Methods and apparatus for measuring analytes using large scale FET arrays

• US 8,306,757 B2
• Filed: 03/10/2010
• Issued: 11/06/2012
• Est. Priority Date: 12/14/2006
• Status: Active Grant

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Rothberg, Jonathan M., Hinz, Wolfgang, Johnson, Kim L., Bustillo, James M.
• Primary Examiner(s): Gross, Christopher M
• Assistant Examiner(s): Flinders, Jeremy C
• Application Number:
  US12/721,458
• Publication Number:
  US 20100197507A1
• Time in Patent Office: 972 Days
• Field of Search: 257/253, 257/E29.3, 702/20, 327/427, 327/434, 438/10, 435/288.4
• US Class Current: 702/20
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2533/101 Primer extension
• CPC Class Codes: C12Q 2565/301 Pyrophosphate (PPi)
• CPC Class Codes: C12Q 2565/607 being a sensor, e.g. electrode
• CPC Class Codes: G01N 27/4145 specially adapted for biomolecules, e.g. gate electrode with immobilised receptors
• CPC Class Codes: G01N 27/4148 Integrated circuits therefor, e.g. fabricated by CMOS processing CMOS processing per se H01L21/82
• CPC Class Codes: G01N 33/54373 involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
• CPC Class Codes: G01N 33/5438 Electrodes
• CPC Class Codes: H01L 27/088 the components being field-effect transistors with insulated gate
• CPC Class Codes: H01L 29/42324 Gate electrodes for transistors with a floating gate
• CPC Class Codes: H01L 29/78 with field effect produced by an insulated gate H01L29/7725, H01L29/775, H01L29/778 take precedence
• CPC Class Codes: H01L 2924/01006 Carbon [C]
• CPC Class Codes: H01L 2924/01013 Aluminum [Al]
• CPC Class Codes: H01L 2924/01015 Phosphorus [P]
• CPC Class Codes: H01L 2924/01073 Tantalum [Ta]
• CPC Class Codes: H01L 2924/14 Integrated circuits
• CPC Class Codes: H01L 2924/1433 Application-specific integrated circuit [ASIC]
• CPC Class Codes: Y10T 436/22 Hydrogen, per se

Abstract

Methods and apparatus relating to very large scale FET arrays for analyte measurements. ChemFET (e.g., ISFET) arrays may be fabricated using conventional CMOS processing techniques based on improved FET pixel and array designs that increase measurement sensitivity and accuracy, and at the same time facilitate significantly small pixel sizes and dense arrays. Improved array control techniques provide for rapid data acquisition from large and dense arrays. Such arrays may be employed to detect a presence and/or concentration changes of various analyte types in a wide variety of chemical and/or biological processes. In one example, chemFET arrays facilitate DNA sequencing techniques based on monitoring changes in hydrogen ion concentration (pH), changes in other analyte concentration, and/or binding events associated with chemical processes relating to DNA synthesis.

Claims

1. A method for nucleic acid sequencing, comprising:

disposing a plurality of template nucleic acids in at least 10⁵ reaction volumes for at least 10⁵ sensors, the sensors comprising field-effect transistors having floating gates coupled to the reaction volumes to detect hydrogen ions liberated through incorporation of a nucleotide into one or more primers that are hybridized to at least one of the plurality of template nucleic acids;

introducing a known nucleotide into the reaction volumes;

coupling circuitry to field-effect transistors of the sensors to measure pH changes within the reaction volumes within a pre-specified pH range that is less than a maximum pH range detectable by the field-effect transistors, the measured pH change based on liberation of hydrogen ions detected by the field-effect transistors resulting from incorporation of the introduced known nucleotide into the one or more primers, and the circuitry further to generate output signals indicating the measured pH changes; and

analyzing the output signals generated by the circuitry to determine at least a portion of sequences corresponding to a portion of the template nucleic acids.

2. The method of claim 1, wherein the sensors respectively occupy an area of 100 μ

m² or less and have a pitch of 10 μ

m or less and wherein the reaction volumes have respective volumes in the range of from 1 μ

m³ to 1500 μ

m³.

3. The method of claim 1, wherein the reaction volumes respectively contain at least 10⁵ copies of one of said template nucleic acids.

4. The method of claim 1, wherein the nucleic acid templates are retained by at least one solid support.

5. The method of claim 1, wherein the reaction volumes and the sensors are each greater than 256,000.

6. The method of claim 4, wherein the at least one solid support comprises at least one particle.

7. The method of claim 6, wherein the at least one particle comprises cellulose, a cellulose derivative, gelatin, an acrylic resin, glass, silica gel, polyvinyl pyrrolidine (PVP), co-polymers of vinyl and acrylamide, polystyrene, polystyrene cross-linked with divinylbenzene, a polyacrylamides, a latex gel, dextran, a crosslinked dextrans, rubber, silicon, a plastic, nitrocellulose, a natural sponge, a metal, agarose gel, or a streptavidin coating.

8. The method of claim 6, wherein the at least one particle is 1-10 μ

m in diameter.

9. The method of claim 1, wherein the known nucleotide is a nucleotide selected from the group consisting of dATP, dCTP, dGTP and dTTP.

10. The method of claim 1, wherein introducing the known nucleotide comprises delivery of a wash buffer solution comprising ATP to the reaction volumes.

11. The method of claim 1, further comprising:

washing unincorporated introduced known nucleotide from the reaction volumes;

introducing a known second nucleotide into the reaction volumes;

coupling the circuitry to the field effect transistors to measure second pH changes within the reaction volume based on liberation of hydrogen ions detected by the field-effect transistors resulting from incorporation of the introduced known second nucleotide into the one or more primers, and the circuitry further generating second output signals indicating the measured second pH changes; and

analyzing the second output signals to determine at least a second portion of sequences corresponding to a second portion of the template nucleic acids.

12. The method of claim 1, wherein the measured pH changes are based on threshold voltages of the field-effect transistors due to the liberated hydrogen ions.

13. The method of claim 1, wherein coupling the circuitry to a given field-effect transistor of a given sensor in the sensors comprises:

coupling a first terminal and a second terminal of the given field-effect transistor to bias circuitry, the bias circuitry applying a read voltage to the first terminal and establishing a voltage difference between the first terminal and the second terminal;

coupling the given field-effect transistor to a current source to provide a bias current through the given field-effect transistor, thereby establishing a voltage level at the second terminal based on a threshold voltage of the given field-effect transistor resulting from a corresponding pH change; and

coupling the second terminal to readout circuitry to read the voltage level at the second terminal to generate a corresponding output signal.

14. The method of claim 13, wherein the given sensor further comprises:

a first switch coupled between the second terminal of the given field-effect transistor and the bias circuitry and the readout circuitry; and

a second switch coupled between the second terminal of the given field-effect transistor and the current source.

15. The method of claim 14, wherein the first switch and the second switch are responsive to a common select signal to couple the second terminal of the given field-effect transistor to the bias circuitry and to the current source.

16. The method of claim 14, wherein the first switch and the second switch are each single-transistor switches having channel types the same as that of the given field-effect transistor.

17. The method of claim 14, wherein the first terminal of the given field-effect transistor is directly connected to the bias circuitry.

18. The method of claim 14, wherein the bias circuitry and the readout circuitry is shared among a plurality of the sensors.

19. The method of claim 18, wherein the sensors are arranged in a plurality of rows and a plurality of columns, and the bias circuitry and the readout circuitry is shared among a column of sensors in the plurality of columns.

20. The method of claim 1, wherein a given sensor of the sensors further comprises:

a first single-transistor switch coupled between a given field-effect transistor of the given sensor and a current source to provide a bias current through the given field-effect transistor to establish a voltage level on a first terminal of the given field-effect transistor based on the pH change; and

a second single-transistor switch coupled between the first terminal of the given field-effect transistor and the circuitry to read the voltage level at the first terminal to generate a given output signal.

21. The method of claim 20, wherein the first single-transistor switch and the second single-transistor switch have channel types the same as that of the given field-effect transistor.

22. The method of claim 20, wherein body connections of the first single-transistor switch, the second single-transistor switch and the given field-effect transistor are each coupled to a common line.

23. The method of claim 1, wherein the pre-specified pH range extends between pH 7 and pH 9.

24. The method of claim 1, wherein the pre-specified pH range corresponds to 2 pH units.

25. The method of claim 1, wherein the floating gates of the field-effect transistors are coupled to the reaction volumes via a passivation layer sensitive to hydrogen ions.

26. The method of claim 1, wherein the disposing includes disposing a template nucleic acid into a reaction volume, and amplifying the template nucleic acid in the reaction volume.

27. The method of claim 26, wherein the amplifying includes isothermally amplifying the template nucleic acid in the reaction volume.