Active CMOS biosensor chip for fluorescent-based detection

• US 7,738,086 B2
• Filed: 05/04/2007
• Issued: 06/15/2010
• Est. Priority Date: 05/09/2005
• Status: Active Grant

• Current Assignee: Trustees Of Columbia University In The City Of New York (Columbia University)
• Original Assignee: Trustees Of Columbia University In The City Of New York (Columbia University)
• Inventors: Patounakis, George, Shepard, Kenneth L., Levicky, Rastislav
• Primary Examiner(s): LAUCHMAN, LAYLA G
• Application Number:
  US11/800,468
• Publication Number:
  US 20080037008A1
• Time in Patent Office: 1,138 Days
• Field of Search: 356/73, 356/244, 356/246, 356/317, 356/318, 435/4, 435/6, 436/172, 422/82.08
• US Class Current: 356/73
• CPC Class Codes: G01N 21/6408 with measurement of decay time, time resolved fluorescence
• CPC Class Codes: 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
• CPC Class Codes: G01N 21/6454 using an integrated detector array
• CPC Class Codes: H01L 27/14621 Colour filter arrangements
• CPC Class Codes: H01L 27/14643 Photodiode arrays; MOS imagers
• CPC Class Codes: H04N 25/51 Control of the gain
• CPC Class Codes: H04N 25/771 comprising storage means other than floating diffusion
• CPC Class Codes: H04N 3/155 Control of the image-sensor operation, e.g. image processing within the image-sensor

Abstract

An active CMOS biosensor chip for fluorescent-based detection is provided that enables time-gated, time-resolved fluorescence spectroscopy. In one embodiment, analytes are loaded with fluorophores that are bound to probe molecules immobilized on the surface of the chip. Photodiodes and other circuitry in the chip are used to measure the fluorescent intensity of the fluorophore at different times. These measurements are then averaged to generate a representation of the transient fluorescent decay response unique to the fluorophores. In addition to its low-cost, compact form, the biosensor chip provides capabilities beyond those of macroscopic instrumentation by enabling time-gated operation for background rejection, easing requirements on optical filters, and by characterizing fluorescence lifetime, allowing for a more detailed characterization of fluorophore labels and their environment. The biosensor chip can be used for a variety of applications including biological, medical, in-the-field applications, and fluorescent lifetime imaging applications.

Claims

1. A method for operating an imager with time-resolved, time-gated fluorescent-based detection comprising:

(a) receiving light from a fluorescent source on a surface layer of a complementary metal oxide semiconductor (CMOS) biosensor chip, wherein the fluorescent source is excited by an external pulsed excitation light source;

(b) directing the light source to turn off after a first time period;

(c) measuring a fluorescent light from the fluorescent source on the surface layer of the CMOS biosensor chip after a second time period measured from when the light source is directed to turn off;

(d) repeating (a)-(c) a number of times; and

(e) averaging results from each measuring.

2. The method of claim 1 further comprising measuring the fluorescent light with a photodiode.

3. The method of claim 1 wherein the CMOS biosensor chip is used as a microarray, in which the measuring comprises:

immobilizing a probe on the surface of the CMOS biosensor chip;

binding a fluorescently labeled target to the probe; and

detecting the fluorescent light from the fluorescently labeled target.

4. The method of claim 1 wherein the CMOS biosensor chip is used as an imager in a widefield epifluorescent microscope for lifetime imaging.

5. The method of claim 1 wherein the second time period changes with each subsequent measuring.

6. The method of claim 1 wherein the second time period is the same with each subsequent measuring.

7. The method of claim 1 wherein the measuring comprises:

driving a photodiode with a reset signal at the end of the second time period;

receiving an integrated photocurrent across the photodiode;

sampling the integrated photocurrent;

converting the sampled integrated photocurrent from an analog format to a digital format; and

averaging the converted integrated photocurrent.

8. A system for time-resolved, time-gated fluorescent-based detection comprising:

an external pulsed excitation light source; and

a complementary metal oxide semiconductor (CMOS) biosensor chip coupled to the light source, wherein the CMOS biosensor chip is operative to;

(a) direct the light source to turn on,

(b) direct the light source to turn off after a first time period,

(c) measure a fluorescent light from a fluorescent source on a surface layer of the CMOS biosensor chip after a second time period measured from when the light source is directed to turn off,

(d) repeat (a)-(c) a number of times, and

(e) average results from each measure.

9. The system of claim 8 wherein the CMOS biosensor chip comprises:

at least one driver operative to direct the light source to turn on and off;

at least one photodiode operative to receive the fluorescent light;

processing circuitry operative to measure the fluorescent light and average results from each measure; and

control circuitry operative to control the operation of the driver, the photodiode, and the processing circuitry.

10. The system of claim 9 further comprising delay circuitry operative to delay a reset signal by the second time period, wherein the output of the delay circuitry is used to time-gate the integration of photocurrent from the photodiode.

11. The system of claim 9 wherein the processing circuitry further comprises:

sample-and-hold circuitry operative to sample the integrated photocurrent from the photodiode;

an analog-to-digital converter operative to convert the integrated photocurrent from an analog format to a digital format; and

an accumulator operative to average the converted integrated photocurrent.

12. The system of claim 8 wherein the second time period changes with each subsequent measuring.

13. The system of claim 8 wherein the second time period is the same with each subsequent measuring.

14. The system of claim 8 wherein the light source is a laser.

15. The system of claim 14 wherein the laser comprises a laser diode, a collimating lens, and a focusing lens held by a lens holder.

16. The system of claim 8 further comprising:

a first printed circuit board on which is mounted the light source;

a second printed circuit board on which is mounted the CMOS biosensor chip; and

at least one cable with a first connector attached to the first printed circuit board and coupled to the light source and a second connector attached to the second printed circuit board and coupled to the CMOS biosensor chip.

17. The system of claim 8 wherein the CMOS biosensor chip is a ceramic quad-flat-pack packaged biochip.

18. The system of claim 8 wherein the CMOS biosensor chip is used as a microarray, in which the CMOS biosensor chip that is operative to measure the fluorescent light is further operative to:

immobilize a probe on the surface of the CMOS biosensor chip;

bind a fluorescently labeled target to the probe; and

detect the fluorescent light from the fluorescently labeled target.

19. The system of claim 8 wherein the CMOS biosensor chip is used as an imager in a widefield epifluorescent microscope for lifetime imaging.

20. Apparatus for time-resolved, time-gated fluorescent-based detection comprising:

a first printed circuit board on which is mounted a pulsed excitation light source;

a second printed circuit board on which is mounted a complementary metal oxide semiconductor (CMOS) biosensor chip; and

at least one cable with a first connector attached to the first printed circuit board and coupled to the light source and a second connector attached to the second printed circuit board and coupled to the CMOS biosensor chip.

21. The apparatus of claim 20 wherein the CMOS biosensor chip is operative to measure a fluorescent decay response of at least one fluorescently labeled target, wherein the target is bound to a probe immobilized on the surface of the CMOS biosensor chip, and wherein the fluorescent decay response is measured at a plurality of different time periods measured from a time when the light source is turned off after a period during which the light source is turned on.

22. The apparatus of claim 21 wherein the CMOS biosensor chip is further operative to average the fluorescent decay response measured at the plurality of different time periods.

23. The apparatus of claim 20 wherein the CMOS biosensor chip is operative to measure a fluorescent decay response of at least one fluorescently labeled target, wherein the target is bound to a probe immobilized on the surface of the CMOS biosensor chip, and wherein the fluorescent decay response is measured a plurality of times at a time period measured from a time when the light source is turned off after a period during which the light source is turned on.

24. The apparatus of claim 23 wherein the CMOS biosensor chip is further operative to average the fluorescent decay response measured at the plurality of times.

25. The apparatus of claim 20 wherein the CMOS biosensor chip is used as a microarray.

26. The apparatus of claim 20 wherein the light source is a laser.