# US 11,110,461 B2 · Integrated nucleic acid analysis

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- Number: US 11,110,461 B2
- Document ID: US11110461B2
- Status: Granted
- Kind: B2
- Application number: 15/894,630
- Filed: 2018-02-12
- Published: 2018-06-21
- Granted: 2021-09-07
- Earliest claimed: 2007-04-04
- Assignees: ANDE Corporation
- Applicants: Ande Corporation
- Inventors: Eugene Tan; Heung Chuan Lam; Valery Leonidovich Bogdanov; Gregory John Kellogg; John A. Wright; Ulrich Hans Thomann; Richard F. Selden
- Web page: https://patentgrep.com/patent/US11110461B2

## CPC

- B01L 7/52: {with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples}
- B01L 3/502715: {characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces}
- B01L 3/50273: {characterised by the means or forces applied to move the fluids}
- B01L 3/502753: {characterised by bulk separation arrangements on lab-on-a-chip devices, e.g. for filtration or centrifugation}
- C12Q 1/686: Polymerase chain reaction [PCR]
- C12Q 1/6869: Methods for sequencing
- G01N 21/6402: {Atomic fluorescence; Laser induced fluorescence}
- G01N 21/6428: {Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" (in vivo A61B5/00; immunoassay G01N33/53)}
- G01N 21/6452: {Individual samples arranged in a regular 2D-array, e.g. multiwell plates}
- G01N 21/6486: {Measuring fluorescence of biological material, e.g. DNA, RNA, cells (G01N21/6428 takes precedence)}
- G01N 27/44726: {using specific dyes, markers or binding molecules}
- G01N 27/44743: {Introducing samples}
- G01N 27/44782: {of a plurality of samples}
- G01N 27/44791: {Microapparatus (sample containers with integrated microfluidic structures B01L3/5027)}
- B01L 2200/0684: Venting, avoiding backpressure, avoid gas bubbles
- B01L 2200/10: Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept
- B01L 2200/147: Employing temperature sensors
- B01L 2300/0627: Sensor or part of a sensor is integrated
- B01L 2300/0654: Lenses; Optical fibres
- B01L 2300/069: Absorbents; Gels to retain a fluid
- B01L 2300/0816: Cards, e.g. flat sample carriers usually with flow in two horizontal directions
- B01L 2300/0819: Microarrays; Biochips
- B01L 2300/0864: comprising only one inlet and multiple receiving wells, e.g. for separation, splitting
- B01L 2300/0887: Laminated structure
- B01L 2300/16: Surface properties and coatings
- B01L 2300/1822: using Peltier elements
- B01L 2300/1844: using fans
- B01L 2300/1894: Cooling means; Cryo cooling
- B01L 2400/0421: electrophoretic flow
- G01N 2021/6441: {with two or more labels}
- G01N 2201/06113: Coherent sources; lasers
- Y10T 436/2575: Volumetric liquid transfer
- B01L: CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- 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
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- Y10T: TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION

## Abstract

The present disclosure provides fully integrated microfluidic systems to perform nucleic acid analysis. These processes include sample collection, nucleic acid extraction and purification, amplification, sequencing, and separation and detection. The present disclosure also provides optical detection systems and methods for separation and detection of biological molecules. In particular, the various aspects of the invention enable the simultaneous separation and detection of a plurality of biological molecules, typically fluorescent dye-labeled nucleic acids, within one or a plurality of microfluidic chambers or channels. The nucleic acids can be labeled with at least 6 dyes, each having a unique peak emission wavelength. The present systems and methods are particularly useful for DNA fragment sizing applications such as human identification by genetic fingerprinting and DNA sequencing applications such as clinical diagnostics.

## Claims

### Claim 1

A method for DNA analysis of at least two samples, comprising:
injecting a first template nucleic acid extracted based on a first sample from a first sample chamber on a biochip through a first channel to a first reaction reservoir in a first region of said biochip;
injecting a second template nucleic acid extracted based on a second sample from a second sample chamber on said biochip through a second inlet to a second reaction reservoir in the first region of said biochip, the second sample chamber being separate from the first sample chamber;
inducing multiplex PCR amplification of nucleic acid fragments, in said first and second reaction reservoirs of the biochip such that in each of the first and second reaction reservoirs primers labeled with six or more fluorescent dyes, each having a unique peak emission wavelength are used to generate amplified nucleic acid fragments labeled with said 6 or more fluorescent dyes, the first region including at least said first reaction reservoir designated for multiplex PCR amplification based on the first sample, and said second reaction reservoir designated for multiplex PCR amplification based on the second sample;
inducing liquid flow to respectively move first amplified DNA fragments from the first reaction reservoir to a first separation unit in a second region of the biochip, and second amplified DNA fragments from the second reaction reservoir to a second separation unit in the second region of the biochip;
inducing electric fields in the first separation unit to separate the first amplified nucleic acid fragments by size in a first separation channel on the biochip;
inducing electric fields in the second separation unit to separate the second amplified nucleic acid fragments by size in a second separation channel on the biochip, the second separation channel being fluidically separated from the first separation channel; and
providing said an optical detector, comprising:
(i) one light source positioned for illuminating a first detection position on said first separation channel and a second detection position on said second separation channel;
(ii) a mirror to scan said light source sequentially between detection positions;
(iii) one or a plurality of first optical elements positioned for collecting and directing light emanating from the detection positions; and
(iv) a light detector positioned to accept light directed from the one or plurality of first optical elements, wherein the light detector comprises a wavelength dispersive element selected from the group consisting of prisms, diffraction gratings, transmission gratings, holographic diffraction gratings and spectrographs, to disperse the light from the one or plurality of first optical elements according to light wavelength into at least six wavelength components over a physical space according to light wavelength to at least six detection elements or to a multi-anode photomultiplier tube, wherein each of the detection elements are in communication with a first control element for simultaneously collecting detection information from each of said detection elements or from said multi-anode photomultiplier tube, wherein said light detector detects fluorescence from at least six dyes labeled to one or more labeled nucleic acid fragments, each dye having a unique peak emission wavelength; and
detecting the separated nucleic acid fragments labeled with said at least 6 florescent dyes in each of said first and second separation channels using said optical detector.

### Claim 2 (depends on 1)

The method of claim 1, further comprising: extracting the first template nucleic acid from the first sample; and extracting the second template nucleic acid from the second sample.

### Claim 3 (depends on 1)

The method of claim 1, further comprising: injecting a third template nucleic acid extracted based on a third sample from a third sample chamber on a biochip through a third channel to a third reaction reservoir in a first region of said biochip, the third sample chamber being separate from the first and second sample chambers;
injecting a fourth template nucleic acid extracted based on a fourth sample from a fourth sample chamber on said biochip through a fourth inlet to a fourth reaction reservoir in the first region of said biochip, the fourth sample chamber being separate from the first, second and third sample chambers;
inducing multiplex PCR amplification of nucleic acid fragments in said third and fourth reaction reservoirs of the biochip such that primers labeled with six or more fluorescent dyes, each having a unique peak emission wavelength are used to generate amplified nucleic acid fragments labeled with said 6 or more fluorescent dyes, the first region including at least the third reaction reservoir designated for multiplex PCR amplification based on the third sample, and the fourth reaction reservoir designated for multiplex PCR amplification based on the fourth sample;
inducing liquid flow to respectively move first amplified nucleic acid fragments from the third reaction reservoir to a third separation unit in said second region of the biochip, and fourth amplified nucleic acid fragments from the fourth reaction reservoir to a fourth separation unit in the second region of the biochip;
inducing electric fields in a third separation unit to separate the third amplified nucleic acid fragments by size in a third separation channel on the biochip;
inducing electric fields in the fourth separation unit to separate the fourth amplified nucleic acid fragments by size in a fourth separation channel on the biochip, the fourth separation channel being fluidically separated from the first, second and third separation channels; and
detecting the separated nucleic acid fragments labeled with said at least 6 florescent dyes in the detection regions of each of said third and fourth separation channels using said optical detector.

### Claim 4 (depends on 2)

The method of claim 2, further comprising: injecting first reagents and the first template nucleic acid into the first reaction reservoir; and injecting second reagents and the second template nucleic acid into the second reaction reservoir.

### Claim 5 (depends on 1)

The method of claim 1, wherein the optical detector has at least 6 detection elements, each being a single-anode photomultiplier tube, and the wavelength dispersive element is composed of one or a plurality of dichroic mirrors positioned to provide at least a portion of the dispersed at least 6 wavelength components from said one or a plurality of first optical elements to each of said at least 6 detection elements, wherein each dichroic mirror reflects and independently predetermined wavelength of light.

### Claim 6 (depends on 5)

The method of claim 5, further comprising a bandpass filter wherein each independently predetermined wavelength of light essentially corresponds to a fluorescence emission maximum of a fluorescent dye present in at least one of the detection positions.

### Claim 7 (depends on 1)

The method of claim 1, wherein inducing liquid flow to respectively move the first amplified nucleic acid fragments from the first reaction reservoir to the first separation unit in the second region of the biochip, and the second amplified nucleic acid fragments from the second reaction reservoir to the second separation unit in the second region of the biochip, further comprises: inducing liquid flow to move a first PCR mixture having the first amplified nucleic acid fragments from the first reaction reservoir to a first dilution reservoir; diluting the first PCR mixture with a first dilutant; inducing liquid flow to move a second PCR mixture having the second amplified nucleic acid fragments from the second reaction reservoir to a second dilution reservoir; and diluting the second PCR mixture with a second dilutant.

### Claim 8 (depends on 1)

The method of claim 1 wherein said light detector detects fluorescence from at least 8 dyes labeled to one or more nucleic acid fragments, each dye having a unique peak wavelength.

### Claim 9 (depends on 1)

The method of claim 1 wherein the light source is a laser, the wavelength dispersive element is a spectrograph, and each of said at least 6 detection elements is a linear multi-anode photomultiplier tube.