# US 8,465,926 B2 · Method and system for real time quantification and monitoring of nucleic acid amplification using electroconductive or electrochemically active labels

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- Number: US 8,465,926 B2
- Document ID: US8465926B2
- Status: Granted
- Kind: B2
- Application number: 12/377,124
- Filed: 2007-08-10
- Published: 2010-07-22
- Granted: 2013-06-18
- Earliest claimed: 2006-08-11
- Assignees: The Hong Kong University of Science and Technology
- Applicants: I Ming Hsing; Thomas Ming Hung Lee; Stephen Siu Wai Yeung
- Inventors: I Ming Hsing; Thomas Ming Hung Lee; Stephen Siu Wai Yeung
- Web page: https://patentgrep.com/patent/US8465926B2

## CPC

- C12Q 1/6825: Nucleic acid detection involving sensors
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES

## Abstract

A method and device for real time electrochemically or electrically monitoring and detecting nucleic acid amplification products, i.e. after each polymerase chain reaction cycle, utilizes electrochemically active or electrically conductive reporter materials. An electric voltage is applied and electric signals are measured during a PCR amplification process to the electrodes that is suitable for quantifying the amplified products of a sample's nucleic acid(s) produced. This technique is suitable for point-of-use applications, e.g. detecting bioanalytes in remote locations.

## Claims

### Claim 1

A real time solid phase method for electrochemically or electrically monitoring or quantifying the amount of nucleic acid(s) by formation of a polymerase chain reaction (PCR) produced polynucleic acid(s) comprising
contacting a sample comprising a target nucleic acid(s), a solid surface bound probe(s) comprising a first primer(s) provided with a sequence(s) that is complementary to at least a portion of one end of the target nucleic acid(s), a second primer(s) in solution that is complementary to at least a portion of the opposing end of the complementary strand of the target nucleic acid(s), and an electrochemically or electrically conductive labeled marker(s) that is(are) adapted for incorporation into a polynucleic acid(s) by chain polymerization and when incorporated thereof produces a signal(s) change(s) if subjected to an electric potential wherein at least one of the probe(s) is(are) bound onto the conductive electrode surface and said probes remain immobilized during chain polymerization;
adding a polymerase chain reaction enzyme(s) under conditions effective for PCR amplification to occur;
applying an electric potential to the sample and detecting or measuring in real time an electric signal(s) produced by the labeled marker(s) incorporated into said solid surface bound probe(s); and
quantifying the amount of nucleic acid(s) present in the sample and the amount of polynucleic acid(s) produced by correlating the change(s) in signal(s) over time with the formation of polynucleic acid(s),
wherein said electric signal(s) is detected or measured with a conductive electrode(s) placed in the sample; said electrode(s) is(are) treated for a sufficient period of time to prevent electron transfer from said un-reacted electrochemically or electronically conductive labeled marker during real time solid phase PCR;
wherein said electrodes are treated with a blocking agent for at least twelve hours; and said blocking agent of said treatment is ethanolamine.

### Claim 2 (depends on 1)

The method of claim 1, further comprising denaturing the target nucleic acid(s) prior to the polymerization reaction; and wherein the polymerase chain reaction enzyme comprises a thermo-stable enzyme.

### Claim 3 (depends on 1)

The method of claim 1, wherein the signal(s) change comprise(s) an intensity change(s) that is proportional to the concentration of the polynucleic acid(s) produced, and of nucleic acid(s) in the sample.

### Claim 4 (depends on 1)

The method of claim 1, further comprising conducting more than one PCR amplification cycles; wherein each signal(s) change(s) is(are) proportional to the formation of polynucleic add(s) in each PCR amplification cycle.

### Claim 5 (depends on 1)

The method of claim 1 wherein a surface of at least one of the conductive electrode(s) comprises indium tin oxide, gold, platinum, carbon or magnetic particles.

### Claim 6 (depends on 1)

The method of claim 1, where in the label(s) comprise(s) ferrocene or ferrocene derivatives.

### Claim 7 (depends on 1)

The method of claim 1, wherein the marker(s) comprise(s) dUTP, dATP, dGTP, or dCTP.