This protocol outlines the fabrication of a large-scale, multiplexed two-dimensional DNA or antibody array, with potential applications in cell signaling studies and biomarker detection.
Antibody microarray as a well-developed technology is currently challenged by a few other established or emerging high-throughput technologies. In this report, we renovate the antibody microarray technology by using a novel approach for manufacturing and by introducing new features. The fabrication of our high-density antibody microarray is accomplished through perpendicularly oriented flow-patterning of single stranded DNAs and subsequent conversion mediated by DNA-antibody conjugates. This protocol outlines the critical steps in flow-patterning DNA, producing and purifying DNA-antibody conjugates, and assessing the quality of the fabricated microarray. The uniformity and sensitivity are comparable with conventional microarrays, while our microarray fabrication does not require the assistance of an array printer and can be performed in most research laboratories. The other major advantage is that the size of our microarray units is 10 times smaller than that of printed arrays, offering the unique capability of analyzing functional proteins from single cells when interfacing with generic microchip designs. This barcode technology can be widely employed in biomarker detection, cell signaling studies, tissue engineering, and a variety of clinical applications.
Antibody microarrays have been widely used in proteomic studies for decades to examine the presence of targeted proteins, including protein biomarkers1-3. Although this field is currently facing great challenges from other high-throughput technologies such as mass spectrometry (MS), there is still plenty of room for the utility of antibody microarrays, mainly because these devices afford simple data interpretation and easy interface with other assays. In recent years, the integration of microarrays into microchip scaffolds has provided the antibody microarray a new opportunity to thrive4-7. For instance, the barcode microarray integrated into a single-cell microchip has been used in cell communication studies8,9. This technology has distinctive advantages over other available microarray technologies. It features array elements at 10-100 μm, much smaller than the typical 150 μm size used in conventional microarray elements. The construction of smaller array elements is achieved using systematic flow-patterning approaches, and this gives rise to compact microarrays that can detect single-cell secreted proteins and intracellular proteins. Another advantage is the use of a simple, instrument-free setup. This is particularly important, because most laboratories and small companies may not be able to access microarray core facilities. Such barcode antibody microarrays feature enhanced assay throughput and can be used to perform highly multiplexed assays on single cells while achieving high sensitivity and specificity comparable with that of conventional sandwich enzyme-linked immunosorbent assay (ELISA8). This technology has found numerous applications in detecting proteins from glioblastoma9-11, T cells12, and circulating tumor cells13. Alternatively, barcode DNA microarrays alone have been utilized in the precise positioning of neurons and astrocytes for mimicking the in vivo assembly of brain tissue14.
This protocol focuses only on the experimental steps and build-up blocks of the two-dimensional (2-D) barcode antibody microarray which has potential applications in the detection of biomarkers in fluidic samples and in single cells. The technology is based on an addressable single-stranded, one-dimensional (1-D) DNA microarray constructed using orthogonal oligonucleotides that are patterned spatially on glass substrates. The 1-D pattern is formed when parallel flow channels are used in the flow-patterning step, and such a pattern appears as discrete bands visually similar to 1-D Universal Product Code (UPC) barcodes. The construction of a 2-D (n x m) antibody array — reminiscent of a 2-D Quick Response (QR) matrix code — needs more complex patterning strategies, but allows for the immobilization of antibodies at a higher density8,15. The fabrication requires two DNA patterning steps, with the first pattern perpendicular to the second. The points of intersection of these two patterns constitute the n x m elements of the array. By strategically selecting the sequences of single-stranded DNA (ssDNA) utilized in flow-patterning, each element in a given array is assigned a specific address. This spatial reference is necessary in distinguishing between fluorescence signals on the microarray slide. The ssDNA array is converted into an antibody array through the incorporation of complementary DNA-antibody conjugates, forming a platform called DNA-encoded antibody library (DEAL16).
This video protocol describes the key steps in creating n x m antibody arrays which include preparing polydimethylsiloxane (PDMS) barcode molds, flow-patterning ssDNA in two orientations, preparing antibody-oligonucleotide DEAL conjugates, and converting the 3 x 3 DNA array into a 3 x 3 antibody array.
Caution: Several chemicals used in this protocol are irritants and are hazardous in case of skin contact. Consult material safety data sheets (MSDS) and wear appropriate personal protective equipment before performing this protocol. The piranha solution used in Step (1.1.1) is highly corrosive and should be prepared by adding the peroxide slowly to the acid with agitation. Handle this solution with extreme caution in a fume hood. Use appropriate eye protection and acid-resistant gloves. Trimethylchlorosilane (TMCS) is a corrosive, flammable chemical used in an optional step after (1.1.6). Handle this chemical in a fume hood.
Note: Perform the barcode slide fabrication and critical flow-patterning procedures in a clean room to minimize contamination by particulate matter. Dust particles may block the ports and microchannels of PDMS molds and interfere with flow-patterning.
1. Construction of the One-dimensional DNA Barcode Slide
2. Validation of the One-dimensional Pattern on the Barcode Slide
Note: This validation protocol may also be adapted for use in assessing the quality of subsequent flow patterning steps.
3. Fabrication of the 2-dimensional (3×3) DNA Array14
4. Conversion of the 3 x 3 DNA Array into an Antibody Array
The designs for the PDMS molds (Figure 1A-1B) were drawn using a CAD program (AutoCAD). Two designs shown feature channels for flow patterning, one horizontal and one vertical. The left and right parts of each design are symmetric; either of them could be inlets or outlets. Each of 20 channels is winding from one end all the way to the other end. Each design is printed on a chrome photomask (Figure 1C). The fabricated SU-8 master on a wafer is shown in Figure 1D. To facilitate the flow-patterning of PLL or DNA, the PDMS mold was coupled to a nitrogen gas flow set-up (Figure 1E).
There are three flow-patterning steps used in this protocol. The first step immobilizes PLL on the glass substrate, while the succeeding steps both introduce oligonucleotide solutions. In Table 1, the oligonucleotide compositions of Solutions 1-3 are given. The working concentration of each oligonucleotide is 50 µM and the total volume of each solution is 39 µl. These solutions are prepared by combining 13 µl of each oligonucleotide stock solution (150 µM).
The patterned DNA microarray units are repetitive and adjacent across the whole glass slide. For 3 x 3 microarrays, the maximum density is about 400,000 spots on one glass slide. Side-by-side comparison with commercial DNA microarray reveals that our technology is about 5 times more sensitive when binding to Cy3 tagged complementary DNAs. The patterned DNAs are relatively uniform with ~5% variation across multiple repeats. Those features promise the high quantification ability of our technology when measuring protein contents from biological samples. In addition, the orthogonality of DNAs in combination with the flow channel design offer the flexibility of patterning in a variety of geometries (Figure 2D).
After converting the DNA array into an antibody array through hybridization with DNA-antibody conjugates (Figure 3B), the resulting antibody panel is used in multiplexed detection, mainly through sandwich ELISA platform as shown in Figure 3C. In sandwich ELISA, biotinylated detection (secondary) antibodies bind to the captured proteins, and subsequent labeling with fluorophore-conjugated streptavidin allows for fluorescence-based detection (Figure 3C-E). Detection of proteins shows <10% variation from one end to the other end of the glass slide (Figure 3D). With the 3 x 3 array, we are able to detect up to 7 proteins, while assigning one array element as reference (Cy3 labeled) and another element as negative control. For instance, in a single-cell experiment performed for tumor studies, the proteins interleukin-6 (IL-6), matrix metallopeptidase 9 (MMP9), hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), interleukin-8 (IL-8), and macrophage migration inhibitory factor (MIF) from a single cell can be detected simultaneously (Figure 3E).
We also calibrated the system using recombinant proteins at various concentrations. In Figure 3F, calibration curves for the recombinant proteins interferon-γ(IFN-γ), tumor necrosis factor α (TNF α), interleukin-13 (IL-13), tumor necrosis factor β (TNF β), granzyme B, interleukin-4 (IL-4), and interleukin-8 (IL-8) are shown. The sensitivity of detection is quite similar to that of conventional sandwich ELISA on well plates even with higher loading of DNAs and possibly antibodies on the substrate.
The barcode antibody microarray can be used to detect biomarkers in fluidic samples as well as in single cells. Since single-cell analysis is not the focus of this protocol, we simply exemplified the application of the 3 x 3 antibody microarray in the detection of cytokines. Through antibody-surface marker interactions, cluster of differentiation 8 (CD8)-positive cells were captured on one of the microarray elements (Figure 3G; a PDMS chip on the top), and the rest of the elements were used to detect secreted cytokines (Figure 3H). With this cell capture method, "cell arrays" can be formed. This array capture technique also allows for control over the number of cells subjected to each ELISA experiment. The cells were physically isolated in the microchambers so that the detected proteins pertained to particular single cells. In Figure 3H, it is shown that each microchamber is equipped with 16 microarray elements to ensure encapsulation of a complete 3 x 3 microarray set.
Figure 1. Design and fabrication of PDMS barcode molds for flow-based DNA patterning. Panel (A) shows the AutoCAD drawings for horizontal and vertical one-dimensional barcode patterns. Panel (B) shows the superimposed horizontal and vertical barcode patterns from (A). Green boxes enclose areas patterned with discrete 3 x 3 arrays. (C) Chrome mask for fabricating the SU-8 master. (D) SU-8 master fabricated on a silicon wafer. (E) Set-up for flow patterning. Please click here to view a larger version of this figure.
Figure 2. Construction and validation of a 3 x 3 DNA array. This figure has been modified from "Quantitating Cell-Cell Interaction Functions with Applications to Glioblastoma Multiforme Cancer Cells," by Wang, J. et al., 2012, Nano Lett 12. Copyright 2012 by the American Chemical Society. Adapted with permission. (A) Scheme for the DNA flow-patterning steps. (B) Fluorescence intensity profile of 20 channels in a selected area (traced by a vertical line) of the 1D barcode. Cy3-conjugated oligonucleotides were hybridized with the DNA arrays for validation. (C) Fluorescence intensity profile of arrays validated with Cy3-conjugated DNA after completing the second DNA patterning step. (D) Alternative fluorescent patterns from different flow-patterning strategies. Please click here to view a larger version of this figure.
Figure 3. Applications of the 3 x 3 microarray. (A) Scheme for the synthesis of antibody-oligonucleotide DEAL conjugates. (B) Scheme for the conversion of the DNA array into an antibody array through hybridization with antibody-oligonucleotide conjugates. (C) Scheme for utilizing the 3 x 3 microarray in a sandwich ELISA platform. This figure has been modified from "Quantitating Cell-Cell Interaction Functions with Applications to Glioblastoma Multiforme Cancer Cells," by Wang, J. et al., 2012, Nano Lett 12. Copyright 2012 by the American Chemical Society. Adapted with permission. Panel (D) shows a fluorescence readout generated under a Cy5 channel. This corresponds to a sandwich ELISA experiment that detected 6 proteins. Panel (E) is a zoomed-in profile of a 3 x 3 array readout under Cy3 and Cy5 channels. Panel (F) shows the sandwich ELISA calibration curves for recombinant proteins. Panel (G) shows a "cell array" formed by capturing cells through binding with antibodies on the array. Panel (H) shows the detection result superimposed with microchambers in a microchip. Please click here to view a larger version of this figure.
Solution | Oligonucleotide Composition |
1 | A’-i, B’-ii, C’-iii |
2 | A’-iv, B’-v, C’-vi |
3 | A’-vii, B’-viii, C’-ix |
Table 1. Composition of Solutions 1-3 for DNA Flow Patterning.
Anchoring sequences | |
A | ATCCTGGAGCTAAGTCCGTA-AAAAAAAAAAAAAAAAAAAA-ATCCTGGAGCTAAGTCCGTA-AAAAAAAAAAAAAAAAAAAA |
B | GCCTCATTGAATCATGCCTA-AAAAAAAAAAAAAAAAAAAA-GCCTCATTGAATCATGCCTA- |
AAAAAAAAAAAAAAAAAAAA | |
C | GCACTCGTCTACTATCGCTA-AAAAAAAAAAAAAAAAAAAA-GCACTCGTCTACTATCGCTA-AAAAAAAAAAAAAAAAAAAA |
Bridging sequences | |
A’-i | TACGGACTTAGCTCCAGGAT-AAAAAAAAAAAAAAAAAAAA-ATGGTCGAGATGTCAGAGTA |
B’-ii | TAGGCATGATTCAATGAGGC-AAAAAAAAAAAAAAAAAAAA-ATGTGAAGTGGCAGTATCTA |
C’-iii | TAGCGATAGTAGACGAGTGC-AAAAAAAAAAAAAAAAAAAA-ATCAGGTAAGGTTCACGGTA |
A’-iv | TACGGACTTAGCTCCAGGAT-AAAAAAAAAAAAAAAAAAAA-GAGTAGCCTTCCCGAGCATT |
B’-v | TAGGCATGATTCAATGAGGC-AAAAAAAAAAAAAAAAAAAA-ATTGACCAAACTGCGGTGCG |
C’-vi | TAGCGATAGTAGACGAGTGC-AAAAAAAAAAAAAAAAAAAA-TGCCCTATTGTTGCGTCGGA |
A’-vii | TACGGACTTAGCTCCAGGAT-AAAAAAAAAAAAAAAAAAAA-TCTTCTAGTTGTCGAGCAGG |
B’-viii | TAGGCATGATTCAATGAGGC-AAAAAAAAAAAAAAAAAAAA-TAATCTAATTCTGGTCGCGG |
C’-ix | TAGCGATAGTAGACGAGTGC-AAAAAAAAAAAAAAAAAAAA-GTGATTAAGTCTGCTTCGGC |
Cy3-conjugated oligonucleotides for validation | |
A’ | TACGGACTTAGCTCCAGGAT |
B’ | TAGGCATGATTCAATGAGGC |
C’ | TAGCGATAGTAGACGAGTGC |
i’ | TACTCTGACATCTCGACCAT |
ii’ | TAGATACTGCCACTTCACAT |
iii’ | TACCGTGAACCTTACCTGAT |
iv’ | AATGCTGGGGAAGGCTACTC |
v’ | CGCACCGCAGTTTGGTCAAT |
vi’ | TCCGACGCAACAATAGGGCA |
vii’ | CCTGCTCGACAACTAGAAGA |
viii’ | CCGCGACCAGAATTAGATTA |
ix’ | GCCGAAGCAGACTTAATCAC |
Table 2. Sequences used in DNA flow-patterning.
Flow pattern design is the first critical step in fabricating the 2-D microarray. To generate two overlapping DNA patterns on a glass substrate, the channel features of the first design should be perpendicular to those of the second (Figure 1A-B). The designs also consider the downstream applications of the microarray. In the case of single cell analysis, the microarray is used to detect proteins from single cells enclosed in microchambers, therefore the channel dimensions are made compatible with the microchambers that align with the 2-D arrays. Each design is rendered on a photomask and standard photolithography techniques are used to fabricate the channel features in SU-8 on a silicon wafer (Figure 1C-D). This serves as the master for molding PDMS. Once the mold has been fabricated, it is bonded to a PLL-coated slide and then coupled with a nitrogen gas flow set-up (Figure 1E). The set-up we use is simple and has the advantage of being easily incorporated into any laboratory with a pressure-regulated nitrogen gas source. We use an assembly of inexpensive multiple 3-way valves connected to a nitrogen gas tank. The air flow for patterning must be maintained at low pressures (0.5-1 psi) to avoid the delamination of the glass slide from the mold. Leaks within the PDMS mold can compromise the integrity of the patterns.
The DNA flow patterning steps in this protocol utilize ssDNA with carefully selected orthogonal sequences (Figure 2A). Prior to flow-patterning, the unique sequences on these oligonucleotides should be tested for the absence of cross-talk. A simple test for cross-talk is done by modifying the validation procedure we introduce in our protocol (Step 2). Instead of using a cocktail of Cy3-tagged complementary DNA, only one type of complementary sequence is used at a time for a selected area of the slide on which multiple DNA sequences are immobilized. In the absence of DNA cross-talk, only one stripe (for the 1-D DNA array) or one spot (for the 2-D DNA array) should be fluorescent under a Cy3 filter. Examples of thoroughly validated orthogonal sequences can be found in the Table of Materials and Equipment. The first DNA patterning step introduces three kinds of "anchor" oligonucleotides. These 80-nt sequences are comprised of two 20-mer poly-A regions that alternate with two unique 20-mer sequences. The 5'-amine modification on these anchor sequences is necessary for amine-to-amine cross-linking17 with PLL mediated by BS3. The second DNA patterning step does not require a cross-linker. This step introduces "bridging" oligonucleotides that partly hybridize with the anchor sequences. Each 60-nt bridging sequence consists of a 20-mer region complementary to one anchor sequence, a 20-mer poly-A spacer, and a unique 20-mer sequence. Validation of the DNA arrays (Figure 2B-C) is performed after every DNA flow-patterning step to assess the quality of the patterning steps and to ensure no cross-contamination14. This is performed by introducing Cy3-conjugated oligonucleotide probes that hybridize with the DNA patterns. With the parameters specified in Step (2.3.2) for analyzing fluorescence intensity, the DNA patterns should exhibit fluorescence intensities of at least 40,000 a.u. to maximize the sensitivity of the downstream immunoassays performed using the microarray. Strategic use of different Cy3-DNA sets during validation gives rise to alternative patterns (Figure 2D), thus demonstrating the flexibility of the DNA patterning steps8. Failure to achieve uniform patterns could result from leaks or mechanical obstructions (such as dust particles) encountered in the flow-patterning steps.
The preparation of antibody-oligonucleotide DEAL conjugates is another critical step in this protocol. The choice of antibodies is dictated by the biological system under study. The oligonucleotides used in conjugation should have sequences complementary to those anchored on the DNA array. Stock solutions of the S-4FB and S-HyNic linkers should be freshly prepared and kept away from moisture to avoid hydrolysis. The incubation times used in our conjugation protocol are long enough to introduce the desired functionalities on the antibodies and DNA. The final coupling reaction is only quenched by removing the unreacted S-4FB-conjugated oligonucleotides via FPLC. Thus, FPLC purification should be performed immediately after completing the conjugation. When performing FPLC with the system described in our protocol, lower flow rates (0.2-0.25 ml/min) may also be used to improve resolution. The conjugates are eluted as a broad peak (elution volume of around 9-15 ml) that appears before a narrower and higher DNA peak (17-20 ml elution volume). We do not recommend storing solutions of conjugates with excess S-4FB-DNA because this can lead to loss of antigen-binding sites in the antibody upon conjugation with excess functionalized DNA. The sensitivity, specificity, and reproducibility of sandwich ELISAs using DEAL conjugates have been demonstrated in previous studies.8,9,11,12,16 To assess the quality of antibody-DNA conjugates, the conjugates may be incubated on DNA arrays to generate the antibody array, which is subsequently used in sandwich ELISA experiments to generate calibration curves for protein detection (Figure 3F). To generate these calibration curves, recombinant proteins provided in a conventional sandwich ELISA kit may be used. The use of high-quality conjugates should give rise to linear ranges and lower limits of detection comparable with those of the kit. As an example, the lower limits for INFγ and TNFα for sandwich ELISA on our antibody array (Figure 3F) are ~50-100 pg/ml, and are consistent with the linear detection range of ~15-1,000 pg/ml indicated in the product datasheet for the conventional sandwich ELISA kit. Poor signal readout obtained in downstream sandwich ELISA experiments could be attributed to the low quality of DNA array, the interference of excess DNA in the conjugate solutions, or alternatively, incomplete conjugation of ssDNAs with antibodies.
Major concerns for performing sandwich ELISA on the antibody array include cross-talk between reagents and whether the signal reflects the real biological events. The orthogonal DNAs we use in this protocol have been thoroughly validated to have less than 0.1% cross-talk. Therefore any cross-talk observed at the immunoassay stage is mainly from the antibodies and ELISA reagents. Testing for cross-talk among antibodies in the array should be performed prior to conducting assays on real samples. Once an antibody panel is constructed, a few sandwich ELISA control experiments should be performed with the following conditions: 1. Without antigens, 2. Without detection antibodies, and 3. Detection antibodies and labeling reagents only. If cross-talk is observed at the immunoassay stage, the antibody pairs and/or ELISA reagents should be replaced. It should be noted that the use of commercially available antibodies from conventional ELISA kits does not guarantee applicability to the array-based sandwich ELISA technique.
The merits of this technology include inexpensive manufacturing, miniature size, and flexibility of design. Our fabrication protocol does not need a microarray spotter that may not be available to many users of antibody arrays. The flow patterning set-up can be easily assembled in simple laboratories. For laboratories that are not equipped with instruments for photolithography, the CAD designs we provide in our protocol may be forwarded to companies that offer microfabrication services. Downsizing the conventional microarray into the compact array fabricated with our protocol allows for compatibility with microchips used in single-cell experiments. Our protocol features the production of the array as an ssDNA array first before conversion to an antibody array. Preliminary patterning with ssDNAs has a number of advantages. First, ssDNAs are chemically more stable compared to antibodies, thus the ssDNA-patterned slides can be stored in a desiccator for at least 6 months without significant degradation8,16. Second, our approach offers an end user the flexibility to choose the assays needed, by simply mixing the selective conjugates together in a solution without modification of the microarray; On the contrary, conventional protein/antibody arrays are pre-designed and fixed on a substrate, so the change of assays would require the patterning/printing of proteins/antibodies from the very beginning. Representative studies illustrate the use of the barcode antibody microarray in the high-throughput detection of multiple signaling proteins from single cells. By varying the flow pattern designs and/or the oligonucleotide patterning solutions used, a multitude of array layouts can be explored. As an example of this application, functional proteins from single cells may be studied. The chip for single-cell analysis encompasses as many as ~8,700 microchambers, each aligned with a complete 3 x 3 antibody array (Figure 3H). Such a set-up allows for high-throughput detection. Proteins secreted by cells cultured in microchambers can be detected by this array. The versatile microarray design also allows for multiplexed detection of proteins from serum, cell lysates, and single cells after combining the microarray with other generic components8,15. In addition to protein detection, the 3 x 3 antibody array is also useful in capturing cells (Figure 3G).
The main disadvantage of this approach is its added complexity compared with the manufacturing of conventional microarrays. Strategies for patterning and for array design must be carefully conceptualized while considering the specific applications of the fabricated array. This approach also requires a number of critical steps including validation procedures and tests for cross-talk. The 3 x 3 array constructed using our protocol is limited to detecting 7 proteins at a time. However, this limit can be surpassed by creating larger arrays. The protocol featured here is not limited to the fabrication of 3 x 3 microarrays. We have been able to successfully fabricate 5 x 5 microarrays and other dimensions of array elements. In principle, other n x m arrays may be fabricated using similar techniques as long as the oligonucleotide sets used in creating the preliminary DNA-patterned array are orthogonal to avoid cross-talk between array elements. The creation of expanded arrays is achieved by flow patterning n types of ssDNA anchors for the first DNA patterning step, followed by n x m bridging ssDNA sequences for the second patterning step. For instance, to create a 5 x 5 array, anchor sequences A to E may be used, while bridging sequences A'-i to E'-xxv are utilized. To automate the flow patterning process, a robotics platform has been developed18 although this has only utilized one DNA flow patterning step. In principle, the same platform can be extended to the second step of perpendicular flow patterning, while the switching between two steps may require manually peeling off the first PDMS mold after the first patterning step, followed by washing and drying the slide (this step would take about 10 min), before mating the slide with another PDMS mold to facilitate the perpendicularly oriented second patterning step.
We have discussed detailed procedures for fabricating an n x m array patterned with antibodies at high density, which holds great promise for future applications in proteomic studies and cell signaling. The protocol presented here may also serve as a guide for the construction of more elaborate DNA/antibody arrays for other purposes.
The authors have nothing to disclose.
The authors would like to acknowledge the startup fund from SUNY Albany and the access of facilities at the University at Albany Cancer Research Center.
Sylgard 184 silicone elastomer base | Dow Corning | 3097366-1004 | |
Sylgard 184 silicone elastomer curing agent | Dow Corning | 3097358-1004 | |
SU-8 2025 photoresist | MicroChem | Y111069 | |
Silicon wafers | University Wafers | 452 | |
Poly-L-lysine coated glass slides | Thermo Scientific | C40-5257-M20 | |
Oligonucleotides | Integrated DNA Technologies | *Custom-ordered from Integrated DNA Technologies, see table below | |
Poly-L-lysine adhesive stock solution | Newcomer Supply | 1339 | |
Bis (sulfosuccinimidyl) suberate (BS3) | Thermo Scientific | 21585 | |
1x Phosphate buffered saline, pH 7.4 | Quality Biological | 114-058-101 | |
Äkta Explorer 100 Fast Protein Liquid Chromatography (FPLC) System | GE (Amersham Pharmacia) | 18-1112-41 | |
Superose 6 10/300 GL column | GE Healthcare Life Sciences | 17-5172-01 | |
Capture antibodies | various | various | *Antibody selection depends on application |
Succinimidyl-6-hydrazino-nicotinamide (S-HyNic) | Solulink | S-1002 | |
Succinimidyl-4-formylbenzamide (S-4FB) | Solulink | S-1004 | |
N,N-dimethylformamide | Sigma-Aldrich | 227056 | |
Citric acid, anhydrous | Acros | 42356 | |
Sodium hydroxide | Fisher Scientific | S318 | |
Amicon Ultra spin filter 10 kDa MWCO | EMD Millipore | UFC201024 | |
Spin coater | Laurell Technologies | WS-650-MZ | |
Biopsy punch with plunger (0.50 mm diameter) | Electron Microscopy Sciences | 57393 | |
Diamond scribe (Style 60) | SPI supplies | 6004 | |
Trimethylchlorosilane | Sigma Aldrich | 92361 |