(12) united states patent us 7,738,086 b2 shepard et al ... · and systemsii: fundamental theory...

(12) United States Patent Shepard et al.

US007738086B2

US 7,738,086 B2 Jun. 15, 2010

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ACTIVE CMOS BIOSENSOR CHIP FOR FLUORESCENT-BASED DETECTION

Inventors: Kenneth L. Shepard, Ossining, NY (US); Rastislav Levicky, Irvington, NY (US); George Patounakis, North Brunswick, NJ (US)

Assignee: The Trustees of Columbia University in the City of New York, New York, NY

(Us)

Notice: Subject to any disclaimer, the term of this patent is extended or adjusted under 35 U.S.C. 154(b) by 398 days.

Appl. No.: 11/800,468

Filed: May 4, 2007

Prior Publication Data

US 2008/0037008 A1 Feb. 14, 2008

Related US. Application Data

Continuation-in-part of application No. 11/431,405, ?led on May 9, 2006, now abandoned.

Provisional application No. 60/679,545, ?led on May 9, 2005, provisional application No. 60/799,408, ?led on May 9, 2006.

Int. Cl.

G01N 21/00 (2006.01) US. Cl. ....................... .. 356/73; 356/317; 356/318;

436/172; 435/4; 435/6 Field of Classi?cation Search ................. .. 356/73,

356/244, 246, 317, 318; 435/4, 6; 436/172; 422/8208

See application ?le for complete search history.

(56) References Cited

U.S. PATENT DOCUMENTS

5,039,219 A 8/1991 James et a1. 5,812,272 A 9/1998 King et a1. 6,078,705 A 6/2000 Neuschafer et a1. 6,117,643 A 9/2000 Simpson et a1. 6,197,503 B1 3/2001 Vo-Dinh et a1. 6,317,207 B2 11/2001 French et a1.

(Continued) OTHER PUBLICATIONS

Schena, M. et a1., “Microarrays: biotechnology’s discovery platform for functional genomics,” Trends in Biotechnology, v01. 16, pp. 301 306, Jul. 1998.

(Continued) Primary ExamineriL. G Lauchman (74) A no rn ey, A gen Z, or F i rmiWi lmer Cutler Pickering Hale and Dorr

(57) ABSTRACT

An active CMOS biosensor chip for ?uorescent-based detec tion is provided that enables time-gated, time-resolved ?uo rescence spectroscopy. In one embodiment, analytes are loaded with ?uorophores 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 ?uorescent inten sity of the ?uorophore at different times. These measure ments are then averaged to generate a representation of the transient ?uorescent decay response unique to the ?uoro phores. In addition to its low-cost, compact form, the biosen sor chip provides capabilities beyond those of macroscopic instrumentation by enabling time-gated operation for back ground rejection, easing requirements on optical ?lters, and by characterizing ?uorescence lifetime, allowing for a more detailed characterization of ?uorophore labels and their envi ronment. The biosensor chip can be used for a variety of applications including biological, medical, in-the-?eld appli cations, and ?uorescent lifetime imaging applications.

26 Claims, 16 Drawing Sheets

FLUORESCENTLY 100 LABELED SAMPLE (a)

FLUORESCENTLY LABELED?gMPLEW) EXCITATION

PROBE MEMISSION 104 ‘ PROBE

BIOPOLYMER LAYER \ 102 ’

5 110\ SENSOR ELECTRONICS

US 7,738,086 B2 Page 2

U.S. PATENT DOCUMENTS

6,331,438 B1 12/2001 Aylottet a1. 6,448,064 B1 9/2002 Vo-Dinh et a1. 6,469,785 B1 10/2002 Duveneck et al. 6,743,581 B1 6/2004 Vo-Dinh 6,784,982 B1 8/2004 Blumenfeldet al. 6,803,238 B1 10/2004 Eggers 6,867,851 B2 3/2005 Blumenfeldet al. 6,975,251 B2 12/2005 Pavicic 7,145,645 B2 12/2006 Blumenfeldet al. 7,179,654 B2 2/2007 Verdonk et al.

2003/0143575 A1 7/2003 Caria 2004/0175821 A1 9/2004 Ehman 2004/0234417 A1 2004/0249227 A1 2005/0136448 A1 2006/0014151 A1 2006/0134644 A1

11/2004 Schienle et al. 12/2004 Klapproth et a1. 6/ 2005 Hartel et al. 1/2006 Ogura et al. 6/ 2006 Hartel et al.

2006/0197960 A1 9/2006 Bazylenko 2007/0121111 A1 5/2007 Blumenfeld et al. 2008/0265177 A1* 10/2008 Connally et a1. ....... .. 250/461.2

OTHER PUBLICATIONS

Daubert, S]. et al., “Current Copier Cells,” Electronic Letters, vol. 24, No. 25, pp. 1560-1562, Dec. 8, 1988.

Mir, K.U. et al., “Sequence Variation in Genes and Genomic DNA: Methods for Large-Scale Analysis,” Annu. Rev. Genomics Hum, Genet. 2000. 1:329-60.

McIntosh, S.L., et al., “Fluorescence Lifetime for On-the-Fly Mul tiple Detection of DNA Restriction Fragments in Capillary Electrophoresis,” Analytical Chemistry, vol. 72, No. 1, pp. 5444 5449, Nov. 1, 2000.

Templin, M.T. et al., “Protein microarray technology,” Trends in Biotechnology, vol. 20, No. 4, pp. 160-166, Apr. 2002. Daubert, S]. et al., “A Transistor-Only Current-Mode EA Modula tor,” IEEE Journal ofSolid-State Circuits, vol. 27, No. 5, pp. 821-830, May 1992.

Hacia, J .G. et al., “Mutational analysis using oligonucleotide microarrays,” J. Med. Genet, pp. 730-736, 1999. Hyun, D. et al., “Limit Cycles and Pattern Noise in Single-Stage Single-Bit Delta-Sigma Modulators,” IEEE Transactions on Circuits and SystemsiI: Fundamental Theory and Applications, vol. 49, No. 5, pp. 646-656, May 2002. Eltoukhy, H. et al., “A 0.18 pm CMOS 106qu Bioluminescence Detection System-on-Chip,” ISSCC 2004 / Session 12 / Biomicrosystems/ 12.3, pp. 1-3.

* cited by examiner

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US Patent Jun. 15, 2010 Sheet 16 0f 16 US 7,738,086 B2

CHIP SPECIFICATIONS AND MEASURED PERFORMANCE

Item Value

Chip

Technology TSMC 0.25 pm

Mixed-Signal CMOS

Die Size 5 mm X 5 mm

Clock Speed 20 MHz

SRAM Size 2048x24-bits

Sensitivity 1.15 >< 10“ photons/(2mg

Linearity 9 bits (w/o calibration)

Delay line resolution

Laser drive maximum current (at 2.7 V)

Time to complete single measurement

Pixel array

Array size

Quantum ef?ciency (at 635 nm)

Pixel size

Photodiode size

Transconductor gain

Pixel gain

Dark signal

Rdiadii

ADC

Architecture

Order

DAC

ADC gain

Cycle Time

Cycles per Sample

Input full scaie

230 ps

130 mA

33 ms

8 x 4

0.45

180 X 4l5 pm2

100 x 100 ,umz

0.22 mS

7.04 >12-bit settling)

4096 (adjustable)

FIG. 19

US 7,738,086 B2 1

ACTIVE CMOS BIOSENSOR CHIP FOR FLUORESCENT-BASED DETECTION

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation-in-part of US. patent application Ser. No. 11/431,405, ?led May 9, 2006, which claims the bene?t under 35 U.S.C. §119(e) of US. Provi sional Patent Application No. 60/679,545, ?led May 9, 2005, which are hereby incorporated by reference herein in its entirety. This application also claims the bene?t under 35 U.S.C. §119(e) of US. Provisional Patent Application No. 60/799,408, ?led May 9, 2006, which is hereby incorporated by reference herein in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with United States Government support under National Science Foundation Grant No. BES 0428544 and National Institutes of Health Grant No. HG003089. The United States Government may have certain rights in this invention.

BACKGROUND OF THE INVENTION

1. Technical Field The present invention relates to ?uorescent-based detec

tion. More particularly, the present invention relates to sys tems and methods for providing time-gated, time-resolved ?uorescent-based detection on an active complementary metal oxide semiconductor (CMOS) biosensor chip.

2. Description of the Related Art An assay is a qualitative and/or quantitative analysis of an

unknown analyte. In one example, an assay can be a proce dure that determines the concentration and sequences of DNA in a mixture. In another example, an assay can be an analysis of the type and concentrations of protein in an unknown sample.

Surface-based sensing assays are typically performed in environmental and biomedical diagnostics. The detection of analytes (targets) in a mixture is often implemented at a solid-liquid interface. Passive solid supports, which include glass substrates or polymer membranes, have probe mol ecules (i.e., “probes”) immobilized on the surface of the solid supports that are used to bind the analytes of interest. Probes include, for example, proteins and nucleic acids. Probes are selected based on the analytes of interest such that there is a strong and speci?c interaction between a particular type of probe and a particular target. More than one analyte can be detected using multiplexed

detection. In multiplexed detection, different types of probes are arranged in an array on the surface of the solid supports. Each type of probe results in a strong and speci?c interaction with a different analyte of interest. For example, in DNA analysis, high density microarrays are used to examine gene expressions at the scale of entire genomes by simultaneously assaying mixtures derived from expressed mRNA against thousands of array sites, each bearing probes for a speci?c gene. Microarrays generally quantify target concentrations in relative terms, for example, in the form of a ratio to hybrid ization signal obtained using a reference target sample. Other biosensing applications are calibrated to provide absolute target concentrations.

Fluorescent-based detection is commonly used for quanti fying the extent of probe-target binding in surface-based

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2 sensing assays. In ?uorescent-based detection, a target is labeled with a ?uorophore molecule, which can cause the target ?uorophore to be ?uorescent. Traditional microarray scanners include an excitation source, such as a laser, that emits light on the bound target ?uorophores. This causes the target ?uorophores to emit ?uorescent light that is focused and collected (through a generally lossy optical path) onto a cooled charge-coupled device (CCD) or a photomultiplier tube (PMT). Optical ?ltering is typically used to improve the signal-to-noise ratio (SNR) by removing background light or re?ected excitation light. In addition, the arrays are generally sensitive to particular ?uorophore concentrations from 108 to 101 l cm_2.

Characteristic lifetimes are associated with each ?uoro phore. The lifetime is de?ned by the transient exponential ?uorescent decay of the ?uorophore once the excitation source is removed. The lifetime, which is typically on the order of nanoseconds for organic dyes, is characteristic of the dye and the environment, and can be used in addition to color and intensity for multiplexed detection. Quantum-dot ?uoro phores can have lifetimes exceeding 15 nanoseconds at the cost of slightly lower quantum yields. Fluorescent lifetime detection, for example, has been employed for capillary elec trophoresis in both the time and frequency domain. Fluores cent lifetime is also sensitive to excited-state reactions, such as ?uorescent resonance energy transfer (FRET), which allows for the detection of macromolecular associations labeled by two different ?uorophores. For micro-arrays, FRET can be used to detect in situ real-time hybridization kinetics in which both the probe and target are ?uorophore labeled.

In most commercial time-resolved systems, PMT detectors use time-correlated single photon counting (TCSPC). In this case, sensitivity is limited by a dark count, which is typically about 400 Hertz (Hz). For a typical peak quantum e?iciency of 25%, this corresponds to a detection limit of approximately 2>

US 7,738,086 B2 3

Accordingly, it is desirable to provide methods and sys tems that overcome these and other de?ciencies of the prior art.

SUMMARY OF THE INVENTION

In accordance with the present invention, systems and methods are provided for providing time-resolved, time gated ?uorescent-based detection on an active complemen tary metal oxide semiconductor (CMOS) biosensor chip. An active CMOS biosensor chip for ?uorescent-based

assays is provided that enables time-gated, time-resolved ?uorescence spectroscopy. Analytes are loaded with ?uoro phores 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 ?uorescent intensity of the ?uoro phore at different times. These measurements are then aver aged to generate a representation of the transient ?uorescent decay response of the ?uorophores, which is unique to the ?uorophores. This data can then be used for further analysis of the analytes.

In addition to its low-cost, compact form, the biosensor chip provides capabilities beyond those of macroscopic instrumentation by enabling time-gated operation for back ground rejection, easing requirements on optical ?lters, and by characterizing ?uorescence lifetime, allowing for a more detailed characterization of ?uorophore labels and their envi ronment. The biosensor chip can be used for a variety of applications including biological, medical, and in-the-?eld applications. The biosensor chip can be used for DNA and protein microarrays where the biomolecular probe is attached directly to the chip surface. The biosensor chip can also be used as a general ?uorescent lifetime imager in a wide-?eld or confocal microscopy system. For example, the biosensor chip can be used as an imager in a conventional wide?eld epi?uo rescent microscope for lifetime imaging.

According to one or more embodiments of the invention, a method is provided for operating an imager with time-re solved, time-gated ?uorescent-based detection comprising: (a) receiving light from a ?uorescent source on a complemen tary metal oxide semiconductor (CMOS) biosensor chip, wherein the ?uorescent source is excited by an external pulsed excitation light source; (b) directing the light source to turn off after a ?rst time period; (c) measuring a ?uorescent light from the ?uorescent source on 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. The biosensor chip can be used as a microarray or as an imager in a wide?eld epi?uorescent microscope for lifetime imaging.

According to one or more embodiments of the invention, a system is provided for time-resolved, time-gated ?uorescent based detection comprising: an external pulsed excitation light source; and a complementary metal oxide semiconduc tor (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 ?rst time period, (c) measure a ?uorescent light from a ?uorescent source on the CMOS biosensor chip after a sec ond 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. The CMOS biosensor chip can include at least one driver, at least one photodiode, processing circuitry (e. g., sample-and-hold circuitry, analog to-digital converter, and accumulator), and control circuitry. The CMOS biosensor can also include delay circuitry. The

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4 biosensor chip can be used as a microarray or as an imager in a wide?eld epi?uorescent microscope for lifetime imaging.

According to one or more embodiments of the invention, an apparatus is provided for time-resolved, time-gated ?uo rescent-based detection comprising: a ?rst printed circuit board on which is mounted a pulsed excitation light source; a second printed circuit board on which is mounted a comple mentary metal oxide semiconductor (CMOS) biosensor chip; and at least one cable with a ?rst connector attached to the ?rst 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. The CMOS bio sensor chip can be operative to measure a ?uorescent decay response of at least one ?uorescently labeled target, wherein the target is bound to a probe immobilized on the surface of the CMOS biosensor chip, and wherein the ?uorescent 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.

There has thus been outlined, rather broadly, the more important features of the invention in order that the detailed description thereof that follows may be better understood, and in order that the present contribution to the art may be better appreciated. There are, of course, additional features of the invention that will be described hereinafter and which will form the subject matter of the claims appended hereto.

In this respect, before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not limited in its application to the details of construction and to the arrangements of the components set forth in the following description or illustrated in the draw ings. The invention is capable of other embodiments and of being practiced and carried out in various ways. Also, it is to be understood that the phraseology and terminology employed. herein are for the purpose of description and should not be regarded as limiting. As such, those skilled in the art will appreciate that the

conception, upon which this disclosure is based, may readily be utilized as a basis for the designing of other structures, methods and systems for carrying out the several purposes of the present invention. It is important, therefore, that the claims be regarded as including such equivalent constructions insofar as they do not depart from the spirit and scope of the present invention.

These together with the other objects of the invention, along with the various features of novelty which characterize the invention, are pointed out with particularity in the claims annexed to and forming a part of this disclosure. For a better understanding of the invention, its operating advantages and the speci?c objects attained by its uses, reference should be had to the accompanying drawings and descriptive matter in which there are illustrated preferred embodiments of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

Various objects, features, and advantages of the present invention can be more fully appreciated with reference to the following detailed description of the invention when consid ered in connection with the following drawings, in which like reference numerals identify like elements.

FIG. 1 is a block diagram of a sensor chip in accordance with an embodiment of the invention.

FIG. 2 is a timing diagram of time-resolved, time-gated ?uorescent-based detection in accordance with an embodi ment of the invention.

(12) united states patent us 7,738,086 b2 shepard et al ... · and systemsii: fundamental theory...

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