# US 2013/0155403 A1 · Integrated Nucleic Acid Analysis

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- Number: US 2013/0155403 A1
- Document ID: US20130155403A1
- Status: Published application
- Kind: A1
- Application number: 13/191,952
- Filed: 2011-07-27
- Published: 2013-06-20
- Earliest claimed: 2007-04-04
- Assignees: NetBio, Inc.
- 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/US20130155403A1

## 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 at position 1

1-46. (canceled)

### Claim 47

A system to detect components separated by electrophoresis comprising:
(i) an integrated biochip comprising a thermal cycling chamber adapted for multiplexed amplification such that primers labeled with 6 or more fluorescent dyes are used to generate amplified nucleic acid fragment labeled with said 6 or more fluorescent dyes, said thermal cycling chamber in fluid communication with at least one channel on said biochip, wherein each channel comprises a detection position;
(ii) one or more light sources positioned for illuminating one or a plurality of detection positions over a physical space on said biochip;
(iii) one or a plurality of first optical elements positioned over said physical space for collecting and directing light emanating from the one or plurality of detection positions on said substrate; and
(iv) a wavelength dispersive element positioned to disperse the wavelength components of the fluorescence over a physical space according to light wavelength, and to provide at least a portion of the dispersed wavelength components to at least six detectors positioned to detect fluorescence from said labeled amplified nucleic acid fragments.

### Claim 48 (depends on 47)

The system of claim 47, wherein the one or more light sources is a laser.

### Claim 49 (depends on 47)

The system of claim 47, wherein each detector is capable of detecting light selected from the group consisting of: ultraviolet light, visible light, infrared light, and combinations thereof.

### Claim 50 (depends on 47)

The system of claim 47, wherein the wavelength dispersive element is selected from the group consisting of a prism, diffraction grating, transmission grating and holographic diffraction grating.

### Claim 51 (depends on 47)

The system of claim 47, further comprising a mirror to scan said one or more light sources sequentially between said detection positions.

### Claim 52 (depends on 47)

The system of claim 47, wherein said one or more light sources illuminate all of said detection positions at the same time.

### Claim 53

A system to simultaneously detect fragments from sequencing of at least two DNA templates in a single separation channel, comprising:
(i) an integrated biochip comprising a thermal cycling chamber adapted for multiplexed amplification such that primers labeled with 8 or more fluorescent dyes are used to generate two sets of nucleic acid fragments labeled with said 8 or more fluorescent dyes, said dyes being members of at least two 4-dye containing sets, such that the emission wavelengths of the dyes in the at least two sets are different and said thermal cycling chamber in fluid communication with at least one channel on said biochip, wherein each channel comprises a detection position;
(ii) one or more light sources positioned for illuminating one or a plurality of detection positions over a physical space on said biochip;
(iii) one or a plurality of first optical elements positioned over said physical space for collecting and directing light emanating from the one or plurality of detection positions on said substrate; and
(iv) a wavelength dispersive element positioned to disperse the wavelength components of the fluorescence over a physical space according to light wavelength, and to provide at least a portion of the dispersed wavelength components to at least eight detectors positioned to detect fluorescence from said labeled amplified nucleic acid fragments.

### Claim 54 (depends on 53)

The system of claim 53, in which the number of sequenced fragments detected simultaneously is at least three, the number of 4-dye sets is at least three, and the number of fluorescent dyes is at least 12.

### Claim 55 (depends on 53)

The system of claim 53, in which the number of sequenced fragments detected simultaneously is at least four, the number of 4-dye sets is at least four, and the number of fluorescent dyes is at least 16.

### Claim 56 (depends on 53)

The system of claim 53, in which the number of sequenced fragments detected simultaneously is at least 5, the number of 4-dye sets is at least five and the number of fluorescent dyes is at least 20.

### Claim 57

A system to detect components separated by electrophoresis comprising:
(i) an integrated biochip comprising a thermal cycling chamber adapted for multiplexed amplification such that primers labeled with 6 or more fluorescent dyes are used to generate amplified nucleic acid fragments labeled with said 6 or more fluorescent dyes, said thermal cycling chamber in fluid communication with at least one channel on said biochip, wherein each channel comprises a detection position;
(ii) one or more light sources positioned for illuminating one or a plurality of detection positions over a physical space on said biochip;
(iii) one or a plurality of first optical elements positioned over said physical space for collecting and directing light emanating from the one or plurality of detection positions on said substrate; and
(iv) a spectrograph positioned to disperse the wavelength components of the fluorescence over a physical space according to light wavelength, and to provide at least a portion of the dispersed wavelength components to a multi-element PMT positioned to detect fluorescence from said labeled amplified nucleic acid fragments.

### Claim 58 (depends on 57)

The system of claim 57, wherein the one or more light sources is a laser.

### Claim 59

A system to detect components separated by electrophoresis comprising:
(i) a biochip comprising a thermal cycling chamber adapted for multiplexed amplification such that primers labeled with 6 or more fluorescent dyes are used to generate amplified nucleic acid fragments labeled with said 6 or more fluorescent dyes, said thermal cycling chamber in fluid communication with at least one channel on said biochip, wherein each channel comprises a detection position, and a second chamber adapted for additional processing, said processing selected from the group consisting of:
(i) nucleic acid extraction;
(ii) nucleic acid purification;
(iii) pre-nucleic acid amplification cleanup;
(iv) post-nucleic acid amplification cleanup;
(v) pre-nucleic acid sequencing cleanup;
(vi) nucleic acid sequencing;
(vii) post-nucleic acid sequencing cleanup;
(viii) reverse transcription;
(ix) pre-reverse transcription cleanup;
(x) post-reverse transcription cleanup;
(xi) nucleic acid ligation;
(xii) nucleic acid hybridization; and
(xiii) quantification; and
(ii) one or more light sources positioned for illuminating one or a plurality of detection positions over a physical space on said biochip;
(iii) one or a plurality of first optical elements positioned over said physical space for collecting and directing light emanating from the one or plurality of detection positions on said substrate; and
(iv) a wavelength dispersive element positioned to disperse the wavelength components of the fluorescence over a physical space according to light wavelength, and to provide at least a portion of the dispersed wavelength components to at least six detectors positioned to detect fluorescence from said labeled amplified nucleic acid fragments.

### Claim 60 (depends on 59)

The system of claim 59, wherein the one or more light sources is a laser.

### Claim 61 (depends on 59)

The system of claim 59, wherein each detector is capable of detecting light selected from the group consisting of: ultraviolet light, visible light, infrared light, and combinations thereof.

### Claim 62 (depends on 59)

The system of claim 59, wherein the wavelength dispersive element is selected from the group consisting of: a prism, diffraction grating, transmission grating and holographic diffraction grating.

### Claim 63 (depends on 59)

The system of claim 59, further comprising a mirror to scan said one or more light sources sequentially between said detection positions.

### Claim 64 (depends on 59)

The system of claim 59, wherein said one or more light sources illuminate all of said detection positions at the same time.

### Claim 65

An integrated biochip system comprising:
(a) a biochip comprising one or a plurality microfluidic systems, wherein each microfluidic system comprises a first reaction chamber in microfluidic communication with a separation chamber, wherein
the first reaction chamber is adapted for
(xiv) nucleic acid extraction;
(xv) nucleic acid purification;
(xvi) pre-nucleic acid amplification cleanup;
(xvii) nucleic acid amplification;
(xviii) post-nucleic acid amplification cleanup;
(xix) pre-nucleic acid sequencing cleanup;
(xx) nucleic acid sequencing;
(xxi) post-nucleic acid sequencing cleanup;
(xxii) reverse transcription;
(xxiii) pre-reverse transcription cleanup;
(xxiv) post-reverse transcription cleanup;
(xxv) nucleic acid ligation;
(xxvi) nucleic acid hybridization; or
(xxvii) quantification; and
the separation chamber comprises a detection position; and
(b) a separation and detection system comprising,
(i) a separation element for simultaneously separating a plurality of target analytes in the separation chamber;
(ii) one or more light sources positioned to illuminate the detection positions on the biochip;
(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 to disperse the light from the one or plurality of first optical elements according to light wavelength into at least 6 wavelength components and, the wavelength dispersive element is positioned to provide at least a portion of the dispersed at least 6 wavelength components to at least 6 detection elements, wherein
each of the detection elements is in communication with a first control element for simultaneously collecting detection information from each of the detection elements; and wherein said light detector detects fluorescence from at least 6 dyes labeled to one or more biological molecules, each dye having a unique peak wavelength.

### Claim 66 (depends on 65)

The integrated biochip system of claim 65, wherein the first reaction chamber is adapted for additional processing selected from the group consisting of nucleic acid extraction, nucleic acid purification, reverse transcription, nucleic acid ligation, nucleic acid hybridization, ultrafiltration, cleanup, quantitation and nucleic acid amplification and the combinations thereof.

### Claim 67

An integrated biochip system comprising:
(a) a biochip comprising one or a plurality microfluidic systems, wherein each microfluidic system comprises a first reaction chamber in microfluidic communication with a separation chamber, wherein
the first reaction chamber is adapted for
(i) nucleic acid extraction;
(ii) nucleic acid purification;
(iii) pre-nucleic acid amplification cleanup;
(iv) nucleic acid amplification;
(v) post-nucleic acid amplification cleanup;
(vi) pre-nucleic acid sequencing cleanup;
(vii) nucleic acid sequencing;
(viii) post-nucleic acid sequencing cleanup;
(ix) reverse transcription;
(x) pre-reverse transcription cleanup;
(xi) post-reverse transcription cleanup;
(xii) nucleic acid ligation;
(xiii) nucleic acid hybridization; or
(xiv) quantification; and
the separation chamber comprises a detection position;
and
(b) a separation and detection system comprising,
(i) a separation element for simultaneously separating a plurality of biological molecules comprising DNA sequences, in the separation chamber;
(ii) one or more light sources positioned to illuminate the detection positions on the biochip;
(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 to disperse the light from the one or plurality of first optical elements according to light wavelength into at least 6 wavelength components, and the wavelength dispersive element positioned to provide at least a portion of the dispersed at least 6 wavelength components to at least 6 detection elements, wherein each of the detection elements is in communication with a first control element for simultaneously collecting detection information from each of the detection elements; and
wherein said light detector detects fluorescence from at least 8 dyes labeled to one or more DNA sequences, each dye having a unique peak wavelength, said dyes being members of at least two 4-dye containing subsets, such that said dye sets are capable of detecting at least two DNA sequences in a single channel, wherein the number of dyes is a multiple of four, and the number of DNA sequences to be detected is equal to one quarter of the multiple, such that each of the different dyes is present in only one subset.

### Claim 68 (depends on 67)

The integrated biochip system of claim 67, wherein the first reaction chamber is adapted for additional processing selected from the group consisting of nucleic acid extraction, nucleic acid purification, reverse transcription, nucleic acid ligation, nucleic acid hybridization, ultrafiltration, cleanup, quantitation and nucleic acid amplification and the combinations thereof.

### Claim 69

A system for multiplexed analysis of at least one nucleic acid fragment in a biological sample, said system comprising:
(a) a biochip comprising one or a plurality microfluidic systems, wherein each microfluidic system comprises at least one reaction chamber in microfluidic communication with: at least one other reaction chamber or an excitation subsystem or an optical subsystem, wherein
said at least one reaction chamber is adapted for multiplexed amplification such that primers labeled with 6 or more fluorescent dyes are used to generate amplified nucleic acid fragments labeled with said 6 or more fluorescent dyes;
(b) an excitation subsystem comprising an excitation source and one or a plurality of optical elements to focus said excitation source; and
(c) a detection subsystem comprising one or a plurality of optical detectors, a wavelength dispersive element and one or a plurality of optical elements to collect emitted fluorescence from said labeled amplified nucleic acid fragments.

### Claim 70 (depends on 69)

The system of claim 69, wherein the amplified nucleic acid fragments generated are labeled with at least 3 of said primers.

### Claim 71 (depends on 69)

The system of claim 69, wherein the amplified nucleic acid fragments generated are labeled with at least 4 of said primers.

### Claim 72 (depends on 69)

The system of claim 69, wherein the amplified nucleic acid fragments generated are labeled with at least 5 of said primers.

### Claim 73 (depends on 69)

The system of claim 69, wherein amplified nucleic acid fragments are generated from all of said labeled primers.

### Claim 74 (depends on 69)

The system of claim 69, wherein said biochip further comprises one or a plurality of additional reaction chambers adapted for one or more of the following:
(i) nucleic acid extraction;
(ii) nucleic acid purification;
(iii) pre-nucleic acid amplification cleanup;
(iv) post-nucleic acid amplification cleanup;
(v) pre-nucleic acid sequencing cleanup;
(vi) nucleic acid sequencing;
(vii) post-nucleic acid sequencing cleanup;
(viii) reverse transcription;
(ix) pre-reverse transcription cleanup;
(x) post-reverse transcription cleanup;
(xi) nucleic acid ligation;
(xii) nucleic acid hybridization; or
(xiii) quantification.

### Claim 75 (depends on 74)

The system of claim 74, wherein the biochip is primarily plastic.

### Claim 76

A system for multiplexed analysis of at least one nucleic acid fragment in a biological sample, said system comprising:
(a) a biochip comprising one or a plurality microfluidic systems, wherein each microfluidic system comprises at least one reaction chamber in microfluidic communication with: at least one other reaction chamber or an excitation subsystem or an optical subsystem, wherein
said at least one reaction chamber is adapted for multiplexed amplification such that primers labeled with 5 or more fluorescent dyes are used to generate amplified nucleic acid fragments labeled with said 5 or more fluorescent dyes and a nucleic acid sizing standard labeled with a sixth fluorescent dye
(b) an excitation subsystem comprising an excitation source and one or a plurality of optical elements to focus said excitation source; and
(c) a detection subsystem comprising one or a plurality of optical detectors, a wavelength dispersive element and one or a plurality of optical elements to collect emitted fluorescence from said amplified labeled nucleic acid fragments and labeled sizing marker.

### Claim 77 (depends on 76)

The system of claim 76, wherein the biochip is primarily plastic.

### Claim 78

A system for multiplexed analysis of at least one nucleic acid fragment in a biological sample, said system comprising:
(a) a biochip comprising one or a plurality microfluidic systems, wherein each microfluidic system comprises at least one reaction chamber in microfluidic communication with: at least one other reaction chamber or an excitation subsystem or an optical subsystem, wherein
said at least one reaction chamber is adapted for multiplexed amplification such that primers labeled with 4 or more fluorescent dyes are used to generate amplified nucleic acid fragments labeled with said 4 or more fluorescent dyes and two nucleic acid sizing standards labeled with a fifth and sixth fluorescent dye
(b) an excitation subsystem comprising an excitation source and one or a plurality of optical elements to focus said excitation source; and
(c) a detection subsystem comprising one or a plurality of optical detectors, a wavelength dispersive element and one or a plurality of optical elements to collect emitted fluorescence from said amplified labeled nucleic acid fragments and labeled sizing marker.

### Claim 79 (depends on 78)

The system of claim 78, wherein said biochip further comprises one or a plurality of additional reaction chambers adapted for one or more of the following:
(i) nucleic acid extraction;
(ii) nucleic acid purification;
(iii) pre-nucleic acid amplification cleanup;
(iv) post-nucleic acid amplification cleanup;
(v) pre-nucleic acid sequencing cleanup;
(vi) nucleic acid sequencing;
(vii) post-nucleic acid sequencing cleanup;
(viii) reverse transcription;
(ix) pre-reverse transcription cleanup;
(x) post-reverse transcription cleanup;
(xi) nucleic acid ligation;
(xii) nucleic acid hybridization; or
(xiii) quantification.

### Claim 80 (depends on 78)

The system of claim 78, wherein the biochip is primarily plastic.

### Claim 81 (depends on 79)

The system of claim 79, wherein the biochip is primarily plastic.

### Claim 82 (depends on 47)

A method of detecting components separated by electrophoresis using the system of claim 47.

### Claim 83 (depends on 53)

A method of simultaneously detecting fragments from sequencing of at least two DNA templates using the system of claim 53.

### Claim 84 (depends on 57)

A method of detecting components separated by electrophoresis using the system of claim 57.

### Claim 85 (depends on 59)

A method of detecting components separated by electrophoresis using the system of claim 59.

### Claim 86 (depends on 65)

A method of separating and detecting the plurality of target analytes using the integrated biochip system of claim 65.

### Claim 87 (depends on 67)

A method of simultaneously separating a plurality of biological molecules using the integrated biochip system of claim 67.

### Claim 88 (depends on 69)

A method of performing multiplexed analysis of at least one nucleic acid fragment in the biological sample using the system of claim 69.

### Claim 89 (depends on 76)

A method of performing multiplexed analysis of at least one nucleic acid fragment in the biological sample using the system of claim 76.

### Claim 90 (depends on 78)

A method of performing multiplexed analysis of at least one nucleic acid fragment in the biological sample using the system of claim 78.