EP1794326A4 - Utilisation de la photopolymerisation pour amplifier et detecter un evenement de reconnaissance moleculaire - Google Patents

Utilisation de la photopolymerisation pour amplifier et detecter un evenement de reconnaissance moleculaire

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Publication number
EP1794326A4
EP1794326A4 EP05725765A EP05725765A EP1794326A4 EP 1794326 A4 EP1794326 A4 EP 1794326A4 EP 05725765 A EP05725765 A EP 05725765A EP 05725765 A EP05725765 A EP 05725765A EP 1794326 A4 EP1794326 A4 EP 1794326A4
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European Patent Office
Prior art keywords
target
polymer
probe
photoinitiator
polymer precursor
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EP05725765A
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German (de)
English (en)
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EP1794326A2 (fr
Inventor
Hadley Sikes
Christopher Bowman
Ryan Hansen
Robert Kuchta
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University of Colorado
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University of Colorado
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Priority claimed from PCT/US2004/029733 external-priority patent/WO2005024386A2/fr
Application filed by University of Colorado filed Critical University of Colorado
Publication of EP1794326A2 publication Critical patent/EP1794326A2/fr
Publication of EP1794326A4 publication Critical patent/EP1794326A4/fr
Withdrawn legal-status Critical Current

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54306—Solid-phase reaction mechanisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips

Definitions

  • This invention is in the field of detection of molecular recognition events, in particular use of photopolymerization for amplification and detection of these events.
  • PCR polymerase chain reaction
  • RT-PCR reverse transcription polymerase chain reaction
  • strand displacement amplification strand displacement amplification
  • Eberwine linear amplification Eberwine linear amplification
  • the branched DNA assay is a solution phase assay that involves a number of probe oligonucleotides that bind to multiple sites on the target viral RNA.
  • Detection is possible because each hybridization event is accompanied by the binding of a fluorophore ( Kern, D., Collins, M., Fultz, T., Detmer, J., Hamren, S., Peterkin, J., Sheridan, P., Urdea, M., White, R., Yeghiazarian, T., Todd, J. (1996) "An Enhanced-sensitivity Branched-DNA Assay for Quantification of Human Immunodeficiency Virus Type 1 RNA in Plasma" Journal of Clinical Microbiology 34:3196-3203).
  • the synthetic effort required for this assay is relatively large: multiple probes are designed for each RNA of interest, and the assay depends on the binding of these probes to multiple preamplifier and amplifier molecules that also must be designed and synthesized.
  • Dendrimer-based DNA detection methods have also been reported (U.S. Patents 5,710,264, 5,175,270, 5,487,973, 5,484,904 and Stears, R. et al., 2000, Physiol. Genomics 3: 93-99).
  • Dendrimers are complexes of partially double-stranded oligonucleotides, which form stable, spherical structures with a determined number of free ends. Specificity of the dendrimer detection is accomplished through specific binding of a capture oligonucleotide on a free arm of the dendrimer. Other arms of the dendrimer are labeled for detection. This method does not require enzymes and can produce amplification of 300 - 400.
  • Rolling circle amplification is capable of detecting as few as 150 molecules bound to a microarray (Nallur, G., Luo, C, Fang, L., Cooley, S., Dave, V., Lambert, J., Kukanskis, K., Kingsmore, S., Lasken, R., Schweitzer, B. (2001) "Signal Amplification by Rolling Circle Amplification on DNA Microarrays” Nucleic Acids Research 29:E118).
  • the main drawback to RCA is the necessity of DNA polymerase.
  • Microfabricated disposable DNA sensors based on enzymatic amplification electrochemical detection was reported by Xu et al. (Xu et al., 2001 , Electoanalysis, 13(10), 882-887).
  • DNA microarrays, or biochips represent promising technology for accurate and relatively rapid pathogen identification (Wang, D., Coscoy, L., Zylberberg, M., Avila, P. C, Boushey, H.A., Ganem, D., DeRisi, J. L. (2002) “Microarray Based Detection and Genotyping of Viral Pathogens," PNAS, 99(24), 15687-15692). Anthony et al. recently demonstrated rapid identification of 10 different bacteria in blood cultures using a BioChip (Anthony, R. M., Brown, TJ. , French, G. L.
  • the invention provides methods to detect molecular recognition events, in particular a relatively small number of molecular recognition events.
  • the methods of the invention are based on amplification of the signal due to each molecular recognition event, rather than amplification of the number of molecular recognition events taking place.
  • the present invention can limit or eliminate the need for techniques which increase the number of recognition events taking place, including PCR and techniques involving culturing of bacteria.
  • the present invention can replace PCR and RT-PCR techniques for microarray applications as a means to achieve acceptable signals.
  • the methods of the invention can be used to generate and amplify a signal due to many types of molecular recognition events that can be described by the following equation: A + B + ln - ⁇ A-B-In (Eqn 1)
  • a and B are the species of interest that undergo molecular recognition and In is a photoinitiator.
  • A is the probe species and B is the target species.
  • the probe A is attached to the substrate.
  • the target species, B, and/or the photoinitiator may comprise a linking group which allows selective binding of the photoinitiator to the target or the A-B complex.
  • the target species may comprise biotin and the initiator avidin.
  • Equation 1 may also be written as.
  • each initiator can lead to the polymerization of as many as 10 2 - 10 6 monomer units.
  • each single molecular recognition event has the opportunity to be amplified by the polymerization of up to 10 6 monomers, each of which may be fluorescent or enable detection of its presence through one of a variety of means.
  • a fluorescent polymer, a magnetic polymer, a radioactive polymer or an electrically conducting polymer can form the basis of detection and amplification.
  • a polymer gel swollen with a fluorescent solution, a magnetic solution, a radioactive solution or an electrically conducting solution can form the basis of detection and amplification.
  • the quantity of polymer formed is sufficient to allow visual detection of polymer formation.
  • the polymer need not be fluorescent, magnetic , radioactive or electrically conducting. This embodiment can be achieved through a synergistic combination of reduction of oxygen content in the polymer precursor solution by purging, utilization of a macroinitiator with an appropriate ratio of initiator to molecular recognition agent, and the identification of the appropriate exposure time. Without wishing to be bound by any particular belief, it is believed that a process having all these attributes can yield much higher degrees of amplification and enable better contrast than is possible with a process having only one of these attributes.
  • the invention provides a method for amplifying a molecular recognition interaction between a target and a probe comprising the steps of: a) contacting the target with the probe under conditions effective to form a target-probe complex; b) removing target not complexed with the probe; c) contacting the target-probe complex with a photoinitiator label under conditions effective to attach the photoinitiator label to the target- probe complex; d) removing photoinitiator label not attached to the target-probe complex; e) contacting the photoinitiator-labeled target-probe complex with a polymer precursor solution; f) exposing the photoinitiator-labeled target-probe complex and the polymer precursor solution to light, thereby forming a polymer; and g) detecting the polymer formed, thereby detecting an amplified target- probe interaction wherein the photoinitiator label is a macroinitiator and the oxygen content of the polymer precursor solution during light exposure is sufficiently low and
  • the invention provides methods for identification of a target species based on its molecular interaction with an array of different probe species, each probe species being attached to a solid substrate at known locations.
  • the probe will be labeled with a polymer. Detection of the polymer-labeled probes allows identification of which probes have undergone the molecular recognition reaction and therefore identification of the target.
  • the invention provides a method for identifying a target comprising the steps of a) providing a probe array comprising a plurality of different probes, wherein the probes are attached to a solid substrate at known locations; b) contacting the probe array with the target under conditions effective to form a target-probe complex; c) removing target not complexed with the probe; d) contacting the target-probe complex with a photoinitiator label under conditions effective to attach the label to the target-probe complex; e) removing photoinitiator label not attached to the target-probe complex; f) contacting the photoinitiator-labeled target-probe complex with a polymer precursor solution; g) exposing the photoinitiator-labeled target-probe complex and the polymer precursor solution to light, thereby forming a polymer; and h) detecting the polymer formed, wherein the polymer location indicates the probe which forms a target-probe complex with the target, thereby identifying the target
  • the methods of the invention can be used to identify target species using DNA microarrays, also commonly referred to as DNA chips or BioChips, to provide the probe array.
  • DNA microarrays also commonly referred to as DNA chips or BioChips
  • the DNA microarray may be an array which allows identification and strain analysis for one or more genera of influenza virus.
  • the methods of the invention provide sufficient amplification that molecular recognition can be detected without instrumentation. In another embodiment, the methods of the invention provide sufficient amplification that molecular recognition can be detected using a relatively inexpensive microarray reader or scanner which may not have the highest instrument sensitivity or resolution.
  • Figure 1 schematically illustrates some of the steps in detection and amplification of hybridization using photopolymerization.
  • Figure 2 illustrates an alternate two-step hybridization scheme for detection and amplification.
  • Figure 3 illustrates fluorescence detection of macroinitiators on a biotin array.
  • Figure 4 illustrates an image of a FluChip developed using photopolymerization for signal enhancement.
  • Figure 5 shows a profilometry scan across a row of polymer spots that were grown from an array following recognition of biotin by a streptavidin macrophotoinitiator.
  • Figure 1 is a conceptual diagram of how photopolymerization of a fluorescent monomer M is used to generate and amplify a signal from a single captured genetic target on a DNA microarray.
  • Figure 1 shows addition of biotin (2) to the target oligonucleotide (10) to form a biotinylated target nucleotide (12).
  • the biotinylated target nucleotide (12) is hybridized to a complementary probe (20), forming a target- probe complex (30) on the surface of the microarray (40).
  • the microarray shown in Figure 1 contains a biotin-labeled probe (22) which acts as a positive control.
  • the microarray After hybridization, the microarray is exposed to a photoinitiator label (50), initiator- functionalized avidin, which interacts with the biotinylated target oligonucleotide (12) and control probe (22) to form an initiator-labeled target-probe complex (35) and an initiator-labeled positive control probe (25).
  • the microarray surface is then exposed to a fluorescent monomer (M) under the appropriate initiation conditions. In the presence of light and a fluorescent monomer (represented by M in Figure 1), a polymerization reaction occurs from sites on the surface where targets have been captured by the probe DNA, forming a polymer-labeled target-probe complex (37) and a polymer-labeled positive control probe (27).
  • M fluorescent monomer
  • Figure 1 the polymer label is denoted by (60). Ideally, since initiators are not bound to sites where hybridization has not occurred, polymerization does not occur from those sites. For brevity, Figure 1 omits several steps which are typically used in the process, including removal of uncomplexed target material prior to exposure of the microarray to initiator functionalized avidin, removal of initiator functionalized avidin not attached to the target-probe complex, and removal of unpolymerized monomer prior to detection.
  • Figure 1 illustrates hybridization of complementary DNA to a DNA microarray as a specific example
  • the detection and amplification scheme generalizes to many other types of molecular recognition events.
  • Agents capable of participating in molecular recognition events include, but are not limited to, agonists and antagonists for cell membrane receptors, toxins and venoms, viral epitopes, hormones (e.g., opiates, steroids, etc.), hormone receptors, peptides, enzymes, enzyme substrates, substrate analogs, transition state analogs, cofactors, drugs, proteins, and antibodies, cell membrane receptors, monoclonal antibodies and antisera reactive with specific antigenic determinants (such as on viruses, cells or other materials), drugs, polynucleotides, nucleic acids, peptides, cofactors, lectins, sugars, polysaccharides, cells, cellular membranes, and organelles.
  • the detection and amplification scheme can be used to detect and amplify the molecular recognition interaction between nucleic acids, an antibody and an antigen, and a first and a second protein.
  • Microarrays can be used to detect hybridization as well as protein-protein interactions, protein drug binding, and enzymatic catalysis (Schena, M., "Microarray Analysis, (2003) John Wiley & Sons, New Jersey, p. 153).
  • molecular recognition interactions are those in which the probe recognizes and selectively binds a target, resulting in a target-probe complex.
  • Molecular recognition interactions also involve the formation of noncovalent bonds between the two species.
  • the binding occurs between specific regions of atoms (molecular domains) on the probe species which have the characteristic of binding or attaching specifically to unique molecular domains on specific target species.
  • Molecular recognition interactions can also involve responsiveness of one species to another based on the reciprocal fit of a portion of their molecular shapes.
  • the target and probe are two species of interest which undergo molecular recognition.
  • the target may also be referred to as a ligand.
  • the probe may also be referred to as a receptor.
  • at least some characteristics of the probe are known.
  • the probe is an oligonucleotide whose sequence is known or partially known. In other embodiment, the sequence of the probe may not be known, but it is known to be complementary to a possible target species.
  • the probe will be selected so that it is capable of selected recognition with the known or suspected identity of the target. In some cases a single probe can be used to detect the presence of a target. In other cases more than one probe will be necessary to detect the presence of or identify a target.
  • the selectivity is a measure of the specificity of the molecular recognition event.
  • “Selectivity” or “hybridization selectivity” is the ratio of the amount of hybridization (i.e., number of second nucleic acids hybridized) of fully complementary hybrids to partially complementary hybrids, based on the relative thermodynamic stability of the two complexes. For the purpose of this definition it is presumed that this ratio is reflected as an ensemble average of individual molecular binding events. Selectivity is typically expressed as the ratio of the amount of hybridization of fully complementary hybrids to hybrids having one base pair mismatches in sequence.
  • Selectivity is a function of many variables, including, but not limited to,: temperature, ionic strength, pH, immobilization density, nucleic acid length, the chemical nature of the substrate surface and the presence of polyelectrolytes and/or other oligomers immobilized on the substrate or otherwise associated with the immobilised film.
  • the homology of the target and probe molecules influences whether hybridization occurs.
  • Cross-hybridization can occur if the sequence identity between the target and the probe is greater than or equal to about 70% (Schena, M., "Microarray Analysis, (2003) John Wiley & Sons, New Jersey, p. 151).
  • either the target or the probe is a nucleic acid.
  • both the target and the probe are a single stranded nucleic acid.
  • the probe is an oligonucleotide, a relatively short chain of single- stranded DNA or RNA.
  • Nucleic acid includes DNA and RNA, whether single or double stranded. The term is also intended to include a strand that is a mixture of nucleic acids and nucleic acid analogs and/or nucleotide analogs, or that is made entirely of nucleic acid analogs and/or nucleotide analogs and that may be conjugated to a linker molecule.
  • Nucleic acid analogue refers to modified nucleic acids or species unrelated to nucleic acids that are capable of providing selective binding to nucleic acids or other nucleic acid analogues.
  • nucleotide analogues includes nucleic acids where the internucleotide phosphodiester bond of DNA or RNA is modified to enhance bio-stability of the oligomer and "tune" the selectivity/specificity for target molecules (Uhlmann, et al., (1990), Angew. Chem. Int. Ed. Eng., 90: 543; Goodchild, (1990), J.
  • nucleic acid analogues also include alpha anomers ( ⁇ -DNA), L-DNA (mirror image DNA), 2'-5' linked RNA, branched DNA/RNA or chimeras of natural DNA or RNA and the above- modified nucleic acids.
  • ⁇ -DNA alpha anomers
  • L-DNA mirror image DNA
  • 2'-5' linked RNA branched DNA/RNA or chimeras of natural DNA or RNA and the above- modified nucleic acids.
  • any nucleic acid containing a "nucleotide analogue” shall be considered as a nucleic acid analogue.
  • Backbone replaced nucleic acid analogues can also be adapted to for use as immobilised selective moieties of the present invention.
  • PNAs peptide nucleic acids
  • Anti-Cancer Drug Design 8: 53; Engels ef a/., (1992), Angew, Chem. Int. Ed. Eng., 31 : 1008
  • carbamate-bridged morpholino-type oligonucleotide analogs Burger, D. R., (1993), J. Clinical Immunoassay, 16: 224; Uhlmann, et al., (1993), Methods in Molecular Biology, 20,. "Protocols for Oligonucleotides and Analogs," ed.
  • the probe and/or target can be an oligomer.
  • Oligomer refers to a polymer that consists of two or more monomers that are not necessarily identical. Oligomers include, without limitation, nucleic acids (which include nucleic acid analogs as defined above), oligoelectrolytes, hydrocarbon based compounds, dendrimers, nucleic acid analogues, polypeptides, oligopeptides, polyethers, oligoethers any or all of which may be immobilized to a substrate . Oligomers can be immobilized to a substrate surface directly or via a linker molecule.
  • the probe is DNA.
  • the DNA may be genomic DNA or cloned DNA.
  • the DNA may be complementary DNA (cDNA), in which case the target may be messenger RNA (mRNA).
  • the DNA may also be an Expressed Sequence Tag (EST) or a Bacterial Artificial Chromosome (BAC).
  • EST Expressed Sequence Tag
  • BAC Bacterial Artificial Chromosome
  • DNA microarrays are known to the art and commercially available.
  • the general structure of a DNA microarray is a well defined array of spots on an optically flat surface, each of which contains a layer of relatively short strands of DNA.
  • microarrays have a spot size less than about 1.0 mm.
  • 15-25 nucleotide sequences are the minimum oligonucleotide probe length (Schena, M., "Microarray Analysis, (2003) John Wiley & Sons, New Jersey, p. 8).
  • the substrate is generally flat glass primed with an organosilane that contains an aldehyde functional group. The aldehyde groups facilitate covalent bond formation to biomolecules with free primary amines via Schiff base interactions. After reaction the chip is cured to form a very stable array ready for hybridization.
  • Protein microarrays are also known to the art and some are commercially available.
  • the general structure of protein microarrays can be similar to that of DNA microarrays, except that array spots can contain antibodies (in particular monoclonal antibodies), antigens, recombinant proteins, or peptides.
  • surface-bound proteins must be correctly folded and fully functional (Constans, A. , 2004, The Engineer, 18(15) 42).
  • the proteins can be protected by use of stabilizing buffers and/or relatively high protein concentrations (Schena, M., "Microarray Analysis, (2003) John Wiley & Sons, New Jersey, p. 154).
  • the functional domains of interest can be arrayed rather than the whole protein, forming domain- based arrays (Constans, 2004, ibid).
  • the target is genetic material from influenza A, B, or C.
  • Influenza is an orthomyxovirus with three genera, types A, B, and C.
  • the types are distinguished by the nucleoprotein antigenicity (Dimmock, N.J., Easton, A.J., Leppard, K.N. (2001) "Introduction to Modern Virology” 5 th edition, Blackwell Science Ltd., London).
  • Influenza A and B each contain 8 segments of negative sense ssRNA.
  • Type A viruses can also be divided into antigenic sub-types on the basis of two viral surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA).
  • H1 through H15 There are currently 15 identified HA sub-types (designated H1 through H15) and 9 NA sub ⁇ types (N1 through N9) all of which can be found in wild aquatic birds (Lamb, R.A. & Krug, R. M., (1996) "Orthomyxoviridae: The Viruses and their Replication, in Fields Virology", B.N. Fields, D. M. Knipe, and P.M. Howley, Editors. Lippincott-Raven: Hagerstown). Of the 135 possible combinations of HA and NA, only four (H 1 N1 , H1 N2, H2N2, and H3N2) have widely circulated in the human population since the virus was first isolated in 1933.
  • H3N2 and H1 N1 The two most common sub-types of influenza A currently circulating in the human population are H3N2 and H1 N1.
  • LI et al. describe a DNA microarray whose probes were multiple fragments of the hemagglutinin, neuraminidase, and matrix protein genes. (Li, J. et al., (2001), J. Clinical Microbio., 39(2), 696-704).
  • the substrate may be treated with an agent to reduce the remaining aldehydes prior to contacting the probe with the target.
  • an agent is sodium borohydride NaBH 4 .
  • Such a treatment can decrease the amount of reaction between the monomer and the aldehyde coating on the glass, thus decreasing the amount of background signal during the detection step.
  • the target Prior to contacting the target with the probe, the target may be biotinylated to allow later attachment of at least one initiator via biotin-avidin interaction.
  • photobiotinylation reagents Pierce, Quanta Biodesign
  • the target may be contacted with the photoinitiator label prior to contacting the target with the probe, so long as use of a photoinitiator-labeled target does not substantially limit its participation in the desired molecular recognition event.
  • the invention provides a method for amplifying a molecular recognition interaction between a target and a probe comprising the steps of contacting a photoinitiator-labeled target with a probe under conditions effective to form a photoinitiator-labeled target-probe complex, removing target not complexed with the probe, contacting the photoinitiator-labeled target-probe complex with a polymer precursor, exposing the photoinitiator-labeled target-probe complex and the polymer precursor to light, thereby forming a polymer, and detecting the polymer formed, thereby detecting an amplified target-probe interaction.
  • the probe is contacted with a solution comprising the target under conditions effective to form a target-probe complex.
  • the conditions effective to form a target- probe complex depend on the target and probe species.
  • suitable hybridization conditions have been described in the scientific literature.
  • this solution also comprises an agent, such as a crowding agent, to limit nonspecific interactions.
  • a crowding agent is an agent that interrupts nonspecific adsorption between nucleic acids that are not complementary.
  • Formamide is one such agent to limit nonspecific interactions (Stahl, D. A., and R.
  • Nonspecific interactions can also be limited by applying a blocking agent to the microarray prior to contacting the target with the probe.
  • Suitable blocking agents include, but are not limited to BSA , nonfat milk, and sodium borohydride.
  • Detergents such as sodium lauroyl sarcosine or sodium dodecyl sulfate can also be added to aldehyde surface hybridization reactions to reduce background (Schena, M., "Microarray Analysis, (2003) John Wiley & Sons, New Jersey, p. 117).
  • the target solution may also be contacted with the probe at higher temperatures in order to limit nonspecific interactions.
  • targets which have not formed target-probe complexes are removed.
  • the unbound targets can be removed through rinsing. Water or an aqueous solution may be used for rinsing away unbound targets.
  • a blocking agent can be applied to the microarray to limit nonspecific interaction of avidin.
  • Suitable blocking agents include, but are not limited to, BSA and nonfat milk.
  • array is incubated with the blocking agent for approximately 20 minutes at about room temperature.
  • the target-probe complex is contacted with a photoinitiator label under conditions effective to attach the photoinitiator label to the target probe complex.
  • the photoinitiator label comprises avidin or streptavidin and at least one photoinitiator.
  • a plurality of photoinitiators are attached to the avidin or streptavidin to form a polymeric photoinitiator label.
  • a plurality of photoinitiators and avidin or streptavidin are attached to a polymer. If the target has been biotin-labeled, interaction between the avidin or streptavidin and the biotin can attach the photoinitiator label to the target, and thus to the target-probe complex.
  • biotin-labeled target-probe complex is contacted with a solution comprising the photoinitiator label for about 20 minutes at room temperature.
  • the photoinitiator label (50) comprises a single strand of DNA attached to at least one photoinitiator (In).
  • the photoinitiator can be attached to the ssDNA through biotinylation of the DNA followed by interaction with avidin or streptavidin with at least one photoinitiator attached.
  • photoinitiators are coupled directly to the end of the oligonucleotide label sequences. Oligonucleotides with 5' amine modifications can be purchased and the reaction conditions in Scheme 2 (EDC coupling) used to form a peptide bond between this amine and the carboxylic acid group of the initiator. The product can be purified by HPLC.
  • the target anchors the initiator, through the label sequence, to the microarray spot. If target viral RNA will not tolerate the presence of the fluorescent monomer and UV light, it is possible to connect the label sequence to the probe oligonucleotide via a treatment with ligase prior to exposure to the photopolymerization reaction conditions. Another method that avoids the use of an enzyme is to place pendant photocrosslinkable groups on the probe oligonucleotide and the label sequence. If 1 however, the polymerization reaction is fast when compared with the timescale of diffusion, these steps will not be necessary even if the target genetic material detaches from the capture strand.
  • Photoinitiators that are useful in the invention include those that can be activated with light and initiate polymerization of the polymer precursor.
  • the photoinitiator is water soluble.
  • Commercially available photoinitiators for example lrgacure 2959 (Ciba), can be modified to improve their water solubility.
  • the photoinitiator is a radical photoinitiator.
  • the photoinitiator is a cationic photoinitiator.
  • the photoinitiator comprises a carboxylic acid functional group. The photoinitiator is selected to be compatible with the wavelengths of light supplied.
  • Photoinitiators include azobisisobutyronitrile, peroxides, phenones, ethers, quinones, acids, formates.
  • Cationic initiators include aryldiazonium, diaryliodonium, and triarylsulfonium salts.
  • the photoinitiator is selected from the group consisting of Rose Bengal (Aldrich), Darocur or lrgacure 2959 (2-hydroxy-1-(4- (hydroxyethoxy)phenyl)-2-methyl-1-propanone, D2959, Ciba-Geigy), lrgacure 651 (2,2-dimethoxy-2-phenylacetophenone, 1651 , DMPA, Ciba-Geigy), lrgacure 184 (1- hydroxycyclohexyl phenyl ketone, 1184, Ciba-Geigy), lrgacure 907 (2-methyl-1-(4- (methylthio)phenyl)-2-(4-morpholinyl)-1 -propanone, I907, Ciba-Geigy), Camphorquinone (CQ, Aldrich), isopropyl thioxanthone (quantacure ITX, Great Lake
  • Photoinitiator molecules can be attached to avidin or streptavidin by modification of avidin or streptavidin lysine residues.
  • the carboxylic functional group of the photoinitiator can be coupled to the amine of the lysine residue in the presence of a coupling agent. The result is the formation of a peptide bond between the initiator and the protein.
  • Suitable coupling agents are known to those skilled in the art and include, but are not limited to, EDC.
  • a polymeric photoinitiator label is formed.
  • a polymeric photoinitiator label can be formed from a polymer which can be coupled with both the photoinitiator and a molecular recognition group such as avidin or streptavidin.
  • the photoinitiator can be attached to the polymer by an ester linkage or by any other kind of linkage known to the art.
  • the avidin or streptavidin can be attached to the polymer by an amide linkage.
  • the polymer comprises carboxylic acid groups and amide groups.
  • the polymer comprises a poly(acrylic acid-co-acrylamide) backbone.
  • a polymeric photoinitiator label can be formed from a polymer which comprises one part of a two-part photoinitiator system.
  • the polymer is coupled to a molecular recognition group such as avidin or streptavidin.
  • avidin or streptavidin When the combination of the polymer and the second part of the initiator system is exposed to the appropriate wavelength of light, the initiator system is capable of capable of initiating polymerization of a polymer precursor.
  • one part of the two-part photoinitiator system is a tertiary amine which is part of the polymeric photoinitiator label.
  • the other part of the photoinitiator system can be camphorquinone. (CQ) This two-part system can be activated by light of approximately 469 nm.
  • the tertiary amine can be incorporated into the polymer label by co-polymerizing acrylic acid with a monomer comprising the tertiary amine and an acrylate group.
  • the polymeric photoinitiator comprises sufficient photoinitiators so that it may be regarded as a macroinitiator (having many initiators present on a single molecule).
  • the number of initiator groups per molecule or chain may vary from one chain to another.
  • the use of a macroinitiator can increase the average initiator concentration by a factor of between about 10 to about 100.
  • the average number of initiators per polymer chain is between about 100 and about 200.
  • the average number of initiators per polymer chain is between about 120 and about 160.
  • the number of molecular recognition groups may also vary from chain to chain. In an embodiment, the average number of molecular recognition groups is between one and three. Without wishing to be bound by any particular belief, it is believed that the incorporation of too may initiator groups can lead to nonspecific interaction between the macroinitiator and the array.
  • the molecular weight of the backbone polymer is selected to be large enough to allow attachment of the appropriate number of initiator and molecular recognition groups. For a poly(acrylic acid-co-acrylamide) backbone, the molecular weight of the backbone is preferably greater than about 50,000.
  • Unattached photoinitiator After contact of the photoinitiator label with the target-probe complex, unattached photoinitiator is removed. Unattached photoinitiator may be removed by rinsing with water or an aqueous solution.
  • the photoinitiator-labeled target-probe complex is contacted with a solution comprising a polymer precursor.
  • a polymer precursor means a molecule or portion thereof which can be polymerized to form a polymer or copolymer.
  • Polymer precursors include any substance that contains an unsaturated moiety or other functionality that can be used in chain polymerization, or other moiety that may be polymerized in other ways. Such precursors include monomers and oligomers.
  • the solution further comprises a solvent for the polymer precursor. In an embodiment, the solvent is aqueous.
  • the amount of oxygen dissolved in the polymer precursor solution is minimized to minimize oxygen inhibition of the polymerization process.
  • the oxygen content of the solution is less than about 1 x 10 "5 moles/liter.
  • the amount of oxygen dissolved in the solution may be minimized by control of the atmosphere under which polymerization takes place, reducing the oxygen content of the polymer precursor solution by flowing a gas through it and/or the addition of oxygen inhibition agents.
  • oxygen inhibition agents such as multifunctional thiol reagents are not used.
  • the oxygen content of the polymer precursor solution during polymerization can be minimized by performing the polymerization in an enclosure and introducing a gas which does not have a substantial oxygen content into the enclosure.
  • the oxygen content of the gas is less than about 10%, less than about 5% and less than about 1 %.
  • gases include, but are not limited to, commercial purity argon and nitrogen.
  • the atmosphere in the enclosure may be obtained by simply filling the enclosure with the desired gas, or by flowing gas through the enclosure.
  • the enclosure can also be evacuated and backfilled with gas.
  • the oxygen content of the polymer precursor solution can also be reduced prior to polymerization by bubbling a suitable gas through the solution, or by any other method known in the art.
  • Suitable gases include those which do not have a substantial oxygen content, such as argon and nitrogen.
  • the solution may further comprise oxygen inhibition agents and/or cross- linking agents.
  • the oxygen inhibition agent is a multithiol (Bhanu, V.A. & Kishore, K. (1991) Role of Oxygen in Polymerization Reactions, Chemical Reviews 91 : 99-117).
  • the amount of oxygen inhibition agent should not be so much that polymerization occurs in the bulk of the solution rather than from the surface.
  • oxygen inhibition agents which can act as chain transfer agents are not recommended for use with radical polymerization processes when it is desired to form sufficient quantities of the polymer for visual detection.
  • a crosslinking agent can stabilize the polymer that is formed and improve the amplification factor (Hacioglu B., Berchtold K.A., Lovell L.G., Nie J., & Bowman CN. (2002) Polymerization Kinetics of HEMA/DEGDMA: using Changes in initiation and Chain Transfer Rates to Explore the Effects of Chain-Length-Dependent Termination. Biomaterials 23:4057-4064). Finally, a small amount of inhibitor can be added to the formulation to limit background polymerization caused by impurities and trace radicals formed by absorption by molecules other than the initiator.
  • the polymer precursor is a photopolymerizable monomer capable of forming a fluorescent polymer, a magnetic polymer, a radioactive polymer or an electrically conducting polymer.
  • the polymer precursor is water soluble.
  • the polymer precursor is a photopolymerizable fluorescent methacrylate monomer.
  • the fluorophore may absorb the light used in the photopolymerization process. To compensate, the exposure time of the polymer precursor to the light and/or the light intensity can be adjusted.
  • the polymer precursor need not be capable of forming a fluorescent, magnetic, radioactive or electrically conducting polymer if sufficient quantities of the polymer can be formed.
  • the polymer precursor can be any photopolymerizable polymer precursor or monomer.
  • the polymer precursor can be an acrylate or a mixture of acrylates.
  • the polymer precursor can also comprise a chromophore.
  • the photoinitiator and chromophore preferentially absorb different wavelengths of light.
  • the polymer precursor is capable of forming a polymer gel.
  • the gel is covalently crosslinked and a cross-linking agent is added to the polymer precursor containing solution.
  • the gel is noncovalently crosslinked.
  • the polymer gel formed is not substantially fluorescent, magnetic, radioactive, or electrically conducting. Instead, detection can occur through absorption of a fluorescent, magnetic, radioactive, or electrically conducting solution by the gel. Detection can also occur through visual inspection of the quantity of gel formed is sufficiently large.
  • the polymer gel is a hydrogel.
  • hydrogel refers to a class of polymeric materials which are extensively swollen in an aqueous medium, but which do not dissolve in water.
  • hydrogels are prepared by polymerization of a hydrophilic monomer under conditions where the polymer becomes cross-linked in a three dimensional matrix sufficient to gel the solution.
  • the hydrogel may be natural or synthetic.
  • a wide variety of hydrogel- forming compositions are known to the art.
  • the monomer used to form the hydrogel is selected from the group consisting of acrylates, methacrylates, acrylamides, methacrylamides, cyclic lactams and monomers with ionic functionality.
  • Monomers with ionic functionality include methacrylate, methacrylamide, and styrene based monomers with acidic or basic functionality.
  • the monomer used to form the hydrogel is an acrylate or methacrylate.
  • the hydrogel-forming monomer is selected from the group consisting of polysaccharides and proteins. Polysaccharides capable of forming hydrogels include alginate, chitin, chitosan, cellulose, oligopeptides, and hyalauric acids. Proteins capable of forming hydrogels include albumin and gelatin.
  • Suitable acrylate mixtures for hydrogel formation include, but are not limited to, mixtures of hydroxyethyl acrylate (HEA) and elthylene glycol dimaethacylate (EGDMA).
  • the monomer is hydroxyl ethyl acrylate (HEA).
  • the photoinitiator-labeled target-probe complex and polymer precursor are exposed to light, thereby forming a polymer.
  • Photopolymerization occurs when polymer precursor solutions are exposed to light of sufficient power and of a wavelength capable of initiating polymerization.
  • the wavelengths and power of light useful to initiate polymerization depends on the initiator used.
  • Light used in the invention includes any wavelength and power capable of initiating polymerization. Preferred wavelengths of light include ultraviolet or visible.
  • Any suitable source may be used, including laser sources.
  • the source may be broadband or narrowband, or a combination.
  • the light source may provide continuous or pulsed light during the process. Both the length of time the system is exposed to UV light and the intensity of the UV light can be varied to determine the ideal reaction conditions.
  • the exposure time and light intensity can be varied to obtain maximal fluorescence signal from spots on a microarray and minimal fluorescence signal from the background.
  • the intensity of UV radiation is selected so that an appropriate dose of UV radiation can be delivered in less than about one-half hour.
  • unpolymerized polymer precursor is removed.
  • the unpolymerized polymer precursor can be removed by rinsing, for example by rinsing with water or an aqueous solution.
  • the unpolymerized polymer precursor need not be removed if formation of the polymer is to be detected by its refractive index or by other means that would not be interfered with by the presence of the unpolymerized polymer precursors.
  • the hydrogel polymer is not substantially fluorescent, magnetic, radioactive, or electrically conducting
  • the hydrogel can be contacted with a detectable solution which is fluorescent, magnetic, radioactive, or electrically conducting so that the hydrogel absorbs a sufficient quantity of the detectable solution. After the detectable solution is absorbed into the hydrogel, the excess solution is removed before detection.
  • the polymer formed is detected by fluorescence, magnetic, radioactive or electrical detection methods as are known to the art. If the probes are part of a DNA microarray, a commercially available microarray scanner and/or imager can be used to detect polymer formation. DNA microarray scanners and/or imagers are commercially available that can detect fluorescent or radioisotopic labels.
  • sufficient quantities of the polymer are formed that polymerization can be detected by visual inspection.
  • Polymerization which is detectable by visual inspection may also be detectable via image analysis of photographs or digital images of part or all of the array or substrate.
  • Polymerization can be detected by visual inspection when there is sufficient contrast between the areas where polymer has formed and the unpolymerized monomer, the other areas of the array or the array substrate.
  • the areas where the polymer has formed appear to be a different color (or shade of gray) than the unpolymerized monomer, the other areas of the array or the array substrate. For example, after unpolymerized monomer is removed, the areas where polymer has formed may appear darker than the array substrate.
  • the areas where the polymer has formed can have a different transparency than the unpolymerized polymer precursor.
  • the unpolymerized polymer precursor may be clear and the polymer more opaque and whitish in color.
  • the amount of polymer required for visual detection of polymer formation may depend upon the polymer.
  • acrylate mixtures such as mixtures of Hydroxyethyl Acrylate (HEA) and Ethylene Glycol Dimethacrylate (EGDMA) 1
  • the thickness of the polymer formed can be greater than about 1 micron, or greater than about 5 microns.
  • Example 1 Synthesis of a Water Soluble Initiator Scheme 1.
  • the lysine residues have been modified with a photoinitiator (rather than a dye) by coupling the carboxylic acid functional group of the photoinitiator to the amine of the lysine residue.
  • a photoinitiator rather than a dye
  • the result is formation of a peptide bond between the initiator and the protein.
  • Avidin and an excess of the initiator (the product in Scheme 1 , represented by In in Scheme 2) were dissolved in an acid aqueous buffered solution in the presence of the water-soluble coupling agent 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) (Hermanson, GT. (1996) Bioconjugate Techniques San Diego, CA: Academic Press, p. 435).
  • Scheme 3 illustrates the formation of a polymer labeled with lrgacure 2959
  • the water-soluble coupling agent 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide (EDC) and the intermediate-stabilizing molecule N-hydroxsuccinimide (NHS) were used to create amide linkages between some of the acrylic acid subunits and streptavidin and to create ester linkages between other acrylic acid subunits and the initiator lrgacure 2959 (I2959). Still other acrylic acid subunits were left unmodified to assure the water solubility of the resulting macroinitiator. Two types of reaction conditions were varied in order to change the number of initiators that coupled per chain.
  • macroinitiators in which many initiators are present on a single molecule, can be used. Synthesis of a macroinitiator can be achieved through a living (or controlled) radical polymerization method prior to utilization on the microarrays (Kamigaito M., Ando T., & Sawamoto M. (2001 ) "Metal- Catalyzed Living Radical polymerization. Chemical Reviews 101 : 3689-3746).
  • Atom transfer radical polymerization (ATRP) schemes can be used to control the macroinitiator molecular weight, composition and architecture (block copolymers, branching, etc.) (Matyjaszewski, K. & Jianhui Xia, J. (2001 ) Atom Transfer Radical Polymerization Chemical Reviews 101 : 2921 -2990).
  • Macroinitiator synthesis can be performed, as presented in Scheme 4, by starting with an oligonucleotide terminated in an amine group.
  • the amine terminus is functionalized with an ATRP initiator, which initiates polymerization of the desired compounds.
  • an initiator As many as three different monomers (an initiator, a spacer, and a
  • photosensitizer may need to be copolymerized.
  • a spacer molecule for example (poly ethylene glycol methacrylate)
  • the spacer may also be used for controlling solubility of the macroinitiator (by changing the hydrophobicity/hydrophilicity) and the macroinitiator molecular weight.
  • Photosensitizing components are incorporated into the macroinitiator in systems where minimization of the background polymerization is necessary.
  • Example 5 Synthesis and photopolymerization of a fluorescent monomer Scheme 5.
  • N-hydroxy succinimide ester of fluorescein was purchased from Pierce Biotechnology (Rockford, IL) and 2 mg were dissolved in 100 ⁇ l_ of DMSO while 0.5 mg of 2-aminoethyl methacrylate was dissolved in 900 ⁇ l_ of sodium bicarbonate buffer, pH 8.2. The two solutions were combined and placed on a shaker for two hours. The solvent was lyophilized off. The structures of the fluorescent monomer and the polymer that results from irradiating the monomer with UV light were verified by NMR.
  • the monomer can be polymerized by irradiating with 365 nm ultraviolet light for one minute.
  • Example 6 Formation of a Hydrogel from a Polymer Labeled with Photoinitiator and Streptavidin and Detection of the Hydrogel Formed.
  • Example 3 The polymer of Example 3 was reacted with a microarray having biotin covalently bound to the microarray substrate.
  • a hydroxyl ethyl acrylate monomer solution was placed in contact with the array by pipetting the solution into a HybriWell (Grace Biolabs) which covers the array.
  • Sixty ⁇ l of an aqueous solution was used which contained hydroxyl ethyl acrylate, initiator (product of Scheme 1 and Scheme 3), cross-linking agent (ethylene glycol dimethacrylate, 3% by volume), and oxygen inhibition agent (mercaptoethanol, 5 x 10 "4 % by volume).
  • the monomer was then photopolymerized to form a hydrogel by irradiating the array with 365 nm light for about 1 minute.
  • the microarray was rinsed with water and then a solution containing Rhodamine B, concentration 100 nM, was pipetted between a glass coverslip and the microarray. The microarray was exposed to the fluorophore containing solution for about 30 minutes. The microarray was then rinsed with water to remove excess fluorophore .
  • Figure 3A is an image of the array after polymerization.
  • the grid of reacted biotin spots (100) are bright and indicate areas of formation of hydrogel swollen with the fluorescent solution.
  • the detector used was an Agilent Microarray Scanner (Fluorescence Ave: 2600 , Std: 1200 Background: 1200Signal to Noise: 2.2).
  • Figure 3B shows negative controls spots (110) on the same array (Fluorescence Ave: 900, Std: 30, Background: 800, Signal to Noise: 1.1 )
  • Example 7 On-Chip Hybridization, Amplification, and Detection on a Flu Chip.
  • On-chip signal amplification by photopolymerization was tested on a custom microarray designed to detect and subtype the influenza virus.
  • the chip had a column of positive control spots (80), spots to capture RNA of influenza C (91), spots to capture RNA of influenza B (93), and spots to capture RNA of influenza A (95). There were three spots in each column.
  • the sequence that is complementary to the positive control spots (but not to the other nine spots on the array) was purchased from Qiagen (Valencia, CA) with a 5' biotin modification. Published protocols were used to hybridize a 1 ⁇ M solution of this oligonucleotide to the microarray. The hybridization procedure was as follows: Pre-hybridization
  • Hybridization 1. Pipette 10 uL of water or buffer into the humidification slots, place microarray in hybridization cassette.
  • Hybridization solution 10 ⁇ l_ 50 micromolar oligo soln (PBS or Tris)
  • the lighter spots represent detected fluorescence. Though the background fluorescence remains relatively high despite the BSA treatment, the positive control spots (80) are definitely more fluorescent than the spots designed to capture the genetic material of influenza A 1 B and C (these spots effectively served as three different negative controls in this experiment).
  • the faint gray vertical lines in Figure 4 are a well understood artifact. Glass slides were placed between the microarray and the UV light source to block high frequency UV light that is harmful to the fluorophore. The vertical lines arise from the seam between two glass slides that were used as a filter in this manner
  • one possible cause of nonspecific binding is reaction of the fluorescent monomer with the aldehyde coating on the glass.
  • alternative attachment chemistries are available, or the unreacted aldehydes outside of the oligonucleotide spots can be passivated or reduced. If macroinitiators containing larger numbers of initiators are used, the irradiation time can be reduced.
  • an inhibitor can be included. This technique will be effective if the number of initiation sites inside the hybridized spots (specific, due to the presence of many initiators) is much greater than the number of possible sites outside the spots (nonspecific, absorption and initiation occurring without initiator, and non-specific binding).
  • False positives can be minimized by the use of macroinitiators, photosensitizers/inhibitors and optimal initiation time and light intensity, combined with judicious choice of probe and label sequence and steps to prevent non-specific binding of the initiator to the array
  • the number of fluorescent molecules incorporated into the polymer and therefore bound to the microarray surface can be quantified.
  • One test is to measure the shape and size of the resulting spot after polymerization is complete using either atomic force microscopy or profilometry. With this information and knowledge of the mass fraction of fluor within the mixture and the polymer density, it is possible to calculate relatively accurately the number of fluors within the polymer. This approach minimizes error due to any fluorescence quenching within the polymer. Quenching is quantified and minimized by conducting a study of fluorescence intensity as a function of mass percent fluor in the polymer. Due to the amorphous nature of the polymer, quenching is not expected to be significant.
  • the reaction conditions for forming the macroinitiator were as given in Table 1.
  • the array was then washed to remove macroinitiator not attached to the biotin array.
  • the photoinitiator-labeled array was then contacted with a monomer solution comprising Hydroxyethyl Acrylate (HEA) and Ethylene Glycol Dimethacrylate (EGDMA).
  • HOA Hydroxyethyl Acrylate
  • EGDMA Ethylene Glycol Dimethacrylate
  • the photoinitiator labeled array and monomer solution were then exposed to 5 mW/cm 2 , 365nm UV light for 20 minutes in an argon atmosphere. Unpolymerized monomer solution was then removed by washing. Details of biotin array preparation, monomer preparation, recognition and amplification procedures are given below.
  • the product of reaction 1 led to the formation of polymer spots that were up to 10 microns thick, as measured with a profilometer.
  • Figure 5 compares a profilometer trace across the hydrogel spots with a control profilometer trace. A digital image of the polymer spots shows that they are darker than the surrounding substrate.
  • the product of reaction 2 led to polymerization everywhere (not just within the spots).
  • the product of reaction 3 led to the formation of polymer spots that were up to 0.1 micron thick, as measured using a profilometer.
  • Vapor-deposit aminopropyltriethoxysilane onto a piranha cleaned silicon (or glass) substrate involves placing substrates in 70% v/v sulfuric acid, 30% v/v 30% hydrogen peroxide at 9O 0 C for 1 hour.
  • Vapor deposition includes placing substrates into a purged Teflon container along with an open vial containing the silane for two hours at 9O 0 C.
  • Blocking of nonspecific binding soak arrays in 1 mg/mL dry milk in water solution for two hours while slowly agitating with shaker.
  • HSA Hydroxyethyl Acrylate
  • EGDMA Ethylene Glycol Dimethacrylate
  • Example 10 Polymerization of a Chromophore-Containing Monomer Using a Macroinitiator Incorporating Tertiary Amines
  • a chromophore -containing monomer is polymerized using a photoinitiator which preferentially absorbs light at a different wavelength than the chromophore.
  • a chromophore which preferentially absorbs UV light can be paired with a photoinitiator which preferentially absorbs visible light.
  • Scheme 6 illustrates formation of an acrylate monomer incorporating the chromophore Cascade Blue Ethylene Diamine, which preferentially absorbs light at approximately 400nm.
  • Scheme 7 illustrates formation of a macroinitiator comprising a polymer incorporating multiple tertiary amines.
  • the photoinitiator for the polymerization of the monomer comprising the tertiary amine and the acrylate group with acrylic acid is (for water solubility and to provide a functional group that streptavidin can be coupled to) lrgacure 184.
  • 1- hydroxycyclohexyl phenyl ketone (Ciba-Geigy) is coupled to at least one molecular recognition group, shown as streptavidin in Scheme 7.
  • the tertiary amines of the macroinitiator and CQ form a two-part initiator system which most strongly absorbs light at 469 nm.
  • the radical species shown in Scheme 8 propagates through the carbon-carbon double bonds of the chromogenic monomer that is the product of Scheme 6 to form a chromogenic monomer.

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Abstract

L'invention concerne des méthodes servant à détecter des événements de reconnaissance moléculaire. Elle concerne également des méthodes servant à détecter la présence d'une espèce ciblée, ou à identifier ladite espèce, en fonction de son interaction avec une ou plusieurs espèces utilisées comme sonde. Ces méthodes consistent à amplifier le signal associé à chaque événement de reconnaissance moléculaire. L'amplification est exécutée par l'intermédiaire d'une photopolymérisation, le polymère obtenu étant associé à l'événement de reconnaissance moléculaire. Dans un mode de réalisation, un polymère fluorescent, un polymère magnétique, un polymère radioactif ou un polymère conducteur d'électricité peuvent constituer la base de la détection et de l'amplification. Dans un autre mode de réalisation, un polymère gel gonflé par une solution fluorescente, une solution magnétique, une solution radioactive ou une solution conductrice peuvent constituer la base de la détection de l'amplification. Dans un autre mode de réalisation, un niveau polymère suffisant peut être détecté par inspection visuelle.
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EP0383126A2 (fr) * 1989-02-17 1990-08-22 OSTER, Gerald Composition d'essai diagnostique de photopolymérisation et méthode d'essai immunologique et d'essai d'acide nucléique
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