molecules, the concentration of the stock solution was adjusted such that a 10 μl solution dispensed about 1×1010 GNPs. This was used in a series of dilutions to obtain GNP-MUA-LUM solutions at concentrations varying over 8 orders of magnitude. The PDMS wells on the test support were loaded with a 10 μl solution of respective concentration and dried in the incubator before chemiluminescent measurements.
It will be appreciated that the mechanism of light production by luminol in different solvents has been previously explored by several researchers. The chemiluminescent reaction of luminol generally utilizes Fe3+ as catalyst and requires two equivalents of base to deprotonate the nitrogen protons, leaving a negative charge which then undergoes resonance to form an enolate ion. Then a cyclic addition reaction of the oxygen at the two carbonyl carbons takes place with the oxygen provided by peroxide (with Fe3+ catalyzing the breakdown of peroxide into oxygen and water), leading to the expulsion of N2 in the gaseous form. This step leads to the formation of 3-aminophthalate (an excited form of luminol) and light emission peaked at the wavelength of μmax=425 nm while electrons return to the ground state. Chemiluminescence of luminol is known to follow a flash mechanism in which chemiluminescence occurs immediately and then decays quickly. The half-life strongly depends on the experimental conditions. It can be seen in
In the experiment with the lowest number of luminol labeled GNPs (i.e., about 1,000 GNPs), the total number of chemiluminescent photons was comparable to the estimated number of luminol molecules (about 1.4×103 luminol/GNP) by assuming the formation of a close-packed thiol monolayer with the same density as on a flat gold surface. But the large variation in the measurement value limited the assessment of exact value of chemiluminescent quantum yield of the attached luminol molecules. In an alternative approach, the chemiluminescent signal measured with 1.0×1010 luminol-labeled GNPs was compared with that from the same number of free luminol molecules that were dispersed in solution (4 μL of 23 μM of luminol in each PDMS well) with all other parameters the same. As shown in
Due to the fast decay in the chemiluminescent signal, it is preferable to use the signal from the first snapshot (i.e., the maximum chemiluminescent signal Imax) instead of the average signal for quantitative analyses.
Log(ΔImax)=0.45 Log(NGNP)+3.23 (1)
with an R2 value of 0.95, where NGNP is the number of GNPs placed in the PDMS well. Even though chemiluminescent signal from 1,000 GNPs can be clearly observed with ΔImax=about 5.0×104 photons/s (see
I
DL
=I
blank+3sblank (2)
where the background signal Iblank is about 2.4×104 photons/s. Therefore, the statistical detection limit is derived to be about 2,600 GNPs. This can be improved by reducing the variation of the background reading which was due to the variation in the experimental setting and the drift of the CCD camera.
The chemiluminescent signal should be, in principle, proportional to the concentration of the luminol. However, the relationship between the background-subtracted maximum chemiluminescent signal (ΔImax) and the number of luminol-attached GNPs (N) was ΔImax∝N0.45 instead of a linear relationship as ΔImax∝N. This might be due to luminol molecules being attached to the surface of GNPs which were deposited at the bottom of the well. It is a pseudo-two-dimensional system instead of the usual dispersion in bulk solution. The mechanism is in further investigation.
GNPs are known to present strong surface plasma resonance (“SPR”), which has been widely utilized to enhance the sensitivity in colorimetric or optical absorption methods. The results suggest that chemiluminescence can provide even higher detection sensitivity. To compare chemiluminescent with absorption approaches,
As illustrated in
Interestingly, the unlysed blood samples showed quite different kinetics in chemiluminescent measurements in both original and diluted samples. As shown in
In short, the foregoing illustrates that the preparation of luminol-functionalized gold nanoparticles with convincing characterization with UV-Vis and IR spectroscopy and transmission electron microscopy. In a preliminary test, luminol-functionalized gold nanoparticles were exposed to blood samples of different concentrations to determine the detection sensitivity which exceeded that of conventional colorimetry assay by about 5 orders of magnitude. It also improves the detection limit of conventional solution-based chemiluminescence by at least 3 orders of magnitude. With the enhancement in signal, detection of blood samples after dilution by 108 times was made—down on single red blood cells.
In this example, the comparison of chemiluminescence signal of luminol molecules in bulk aqueous solutions and equivalent amount of luminol molecules covalently attached to 10 nm diameter gold nanoparticles was made. The number of luminol on each GNP (d=10 nm) was calculated by assuming a close-packed monolayer at a density of 5.0×104 luminol/cm2 on the outer surface (πd2) of each GNP, giving 1.6×103 luminol/GNP. The concentration of GNPs was varied over many orders of magnitude in these measurements. The straight lines are linear fitting of the chemiluminescence signal (above the background) vs. the luminol concentration in log-log scale.
Chemiluminescence Measurement Conditions:
Chemiluminescence experiments were carried out using luminescent mode from GloMax-Multi+ Microplate Multimode Reader. Round bottom 96 wells white polystyrene plate was used in all the luminescent measurements. In the luminol bulk solution experiment, 25 μL of 0.1 M NaOH, 25 μL of 1.408 M H2O2 and 25 μl, of 1 mM K3Fe(CN)6 solution were preloaded in one well of the 96 well plate. Then 25 μL of luminol solution in varying concentration (10−14 to 10−5 M) was added by the injector from the instrument into the above mixed solution to initialize the chemiluminescence reaction. The injection speed is 200 μL/sec. The chemiluminescence signal was recorded for about 8 minutes after injection of the reagents. In the GNP-MUA-LUM solution experiment, 25 μL of 0.1 M NaOH, 25 μl of 1.408 M H2O2 and 25 μL of GNP-MUA-LUM solution in varying number of the GNPs (1.82×102 to about 1.82×1010 GNPs) were preloaded in the 96 well plate. Then 25 μL of 1 mM K3Fe(CN)6 solution was added into the mixture solution by the injector to start the reaction. The chemiluminescence signal was recorded for about 8 minutes after injection of the reagents. In both experiments, the background signal (after 8 minutes) was deducted from the highest chemiluminescence signal (i.e., the first data point) to give ΔI which was used as the corrected signal for each measurement.
As shown in
The foregoing results can be used to extrapolate the detection limit of the invented test strip. The current instrument only measures a small portion of the LUM-GNPs in the 100 μL volume. With the inventive devices, the detection efficiency can be increased by a factor of at least 100 on the test strip. Thus, the detection limit in terms of number of LUM-GNPs is (about 2.0×10−14 M)×(100×10−6 L)×(6.03×1023/mole)/100=about 6.8×103. In principle, about one target nucleic acid is needed to capture on LUM-GNP onto the test strip. Thus, detection down to about 10,000 copies of virus DNAs or RNAs can be made. With further optimization with larger GNP and chemiluminescence enhancers, the detection limit can be further reduced to about 1,000. This is sufficient for detecting virus or bacterial without PCR amplification.
In this example, as shown in
The chemiluminescence experiments were carried out using luminescent mode from GloMax-Multi+Microplate Multimode Reader. Round bottom 96 wells white polystyrene plate was used in all the luminescent measurements. At first, 25 μL of 0.1 M NaOH, 25 μL of 1.408 M H2O2 and 25 μL of K3Fe(CN)6 solution at varied concentrations were preloaded in the wells of a 96 well plate. Then 25 μL of luminol solution at 1 mM concentration was added by the injector into the above mixed solution to initialize the chemiluminescence reaction. The injection speed was 200 μL/sec. The chemiluminescence signal was recorded for about 8 minutes after injection of the reagents. The background signal (after 8 minutes) was deducted from the highest chemiluminescence signal (i.e., the first data point) to give DI which was used as the corrected signal for each measurement.
The foregoing illustrates that the dynamic range for Fe3+ detection using chemiluminescence spanned about 5 orders of magnitude from 1×10−9 M to 1×10−4 M. The detection limit for Fe3+ is about 1×10−9 M. Thus, a single red blood cell using chemiluminescence may be detected.
In this example, Chlamydia specific oligonucleotides were selected based upon unique open reading frames (ORFs) identified in a large-scale comparative genomic analysis. “BLAST screening can be used chlamydial genomes to identify signature proteins that are unique for the Chlamydiales, Chlamydiaceae, Chlamydophila and Chlamydia groups of species” Table 4 of Griffiths et al. BMC Genomics (2006), which is incorporated by reference, identified Chlamydia trachomatis specific proteins. These proteins are uniquely found in species belonging to the Chlamydia genus and are absent in Chlamydophila and Protochlamydia.
The DNA sequence of these proteins were used to search the existing NCBI database and identify regions of 100% DNA sequence identity within Chlamydia trachomatis. Sequence were blasted again using somewhat dissimilar sequences to eliminate any human matching sequences (or expected human associated organisms).
The following sequence is from ORF CT135 and is 100% identical for all deposited Chlamydia trachomatis DNA sequences (nt 63-127).
The following sequence is from ORF CT326.2 and is 100% identical for all deposited Chlamydia trachomatis DNA sequences.
The following sequences is from ORF 115 and is 100% identical for ALL deposited Chlamydia trachomatis DNA sequences.
From the foregoing it will be seen that this invention is one well adapted to attain all ends and objectives herein-above set forth, together with the other advantages which are obvious and which are inherent to the invention. Since many possible embodiments may be made of the invention without departing from the scope thereof, it is to be understood that all matters herein set forth or shown in the accompanying drawings are to be interpreted as illustrative, and not in a limiting sense. While specific embodiments have been shown and discussed, various modifications may of course be made, and the invention is not limited to the specific forms or arrangement of parts and steps described herein, except insofar as such limitations are included in the following claims. Further, it will be understood that certain features and subcombinations are of utility and may be employed without reference to other features and subcombinations. This is contemplated by and is within the scope of the claims.
All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.
Silverstein et al., Spectrometric Identification of Organic Compounds, 4th ed. John Wiley and Sons, New York (1981).
This application is based on and claims priority to U.S. Provisional Application Ser. No. 61/595,958, filed on Feb. 7, 2012, which is hereby incorporated herein by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2013/025120 | 2/7/2013 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 61595958 | Feb 2012 | US |
