p-Chips were provided by PharmaSeq, Inc. (Monmouth Junction, NJ, www.pharmaseq.com
). The properties of the p-Chips and two types of p-Chip readers (flow-based bench-top analyzer and hand-held ID reader) have been described [1
]. The 10-bit p-Chips used in the work described allowed a maximum 1,024 different IDs.
2.2. Coating p-Chips with polymer
The p-Chips were coated by aminopropyltriethoxysilane (APTS) and 3-glycidoxypropyl-trimethoxysilane (GPTS) as previously reported [2
] with several modifications. Briefly, p-Chips were washed with 99.5% methyl alcohol at room temperature (RT) for 5 min three times. The p-Chips were then rinsed with toluene/dimethylformamide (DMF) mixture with 0.01% distilled water and 0.9% APTS at RT four times. After rinsing, p-Chips were immediately treated with a coating solution (mixture of toluene and DMF with 0.01% distilled water, 0.9% APTS, and 0.3% GPTS) at 80°C for 20 min. After the coating reaction, p-Chips were washed once with toluene, three times with DMF, and three times with acetonitrile at RT, followed by air drying. The procedure places both amino and hydroxyl groups within the polymer and on the surface of p-Chips.
To introduce the carboxyl groups, the derivatized p-Chips were treated with 10% succinic anhydride in dry pyridine:DMF (1:9) and placed on a tissue culture rotator at RT for 30 min. This step was repeated once using fresh reagents. After the reaction, the carboxylated p-Chips were washed with DMF four times and acetonitrile twice, followed by air drying.
All chemicals used for coating and carboxyl conversion were purchased from Sigma-Aldrich, St. Louis, MO.
2.3. Deposition of SIF on p-Chips
In order to make SIF-coated p-Chips, the chips were coated with polymer first as described above. Then SIF was deposited on the surface of polymer-coated p-Chips as reported previously [2
] with several modifications. Briefly, 60 µL of 5% NaOH was slowly added to 18 mL of 0.83% AgNO3
solution with intensive stirring at RT in a 50 mL reaction tube. Then 400 µL of 30% NH4
OH was added with intensive stirring at RT. The clear solution was incubated in an ice bath for 10 min, followed by the addition of 4.5 mL of a fresh 4.8% glucose solution with intensive stirring. Polymer-coated p-Chips were incubated in this solution in a 1.5 mL Eppendorf tube on a tissue culture rotator at RT for 50 min. After the silver deposition, the p-Chips were immediately washed with distilled water three times followed by air drying. The dried SIF-deposited p-Chips were coated with another polymer layer and carboxylated as described above to seal the SIF layer.
2.4. IL-6 immunoassay
We performed a standard Il-6 assay on polymer coated p-Chips and SIF/polymer coated p-Chips in order to test the two platforms under identical conditions. To conjugate an antibody, the carboxylated p-Chips were incubated with 100 µL 261 mM N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDC) and 230 mM N-hydroxysulfosuccinimide (NHSS) in 0.1M HEPES buffer (pH 7.5) on a rotator for 30 min at RT. The p-Chips were then washed with 200 µl PBS three times and incubated with 30 µL 200 µg/mL monoclonal anti-human IL-6 antibody (R&D Systems, Minneapolis, MN) for 2 hrs at RT on a tissue culture rotator. The p-Chips were washed with PBS three times and blocked with SuperBlock solution (Thermo Fisher Scientific, Waltham, MA) for 5 min at RT on a rotator three times. The p-Chips were then washed with PBS three times and stored in TBS with 3% BSA at 4 °C.
Anti-IL-6-conjugated p-Chips were incubated with 50 µL 100ng/mL recombinant human IL-6 protein standard (R&D Systems) in TBS with 3% BSA for 1 hour at RT on a rotator. After incubation, the p-Chips were washed with 200 µL of Tris-buffered saline with 0.05% Tween-20 (TBST) three times.
The detection antibody solution was prepared by diluting biotinylated anti-human IL-6 antibody (R&D System) to 5.0 µg/mL with PBS. The p-Chips were then incubated with 50 µL of this solution for 1 hour, followed by washing with TBST three times. The p-Chips were pooled and incubated with 50 µL of 8 µg/mL streptavidin-Alexa Fluor 555 conjugate in TBST for 30 min at RT in the dark. After incubation, the p-Chips were washed with TBST three times and with distilled water twice and were stored in PBS at 4°C between measurements. All measurements except SEM were conducted with the p-Chips immersed in PBS.
Samples were imaged with an FEI model XL30 (Hillsboro, OR) scanning electron microscope (SEM). The SEM is equipped with a tungsten filament operating at accelerating voltages up to 30 kV. The samples were fixed to aluminum sample stubs via carbon conductive tape, which provided a continuous, conductive path from the sample through the stage. When p-Chip cross-sections were imaged, the p-Chip was first cut in half with a razor blade before being attached to the carbon tape, cut side up towards the primary electron beam. The chips were then placed into the sample chamber, where they were measured under vacuum with pressures always below 1 microtorr. The working distance between the bottom of the column and the sample was 10 mm. The spot size and accelerating voltage were varied as needed, and these values are indicated on each particular image. As is typical for SEM imaging, all images reported here were collected from the detection of secondary electrons, emitted as the primary electron beam rasters across the sample surface.
2.6. Elemental analysis in SEM
The SEM was equipped for energy dispersive spectroscopy (EDS) with a detector manufactured by EDAX (Mahwah, NJ). Because semi-conductive and nonconductive materials exhibit very low signals in EDS, the accelerating voltage used for EDS was always 30 kV so as to provide the highest possible count rate.
2.7. Fluorescence microscopy: FLIM and intensity data
Fluorescence lifetime imaging (FLIM) data were collected by the MT200 system (PicoQuant GmbH, Berlin, Germany). This system was coupled to an Olympus (Shinjuku, Tokyo, Japan) IX71 confocal microscope with a 100× oil objective, unless it was noted that a 60× water objective was used. A single photon avalanche detector (SPAD) manufactured by Micro Photon Devices (Bolzano, Italy), model PD1CTC, was chosen for its sensitivity. A 470 nm pulsed laser diode with a 20 MHz repetition rate was utilized for excitation. The emission was filtered by a 470 long pass liquid filter as well as a 560/40 band pass filter. For reflection images, the laser power was reduced and a 470/10 band pass filter was used. The system is capable of picosecond time resolution, and the piezo stage is accurate to within 1 nm.
Symphotime (version 5.2.4) software by Picoquant was used to process all FLIM data and to extract intensity information from the FLIM data. FLIM images are produced with grayscale intensity data and false color overlaid to indicate the lifetime calculated for every pixel. The enhancement in fluorescence intensity was measured with the MT200 simultaneously with FLIM data.
2.8. Fluorescence lifetime measurements
Fluorescence lifetime was measured with a Fluorotime 200 fluorometer, also from Picoquant. The FT200 was equipped with a 470 nm pulsed laser diode, a microchannel plate photomultiplier ultrafast detector, and a monochromator. The p-Chips were placed between two coverslips, a drop of buffer was added, and the coverslips were taped together. The sample was then arranged in a front face attachment with a 495 nm long pass filter on emission. The monochromator was set to 565 nm. The response was measured with the monochromator set to 470 nm along with a 470/10 band pass filter.
2.9. Raman scattering measurements
Raman scattering measurements were collected on a custom-built system consisting of a dispersive spectrometer from Kaiser Optical Systems, Inc. (Ann Arbor, MI) with 4.0 cm−1 resolution and a front-illuminated CCD detector from Roper Scientific (Sarasota, FL). The p-Chips were illuminated with an argon-ion laser (514 nm) emitting 220 mW of power. The light was focused by an Olympus BH-2 microscope with a 40× objective, resulting in a 1.5 µm collection area. The system was capable of measuring Raman shifts up to 2000 cm−1, corresponding to the 572 nm wavelength. The experiments were conducted on p-Chips coated with SIF and polymer in the manner described above but using a different assay. Briefly, the polymer coating the SIF and non-SIF p-Chips was carboxylated and further derivatized by conjugating streptavidin to the carboxyl groups (see sections 2.3 and 2.4 for the conjugation methods). The streptavidin-conjugated p-Chips were then incubated in 50 µL of 10 µg/ml Cy3-labeled biotinylated oligonucleotide at RT in a dark enclosure for 30 min. The p-Chips were washed with TBST three times and air dried. The sequence of the oligo was 5'-Cy3-TTT TTT TTT T-biotin-3'.