METHODS AND APPARATUS FOR MEASURING ANALYTES USING LARGE SCALE FET ARRAYS

• US 20120247977A1
• Filed: 07/28/2011
• Published: 10/04/2012
• Est. Priority Date: 06/25/2008
• Status: Active Grant

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: ROTHBERG, Jonathan, HINZ, Wolfgang, JOHNSON, Kim, BUSTILLO, James, LEAMON, John, SCHULTZ, Jonathan
• Granted Patent: US 8,524,057 B2
• Time in Patent Office: Days
• US Class Current: 205/780.5
• CPC Class Codes: B01L 2300/046 Function or devices integrated in the closure
• CPC Class Codes: B01L 2300/0663 Whole sensors
• CPC Class Codes: B01L 2300/0851 Bottom walls
• CPC Class Codes: B01L 2400/086 using baffles or other fixed flow obstructions
• CPC Class Codes: B01L 3/502761 specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules investigating characteristics of particles G01N15/00
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2565/301 Pyrophosphate (PPi)
• CPC Class Codes: C12Q 2565/607 being a sensor, e.g. electrode
• CPC Class Codes: C12Q 2565/629 being a microfluidic device
• 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

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 the concentration of inorganic pyrophosphate (PPi), hydrogen ions, and nucleotide triphosphates.

Claims

1. An apparatus comprising:

a chemical-sensitive field effect transistor (chemFET) array formed in a semiconductor substrate, the chemFET array having an overlayed array of reaction chambers wherein each reaction chamber is in contact with or capacitively coupled to at least one chemFET, wherein the chemFETs each have a floating gate with zero or substantially zero trapped charge, so that chemFETs of the array have output levels without appreciable variation across the array; and

, a flow cell member configured and arranged for receiving and exposing the array of reaction chambers to a fluid flow of reagents, the flow cell member being configured so that such reagents are delivered transversely over an open portion of each reaction chamber with uniform transit times across the array of reaction chambers, wherein the flow cell member has an inlet and an outlet positioned at opposite corners of the flow cell.

2. The apparatus of claim 1 wherein said flow cell is configured to provide a laminar flow of said reagents.

3. The apparatus of claim 2 wherein each reaction chamber is no greater than one of 100, 70, 50, or 30 μ

m² in open surface area.

4. The apparatus of claim 1 wherein said chemFET array is an array of CMOS-fabricated sensors, each sensor comprising a chemFET.

5. The apparatus of claim 4 wherein said chemFET array includes a two-dimensional array of at least 512 rows and at least 512 columns of said CMOS-fabricated sensors.

6. The apparatus of claim 5 wherein each of said CMOS-fabricated sensors consists of three field effect transistors including a chemFET and each such sensor includes a plurality of electrical conductors electrically connected to the three FETs.

7. The apparatus of claim 1 further comprising control circuitry coupled to said chemFET array and configured to generate at least one array output signal to provide multiple frames of data from the array at a frame rate of at least 20 frames per second.

8. The apparatus of claim 1 wherein each of said reaction chambers has a volume of less than 1 pL.

9. The apparatus of claim 1 wherein said reaction chamber array comprises one of at least 100, 1,000, 10,000, 100,000, or 1,000,000 reaction chambers.

10. A method for sequencing a nucleic acid comprising:

disposing a plurality of template nucleic acids into a plurality of reaction chambers, wherein the plurality of reaction chambers is in contact with a chemFET array of more than 256 chemFETs formed in a semiconductor substrate, the chemFET array comprising at least one chemFET for each reaction chamber, such chemFET having a floating gate with zero or substantially zero trapped charge, so that chemFETs of the array have output levels without appreciable variation across the array, and wherein each of the double stranded DNA template nucleic acids are treated with a nickase thereby creating a nick where nucleotides can be incorporated by a polymerase,

synthesizing a new nucleic acid strand by incorporating one or more known nucleoside triphosphates sequentially at the nick, and

detecting the incorporation of the one or more known nucleoside triphosphates by a change in voltage and/or current at the at least one chemFET within the array in response to a presence and/or concentration of a sequencing reaction byproduct proximate thereto, wherein a center-to-center distance between adjacent reaction chambers is within a range of 1-10 μ

m.

11. The method of claim 10 wherein said sequencing reaction byproduct is hydrogen ion concentration and wherein said reaction takes place in a weak buffer.

12. The method of claim 11 wherein said output of each of said sensors represents changes in said hydrogen ion concentration within a range of from pH 7 to pH 9.

13. The method of claim 10 wherein each of said reaction chambers contains at least 10⁵ copies of one of said template nucleic acids.

14. The method of claim 10 wherein said nucleic acid templates are attached to a solid support.

15. The method of claim 12 wherein each of said reaction chambers contains at least 10⁵ copies of one of said template nucleic acids.

16. The method of claim 14 wherein said solid support is a bead.

17. A method for sequencing a nucleic acid comprising:

(a) disposing a plurality of beads into a plurality of reaction chambers on a sensor array of more than 256 sensors formed in a semiconductor substrate, each reaction chamber comprising a single bead, each bead attached to a plurality of identical template nucleic acids, each of the template nucleic acids hybridized to a sequencing primer and bound to a polymerase immobilised to the bead, and each reaction chamber in contact with or capacitively coupled to at least one chemical-sensitive field effect transistor (chemFET) of a sensor, each such chemFET having a floating gate with zero or substantially zero trapped charge, so that chemFETs of the sensor array have output levels without appreciable variation across the array, and each such chemFET being configured to provide at least one output representing a presence and/or concentration of a sequencing reaction byproduct proximate thereto, and wherein each sensor of the array occupies an area of less than 100 μ

m²,

(b) introducing a known nucleoside triphosphates into each reaction chamber,

(c) detecting sequential incorporation at the 3′

end of the sequencing primer of or more nucleoside triphosphates by the generation of a sequencing reaction byproduct if the known nucleoside triphosphate is complementary to corresponding nucleotides in the template nucleic acid,

(d) washing unincorporated nucleoside triphosphates from the reaction chambers, and

(e) repeating steps (b) through (d) until the nucleic acid is sequenced.

18. The method of claim 17 wherein said reaction chambers have a center-to-center spacing within a range of 1-10 μ

m.

19. A method for sequencing a nucleic acid comprising:

fragmenting a target nucleic acid to generate a plurality of fragmented nucleic acids, attaching each of the plurality of fragmented nucleic acids to individual beads to generate a plurality of beads each attached to a single fragmented nucleic acid,

amplifying the fragmented nucleic acids on each bead resulting in a plurality of identical fragmented nucleic acids on each bead,

immobilising polymerase on each bead,

delivering a plurality of beads attached to fragmented nucleic acids to an array of reaction chambers disposed on a sensor array having more than 256 sensors formed in a semiconductor substrate, each reaction chamber being in a sensing relationship with an electrochemical, non-optical sensor of the array, wherein only one bead is situated in each reaction chamber and wherein each sensor occupies an area of less than 100 μ

m² and is a chemFET having a floating gate with zero or substantially zero trapped charge, so that chemFETs of the sensor array have output levels without appreciable variation across the array, each such chemFET being configured to provide at least one output representing a presence and/or concentration of a sequencing reaction byproduct proximate thereto, and

performing sequencing reactions with the immobilised polymerase simultaneously in the plurality of reaction chambers.

20. The method of claim 19 wherein said reaction chambers have a center-to-center spacing within a range of 1-10 μ

m.

21. The method of claim 19 wherein said sensor array has at least 100,000 sensors.

22. The method of claim 19 wherein said plurality of fragmented nucleic acids is individually amplified using a water in oil emulsion amplification method.

23. The method of claim 19 wherein each of said reaction chambers is in contact with a single said chemFET.