This disclosure provides a method of determining a sequence of nucleotides for a nucleic acid template. The method can include the steps of contacting the nucleic acid template with a conformationally labeled polymerase and at least four different nucleotide species under conditions wherein the conformationally labeled polymerase catalyzes sequential addition of the nucleotide species to form a nucleic acid complement of the nucleic acid template, wherein the sequential addition of each different nucleotide species produces a conformational signal change from the conformationally labeled polymerase and wherein the rate or time duration for the conformational signal change is distinguishable for each different nucleotide species; detecting a series of changes in the signal from the conformationally labeled polymerase under the conditions; and determining the rates or time durations for the changes in the signal, thereby determining the sequence of nucleotides for the nucleic acid template.

This disclosure provides a method of determining a sequence of nucleotides for a nucleic acid template. The method can include the steps of contacting the nucleic acid template with a conformationally labeled polymerase and at least four different nucleotide species under conditions wherein the conformationally labeled polymerase catalyzes sequential addition of the nucleotide species to form a nucleic acid complement of the nucleic acid template, wherein the sequential addition of each different nucleotide species produces a conformational signal change from the conformationally labeled polymerase and wherein the rate or time duration for the conformational signal change is distinguishable for each different nucleotide species; detecting a series of changes in the signal from the conformationally labeled polymerase under the conditions; and determining the rates or time durations for the changes in the signal, thereby determining the sequence of nucleotides for the nucleic acid template.

The present invention pertains to an enzymatic measurement method using an antibody-enzyme complex or a nucleic acid probe measurement method using an enzyme-labeled nucleic acid probe, in both of which the quantification of a product of a reaction by an enzyme in the antibody-enzyme complex or the enzyme-labeled nucleic acid probe is performed by generating thio-NAD(P)H by an enzymatic cycling reaction using NAD(P)H, thio-NAD(P), and a dehydrogenase (DH), and measuring the amount of the generated thio-NAD(P)H or measuring a change in color caused by the generated thio-NAD(P)H. An enzymatic reaction system in which NAD(P) generated from NAD(P)H by the enzymatic cycling reaction is selectively reduced, is caused to coexist with the enzymatic cycling reaction. The present invention also pertains to a kit for enzyme immunoassay, and a kit for nucleic acid probe measurement. In the enzymatic cycling reaction, the detection sensitivity is increased by increasing the amount of thio-NAD(P)H generated per unit time with respect to a predetermined amount of a substrate (reduced), and combining the same with an enzyme immunoassay, etc., enables quantification, etc., of a protein or nucleic acid with high sensitivity.

The present invention pertains to an enzymatic measurement method using an antibody-enzyme complex or a nucleic acid probe measurement method using an enzyme-labeled nucleic acid probe, in both of which the quantification of a product of a reaction by an enzyme in the antibody-enzyme complex or the enzyme-labeled nucleic acid probe is performed by generating thio-NAD(P)H by an enzymatic cycling reaction using NAD(P)H, thio-NAD(P), and a dehydrogenase (DH), and measuring the amount of the generated thio-NAD(P)H or measuring a change in color caused by the generated thio-NAD(P)H. An enzymatic reaction system in which NAD(P) generated from NAD(P)H by the enzymatic cycling reaction is selectively reduced, is caused to coexist with the enzymatic cycling reaction. The present invention also pertains to a kit for enzyme immunoassay, and a kit for nucleic acid probe measurement. In the enzymatic cycling reaction, the detection sensitivity is increased by increasing the amount of thio-NAD(P)H generated per unit time with respect to a predetermined amount of a substrate (reduced), and combining the same with an enzyme immunoassay, etc., enables quantification, etc., of a protein or nucleic acid with high sensitivity.

This disclosure is generally directed to electrochemical readout of rapid diagnostics related to use of CRISPR effector systems. In one aspect, the disclosure provides a nucleic acid detection system. Generally, the system comprises: (1) a detection CRISPR system comprising an effector protein and one or more guide nucleic acid (gNA) strands designed to bind to corresponding target nucleic acid molecules; (2) an effector strand and (3) an electrode.

This disclosure is generally directed to electrochemical readout of rapid diagnostics related to use of CRISPR effector systems. In one aspect, the disclosure provides a nucleic acid detection system. Generally, the system comprises: (1) a detection CRISPR system comprising an effector protein and one or more guide nucleic acid (gNA) strands designed to bind to corresponding target nucleic acid molecules; (2) an effector strand and (3) an electrode.

The present application provides for systems and methods for detecting and estimating signals corresponding to one or more biomarkers in biological samples stained for the presence of protein and/or nucleic acid biomarkers. On particular aspect is directed to a method of estimating an amount of signal corresponding to at least one biomarker in an image of a biological sample. The method includes detecting isolated spots in a first image, deriving an optical density value of a representative isolated spot based on signal features from the detected isolated spots, estimating a number of predictive spots in signal aggregates in each of a plurality of sub-regions based on the derived optical density value of the representative isolated spot, and storing the estimated number of predictive spots and detected isolated spots in each of the plurality of generated sub-regions in a database.

The present application provides for systems and methods for detecting and estimating signals corresponding to one or more biomarkers in biological samples stained for the presence of protein and/or nucleic acid biomarkers. On particular aspect is directed to a method of estimating an amount of signal corresponding to at least one biomarker in an image of a biological sample. The method includes detecting isolated spots in a first image, deriving an optical density value of a representative isolated spot based on signal features from the detected isolated spots, estimating a number of predictive spots in signal aggregates in each of a plurality of sub-regions based on the derived optical density value of the representative isolated spot, and storing the estimated number of predictive spots and detected isolated spots in each of the plurality of generated sub-regions in a database.

Methods for labeling biological samples include (a) contacting a biological sample featuring a target RNA with a probe, where the probe includes a capture moiety that specifically binds to the target RNA, and a plurality of reporter moieties, and (b) for each reporter moiety of the plurality of reporter moieties: contacting the reporter moiety with a catalytic agent that includes an oligonucleotide that specifically binds to the reporter moiety, and a reactive species linked to the oligonucleotide, and contacting the biological sample with a labeling agent that reacts with the reactive species to deposit the labeling agent or a derivative thereof in the sample in proximity to the target RNA.

Methods for labeling biological samples include (a) contacting a biological sample featuring a target RNA with a probe, where the probe includes a capture moiety that specifically binds to the target RNA, and a plurality of reporter moieties, and (b) for each reporter moiety of the plurality of reporter moieties: contacting the reporter moiety with a catalytic agent that includes an oligonucleotide that specifically binds to the reporter moiety, and a reactive species linked to the oligonucleotide, and contacting the biological sample with a labeling agent that reacts with the reactive species to deposit the labeling agent or a derivative thereof in the sample in proximity to the target RNA.