WO2005073407A1

WO2005073407A1 - Advanced integrated circuit biochip

Info

Publication number: WO2005073407A1
Application number: PCT/US2004/032984
Authority: WO
Inventors: Tuan Vo-Dinh; Alan L. Wintenberg
Original assignee: Ut-Battelle, Llc
Priority date: 2003-10-07
Filing date: 2004-10-07
Publication date: 2005-08-11

Abstract

A biochip-based detection system a biosensor platform including at least one receptor probe, the receptor probe providing an intensity modulated electromagnetic detection signal when combined with a target in response to irradiation by an intensity modulated electromagnetic excitation signal. An integrated circuit microchip is optically coupled with the biosensor platform and includes at least one photodetector for converting the electromagnetic detection signal into an electrical signal. Signal processing circuitry is provided for receiving the electrical signal and determining spectroscopic data including at least one lifetime of the target to permit its identification. The system supports the methods of both time resolved spectroscopy and phase resolved spectroscopy.

Description

ADVANCED INTEGRATED CIRCUIT BIOCHIP

FIELD OF THE INVENTION

[0001] The invention relates to methods and integrated circuit based apparatus for detection of chemical and biological molecules, more particularly to systems and methods of spectroscopic detection of such molecules using lifetime differentiation.

BACKGROUND OF THE INVENTION

[0002] There is a strong interest in the development of improved biosensors for environmental and biomedical diagnostics. Whereas there has been active research and development in various laboratories regarding biosensors using DNA probes, there has been very limited work on integrated circuit (IC) gene probe-based microchip sensors. Previous sensor systems have generally used commercially available photomultipliers or 2- dimensional detectors, such as charge-coupled device (CCD) based systems, which although being sensitive require bulky electronic and data conditioning accessories. [0003] The inventor has disclosed biochips including improved integrated circuit based sensors, such as CMOS process compatible photodiode and phototransistor arrays in U.S. Patent No. 6,197,503 (2001) entitled "Integrated Circuit Biochip Microsystem Containing Lens," to Vo-Dinh, et al. U.S. Patent No. 6,197,503 (Vo-Dinh '503) discloses an integrated circuit microchip sensor that detects a target biomolecule, the integrated circuit microchip sensor including a probe that specifically binds to target biomolecules. Vo-Dinh '503 is incorporated by reference in its entirety into the present application. At least one sensor is connected to the probe, wherein when the probe is bound to the target and irradiated with suitable radiation a detection signal is generated which is detected by the sensor. Detection circuitry connected to the sensor is provided for processing the detection signal, and for generating an output signal, wherein the detection signal indicates the presence of the target biomolecule in the sample.

SUMMARY OF INVENTION [0004] A biochip-based detection system includes a biosensor platform including at least one receptor probe, the receptor probe providing an intensity modulated electromagnetic detection signal when combined with a target in response to irradiation by an intensity modulated electromagnetic excitation signal. An integrated circuit microchip is optically coupled with the biosensor platform which includes at least one photodetector for converting the electromagnetic detection signal into an electrical signal. The biosensor platform can be affixed to the microchip. The electromagnetic detection signal can comprises an absorption signal, a fluorescent signal, a phosphorescent signal, an elastic scattering signal or a Raman scattering signal.

[0005] Signal processing circuitry is provided for receiving the electrical signal and determining spectroscopic data including at least one lifetime of the target to permit its identification. The system can support the methods of time resolved spectroscopy and phase resolved spectroscopy.

[0006] The system can include a source of intensity modulated electromagnetic excitation signals. The excitation signal can comprise at least one radiation pulse, the radiation pulse having a pulse width shorter than the lifetime of the target, wherein the lifetime is determined by the signal processing circuitry provided using time resolved spectroscopy. In another embodiment of the invention, the excitation source provides excitation signals modulated at a frequency greater the lifetime, wherein the lifetime of the target is determined by the signal processing circuitry using phase resolved spectroscopy. The signal processing circuitry can include a demodulator for demodulating the electrical signal or an amplified version of the electrical signal, where the excitation signal is used as a phase input for the demodulator circuit, thus providing synchronous demodulation.

[0007] The system can include an on-chip source of excitation signals and on chip mirrors for directing the excitation signals to the receptor probe. The on-chip source of excitation signals can comprise a laser. The system can include an RF transmitter for transmitting the spectroscopic data.

[0008] The biosensor platform preferably includes a plurality of receptor probes. In this embodiment, a plurality of photodetectors are provided, wherein the plurality of probe elements are each provided a dedicated detection channel, the dedicated detection channel comprising one of the plurality of photodetectors and a dedicated amplifier.

[0009] The signal processing circuitry can include an analog to digital converter. The signal processing circuitry can comprise a band pass filter whose pass band includes a modulation frequency of the excitation signal.

[0010] A method for detecting target analytes comprises the steps of providing a biochip, the biochip including a biosensor platform including at least one receptor probe for combining with at least one target molecule. The receptor probe provides an intensity modulated electromagnetic detection signal when combined with the target in response to irradiation by an intensity modulated electromagnetic excitation signal. The receptor probe is contacted with a sample suspected of containing the target. The probe or a probe/target combination (if the target is bound to the probe) is irradiated with the intensity modulated excitation signal to produce detection signals if the target is bound to the probe. Spectroscopic data derived from the detection signal is then used to identify whether the target is present in the sample, such as by determining at least one lifetime of the target from the detection signal. The method can comprise time resolved spectroscopy or phase resolved spectroscopy.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011] A fuller understanding of the present invention and the features and benefits thereof will be accomplished upon review of the following detailed description together with the accompanying drawings, in which:

[0012] FIG. 1 illustrates a integrated circuit biochip which uses time varying electromagnetic excitation radiation in the form of pulsed light or other modulated light to identify target analytes using lifetime differentiation, according to an embodiment of the invention.

[0013] FIG. 2A illustrates an exemplary excitation pulse and the resulting decay curve response.

[0014] FIG. 2B illustrates a train of periodic excitation pulses and the resulting decay curve responses.

[0015] FIG. 2C illustrates a sinusoidal excitation signal and the resulting phase shifted sinusoidal emission signal.

[0016] FIG. 3 illustrates a block diagram of biochip system for processing responses produced by continuous wave (CW) excitation, according to an embodiment of the invention.

[0017] FIG. 4 illustrates a block diagram of biochip electronics for non-synchronous detection for processing responses produced by modulated excitation, according to an embodiment of the invention.

[0018] FIG. 5 illustrates a block diagram of biochip electronics for synchronous detection for processing responses produced by modulated excitation, according to another embodiment of the invention. [0019] FIG. 6 illustrates a block diagram of biochip electronics which can be used with pulsed excitation and gated detection, according to an embodiment of the invention.

[0020] FIG. 7 illustrates a block diagram of biochip electronics which can be used with modulated excitation, synchronous detection and RF data transmission, according to yet another embodiment of the invention.

[0021] FIG. 8 illustrates a block diagram of an exemplary integrating amplifier which may be used with the invention, according to an embodiment of the invention.

[0022] FIG. 9 illustrates a block diagram of a synchronous demodulator and low-pass filter which may be used with the invention, according to another embodiment of the invention.

[0023] FIG. 10 illustrates typical waveforms for a biochip used with an on/off modulated light source and the integrating preamplifier shown in Fig. 8 and the synchronous demodulator and low-pass filter shown in Fig. 9.

[0024] FIG. 11 illustrates a block diagram of a programmable timer which may be used with the invention, according to an embodiment of the invention.

[0025] FIG. 12 illustrates a block diagram of an exemplary frequency generator and programmable phase shifter which may be used with the invention, according to another embodiment of the invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0026] The invention includes a self-contained advanced integrated circuit (AIC) based biochip and related methods which permit target identification based on lifetime differentiation. Target lifetime can be determined using either time-resolved and/or phase- resolved detection by applying time- varying excitation radiation and measuring the resulting detection signal generated when one or more targets are present in a sample. Different targets provide one or more characteristic lifetimes which permit their positive identification. [0027] The AIC biochip according to the invention includes bioreceptor-based methods for bioanalysis, such as DNA, antibody, and/or protein-based receptor probes, spectroscopy- based detection using lifetime differentiation (time- and phase-resolved detection), and integrated circuits sensors (e.g. CMOS-based). The AIC biochip electronics can be produced using modern microelectronics processing which permits low cost fabrication of multiple optical sensing elements and supporting electronic circuitry on a single chip. [0028] Conventional photoluminescent techniques use a fixed amplitude (non-modulated) excitation signal. Although the bioreceptor probes (e.g. DNA or antibody) generally used with the invention are highly selective even when used with conventional photoluminescent techniques which use a fixed amplitude (non-modulated) excitation signal, lifetime differentiation permits improved detection sensitivity. First, lifetime differentiation provides an enhanced signal to noise ratio (S?NR) as compared to conventional techniques by removing or at least substantially decreasing interference from other signals, such as forward scattered excitation light, background radiation and other interferences. Second, even with probes such as DNA and antibody probes, there can be cross reactivity between similar structures which can be removed using lifetime differentiation, but not with conventional systems and related techniques.

[0029] Figure 1 illustrates an advanced integrated circuit (AIC) biochip 100 which uses time-varying excitation radiation to distinguish target analytes using lifetime differentiation, according to an embodiment of the invention. Biochip 100 includes excitation light source 102, such as a LED or laser. Light modulator 104 or equivalent receives light from light source 102 and provides a pulsed light output. Alternatively, the light source provided can itself be a modulated light source. The modulated light is filtered by optional band-pass filter 106 and is then diffracted by diffracting optic/focusing lens 108. Diffracting optic/focusing lens 108 can provide a plurality of excitation light beams, such as sixteen (16) to provide one light beam per probe, the respective light beams having an area to match the area of the respective receptor probes, such as probes 116-119 (as well as 12 other unnumbered probes) on microarray 121, referred collectively herein as probes 116-119 et al. Probes 116-119 et al. shown in FIG. 1 include cell-based probe 116, enzyme probe 117, DNA probe 118, and antibody probe 119. Reflective optic 115 directs the light beams produced by diffracting optic/focusing lens 108 towards probes 116-119 et al. on microarray 121. [0030] Assuming fluorescent spectroscopy is used, the resulting fluorescence signals produced if binding events take place at any of the respective probes 116-119 et al. is directed via the GRIN lens array 131 toward integrated electrooptic chip 140. A detection wavelength selection filter 132 is preferably included to pass the detection signal of interest and to eliminate background signals as well as the laser (or LED) scattered light. Following wavelength selective filtering, the detection (e.g. fluorescent) signal reaches chip 140. [0031] Chip 140 includes integrated electrooptics, such as a photosensor microarray 142 based on an array of optoelectronic transducers, such as photodiodes, phototransistors or avalanche diodes. As shown in FIG. 1, the photodetector microarray 142 includes sixteen (16) photosensors 143, one sensor 143 for each receptor probe 116-119 et al. on microarray 121. This arrangement permits each detection channel to have customizable characteristics to match the associated bioreceptor, such as a high gain amplifier (not shown) to accompany photodetector channels which are expected to operate at low signal levels. Although generally preferable to have one photosensor 143 and optional amplifier (not shown) and filter (not shown) for each receptor probe 116-119 et al, the invention can clearly be practiced with an unequal number, such as a single photosensor 142 to service all detection signals originating from probes 116-119 et al. using a multiplex switch (not shown). It is noted that circuit components including amplifiers, filters, multiplex switches, and analog to digital converters can all be provided on microchip 140.

[0032] Chip 140 can provide a synchronous or gated detection system 155. In the case of synchronous detection, the excitation signal which modulates the intensity of light source 102 can also be used as a phase input for a demodulator circuit to provide synchronous detection (See FIG. 5). In the case of gated detection, the electrical signal derived from the detection signal is provided to a gated amplifier. The gated amplifier opens to detection gate only when it receives the detection pulse after a user defined delay time, (see FIG. 6). [0033] In addition, chip 140 can provide a microprocessor 150 or equivalent to compile spectroscopic data from detection signals received including determination of lifetimes of the various targets presented to system 100 in a given sample. Lifetime data extraction can be performed using standard techniques. For example, see " Basic Instrumentation in Photonics" by T. Vo-Dinh in Biomedical Photomcs Handbook, T. Vo-Dinh, Editor, CRC Press, Boca Raton, Florida, 2003, Chapter 6, pp. 6-25-6-27.

[0034] A wireless transmission system 162 is an optional feature that is also in system 100, such as on chip 140. Wireless transmission system 162 can include a digital signal processor (not shown) to encode the spectroscopic data onto an RF carrier and a transmitter and antenna (not shown) for transmitting the modulated RF carrier. The antenna is generally located off chip, unless the RF carrier is at more than about 2 to 4 GHz due to the short length of the antenna as compared to the wavelength of the carrier. In the case where the chip size is large, such as several square centimeters or more, an on-chip (integrated) transistor may be practical for RF carrier frequencies of at least about 2.4 GHz. In this embodiment, microprocessor 150 is generally not required. [0035] Although not shown in FIG. 1, the light source 102 can be an on-chip light source. In this embodiment, one or more MEMS mirrors (not shown) can be provided on-chip to direct light from the on-chip light source toward the microarray 121.

[0036] Recently there has been an increasing interest in biosensor technology. Biosensors combine two important concepts that integrate "biological recognition" and "sensing". The basic principle of a biosensor is to detect this molecular recognition and to transform it into another type of signal using a transducer. The selected transducer may produce either an optical signal (i.e., optical biosensors) or an electrochemical signal (i.e., electrochemical biosensors).

[0037] Construction of a biosensor generally involves the integration of several basic elements of very different natures. The basic steps include selection or development of the bioreceptor, selection of the excitation source, selection or development of the transducer, and integration of the excitation source-bioreceptor-transducer system. [0038] The role of the bioreceptor is to identify the target compounds via molecular recognition. Bioreceptors generally determine the specificity provided by the biochip system. Bioreceptors can take many forms and the different bioreceptors that have been used are as numerous as the different analytes that have been monitored using biosensors. However, bioreceptors can generally be classified into several different major categories. These categories include enzymes, antibodies, a gene fragments, chemoreceptors, tissues, organelles or a microorganism, and biomimetics.

[0039] The operation of gene probes is based on the hybridization process. Hybridization involves the joining of a single strand of nucleic acid with a complementary probe sequence. Hybridization of a nucleic acid probe to DNA biotargets (e.g., gene sequences, bacteria, viral DNA) offers a very high degree of accuracy for identifying DNA sequences complementary to that of the probe. Nucleic acids strands tend to be paired to their complements in the conesponding double-stranded structure. Therefore, a single-stranded DNA molecule will seek out its complement in a complex mixture of DNA containing large numbers of other nucleic acid molecules. Hence, nucleic acid probe (i.e., gene probe) detection methods are very specific to DNA sequences. Factors affecting the hybridization or reassociation of two complementary DNA strands include temperature, contact time, salt concentration, and the degree of mismatch between the base pairs, and the length and concentration of the target and probe sequences. [0040] Biologically active DNA probes can be directly or indirectly immobilized onto a transducer detection surface to ensure optimal contact and maximum detection. When immobilized onto a substrate, the gene probes are stabilized and, therefore, can be reused repetitively. In the simplest procedure, hybridization is performed on an immobilized target or a probe molecule attached on a solid surface such as a nitrocellulose, a nylon membrane or a glass plate.

[0041] Several methods can be used to bind DNA to different supports. The method commonly used for binding DNA to glass involves silanization of the glass surface followed by activation with carbodiimide or glutaraldehyde. The silanization method can be used for binding to glass surfaces using 3-glycidoxypropyltrimethoxysilane (GOP) or aminopropyltrimethoxy-silane (APTS) and covalently link DNA via amino linkers incorporated either at the 3' or 5' end of the molecule during DNA synthesis. [0042] Another approach consists of immobilizing the gene probe onto a membrane and subsequently attaching the membrane to the transducer detection surface. This approach avoids the need of binding the bioreceptor onto the transducer and could possibly allow easier large-scale production. Several types of membranes are available for DNA binding include nitrocellulose and charge-modified nylon. The gene probe is then bound to the membrane using ultraviolet activation.

[0043] The AIC biochip 100 is designed to be compatible to a wide variety of amplification techniques such as polymerase chain reaction (PCR) which is an important technique allowing replication of defined DNA sequences, thereby amplifying the detection of these sequences, the strand displacement amplification (SDA) technique [developed by BD Sciences], immuno-PCR. techniques, and other hybrid techniques.

[0044] Antibodies are biological molecules that exhibit very specific binding capabilities for specific structures. This is important due to the complex nature of most biological systems. An antibody is a complex biomolecule, made up of hundreds of individual amino acids arranged in a highly ordered sequence. For an immune response to be produced against a particular molecule, a certain molecular size and complexity are necessary; proteins with molecular weights greater then 5000 Da are generally immunogenic. The way in which an antigen and its antigen-specific antibody interact may be understood as analogous to a lock and key fit, by which specific geometrical configurations of a unique key enables it to open a lock. In the same way, an antigen-specific antibody "fits" its unique antigen in a highly specific manner. This unique property of antibodies is the key to their usefulness in immunosensors where only the specific analyte of interest, the antigen, fits into the antibody binding site.

[0045] Enzymes are often chosen as bioreceptors based on their specific binding capabilities as well as their catalytic activity. In biocatalytic recognition mechanisms, the detection is amplified by a reaction catalyzed by macromolecules called biocatalysts. With the exception of a small group of catalytic ribonucleic acid molecules, all enzymes are proteins. Some enzymes require no chemical groups other than their amino acid residues for activity. Others require an additional chemical component called a cofactor, which may be -l. J- _*1- J- either one or more inorganic ions, such as Fe , Mg , Mn , or Zn , or a more complex organic or metalloorganic molecule called a coenzyme. The catalytic activity provided by enzymes allows for much lower limits of detection than would be obtained with common binding techniques. The catalytic activity of enzymes depends upon the integrity of their native protein conformation. If an enzyme is denatured, dissociated into its subunits, or broken down into its component amino acids, its catalytic activity is destroyed. Enzyme- coupled receptors can also be used to modify the recognition mechanisms. [0046] The AIC system 100 can measure optical signals from a wide variety of spectroscopic processes, including absorption, fluorescence, phosphorescence, elastic scattering, and Raman scattering. One important parameter of the signal emanating from samples of interest is the lifetime of the radiation emanated. The lifetimes of selected various processes are generally as follows: (1) absorption: instantaneous with excitation (2) fluorescence: 10^"10 sec to 10^"8 sec (3) phosphorescence: 10^"6 to 10^"3 sec (4) scattering: almost instantaneous with excitation

[0047] Two methods of measuring emanated signals that permit determination of lifetimes comprise time-resolved and phase-resolved methods. Time-resolved and phase- resolved methods can improve the signal-to-noise values mainly by differencing the actual signal of interest from the background noise (a DC signal).

[0048] hi the time-resolved method, a pulsed excitation signal is used. The width of the excitation is generally much shorter than the emission or other process of interest, so that the excitation width is much shorter than the lifetime or lifetimes (also referred to as decay time(s)) of the samples. If it is desired to measure the lifetime, the time-dependent emanated intensity I(t) can be measured following the excitation pulse. The decay time (τ) can then be calculated from the slope of a plot of log I(t) versus t, where I is the intensity and t is time, or from the time at which the emanated signal intensity (I) decreases to 1/e (about 37%) of the initial emanated intensity value l(t=0).

[0049] To measure the emission (or other emanated signal) intensity free from influence from the excitation pulse 215, the detection process can begin after a delay time (dt) sufficiently after the excitation pulse such that the excitation pulse intensity has decreased close to zero as shown in FIG. 2 A. In this method, the decay curve 210 represents the detection signal to be analyzed. Different compounds generally provide different characteristic decay time(s). Thus, compounds present in samples can be identified on the basis of their decay times, such as the time to reach 1/e of the initial emanated intensity value at the end of the excitation pulse 215.

[0050] Alternatively, different compounds having different decay times can be differentiated by using different delay times (dT) 225 and gate times (ΔT) 230 as shown in FIG. 2B. The gate time (ΔT) corresponds to the time window (portion) of the decay curve 245 in which detection takes place. For example, the emission of a compound having a short decay time could be detected using a short gate time, while a longer decay time sample would require a longer gate to properly register to provide a good signal-to-noise value. To distinguish between two compounds with only one being present, but not knowing which one, two measurements can be performed using two different (short and long) gate times. If the measurements using two gate times show the same results (same signal intensities), it could be concluded that the compound with the short decay time is present, as all the short- decay emission fits in the two gate windows. However, if the two gate times produce different results, such as the signal obtained with the short gate time is lower than the signal with the longer gate time, it could be concluded that the compound present was the one having a long gate time. Similar variations could be performed using a fixed gate time and varying the delay time. A long delay time would cause the measurement to miss short-decay emissions, but register long-decay emissions. A short delay time would register both emissions.

[0051] An important source of noise in many measurement situations is DC noise from the background. Improvement in signal-to-noise can be achieved by using multiple periodic excitation pulses, and by applying the "boxcar" method by integrating the emission signal during a gate time (ΔT) after each excitation pulse 240 as also shown in FIG.2B. [0052] Another method that can be used to determine lifetimes involves phase-resolved techniques, which are often referred to as frequency domain techniques, hi the phase-resolved technique, the sample is again excited with intensity-modulated light. However, rather than a series of square wave pulses used in the time resolved method, the intensity of the incident light changes with a single very high frequency (ω = 2πf, f being the frequency in hertz) as compared to the reciprocal of the target decay time(s) τ in the sample. Following excitation with the high frequency modulation signal, the emission or other signal emanation becomes intensity-modulated at the same modulation frequency. However, since the emission or other emanation follows a decay time, there is a certain delay in the emission relative to the excitation as shown in FIG. 2C. This delay is generally measured as a phase-shift (φ), which can be used to calculate the decay time. At each modulation frequency TΠ, the delay is described as a phase shift §_w, which increases from 0° to 90° with increasing modulation frequency w.

[0053] The finite time response of targets in the sample also results in demodulation of the emission by a factor m_w. This factor decreases from 1.0 to 0 with increasing modulation frequency. At low frequency, the emission or other signal emanated closely follows the excitation signal. Accordingly, the phase angle is near zero and the modulation is near 1. As the modulation frequency is increased, the finite lifetime of the emission or other emanation process prevents the emission from closely following the excitation. This results in a phase delay of the emission, and a decrease in the peak-to-peak amplitude of the modulated emission or other signal emanated.

[0054] The shape of the frequency response is determined by the number of decay times displayed by the sample. If the decay is a single exponential, the frequency analysis is simplified, h this case, the phase angle or modulation at any frequency can be used to calculate the lifetime. For single-exponential decay, the phase and modulation are related to the decay time (τ) by the following relations: tan φ_TO = τσ τ ; and m_ω = (1 + ω² τ²) ^_1/2 [0055] Therefore, one can differentiate and thus identify various emissions or other signal emanations having different decay times by selecting the phase shift (φ) optimized to the decay time (τ) of interest. This method is generally referred to phase-resolved detection. [0056] Figure 3 shows a block diagram of exemplary biochip system 300 for operation with a CW excitation source 305 which irradiates sample/probe 310. Optical emanations (e.g., fluorescence, fluorescence, Raman) from sample/probe 310 are detected by the CMOS sensing elements 320, such as phototransistors, photodiodes or avalanche photodiodes on the biochip, and the photocurrent is integrated with the integrating amplifier 330. Each CMOS sensor 320 can have its own amplification circuit, thus allowing individual amplification gain to be set for each type of probe. For example, some DNA probes need strong amplification whereas other probes do not need high amplification. Lower gain permits lower noise operation. By setting each individual gain for each probe, one can optimize the biochip in the dynamic range of specific target agents.

[0057] Alternatively, all CMOS sensing elements 320 can also be interfaced to a common amplification unit. In this anangement, the integrator 330 can be periodically reset using a stable frequency derived from a crystal oscillator 326 and a programmable timer 327. The integrating amplifier 330 is reset through an appropriate signal sent to turn on switch 328, which is embodied as a transistor. This process can take place over and over again. [0058] The output of the integrator 330 therefore is a "sawtooth" waveform whose period depends upon the timer period and whose amplitude depends upon the magnitude of the photocurrent and inversely upon the size of the integrating capacitor 322. Thus, the transimpedance gain (voltage out/current in) of integrator 330 can be programmed either by setting the timer period or by the selection of the value of the integrating capacitor 322. The use of a long integration period and a small capacitor 322 allows a very large gain, thus providing high sensitivity.

[0059] There are a number of ways to further process the signal output from the integrator 330. Figure 3 illustrates one way. The integrating amplifier output is shown fed to a low-pass filter 340 which is then converted to a digital format using an analog-to-digital converter (ADC) 350. This arrangement provides "averaging" of the integrated signal which permits removal of the background signal. Thus, electronics 300 provides signal-to-noise ratio values better than previous systems.

[0060] The digitized result can be stored, averaged, or otherwise manipulated. Another anangement could make additional use of a programmable timer to trigger the ADC to convert the integrator output immediately before the reset signal. In this case, the low-pass filter 340 would not be needed.

[0061] Figure 3 illustrates only one CMOS sensor 320 (e.g. photodiode) and one integrating amplifier 330. In practice, sensors (e.g. photodiodes) will generally be part of an array of sensors. There may be one integrating amplifier 330 and other circuitry per sensor (e.g. photodiode), or there may be multiplexing between a plurality of photodiodes and a lower number of integrating amplifiers. Also, other photodetectors 320, such as phototransistors or avalanche photodiodes (APD) could also be used, rather than photodiodes. [0062] There is at least one significant limitation to using a CW laser light source for the biochip. One obvious limitation is that there is no frequency difference between the desired emissions and a portion of the essentially continuous background spectrum due to room light. As a result, it is not possible to electronically separate the background from the signal of interest. Also, for a dc signal, the only practical filter is a low-pass filter (either as an analog filter or as digital averaging), and that makes the measurement subject to enors due to dc drifts due to thermal effects, 1/f noise or other low-frequency random noise. An alternative to CW excitation is to modulate the laser output, such as on-off to produce a square wave or other amplitude modulation and use frequency sensitive detection.

[0063] Figure 4 shows the design of an AIC System 400 with modulated excitation and non-synchronous detection using a band-pass filter. A crystal oscillator 405 provides the modulation frequency to a frequency selector (programmable timer) 410, which selects the frequency used to run the laser driver module 415. The laser driver unit modulates the intensity of the laser light emitted by laser 420 w?hich is used to excite the sample/bioprobe 425 on a microarray sampling platform or other substrate.

[0064] For example, for biosensor systems which include DNA probes, light from laser 420 can be used to excite fluorescent labels on the DNA probes. If the target DNA is present, fluorescence emission from the probe labels responsive to the excitation radiation is detected via optics/filter system 430, which is used to isolate the fluorescence emission which is shifted in frequency from the excitation frequency from other interferences such as laser scattering and background noise. The isolated fluorescence emission is detected by CMOS sensor(s) 435, such as a photodiode(s), which converts the luminescence signal into an electrical signal.

[0065] The electrical signal is preferably amplified by amplifier 440 and fed to a bandpass filter 445 and then to a power averaging circuit 450. An amplifier 440 whose pass-band includes the modulation frequency of the excitation signal, such as 10 kHz, is used. The amplified signal from photodiode 435 is sent through a band-pass filter 440 that is centered about the modulation frequency of the excitation signal. This filter 440 blocks the dc term due to background light.

[0066] Following the band-pass filter 445, the system 400 preferably includes power level averaging circuit 450 or peak-power detecting circuit and then an ADC 455, which provides the data in digital form. Since system 400 uses band-pass amplifiers and filters, it also avoids some of the problems that are encountered with dc-amplifiers and filters. Similar signal processing chains are commonly used in communications.

[0067] It is possible to improve frequency-sensitive detection by using synchronous detection. Since in the single chip embodiment the electronics for the biochip drive both the laser as well as detect the signal from the photodetectors, these two signals can easily be made synchronous. This allows using the emission's frequency (or waveshape), along with the phase of the emission or time of occurrence.

[0068] Figure 5 shows an AIC System 500 with modulated excitation and synchronous detection using analog modulation. A crystal oscillator 505 provides the modulation frequency to a frequency selector (programmable timer) 510, which selects the frequency used to run a laser driver module 515. The laser driver unit 515 modulates the intensity of the laser light emitted by laser 520 which is used to excite the target sample/bioprobe 525 on the microanay sampling platform provided or other substrate. The emission or other detection signal filtered by optics/filter 530 to remove forward scattered laser light and background radiation and is detected by the CMOS sensing 535, such as photodiode, which converts the luminescence signal into an electrical signal. The electrical signal is amplified by the amplifier 540 and fed into a synchronous demodulator 545.

[0069] The laser 520 can be modulated with selectable duty cycles, such as a 50% duty cycle which represents 50% on-time and 50% off-time. The photodiode 535 output current is amplified using an amplifier 540 having sufficient bandwidth to pass the modulation frequency. The excitation signal is also used as the phase input though connection to phase shift selector 548 which passes the excitation signal on to the synchronous demodulator circuit 545. This synchronously rectifies the amplifier output, and a low-pass filter 550 is used to average the output of the synchronous demodulator 545. To the first order, signals not having the same frequency as the modulation excitation frequency are averaged to zero. Accordingly, this approach would reject dc and other interfering signals. This is approximately equivalent to using the circuit of FIG. 4, except it is easier to implement a very narrow-band system due to the phase locking. The output signal from the synchronous demodulator 545 is then fed to a low-pass filter 550 and then to an analog-to-digital converter (ADC) 555, which provides the data out 560 in digital form. [0070] Figure 6 shows an AIC Biochip System 600 with pulsed excitation and gated detection using analog gated integration. A crystal oscillator 605 provides the modulation frequency to a frequency selector (programmable timer) 610, which selects the frequency used to run laser driver module 615. The laser driver module 615 triggers laser 620 to produce laser pulses which are used to excite the target sample/bioprobe 625 on a microarray sampling platform or other substrate. Optics/filter 630 is used to isolate the fluorescence emission from other interferences such as laser scattering and background noise. The isolated emission is detected by the CMOS sensing element 635, which converts the luminescence signal into an electrical signal. The electric signal is amplified by amplifier 640 and fed into a gated integrator 645. The gated integrator 645 opens "the detection gate" only when it receives a signal from a gate generator 647, which is triggered by a delay generator 648. The delay generator 648 is run by the same excitation pulses (that triggers the pulsed laser light), but provides a time delay (δt) between the excitation pulses and the opening of the detection gate. Therefore, it is possible to eliminate excitation from the laser light scattered and background radiation by setting the delay period after the excitation pulse is off. Fluorescence emission from different probes having different decay times τ can also be discriminated by using different δt values optimized to the target probe decay times.

[0071] The detection time Δt during which the detection gate is open is set by the gate generator 647. Since fluorescence decay time is in the nanosecond range, the detection time can be selected to be large enough to collect most to the fluorescence (or other) signals of interest, but avoiding most of the DC background noise. The gate width can be adjusted to integrate as much signal as possible while integrating as little noise as possible. For example, if the laser pulse is 5 nanosecond wide and the fluorescence decay is 100 nanoseconds, the delay may be set to 8 nanoseconds and the integration gate set to 150 nanoseconds. Also, the Δt values can be selected to be optimized with the fluorescence (or other) decay time(s) δt of interest.

[0072] The signal from the gated integrator 645 is fed to a band-pass filter 650 and then to a power averaging circuit 655. Finally the analog signal is fed into an analog-to-digital converter (ADC) 660, which provides the data out 665 in digital form. [0073] Figure 7 shows an AIC Wireless Biochip 700 with modulated excitation, synchronous detection and RF data transmission. A crystal oscillator 705 provides the modulation frequency to a frequency synthesizer (programmable timer) 710, which selects the frequency used to run a laser driver module 715. The laser driver unit 715 modulates the intensity of the laser light emitted by laser 720 which is used to excite the target sample 725. Optics/filter system 730 is used to isolate the desired emanated signal (e.g. fluorescence emission) from other interferences such as laser scattering and background noise. The isolated emission or other detection signal is detected by the CMOS sensing element 735, which converts the luminescence signal into an electrical signal. The electrical signal is amplified by amplifier 740 and fed to a synchronous demodulator 745. [0074] In system 700 the sample is excited with intensity-modulated light which results in an emission which is also intensity-modulated at the same modulation frequency. The delay in the emission relative to the excitation is measured as a phase-shift (φ), which can be used to calculate the decay time. At each modulation frequency, the delay is described as the phase shift φc, which increases from 0° to 90° with increasing modulation frequency TO. [0075] The synchronous demodulator 745 is operated by a phase-shift selector 747, which is synchronized with the laser excitation modulation frequency. The finite time response of the sample also results in demodulation of the emission (or other detection signal) by a factor m. Therefore, differentiation of various emissions having different decay times can be accomplished by selecting the phase shift optimized to the decay time of interest. [0076] The detected signal from synchronous demodulator 745 after filtering by low pass filter 750 has low frequency (near dc) and is used to modulate the ? F carrier via mixer 755. The RF carrier signal provided to mixer 755 in FIG. 7 is the same as the signal used to used to modulate the laser 720. However, this anangement is not required as a separate signal can be used to provide a earner to mixer 755. System 700 also includes RF amplifier, transmitter and antenna 760.

[0077] In system 700 shown in FIG. 7 the detection signal is not digitized on chip, but is transmitted over the air, using the radio-frequency (RF) wireless link comprising mixer 755 and RF amplifier, transmitter and antenna 760. RF amplifier 760 amplifies the modulated carrier signal including the detection signal prior to transmission. The microchip which provides other system components such as sample/bioprobe 725, CMOS sensor 735 and amplifier 740 can also include an integrated antenna for this purpose. A remotely located receiver (not shown) demodulates the transmitted signal and returns the low-frequency signal of interest (detection signal) for digitization (e.g. using ADC) and other data processing circuitry.

[0078] Whereas the previous sections describe overall detection systems, various exemplary electronic circuits which can be used in the systems described earlier are now described. Figure 8 shows a simplified schematic diagram of a low-noise integrating amplifier 800 which can be used as an amplifier in biochip systems, including those described herein. The designs described can be realized using convention CMOS processing and as a result can preferably be provided on a microchip which provides a plurality of biochip components.

[0079] MOSFETs Ql (802) and Q2. (804) implement a folded cascade circuit, which provides gain and low input-referred noise. Q3 (806) is a common source stage, which provides approximately unity gain and low output impedance, while Q4 (808) is a current source that sets the quiescent current for Q3(806). Values for Rl (822) and R2 (824) are selected to set the biasing of Q3 (806). Rl (822) sets the supply drain current to Ql (802) and Q2 (804). The cunent passing through Q2 (804) develops a voltage across ? 2 (824), which sets the biasing for Q3 (806).

[0080] CI (810) is an integrating capacitor, which provides negative feedback from the amplifier's output 815 to its input 820. The effect of the feedback provided by CI (810) is to maintain an essentially constant voltage at the input 820 such that cunent from photodiode 830 (resulting from the luminescent detection signal) is forced onto CI (810), where it is integrated until discharged by ?NMOS transistor M5 (840). M5 (840) acts as a switch, which when turned on discharges CI (810).

[0081] Figure 9 shows an exemplary design of a synchronous demodulator 910 and low- pass filter (or lock-in amplifier) 960. The signal from a preamplifier, such as amplifier 800, is input to two amplifiers 912 and 914 which have the same gain magnitude but provide opposite gain polarity, shown as xi and x_ι, respectively. Amplifiers 912 and 914 are connected to a double-pole, single-throw switch, shown as a CMOS 2:1 analog multiplexer switch 930. The switch 930 is controlled by a phase signal 924 of the same frequency as the original modulation signal used to modulate the excitation source, such as a laser. For the case of on-off (pulsed) modulation with little phase shift occurring in the detection process, the switch 930 would be operated to connect the Xi amplifier (912) when the excitation is on and to the x.ι amplifier (914) when the excitation is off. This is the process of synchronous demodulation. The net effect is that the signal due to the excitation plus any background is amplified with one polarity for half the time, while the background alone is amplified with the other polarity for the other half of the time. The output of the switch 930 is connected to a low-pass filter 960 implemented using operation amplifier 965. Low pass filter 965 averages the signal received from the synchronous demodulator 910, allowing the unwanted background to be removed. The output from low pass filter can be provided to an A/D converter (not shown). [0082] Figure 10 shows typical waveforms for the case of a biochip used with an on/off modulated light source and an integrating preamplifier (e.g. Fig. 8) and a synchronous demodulator and low-pass filter (e.g. Fig. 9). As the light source (e.g. laser) is modulated off and on, the preamplifier is reset once during each off cycle and once during each on cycle. During the off cycle, the preamp integrates the background signal (due to background light or photodiode leakage), and during the on cycle, the preamp integrates the stimulated signal (due to the effects of the light source, i.e., fluorescence) plus the background signal. The same signal as the on/off modulation is used as the phase input for the demodulator, and this produces the demodulator output. It can be seen from FIG. 10 that the off cycle portion of the signal (integrated background) has been inverted by this process. When the demodulator signal is low-pass filtered (or averaged), the background from the off cycles is essentially subtracted from the background of the on cycles and the result is the average of integrated stimulated signal.

[0083] Figure 11 shows an exemplary implementation of a programmable timer 1100. A crystal or other type oscillator 1110 is generally used as a base frequency source. Typically, the oscillator 1110 provides a signal frequency in the 1 to 100 MHz range. This signal frequency is divided by 2^N (1115) where N is a programmable value that can be supplied by data register 1118. For example, a 1 ?MHz signal frequency could be divided by 2⁴ or 16 to provide a 62.5 kHz frequency. For more flexibility, a divide by M circuit 1120 is added. A divide by M circuit includes a counter 1125 and an equality detector 1130. Initially the counter 1125 is reset (output zeroed), and it then begins to count. When the output of counter 1125 reaches a value of M, the equality detector 1130 generates a pulse and resets the counter 1125. The pulse from the equality detector 1130 is used as the clock for a divide by 2 circuit (1135), and the output of the divide by 2 circuit (1135) is a square wave of the programmed frequency. The output frequency 1140 is given by 2^N+1 times M.

[0084] Figure 12 shows an exemplary implementation of a phase-shift selector 1200 which is implemented using a programmable delay circuit. The relation between delay and phase shift is given by Θ = 2πτ/T, where Θ is the phase shift in radians, τ is the delay and T is the period of the waveform. By controlling the delay of the output with respect to the input, the phase for the demodulator circuit can be controlled. The circuit uses two divide by 8 elements (1205 and 1210) to reduce a 1 ? Hz input (such as from a crystal oscillator) to a 15.625 kHz output. This is a simplified version of the programmable timer 1100 shown in FIG. 11. Shift registers 1215 and 1220 are used to provide delays of integer numbers of clock cycles. In this example, the frequency output is introduced to a shift register 1215 that is clocked by the 125 kHz output of the first divide by 8 circuits (1205). This allows the output 1230 to be delayed by 0, 8, 16, 24, 32, 40, 48 or 56 microseconds. The output shift register 1215 is the data input to shift register 1220 that is clocked by the 1 MHz clock. This allows the output to be delayed in steps of 1 microsecond over the range of 0 to 7 microseconds. The combination allows the phase shifted output 1230 to be delayed in steps of 1 microsecond from 0 to 63 microseconds. This conesponds to phase shifts from 0 to 2π in steps of π/32. Phase-shift selector 1200 can operate using a wide range of input frequencies, and similar circuits could be used with other types of dividers, including programmable ones.

[0085] While the preferred embodiments of the invention have been illustrated and described, it will be clear that the invention is not so limited. Numerous modifications, changes, variations, substitutions and equivalents will occur to those skilled in the art without departing from the spirit and scope of the present invention as described in the claims.

Claims

What is claimed is: 1. A biocbip-based detection system, comprising: a biosensor platform including at least one receptor probe, said receptor probe providing an intensity modulated electromagnetic detection signal when combined with a target in response to inadiation by an intensity modulated electromagnetic excitation signal; an integrated circuit microchip optically coupled with said biosensor platform which includes at least one photodetector, said photodetector converting said electromagnetic detection signal into an electrical signal, and signal processing circuitry for receiving said electrical signal and determining spectroscopic data including at least one lifetime of said target.

2. The system of claim 1, wherein said system further comprises a source of said excitation signal.

3. The system of claim 2, wherein said excitation signal comprises at least one radiation pulse, said radiation pulse having a pulse width shorter than said lifetime, wherein said lifetime is determined by said signal processing circuitry using time resolved spectroscopy.

4. The system of claim 3, wherein said at least one radiation pulse comprises a plurality of periodic pulses.

5. The system of claim 2, wherein said excitation signal is modulated at a frequency greater said lifetime, wherein said lifetime is determined by said signal processing circuitry using phase resolved spectroscopy.

6. The system of claim 1, wherein said signal processing circuitry includes a demodulator for demodulating said electrical signal or an amplified version of said electrical signal, wherein said excitation signal is used as a phase input for said demodulator circuit, whereby synchronous demodulation is provided.

7. The system of claim 1, further comprising an on-chip source of said excitation signal and on-chip mirrors for directing said excitation signal to said receptor probe.

8. The system of claim 1, wherein said on-chip source of said excitation signal comprises a laser.

9. The system of claim 1, further comprising a RF transmitter for transmitting said spectroscopic data over the air.

10. The system of claim 1, wherein said biosensor platform includes a plurality of said receptor probes.

11. The system of claim 10, wherein said at least one photodetector comprises a plurality of photodetectors, wherein said plurality of probe elements are each provided a dedicated detection channel, said dedicated detection channel comprising one of said plurality of photodetectors and a dedicated amplifier.

12. The system of claim 1, wherein said biosensor platform is affixed to said microchip.

13. The system of claim 1, wherein said signal processing circuitry includes an analog to digital converter.

14. The system of claim 1, wherein said electromagnetic detection signal comprises an absorption signal, a fluorescent signal, a phosphorescent signal, an elastic scattering signal or a Raman scattering signal.

15. The system of claim 1, wherein said signal processing circuitry comprises a band pass filter whose pass band includes a modulation frequency of said excitation signal.

16. A method for detecting target analytes, comprising the steps of: providing a biochip, said biochip including a biosensor platform including at least one receptor probe for combining with at least one target molecule, said receptor probe providing an intensity modulated electromagnetic detection signal when combined with said target in response to inadiation by an intensity modulated electromagnetic excitation signal; contacting said receptor probe with a sample suspected of containing said target; inadiating said probe or a probe/target combination with said excitation signal to produce said detection signal, and identifying whether said target is present in said sample using spectroscopic data derived from said detection signal.

17. The method of claim 16, further comprising the step of determining at least one lifetime of said target from said detection signal.

18. The method of claim 16, further comprising the step of converting said detection signal to an electrical signal.

19. The method of claim 16, wherein said excitation signal comprises at least one radiation pulse, said radiation pulse having a pulse width shorter than a lifetime of said target, wherein said lifetime is determined by time resolved spectroscopy process.

20. The method of claim 19, wherein said at least one radiation pulse comprises a plurality of periodic pulses.

21. The method of claim 16, wherein said excitation signal is modulated at a frequency greater than a lifetime of said target, wherein said lifetime is determined using phase resolved spectroscopy.

22. The method of claim 18, wherein said identification step includes the step of demodulating said electrical signal or an amplified version of said electrical signal, wherein said excitation signal is used as a phase input for said demodulation step, whereby synchronous demodulation is provided.

23. The method of claim 16, further comprising the step of transmitting said spectroscopic data over the air on a RF carrier.

24. The method of claim 16, wherein said at least one receptor probe comprises a plurality of receptor probes, further comprising the step of processing detection signals from said receptor probes separately.

25. The method of claim 16, wherein said detection signal comprises an absorption signal, a fluorescent signal, a phosphorescent signal, an elastic scattering signal or a Raman scattering signal.

26. The method of claim 16, further comprising the step of band pass filtering said detection signal or an amplified version of said detection signal with a band pass filter whose pass band includes a modulation frequency of said excitation signal.