2.1. Integrated CMOS time-resolved fluorescence detector design
The CMOS chip that was employed for the active substrate of this integrated microarray is shown packaged in . The optical sensor array consists of 64-by-64 pixels with each pixel area being 2500 μm2
. It differs from conventional CMOS imagers (Kwang-Bo, Chiajen et al. 2007
) in that it has both a differential photodiode (PD) design as well as support for pixel-level time-gating (Huang, Sorgenfrei et al. 2009
) as shown in . In conventional CMOS PDs, the photocurrent impulse response is determined by minority carriers generated within a diffusion length of the depletion region of the PD, which create a long “tail” as they diffuse and are collected. When trying to use such PDs for fluorescence detection in the absence of optical filtering, this degraded impulse response means that deeply-generated minority carriers from the excitation source interfere with the carriers produced by the fluorescence emission, even after excitation has been turned off. To attenuate this slow diffusive component of the photocurrent response, a fingered differential PD is used in which alternating fingers are covered with metal, rendering the diffusive component of the photocurrent common-mode. This allows a faster-than-800-ps photocurrent impulse response time to be achieved. The PD is formed between the n-type well diffusion and the p-type substrate.
The differential pixel circuitry, which performs the required common-mode rejection, is shown in . Photocurrent (iph) collected within the integration period (Tint) beginning at tR appears as a differential voltage between node a (Va) and node b (Vb), where Vb-Va=iphTint/Cpar. Cpar is the total capacitance at node a and node b, and is approximately 198.7 fF for this pixel design. Active reset-noise suppression is performed by the pixel differential amplifier through the negative feedback electronic switches (R1, R2) during the reset stage. Fast time gating to define the integration window is accomplished through the action of both reset (R1, R2) and row-select switches (S1, S2). Time-gate resolution is less than 100 ps, synchronized to a laser excitation turn-off time with an accuracy exceeding 150 ps. On-chip analog-to-digital conversion allows the multiplexed data to be retrieved from the 64-by-64 array in digital form.
The effectiveness of time-gating depends primarily on the lifetime of the fluorophore and the impulse response of the detector. The signal-to-background ratio (SBR) ratio (in dB) can be approximated by (see Supplementary Information
is the fluorescence lifetime, τdet
is the detector impulse response, and tR
is the time when time-gating occurs, as measured from the excitation turn-off. In this work, we use quantum dots (q-dots) (Han, Gao et al. 2001
) as the fluorophore which, in addition to improved photostability and enhanced quantum yields relative to organic dyes, have lifetimes typically exceeding 10 ns. With a detector impulse response of 800 ps, this yields a SBR of more than 70 dB (see Supplementary Information
), equivalent to an optical filter of more than OD 10.
2.2. Chip packaging and surface modification
Attachment of DNA capture strands requires surface modification of the CMOS microarrays compatible with their processing limitations. Modification schemes (e.g. organosilylation) used for common solid supports require harsh etches to free hydroxyl groups, deposition of thin films from solvents such as ethanol, and subsequent high-temperature anneals, all of which are damaging to CMOS chips. Instead, we deposit amino-functionalized parylene, aminomethyl-[2,2]paracyclophane (diX AM), directly on the silicon nitride passivation layer of the CMOS microarray through chemical vapor deposition at room temperature, providing a thin (~0.5 μm), stable polymer “blanket” with a functional amino group coverage exceeding 6.2×1013
(Miwa, Suzuki et al. 2008
). Before deposition of functionalized parylene, all CMOS microarrays were cleaned with 1M HCl followed by 10M NaOH for 2 min each. The chips were rinsed in ultrapure (18.2 MΩ cm) Millipore water and dried with a stream of N2
. Coating of the chips was performed using a commercial parylene deposition system (PDS 2010, Specialty Coating Systems, Indianapolis, IN, USA). The dimer was vaporized at 175 °C and pyrolyzed into monomers at 690 °C. The monomers polymerize on the microarray surface at room temperature.
2.3. Immobilization of DNA strands on chip surface
DNA capture strands end-modified with carboxyl groups, catalyzed through the coupling agents EDC and NHS, are covalently attached to the amino-functionalized parylene after non-contact spotting. Capture DNA was printed on the microarrays with a Piezorray piezoelectric non-contact robotic printer (Perkin Elmer, Waltham, MA, USA). The oligo-nucleotide print concentration was 20 μM in 0.16 mM sodium phosphate buffer with 15mM NHS (N-hydroxysuccinimide) and 60mM EDC (1-(3-Dimethyl-aminopropyl)-3-ethylcarbodiimide hydrochloride). After printing, the chips were incubated in 75% humidity chamber for 12 hours, followed by vacuum storage until hybridization.
2.4. Hybridization protocols for CMOS microarrays
Silicone isolation chambers (Grace Bio-labs, Inc., Bend, OR, USA) were used in conjunction with CMOS microarrays. Total reaction volume required to fill a chamber is 60 μl. Before hybridization, microarrays were first incubated in H2O at 65° C for 15 min and then pre-hybridization solution, consisting of 6X SSPE, 0.05% Tween-20, 0.05% SDS, 20mM EDTA, 5X Denhardt’s solution, and 100ng/μl heat-treated salmon sperm DNA, for 30 min at 50° C. Hybridization solution was prepared with 6X SSPE, 0.05% Tween-20, 0.04% SDS, 20mM EDTA, 100ng/μl heat-treated salmon sperm DNA, and either synthetic oligo-nucleotides or heat-treated PCR amplified products. Hybridization was performed at room temperature for 6 hours for synthetic oligonucleotides and at 50° C overnight for PCR amplified products.
After hybridization, the active substrate was washed with 6X SSPE and 0.05% Tween-20 for 5 min at 50° C, 3X SSPE and 0.05% Tween-20 for 1 min at room temperature, 0.5X SSPE and 0.05% Tween-20 for 1 min at room temperature, and 2X PBS and 0.1% Tween-20 for 1 min at room temperature.
A pre-labeling blocking solution was prepared consisting of 2X PBS, 0.1% Tween-20, and 1% BSA. The labeling solution was prepared through the addition of 5 nM streptavidin-conjugated QD-625 solution (Invitrogen, Inc., Carlsbad, CA, USA). The chips were treated with the blocking solution for 15 min and the labeling solution for 30 min. After labeling, the chips were washed twice with 2X PBS and 0.1% Tween-20 for 1 min, and blow-dried with a stream of N2.
2.5. Preparation of target analyte from physiological samples
Peripheral blood is drawn into 0.105 mol/l sodium citrate Vacutainer tube (Becton Dickinson and Company, Franklin Lakes, NJ) from healthy volunteers with informed consent. The blood is exposed at a rate of 0.82 Gy/min to 8 Gy γ-rays using a Gammacell-40 137Cs irradiator (AECL, Ottawa, Ontario, Canada). As control, sham-irradiated blood samples are used. Following irradiation, blood samples are diluted 1:1 with RPMI 1640 medium (Mediatech, Herndon, VA, USA) supplemented with 10% heat-inactivated fetal bovine serum (HyClone, Logan, UT, USA), and are incubated for 6 hours at 37°C in a humidified incubator with 5% CO2.
The RNA was prepared using the PerfectPure RNA Blood Kit (5 Prime Inc., Gaithersburg, MD, USA) according to the manufacturer’s recommendations. This protocol depletes globin m-RNA by differentially lysing red and white blood cells in whole blood. First the red blood cells are lysed by RBC lysis solution and the nucleic acids released are washed away followed by lysis of the white blood cells for purification of RNA. The remaining globin mRNA was further reduced using the GLOBINclear Human Kit (Ambion Inc., Austin, TX) that specifically removes both α- and β-globin mRNA. The RNA was quantified using a NanoDrop-1000 spectrophotometer, and quality was monitored with the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).
2.6. Polymerase Chain Reaction (PCR) and quantitative real-time PCR
Gene-specific primers and probe sets were designed with the aid of Primer Express software (Applied Biosystems, Foster City, CA) and TaqMan Primer Design software (GenScript Corp., Piscataway, NJ, USA). Primers-probes were synthesized with the probes containing 6-FAM (carboxyfluorescein) at 5’-end and BHQ1 (Black Hole Quencer 1) at 3’-end. 500-ng total RNA was reverse transcribed to cDNA using the High-Capacity cDNA Archive Kit (Applied Biosystems, Foster City, CA) according to the manufacturer’s instructions. Regular PCR reactions were performed (MiniOpticon, CFD-3120, Bio-Rad Laboratories, Inc., Hercules, CA, USA) a reaction mixture (50 μl) composed of 1X Taq buffer, 0.5 μM of each primer, 200 μM each of dATP, dGTP and dTTP, 135 μM of dCTP, 65 μM of biotinylated dCTP (ChemCyte, Inc., San Diego, CA, USA), two units of DNA polymerase, and 100 ng of cDNA template. PCR was performed with initial heating at 95° C for 2 min, followed by 25 cycles of denaturing at 94° C for 30 sec, annealing at 55° C for 45 sec and extension at 72° C for 30 sec. A final extension at 72° C was performed for 7 min. The real-time PCR reactions were performed with the ABI 7900HT Fast Real-Time PCR System (Applied Biosystems Inc., Foster City, CA, USA) using ABI’s Universal PCR Master Mix following manufacturer’s recommendations. Standard curves were generated to optimize the amount of input cDNA for measurement of each gene (5 or 10 ng). All samples were run in duplicate and repeated 3 times on different days for each gene. The relative fold induction of target genes was calculated by the ΔΔCT method with ACTB used for normalization.
2.7. Setup for fluorescence lifetime measurement and chip readout
Quantum dots on the active substrate surface were excited at a wavelength of 408 nm with a high-speed narrow-pulsed laser (EIG1000D with PIL040, Advanced Laser Diode Systems A.L.S. GmbH, Berlin, Germany). On-chip data were acquired by the chip’s photodiode circuits, using a custom-interfaced printed-circuit board to mount the socketed chip. Digital data output from the CMOS chip are stored on a PC and processed using MATLAB (The Mathworks, Natick, MA, USA).
2.8. DNA oligonucleotide sequences
The sequences of the 5’-end carboxylated 25-mer DNA oligonucleotide probes (Trilink Biotechnologies, San Diego, CA, USA) used in our CMOS microarray experiments are as follows: P1 5’-GGA GCT GGA AGC AGC CGT GGC CAT C -3’, P2 5’-TGT CTG GCC CAC ACC TTC TTT AGT C and P3 5’-GTA TTT TAA GTG TCC CAT ATC CGC A-3’. The target sequence for microarray platform characterization is: T1 5’-GAT GGC CAC GGC TGC TTC CAG CTC C-3’. Sequences of forward and reverse primers (Operon Biotech, Inc., Huntsville, AL, USA) for PCR and qRT-PCR are: F1 5’- CAC TCT TCC AGC CTT CCT TC-3’, R1 5’- GGA TGT CCA CGT CAC ACT TC-3’, F2 5’- CAG GAC ACG GAA GTG AGA GA-3’, R2 5’- CAG GAC ACG GAA GTG AGA GA-3’, F3 5’-ATT GCG GAT ATG GGA CAC TT-3’, and R3 5’-GCTGGCATCTTAGAAGCAGTTC-3’. Sequences of Taqman probes (Operon Biotech, Inc., Huntsville, AL, USA) are: P1: 5’- TGCCACAGGACTCCATGCCC-3’, P2: 5’- TCCAAGGCCTTGTCTGGCCC-3’, and P3: 5’-TCATCCTCGATCTTGGGAGCCA-3’.