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

Abstract

Composition 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 in the above described methods.

1 .- 149 . (canceled) 
     
     
         150 . An isothermal amplification kit for detecting the presence or absence of a polynucleotide target in a sample, comprising:
 a. a coated, solid support comprising (i) a solid support, (ii) a coating layer comprising at least one polycationic polymer, and (iii) a plurality of target-specific probes attached to the coating layer;   b. one or more reaction medium components selected from a helicase enzyme, DNA polymerase, target-specific DNA primers, and dNTPs, wherein the reaction medium components are combined, the monovalent cation concentration is between about 10 mM and about 100 mM.   
     
     
         151 . The kit of  claim 150 , further comprising one or more labelling agents. 
     
     
         152 . The kit of  claim 151 , further comprising an anti-label, antibody-enzyme conjugate capable of biding to a label from the one or more labelling agents. 
     
     
         153 . The kit of  claim 150 , wherein the solid support is formed from a member of the group consisting of silicon, plastic, glass, and membrane. 
     
     
         154 . The kit of  claim 150 , wherein the solid support comprises a silicon wafer or chip. 
     
     
         155 . The kit of  claim 150 , 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). 
     
     
         156 . The kit of  claim 150 , wherein the polycationic polymer is attached to the solid support. 
     
     
         157 . The kit of  claim 150 , wherein the coating 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. 
     
     
         158 . The kit of  claim 150 , wherein the coating layer comprises or is attached to a compound selected from one or more of the group consisting of aminopropyltri-methoxysilane, aminopropyltriethoxysilane, amino-functional organopolylsiloxanes, and amino-functional siloxane alkoxylates. 
     
     
         159 . The kit of  claim 150 , wherein the coating layer comprises an attachment layer. 
     
     
         160 . The kit of  claim 150 , wherein the plurality of target-specific probes are attached to an attachment layer comprising a coating of coupling agents deposited on a surface of the solid support. 
     
     
         161 . The kit of  claim 150 , wherein the plurality of target-specific probes comprise at least one probe having at least one terminus blocked to reduce recognition by an isothermal amplification enzyme. 
     
     
         162 . The kit of  claim 150 , wherein the plurality of target-specific probes comprise at least one probe modified or blocked to reduce recognition by the helicase enzyme. 
     
     
         163 . The kit of  claim 150 , wherein the plurality of target-specific probes comprise at least one probe comprising a backbone having at least one neutral charge. 
     
     
         164 . The kit of  claim 150 , wherein the plurality of target-specific probes comprise at least one probe comprising a methylphosphonate backbone. 
     
     
         165 . The kit of  claim 150 , wherein the plurality of target-specific probes comprise at least one probe comprising a 2′-O-methyl linkage. 
     
     
         166 . The kit of  claim 150 , wherein the reaction medium components include a single-stranded binding protein. 
     
     
         167 . A coated biosensor for visual detection of a polynucleotide target in a sample, comprising: a coated, solid support comprising (i) a solid support, (ii) a coating layer comprising at least one polycationic polymer, and (iii) a plurality of target-specific probes attached to the coating layer;
 wherein the coated, solid support does not comprise a layer that produces a thin-film effect and is also configured to enable optical detection under visible light of polynucleotide targets in the sample by immersion in a reaction medium comprising a helicase enzyme, DNA polymerase, target-specific DNA primers, and dNTPs having a monovalent cation concentration between about 10 mM and about 100 mM.   
     
     
         168 . The biosensor of  claim 167 , further comprising one or more labels that associate with the target-specific probes. 
     
     
         169 . The biosensor of  claim 168 , further comprising an anti-label, antibody-enzyme conjugate capable of biding to the one or more labels. 
     
     
         170 . The biosensor of  claim 167 , wherein the solid support is formed from a member of the group consisting of silicon, plastic, glass, and membrane. 
     
     
         171 . The biosensor of  claim 167 , wherein the solid support comprises a silicon wafer or chip. 
     
     
         172 . The biosensor of  claim 167 , 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). 
     
     
         173 . The biosensor of  claim 167 , wherein the polycationic polymer is attached to the solid support. 
     
     
         174 . The biosensor of  claim 167 , wherein the coating 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. 
     
     
         175 . The biosensor of  claim 167 , wherein the coating layer comprises or is attached to a compound selected from one or more of the group consisting of aminopropyltri-methoxysilane, aminopropyltriethoxysilane, amino-functional organopolylsiloxanes, and amino-functional siloxane alkoxylates. 
     
     
         176 . The biosensor of  claim 167 , wherein the coating layer comprises an attachment layer. 
     
     
         177 . The biosensor of  claim 167 , wherein the plurality of target-specific probes are attached to an attachment layer comprising a coating of coupling agents deposited on a surface of the solid support. 
     
     
         178 . The biosensor of  claim 167 , wherein the plurality of target-specific probes comprise at least one probe having at least one terminus blocked to reduce recognition by an isothermal amplification enzyme. 
     
     
         179 . The biosensor of  claim 167 , wherein the plurality of target-specific probes comprise at least one probe modified or blocked to reduce recognition by the helicase enzyme. 
     
     
         180 . The biosensor of  claim 167 , wherein the plurality of target-specific probes comprise at least one probe comprising a backbone having at least one neutral charge. 
     
     
         181 . The biosensor of  claim 167 , wherein the plurality of target-specific probes comprise at least one probe comprising a methylphosphonate backbone. 
     
     
         182 . The biosensor of  claim 167 , wherein the plurality of target-specific probes comprise at least one probe comprising a 2′-O-methyl linkage.