3-aminopropyltriethoxysilane (APTES) was obtained from Gelest. Glutaraldehyde (70%), water soluble 1-ethyl-3-[3-dime-thylaminopropyl]carbodiimide (EDC), and 200 proof anhydrous ethanol were obtained from Sigma. Reagents were diluted to the indicated concentrations in the following procedures, but otherwise were used as received. High resistivity water (18.2 MΩ) used for rinsing or diliutions was obtained from a Barnstead Nanopure purification system. Reducing agent sodium tri-acetoxyborohydride (STAB) was obtained from BASF and diluted in water as indicated. Polyethylene glycol (PEG, MW = 44) linker and blocker molecules amino-dPEG™8
-COOH), and amino-dPEG™4
-OH) were obtained from Quanta Biodesign Ltd. and diluted in water or buffer, as indicated. The synthesis and storage of the amine-functionalized chelator designed for PPi capture was described in a separate publication from our group.5
Buffers used in these procedures included 1x phosphate buffered saline (PBS) at pH 8, 0.1 M 2-(N
-morpholino)ethanesulfonic acid (MES) buffer at pH 5, and 0.2 M sodium borate buffer at pH 8. For rinsing non-specifically bound components such as avidin and storage of DNA colonies attached to device surfaces, we typically used a custom 1x ‘TEST’ (Tris, EDTA, Salt, Triton) buffer with composition 10 mM TrisHCl at pH 8, 1 mM EDTA, 50 mM NaCl and 0.01% Triton X-100. In addition we typically used a Tris-based buffer with composition 6.25 mM NaCl, 1.25 mM MgCl2
,10 mM Tris pH 8 and 1 mM DTT as a blank measurement control before and after polymerase reactions.
Immunopure avidin was obtained from Pierce and diluted in 1x PBS buffer. Unless otherwise specified in the RCA DNA preparation section, all DNA oligonucleotides were synthesized by IDT (Integrated DNA Technologies). Taq DNA polymerase was obtained from Applied Biosystems. The kits used for DNA purification were from Qiagen. Phi29 and buffers used with Phi29 were obtained from New England Biolabs (NEB).
Devices and measurement apparatus
The nanoplate SiO2
/Si field effect devices were fabricated using standard lithographic and microfabrication techniques as previously described.28
Optical microscopy images (), a nanoplate cross-section diagram () and a high resolution scanning electron microscopy (SEM, ) cross-section image of devices used in this study are presented in . Briefly, the silicon field effect devices used to electrically detect PPi were fabricated using standard microfabrication techniques from a silicon-on-insulator (SOI) wafer (SOITEC). The FET devices had active areas with 1–2 μm widths and variable lengths: 10, 15 or 20 μm. The majority of the results reported here were obtained on devices with 1 (or 2) × 20 μm active areas. Full details of the device response to pH and other surface modifications are described in previous publications from the Bashir group.29
Devices were mounted on a probe station (Signatone) and characterized using a Keithley Semiconductor Characterization System (SCS) Model 4200. Devices were connected to source, drain, backgate and top-gate (fluid-gate) voltage sources. Field effect devices used for the detection were operated in accumulation mode and the source–drain current was monitored as a function of the backgate bias (Vbg = −10 V to 2 V) at a fixed source–drain potential (Vsd = 100 and 30 mV). The I–V transfer curves reported in this work were collected with Vsd = 30 mV and Vsd = 100 mV, but all data presented in this work was collected with Vsd = 100 mV. Each device used in pyrosphosphate detection measurements was characterized in a variety of control conditions, including dry, in buffer with chelator-modified surface, and in buffer with unmodified surface. Some examples are presented in –V transfer curve graph. A probe station enclosure protected the devices from light, sound and physical vibration during measurements.
Fig. 3 Current–voltage transfer characteristics of a p-type SOI-FET device with Vds = 0.1V in accumulation mode with different surface modifications (Unmodified, Chelator and silane modified and Avidin-only modified) and different solution exposures (more ...)
For device measurements in solution, polydimethylsiloxane (PDMS, Dow Sylgard) was used to create a small well around many devices. These devices were then probed serially on a Signatone probe station. To this was added 2 to 3 μL of the indicated solutions, such as control buffer (1x PBS), control buffer with zinc (1x PBS with Zn(NO3)2, PPi in buffer with zinc, and control buffer with Zn2+ after an incubation and rinse in 0.1M acetic acid to remove Zn2+ complexed to the chelator and any PPi bound to the chelator coordination sites.
An overview of the initial surface modification is illustrated in , Steps 1 and 2. Substrates were cleaned in fresh, hot piranha (1 : 3 hydrogen peroxide:sulfuric acid) for at least 30 min. Caution: Piranha solution is highly corrosive and should be used with extreme caution. Following piranha cleaning, samples were rinsed thoroughly in water and dried in flowing nitrogen gas. Substrates were added to anhydrous ethanol with 1% 3-aminopropyltriethoxysilane (APTES). Then water was added to this solution at a final concentration of 2% by volume and substrates were incubated at room temperature for less than 10 min for monolayer silane deposition. Incubation times much longer than this often resulted in multilayer rather than monolayer formation, as measured by ellipsometry and AFM. After silane deposition, the chips were rinsed with ethanol and water, dried under nitrogen, and annealed for 10 min at 110 °C in order to promote cross-linking of the silane monolayer. For further functionalization, the amine-functionalized chips were immersed in a well-mixed solution of 2% glutaraldehyde in 1 : 1 ethanol:-water. After 2 h, the chips were rinsed with copious amounts of water to remove non-specifically bound material on the surface and dried under nitrogen.
Pyrophosphate chelator and DNA colony silicon oxide surface modification.
The PPi specificity and solution binding characteristics of the recently developed chelator have been previously reported.5
The chelator only modification step is depicted in , Step 3a. To attach amine-functionalized chelator molecules covalently on surfaces previously modified with glutaraldehyde, the surface was incubated in a solution of 1 mM chelator solution in 100 mM sodium borate buffer (pH 8), forming a Schiff base. After an hour, an equal volume of ~0.2 mM sodium triacetoxyborohydride (STAB) solution in water was added to the chelator solution on chip and incubated overnight for reductive amination. Then the device surface was washed with 10 mM sodium borate buffer (pH 8) and with Tris control buffer (6.25 mM NaCl, 1.25 mM MgCl2
,10 mM Tris pH 8 and 1 mM DTT) and stored in control buffer prior to experiments.
The co-deposition of chelator with PEG linkers that serve as avidin attachment sites is depicted in , Step 3b. The attachment chemistry is similar to that for chelator only. Typically, the chelator and PEG linker, H2N-PEG12-COOH (Quanta Biodesign Ltd.), solutions were mixed in a 1 : 5 ratio, approximately 200 uM to 1 mM in 100 mM borate buffer and deposited on silicon pieces or devices. After an hour, ~0.2 mM STAB solution was added to the chip surfaces and incubated overnight. Then the device surface was washed as described in the previous section and stored in the control buffer before measurements. For surface characterization with ellipsometry, AFM or TOF-SIMS, devices and silicon pieces were dried under nitrogen gas and stored in vacuum packed containers until analysis. Devices used for electrical measurements were modified in parallel.
Dipole moment calculations
Molecular dipole moments were calculated using the GAMESS computational chemistry package in Chem3D Pro using the RHF/3–21G level of theory and MM2 energy minimization and these results are presented in the ESI section.†
Prior to DNA exposure, avidin was attached to carboxylic acid terminated PEGn=8 or 12
linkers on the surface as shown in , Step 4. The carboxylic acid terminated linkers were activated by adding 0.1 mM EDC in MES pH 5 to the substrate surface. After a 30 min incubation, an equal volume of 50 μg mL−1
avidin in 1x PBS was added to the surface followed by incubation for 2 h at room temperature. Free avidin was removed by washing the wells 5 times with 1x TEST buffer (10 mM TrisHCl, pH 8, 1 mM EDTA, 50 mM NaCl and 0.01% Triton X-100). Similar procedures have been reported to control avidin orientation on surfaces and bind one biotinylated molecule per avidin binding site.30
DNA preparation and surface attachment
The general scheme for DNA attachment on FET surfaces is depicted in , Step 5 and in . Circular DNA was attached to the surface and one strand of the circular DNA was amplified in order to produce multiple copies of the same sequence on individual binding sites using a technique known as rolling circle amplification (RCA).25
Each DNA colony formed in this manner was a concatemer of a single stranded DNA (ssDNA) formed by amplifying one strand of a circular, doubled stranded DNA (dsDNA). For the surface-immobilized RCA-DNA used in this work, we used PCR to amplify a 260 bp fragment of the pUC19 plasmid using an upstream primer of 5′-pCTGCAATGATACCGCGAGACCCA-3′ and a downstream primer of 5′-pCCTTGATCGTTGGGAACCGGAG-3′. To ensure efficient ligation, purified PCR DNA was treated with Taq DNA polymerase (0.1 U/μl) in the presence of 500 μM dATP for 30 min. This treatment resulted in a single base A protruding from the 3′ end of each DNA strand (A-tail). The treated DNA was purified and quantified using UV absorption (Nanodrop) before ligation. Although the results described in this work were from PCR amplified DNA, we have also demonstrated RCA DNA colony formation on silicon oxide test surfaces and devices from total genomic DNA in parallel work.
Fig. 2 DNA colony formation process and characterization on surface: a) schematic of DNA sample preparation, colony formation and Cy5 dye labeling, b) relatively high density DNA colony modification of selectively patterned test substrates using APTES + GA + (more ...)
In principle, any DNA fragment with phosphate groups at the 5′ ends and hydroxyl group at the 3′ ends can be circularized by the following ligation procedure. A multi-purpose adaptor was assembled from a set of five DNA oligonucleotides present at equimolar ratio (1 : 1 : 1 : 1 : 1): RC1 (5′-pAGCTCGGCGGCC GCTTAAGT-3′), RC2Tb (5′-biotin-spacer-CTCCTATCACT TAAGCGGCCGCCGAGCTT-3′), RC3T (5′-pACGTCCG TACGTTCGGAACCT-3′), RC4 (5′-pGGTTCCGAACGTA CGGACGTCCAGCTGAG-3′, 3′ locked nucleotide, resistant to 3′ → 5′ exonuclease), and RC5 (5′-pGATAGGAGATCTCA GCTGG-3′). The oligonucletide sequence information is also tabulated in the ESI section.†
The oligo mixture was stored at −20 °C before use. The adaptor was designed so that after ligation each 5′ end was phosphorylated and the 3′ end had a single base T (T-tail). In the middle of the adaptor, there was a gap and the 5′ terminus of the gap was tagged with biotin and the 3′ terminus in the gap was rendered resistant to exonuclease digestion by use of a locked nucleotide at the −1 position. To form a circular DNA structure presented in , the A-tailed target DNA and the T-tailed adaptor DNA were mixed at equimolar ratio (1 : 1), typically 0.1–0.5 μM in a 50 μl ligation reaction. After ligation with T4 DNA ligase (0.2 U/uL) at 16 °C overnight, T4 DNA polymerase (10 U) was added to the sample directly to digest non-circular DNA molecules at 37 °C for 30 min. Purified DNA was quantified based on UV absorption.
DNA oligonucleotides assembled in biotinylated DNA circle for Rolling Circle Amplification (RCA).
Circularized DNA (biotin-functionalized) was diluted in 1x TEST buffer to a concentration of ~107 copies/μL. In order to confine the solution to the FET device chip, a 2 mm × 1 mm custom PDMS well was trimmed to fit inside the pad area of the 3 mm × 5 mm chips. The diluted biotinylated DNA samples were incubated with the chelator with avidin on linker surface for at least 2 h, as depicted in , Step 5. Free DNA molecules were then removed by washing the wells 5 times with 1x TEST buffer.
Before rolling circle amplification (RCA) amplification, the wells were conditioned with 1x Phi29 buffer (NEB). For RCA amplification, about 10 μL of the reaction solution (1x Phi29 buffer, 200 μM dNTPs, and 0.5 U/μl Phi29 DNA polymerase) was added to each surface followed by incubation at 30 °C for at least 4 h or overnight to increase the number of DNA fragment copies per colony. Surface reactions were stopped by removing the reaction solution and washing the wells several times with 1x TEST buffer.
Surface analysis tools
The thickness of deposited films was measured using a Variable Angle Spectroscopic Ellipsometer (M2000FI VASE, J.A. Woollam, Lincoln, NE) scanning 685 wavelengths between 240 nm and 1100 nm at 65°, 70° and 75°.31
A Cauchy layer was used to model the organic monolayer on a surface. When possible, measurements were made on the same thermally oxidized silicon substrates before and after surface modification.
The morphology of the sample surfaces was observed by AFM, as in . AFM was performed using a Dimension V Atomic Force Microscope (Veeco, Santa Barbara, CA). Scan rate was set to 1 Hz and the scan area was set between 1 μm2 and 100 μm2. TESPA silicon tips (Veeco) with 20–80 N m−1 stiffness and response frequency of ~250–300 kHz were used. The instrument was operated in tapping mode. Images were flattened to adjust the plane and to remove scan lines. The height scale was adjusted to 15 nm. Feedback controls such as integral gain, proportional gain and amplitude setpoint were modulated in real time as the image was being generated in order to optimize image quality. Integral and proportional gains were typically set between 0.09 and 1.4.
Fig. 4 Fluorescence image and AFM topography of avidin and RCA DNA colonies on a flat SiO2 on Si test substrate: a) fluorescence image of Cy5-dU labeled DNA colonies with a region on the right with few DNA colonies; b) 100 μm2 area equivalent to small (more ...)
Fluorescence images ( and ) of Cy5-dUTP labeled RCA DNA colonies on substrates were obtained on a custom Nikon Eclipse ME600 microscope using a 10x or 50x Nikon objective with an EXFO X-Cite mercury halide lamp, a Cy-5 compatible Omega Optical filter cube, and an Andor iXon+ electron-multiplying (EM) CCD camera. Custom Andor software was used to process and adjust images. Acquisition times were typically 1s.
DNA polymerase reactions
The general scheme for PPi detection from DNA polymerase reactions conducted off-chip (ex situ) in a reaction tube and DNA polymerase reactions performed on surface-immobilized DNA colonies on FET surfaces (in situ) is depicted in .
Fig. 5 Label-free detection of PPi on chelator- and DNA-modified FET devices: a) schematic of a simplified chelator + DNA colony device surface as it is exposed to three different PPi sources including PPi standard solutions (top row), PPi generated in a reaction (more ...)
For ex situ T4 polymerase reactions, the control buffer (T4 buffer) consisted of 10mM Tris pH 8 with 6.25 mM NaCl, 1.25 mM MgCl2, and 1mM DTT. The negative control solution consisted of the T4 buffer solution with 0.2 mM dTTP (mismatch base), 0.1 Unit T4 polymerase, 0.05 μM ‘DNA2’ hairpin DNA (5 ′-/5amMC6/GTC GCG CAA AAA TAC CTA GTC G + AC GTG GTC CTT/iBiodT/TT GG + A CCA CGT CGA CT + A G-3′). The positive control solution consisted of the T4 buffer solution with 0.2 mM dGTP (matching base), 0.1 Unit T4 polymerase, and 0.05 μM hairpin DNA2. Base extension reactions were conducted in small PCR tubes at 37 °C for 1 h and then incubated at 70 °C for 20 min, prior to cool down and storage at 4 °C. For testing, small aliquots of the sample solutions were added to PDMS wells on the chip surfaces for PPi measurement on a Signatone probe station, as described in the measurement apparatus section. All samples were measured at room temperature (22 °C).
For in situ polymerase reactions, RCA DNA colonies were formed on the surface in the manner described previously with chelator deposited on the surface for PPi capture. For on-chip polymerase and control reactions, sample solutions were added to PDMS wells on chip surfaces and were incubated at 37 °C for 30 min in a high humidity enclosure. A number of control samples were characterized prior to proceeding with a series of polymerase reactions, including test solutions containing all base incorporation reaction ingredients before incubation at 37 °C, T4 buffer with PPi standards (25, 10, and/or 1 μM), T4 buffer only, buffer containing nucleotides but no polymerase, and T4 buffer with active or denatured polymerase among others as indicated. When present in the T4 buffer sample, nucleotides and T4 polymerase concentrations were 0.2 mM (each) and 0.1 U/μL, concentrations similar to those described for the off-chip reactions. All samples were measured at room temperature (22 °C) on a Signatone probe station.