Systems and methods for point-of-care amplification and detection of polynucleotides

Abstract

Compositions and methods for amplifying and detecting solution-state polynucleotide targets in a single device are described. In one aspect, a method for a coupled isothermal amplification and detection process utilizes a coated solid support, including a solid substrate, a cationic layer, and a plurality of target-specific probes attached to the coated solid support. Polynucleotide targets in the sample are amplified by an isothermal amplification process involving in situ hybridization onto the coated solid support. The entire process can be carried out with a high degree of specificity under low salt conditions in less than one hour. Further aspects of the present invention include methods for coupled hybridization/detection of polynucleotide targets, coated silicon biosensors optimized for use with the coupled detection systems to provide visual detection of polynucleotide targets under visible light conditions, and kits for practicing the above described methods.

1 - 39 . (canceled) 
     
     
         40 . A method for detecting the presence or absence of a polynucleotide target in a sample, comprising:
 a. providing a coated silicon support comprising a silicon substrate, a cationic layer, and a plurality of target-specific probes attached to the cationic layer;   b. applying to the coated solid support the sample and a reaction medium;   c. subjecting the sample to an isothermal amplification process capable of amplifying a polynucleotide target;   d. subjecting reaction products from the isothermal amplification process to conditions suitable for hybridizing amplified polynucleotide targets to the probes; and   e. optically detecting under visible light the presence or absence of polynucleotide targets bound to the probes.   
     
     
         41 . The method of  claim 40 , wherein the isothermal amplification process is selected from the group consisting of helicase-dependent amplification, strand displacement amplification, recombinase polymerase amplification, and loop-mediated isothermal amplification. 
     
     
         42 . The method of  claim 40 , wherein the isothermal amplification process comprises helicase-dependent amplification. 
     
     
         43 . The method of  claim 40 , wherein the amplification and hybridization steps are performed concurrently in the reaction medium. 
     
     
         44 . The method of  claim 40 , wherein the isothermal amplification and hybridization steps are performed under low salt conditions. 
     
     
         45 . The method of  claim 40 , wherein each of the isothermal amplification, hybridization, and detection steps is performed on the coated solid support. 
     
     
         46 .- 47 . (canceled) 
     
     
         48 . The method of  claim 40 , wherein the hybridization step further comprising the step of subjecting reaction products from the isothermal amplification step to an additional hybridization step under high salt conditions for a period of time less than or equal to about 30 minutes, followed by one or more low salt washes. 
     
     
         49 .- 51 . (canceled) 
     
     
         52 . The method of  claim 40 , wherein the coated silicon support does not comprise a coating layer capable of mediating eye-visible detection of polynucleotide targets by thin-film interference phenomena. 
     
     
         53 . The method of  claim 40 , wherein the coated silicon support comprises an antireflective optical thin-film layer coating on the silicon substrate, the thin-layer coating promoting eye-visible detection of polynucleotide targets by thin-film interference phenomena. 
     
     
         54 . The method of  claim 40 , wherein a surface of the coated solid support is blocked or aged to reduce surface passivation of reaction components interfering with detection of polynucleotide targets bound to the probes. 
     
     
         55 . The method of  claim 40 , wherein amplified polynucleotide targets in the sample are linked to a label. 
     
     
         56 . The method of  claim 55 , wherein the detection step comprises binding an enzyme to the label and generating reaction products that are detectable on the surface of the coated solid support. 
     
     
         57 . The method of  claim 56 , wherein the detection step comprises binding an enzyme to the label and generating precipitable reaction products that are detectable on the surface of the coated solid support under visible light. 
     
     
         58 . The method of  claim 55 , wherein the detection step comprises binding of an anti-biotin antibody/horseradish peroxidase conjugate to a biotin label and addition of a tetramethylbenzidine substrate. 
     
     
         59 . The method of  claim 40 , wherein the cationic layer comprises at least one polycationic polymer. 
     
     
         60 . The method of  claim 59 , wherein the polycationic polymer is selected from one or more of the group consisting of polylysine, poly(lys-phe), poly(lys-tyr), poly(lys-trp), poly(arg-trp), and poly(arg-pro-tyr). 
     
     
         61 . The method of  claim 59 , wherein the polycationic polymer is attached to the silicon substrate. 
     
     
         62 . (canceled) 
     
     
         63 . The method of  claim 40 , wherein the cationic layer comprises a compound selected from one or more of the group consisting of a cationic silane, a cationic siloxane, or a cationic derivative thereof. 
     
     
         64 . The method of  claim 40 , wherein the cationic layer comprises or is attached to a compound selected from one or more of the group consisting of aminopropyltrimethoxysilane, aminopropyltriethoxysilane, amino-functional organopolylsiloxanes, and amino-functional siloxane alkoxylates. 
     
     
         65 . The method of  claim 40 , wherein the cationic layer comprises a probe density enhancing material selected from the group consisting of one or more of the following: latex particle, silica particle, nanoparticle, dextran, dextran sulfate, dendrimer, acrylic acid, polyvinyl pyrolidone., polyethylene glycol, polyvinyl sulfate, polyvinyl alcohol, polyacrylic acid, poly(acrylamide) acrylic acid copolymer, and modified derivatives thereof, 
     
     
         66 . The method of  claim 40 , wherein the coated solid support comprises an anti-reflective layer. 
     
     
         67 . The method of  claim 40 , wherein the coated solid support comprises a T-structure aminoalkyl polydimethylsiloxane layer. 
     
     
         68 . The method of  claim 40 , wherein at least one target-specific probe comprises a modified oligonucleotide containing a structural element reducing binding of an isothermal amplification enzyme to an oligonucleotide bound to the polynucleotide target. 
     
     
         69 . The method of  claim 40 , wherein at least one target-specific probe comprises a modified oligonucleotide containing a structural element reducing enzymatic denaturation of the oligonucleotide bound to the polynucleotide target. 
     
     
         70 . The method of  claim 40 , wherein at least one terminus of the probe is blocked to reduce recognition by an isothermal amplification enzyme. 
     
     
         71 . The method of  claim 40 , wherein at least one target-specific probe is modified or blocked to reduce recognition by a helicase. 
     
     
         72 . The method of  claim 40 , wherein at least one target-specific probe comprises a backbone having at least one neutral charge. 
     
     
         73 . The method of  claim 40 , wherein at least one target-specific probe comprises a methylphosphonate backbone. 
     
     
         74 . The method of  claim 40 , wherein at least one target-specific probe comprises a peptide nucleic acid. 
     
     
         75 . The method of  claim 40 , wherein at least one target-specific probe comprises a locked nucleic acid. 
     
     
         76 . The method of  claim 40 , wherein at least one target-specific probe comprises a cleavable linker. 
     
     
         77 . The method of  claim 40 , wherein at least one target-specific probe is linked to a non-cleavable spacer at one or both ends. 
     
     
         78 . The method of  claim 40 , wherein the silicon substrate is blocked or aged to reduce surface passivation of reaction components interfering with detection of polynucleotide targets bound to the probes. 
     
     
         79 . The method of  claim 40 , wherein the coated silicon support is configured in the form of a microchip, microarray, microtiter plate, or dipstick. 
     
     
         80 . The method of  claim 40 , wherein the sample comprises a purified polynucleotide or extract from an animal cell, microbial cell, or combinations thereof. 
     
     
         81 . (canceled) 
     
     
         82 . A method for detecting the presence or absence of a polynucleotide target in a sample, comprising:
 a. providing a coated solid support comprising a solid support and a plurality of target-specific probes attached to the coated solid support;   b. applying to the coated solid support the sample and a reaction medium;   c. providing conditions and reagents suitable for denaturing polynucleotide targets in the sample by an enzymatic process;   d. providing conditions suitable for hybridizing enzymatically denatured polynucleotide targets in the sample to the target-specific probes;   e. detecting the presence or absence of polynucleotide targets bound to the probes.   
     
     
         83 .- 119 . (canceled) 
     
     
         120 . A coated biosensor for visual detection of a polynucleotide target in a sample, comprising:
 a coated silicon support comprising a silicon substrate, a cationic layer, and a plurality of target-specific probes attached to the silicon substrate, wherein the silicon support does not comprise a layer that produces a thin-film effect and is configured to enable optical detection under visible light of polynucleotide targets in the sample.   
     
     
         121 .- 149 . (canceled) 
     
     
         150 . The method of  claim 82 , wherein step (c) further includes subjecting the sample to an isothermal amplification process capable of amplifying a polynucleotide target. 
     
     
         151 . The method of  claim 82 , wherein the coated solid support comprises a silicon substrate and a cationic layer, and wherein the coated solid support does not comprise a coating layer capable of mediating eye-visible detection of polynucleotide targets by thin film interference phenomena. 
     
     
         152 . A kit comprising the coated solid support of  claim 82  and reagents suitable for denaturing polynucleotide targets by an enzymatic process or one or more enzymes sufficient for an isothermal amplification process. 
     
     
         153 . A kit comprising the coated silicon support in claim  1  and one or more enzymes sufficient for an isothermal amplification process.