APPARATUS AND METHODS FOR PERFORMING ELECTROCHEMICAL REACTIONS

• US 20100300895A1
• Filed: 05/24/2010
• Published: 12/02/2010
• Est. Priority Date: 05/29/2009
• Status: Active Grant

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: ION Torrent Systems Incorporated (Thermo Fisher Scientific Incorporated)
• Inventors: Roth, Thomas, Nobile, John, Schultz, Jonathan, Marran, David, Rothberg, Jonathan, Rearick, Todd
• Granted Patent: US 8,673,627 B2
• Time in Patent Office: Days
• US Class Current: 205/775
• CPC Class Codes: B01J 2219/00315 Microtiter plates
• CPC Class Codes: B01J 2219/00317 Microwell devices, i.e. having large numbers of wells
• CPC Class Codes: B01J 2219/005 Beads
• CPC Class Codes: B01J 2219/00653 the compounds being bound to electrodes embedded in or on the solid supports
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: B01L 2200/0668 Trapping microscopic beads
• CPC Class Codes: B01L 2300/0636 Integrated biosensor, microarrays
• CPC Class Codes: B01L 2300/0645 Electrodes
• CPC Class Codes: B01L 2300/0877 Flow chambers
• 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: G01N 27/301 Reference electrodes
• CPC Class Codes: G01N 27/4145 specially adapted for biomolecules, e.g. gate electrode with immobilised receptors

Abstract

The invention is directed to apparatus and methods for delivering multiple reagents to, and monitoring, a plurality of analytical reactions carried out on a large-scale array of electronic sensors underminimal noise conditions. In one aspect, the invention provides method of improving signal-to-noise ratios of output signals from the electronic sensors sensing analytes or reaction byproducts by subtracting an average of output signals measured from neighboring sensors where analyte or reaction byproducts are absent. In other aspects, the invention provides an array of electronic sensors integrated with a microwell array for confining analytes and/or particles for analytical reactions and a method for identifying microwells containing analytes and/or particles by passing a sensor-active reagent over the array and correlating sensor response times to the presence or absence of analytes or particles. Such detection of analyte- or particle-containing microwells may be used as a step in additional noise reduction methods.

Claims

1. An apparatus for performing multi-step electrochemical reactions, the apparatus comprising:

one or more reaction vessels each coupled to an electronic sensor for monitoring products in the reaction vessel, the electronic sensor generating an output signal related to a concentration or presence of a product, the output signal depending on a reference voltage;

a fluidics system for sequentially delivering a plurality of electrolytes to the reaction vessel one at a time; and

a reference electrode in contact with a selected electrolyte of the plurality, the reference electrode being in fluid communication with the reaction chamber and providing the reference voltage to each electronic sensor without the reference electrode contacting any of the non-selected electrolytes.

2. The apparatus of claim 1 wherein said one or more reaction vessels is an array of microwells disposed on an array of chemFET sensors and wherein said fluidics system comprises a flow cell in fluid communication with the microwells and configured to deliver reactant electrolytes to each microwell at substantially the same flow rate.

3. The apparatus of claim 1 further including a reservoir for holding said selected electrolyte, the reservoir being connected by a branch passage to a common passage for delivering said electrolytes to said reaction vessel, the branch passage having a valve disposed between the reservoir and a junction with the common passage, wherein said reference electrode is disposed in the branch passage between the valve and the junction such that said reference electrode is in fluid communication with said reaction vessel and such that whenever the valve is shut and fluid within the branch passage is stationary and substantially no said non-selected electrolyte contacts said reference electrode.

4. The apparatus of claim 1 wherein said fluidics system comprises a plurality of passages delivering said electrolytes from reservoirs to said reaction chamber and to at least one waste reservoir, each of the plurality of passages being connected to an electrode that is capacitively connected to said reference electrode.

5. An apparatus for performing analytical reactions on a plurality of analytes, the apparatus comprising:

a sensor array comprising a plurality of sensors formed in a circuit-supporting substrate, each sensor of the array being configured to generate at least one electrical signal related to a concentration or presence of one or more reaction products proximate thereto and a microwell array disposed on the circuit-supporting substrate such that each microwell is disposed on at least one sensor, wherein one or more microwells contain analyte; and

a fluidics system for sequentially delivering a plurality of reagents to the microwell array and for providing a reference voltage to the sensors of the sensor array, the reference voltage being provided by a reference electrode upstream of and in continuous fluid communication with, the microwell array, the reference electrode being in contact with only a single one of the plurality of reagents during the delivery of different reagents to the flow cell.

6. The apparatus of claim 5 wherein said single reagent in contact with said reference electrode is a wash solution that contains no reactants of said analytical reaction.

7. An apparatus for performing analytical reactions on a plurality of analytes, the system comprising:

a sensor array comprising a plurality of sensors formed in a circuit-supporting substrate, each sensor of the array comprising a chemically sensitive field-effect transistor (chemFET) having a floating gate, the chemFET being configured to generate at least one electrical signal related to a concentration or presence of one or more reaction products proximate thereto and a microwell array disposed on the circuit-supporting substrate such that each microwell is disposed on at least one sensor, wherein one or more microwells contain analyte; and

a fluidics system for delivering reagents to the microwell array, the fluidics system comprising a flow cell having an inlet, an outlet and a flow chamber that defines a flow path of reagents as they pass from the inlet to the outlet, wherein the flow chamber is configured to deliver the reagents transversely over open portions of the microwells in the flow path, and wherein the floating gates of sensors outside of the flow path are electrically connected and held at a common voltage.

8. The system of claim 7 wherein said sensor array is a CMOS device and wherein said floating gates of said sensors outside of said flow path are electrically connected by forming such floating gates as a continuous metal layer.

9. An apparatus comprising:

a sensor array for monitoring a plurality of multi-step reactions on the surface thereof, the sensor array being formed in a semiconductor substrate and having an active region and an inactive region, the sensors of the inactive region each comprising a chemically-sensitive field effect transistor (chemFET) having a floating gate electrically connected to the gates of every other sensor of the inactive region and the sensors of the active region each comprising a chemFET having a floating gate and being configured to provide at least one output signal related to a concentration or presence of a chemical or biological sample proximate thereto, wherein the active region of the sensor array corresponds to a flow path of reagents delivered to the reactions on the surface of the sensor array.

10. The apparatus of claim 9 further comprising a flow cell having an inlet, an outlet and a flow chamber that defines said flow path of reagents as they pass from the inlet to the outlet;

and the flow chamber being configured so that said reagents are delivered in a flow parallel to a surface of said sensor array across said active region.

11. A method for locating analytes distributed among a plurality of microwells, the method comprising the steps of:

providing a plurality of microwells disposed on an array of sensors, wherein each microwell has an opening in fluid communication with a flow chamber and is capable of retaining at least one analyte, and wherein each microwell is disposed on at least one sensor configured to provide at least one output signal in response to reagents proximate thereto;

changing reagents in the flow chamber from a first reagent in response to which sensors generate a first output signal to a second reagent in response to which sensors to generate a second output signal; and

correlating a time delay of a second output signal from a sensor in response to said changing with the presence of an analyte in its corresponding microwell.

12. The method of claim 11 wherein said analyte comprises a particles.

13. The method of claim 11 wherein said first reagent is at a first pH and said second reagent is at a second pH different from the first pH and wherein at least one of said sensors generates an output signal related to pH proximate thereto.

14. The method of claim 11 wherein said step of changing includes changing pH at at least one of said sensors by at least 0.1 pH units.

15. An article of manufacture comprising:

a sensor array comprising a plurality of sensors formed in a circuit-supporting substrate, each sensor of the array being configured to generate at least one electrical signal related to a concentration or presence of one or more predetermined species proximate thereto and a microwell array disposed on the circuit-supporting substrate such that each microwell thereof has an opening on a surface of the microwell array and is disposed on at least one sensor; and

a plurality of analytes randomly distributed in the microwells at locations determinable by an output signal generated by its corresponding sensor.

16. The article of claim 15 wherein said location of each of said analytes is determined by a delay in a change of said output signal in response to a change in concentration of said one or more predetermined species at said surface of said microwell array.

17. The article of claim 16 wherein said one or more predetermined species is hydrogen ion and wherein said change in said concentration thereof is a step function having a change in magnitude of pH 1.0 or less.

18. The article of claim 15 wherein said analytes each comprise a particle having attached thereto a clonal population of nucleic acid fragments.

19. A method of reducing noise in output signals from a sensor array having disposed thereon a microwell array, such that each microwell has at least one sensor in a sensing relationship therewith, and each sensor is capable of generating an output signal in response to an analyte or an analyte reaction byproduct proximate thereto, the method comprising the steps of:

disposing analyte onto the microwell array such that a portion of the microwells contain analyte;

obtaining an output signal generated by a microwell containing, analyte or reaction byproduct and subtracting therefrom an average of output signals from a plurality of neighboring microwells where analyte and/or a reaction byproduct is absent.

20. The method of claim 19 wherein said plurality of said neighboring microwells are within the same 200 by 200 microwell region of said microwell array as said microwell containing said analyte.

21. The method of claim 19 wherein said plurality of said neighboring microwells are located within a circular region of said microwell array centered on said microwell containing said analyte and having a radius of 200 μ

m or less.

22. The method of claim 19 wherein said output signals of said average are generated by sensors of empty microwells.

23. The method of claim 19 wherein said output signals of said average are generated by sensors of microwells without a reaction byproduct.

24. The method of claim 23 wherein said average describes reagent change noise.

25. The method of claim 19 wherein said portion of microwells containing said analyte is ninety percent or less of said microwells of said microwell array.

26. The method of claim 25 wherein said analyte are solid supports each having a clonal population of nucleic acid templates attached thereto.

27. The method of claim 26 wherein said solid supports are particles.

28. The method of claim 27 wherein said particles are randomly disposed in said microwells.

29. A flow cell member for forming a fluidics interface with sensor arrays of different rectilinear sizes disposed in a rectilinear interface package, the flow cell member comprising:

a rectilinear body having an upper face and a lower face and a shape matched with that of the rectilinear interface package so that the lower face of the rectilinear body may be bonded to the rectilinear interface package to form a fluid-tight enclosure for a sensor array, wherein an inlet is disposed at one end of the upper face, and an outlet is disposed at an opposite end of the upper face;

an inlet passage interior to the rectilinear body providing a fluid passage from the inlet to the fluid-tight enclosure forming an inlet port in the lower face of the rectilinear body positioned above and at one end of the sensor array; and

an outlet passage interior to the rectilinear body providing a fluid passage from the outlet to the fluid-tight enclosure forming an outlet port in the lower face of the rectilinear body positioned above and at an end of the sensor array opposite of that of the inlet port.

30. The flow cell member of claim 29 wherein said inlet is concentrically disposed with an inlet collar in a corner of said upper face and said outlet is concentrically disposed with an outlet collar in a diagonally opposite corner of said upper face as said inlet and inlet collar.

31. The flow cell member of claim 30 wherein said inlet and outlet collars each have a radius and wherein said inlet port and said outlet port are each positioned within perpendicular projections of the radii of said inlet and outlet collars, respectively, onto said lower face of said rectilinear body.