# US 2013/0154671 A1 · Real-time, label-free detection of nucleic acid amplification in droplets using impedance spectroscopy and solid-phase substrate > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2013/0154671 A1 - Document ID: US20130154671A1 - Status: Published application - Kind: A1 - Application number: 13/573,750 - Filed: 2012-10-03 - Published: 2013-06-20 - Earliest claimed: 2011-10-04 - Assignees: The Regents of the University of California - Inventors: Abraham P. Lee; Javier Lopez-Prieto; Robert Lin; Mindy Simon; Nick Martin - Web page: https://patentgrep.com/patent/US20130154671A1 ## CPC - G01R 27/02: Measuring real or complex resistance, reactance, impedance, or other two-pole characteristics derived therefrom, e.g. time constant (by measuring phase angle only G01R25/00) - C12Q 1/686: Polymerase chain reaction [PCR] - G01R: MEASURING ELECTRIC VARIABLES; MEASURING MAGNETIC VARIABLES (indicating correct tuning of resonant circuits H03J3/12) - 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 for detecting presence of nucleic acid amplification in a test droplet. A set of detection electrodes are provided in contact with a fluidic channel. The test droplet is provided in vicinity of the detection electrodes through the fluidic channel. An alternate current (AC) power at a first frequency is applied across the set of detection electrodes. A first measurement value that reflects electrical impedance of the test droplet at the first frequency is obtained. This value is compared with a corresponding reference value, wherein the corresponding reference value is obtained by measuring a reference droplet containing known amplified nucleic acid or known unamplified nucleic acid at the first frequency. The presence of amplified nucleic acid in the test droplet is thus determined based on the comparison. ## Claims ### Claim 1 A method for detecting presence of nucleic acid amplification in a test droplet comprising: providing a set of detection electrodes in contact with a fluidic channel, providing the test droplet in vicinity of said set of detection electrodes through the fluidic channel; applying an alternate current (AC) power at a first frequency across said set of detection electrodes; obtaining a first measurement value reflecting electrical impedance of the test droplet at said first frequency; comparing said first measurement value reflecting electrical impedance of the test droplet with a first corresponding reference value, wherein said first corresponding reference value is obtained by measuring a reference droplet containing known amplified nucleic acid or known unamplified nucleic acid at said first frequency; determining presence of amplified nucleic acid in the test droplet based on said comparison. ### Claim 2 (depends on 1) The method as described in claim 1, wherein said set of electrodes comprises three planar gold electrodes. ### Claim 3 (depends on 1) The method as described in claim 1, wherein said reference droplet contains amplified DNA as a result of PCR. ### Claim 4 (depends on 1) The method as described in claim 1, wherein obtaining a first measurement value comprises utilizing an impedance scope in combination with a current amplifier. ### Claim 5 (depends on 4) The method as described in claim 4, wherein both said first measurement value and said first corresponding reference value are peak-to-peak voltages in respective measuring periods and derived by utilizing said impedance scope in combination with said current amplifier. ### Claim 6 (depends on 5) The method as described in claim 5, wherein said peak-to-peak voltages are derived from respective real time voltage˜time plots produced by said impedance scope in combination with said current amplifier. ### Claim 7 (depends on 6) The method as described in claim 6, wherein said set of detection electrodes comprise two groups of electrodes wherein the two groups share a common detection electrode; and wherein obtaining said first measurement value further comprises a differential impedance measurement conducted across said two groups of detection electrodes respectively. ### Claim 8 (depends on 7) The method as described in claim 7 further comprises performing at least three additional differential impedance measurements on the test droplet at three additional frequencies conducted across said set of detection electrodes; comparing peak-to-peak voltage values obtained from said three additional measurements with three corresponding reference values respectively, wherein said three corresponding reference values are obtained by measuring said reference droplet at said three additional frequencies respectively; and determining presence of amplified nucleic acid in the test droplet by incorporating results from said three additional measurements. ### Claim 9 (depends on 8) The method as described in claim 8, wherein said first frequency and said additional frequencies are equal to 1.2 MHz, 5.2 MHz and 8 MHz and 10 MHz respectively. ### Claim 10 (depends on 6) The method as described in claim 6, wherein determining the presence of amplified nucleic acid in the test droplet is performed at substantially the same time as said obtaining said first measurement value and said performing three additional differential impedance measurements. ### Claim 11 (depends on 1) The method as described in claim 1, wherein the reference droplet is of substantially the same size of the test droplet. ### Claim 12 (depends on 1) The method as described in claim 1, wherein said first measurement value reflecting electrical impedance of the test droplet is independent of the droplet size. ### Claim 13 (depends on 1) The method as described in claim 1 further comprising comparing said first measurement value reflecting electrical impedance of the test droplet with a second reference value obtained by measuring another reference droplet containing known non-amplified nucleic acid at the same AC power with said first frequency. ### Claim 14 (depends on 1) The method as described in claim 1, wherein a series of test droplets can be provided through the fluidic channel in sequence after being subject to thermocycling in a PCR procedure. ### Claim 15 A system for detecting presence of nucleic acid amplification in a series of droplets comprising: a fluidic channel providing a plurality of test droplets of known sizes a set of detection electrodes disposed in contact with the fluidic channel; an AC power supply operable to apply electrical powers across the set of detection electrodes at a plurality of known frequencies; an impedance measurement device operable to measure electrical impedance of each of said series of test droplets; and a processor operable to compare measured electrical impedance of each of said series of test droplets with corresponding reference electrical impedances, wherein the corresponding reference electrical impedances are obtained by measuring a reference droplet at said plurality of known frequencies respectively, and wherein the corresponding reference droplet is of substantially the same size as the testing droplets and contains known amplified nucleic acid or known non-amplified nucleic acid. ### Claim 16 (depends on 15) The system described as in claim 15 further comprising a thermocycling region for implementing PCR on said plurality of test droplets. ### Claim 17 (depends on 15) The system described as in claim 15 wherein the impedance measurement device comprises an impedance scope in combination with a current amplifier. ### Claim 18 (depends on 15) The system described as in claim 15 wherein the AC power supply is capable to supply AC powers to the set of electrodes at frequencies from 1 MHz to 10 MHz. ### Claim 19 (depends on 15) The system described as in claim 15 wherein the processor is capable of determining the presence of amplified nucleic acid in each of the test droplet in substantially real time based on the comparison between electrical impedances of said series of test droplets with corresponding reference electrical impedances. ### Claim 20 A method for detecting presence of nucleic acid amplification in a test droplet comprising: providing a set of detection electrode within a fluidic channel, providing said test droplet in vicinity of said set of electrodes through the fluidic channel; applying high frequency power across said set of detection electrodes at four different frequencies in sequence; measuring electrical impedances of the test droplet at said four frequencies by differential impedance measurements across said detection electrodes; comparing the measured electrical impedances with corresponding reference electrical impedances obtained by measuring a reference droplet at said four different frequencies respectively, wherein the reference droplet is of substantially the same size as the testing droplet and contains known amplified nucleic acid or known unamplified nucleic acid; determining the presence of amplified nucleic acid in the test droplet based on the comparison.