The overall goal of this methodology is to fabricate an antibody array with applications in single-cell analysis. This method can help answer key questions in the field of single-cell analysis such as how can we detect secret proteins and intracellular proteins simultaneously from the same single cell. And how can we fabricate a micro-array platform to accomplish that objective.
The main advantage of this technique is that it is customizable. By varying the flow patterning strategies, arrays of various dimensions may be created. In advance, generate the SU-8 master for the barcode flow patterns.
Instructions are provided in the text protocol. This involves designing, and making a chrome photo-mask. Placing the mask on a photo-resist coat and applying UV provides the pattern.
To start preparing the PDMS barcode mold, combine 40 grams of silicon elastomer base with four grams of curing agent, then stir vigorously for 10 minutes. Then, de-gas the pre-polymer solution for 20 minutes under vacuum. Next, load the solution into a petri dish containing a silicon wafer with the SU-8 master of the barcode pattern.
Pour the solution to a depth of 7.45 millimeters. Then, de-gas the mixture in the dish to remove any remaining bubbles. Follow the de-gassing by baking the mixture for an hour at 75 Degrees Celsius to cure the PDMS.
Next, cutting with a scalpel, carefully remove the barcode mold. Then, carefully peel the slab from the wafer. Next, trim the edges of the slab to make the desired shape for the mold.
Finish the mold by using a biopsy punch with plunger to punch out half millimeter holes at the circular features on the pattern which represent inlets and outlets. After blowing any dust off a poly-L-lysine coated glass slide with a nitrogen blowgun, attach the PDMS mold to the slide. Carefully line up the edges of the mold to the slide.
Now, bake the assembly for 90 minutes at 75 degrees Celsius to strengthen the bond between the PDMS and the slide. Meanwhile, prepare 20 pieces of flexible polyethylene tubing for the inlets and outlets. On each piece of tubing, attach a stainless steel hollow pin with a one millimeter diameter.
Then, via the pins, fill the lengths with a centimeter or more of poly-L-lysine. Once bonded to the glass, carefully fasten the pins to the inlets of the mold. This is the most difficult step of the preparation.
Do not rush the process. Attach the other end of the tubes to a pressure-regulated nitrogen tank setup which consists of a network of 20 three-way valves and can handle two molds at once. Using the setup, blow solution through the mold at 0.5 to one PSI for at least six hours.
Begin by aspirating freshly prepared single-stranded DNA solutions in BS-3 through the stainless steel pins and into the polyethylene tubing. Then, couple the PDMS mold to the nitrogen source and flow the solution through the barcode mold for about 40 minutes or until all the channels are filled. Then, stop the flow and let the mold incubate at room-temperature for two hours.
After a couple of hours, bake the barcode mold at 75 degrees Celsius for an hour. Once baked, detach the mold from the slide and gently wash the slide with 01%SDS. Then, wash the slide three times with ultra-pure water.
Follow the washes by drying the barcode slide using a slide-spinner. Once dry, store the barcode slide in a clean 50 milliliter tube. To proceed, follow the text protocol to validate the one-dimensional pattern.
To perform the validation procedure, block the slide, hybridize cyanine three complementary DNA to it and measure the results. The SU-8 master for a three by three array has a flow pattern perpendicular to that of the first design. Use the previously described method to make a new PDMS mold of this pattern.
To start, make fresh working concentrations of the nine oligonucleotides. Now, flow a three percent BSA blocking solution into all 20 channels of the array slide for an hour at 0.5 to one PSI. Next, load the DNA solutions into their respective channels.
After flowing them for about 40 minutes, incubate the slide at room-temperature for two hours to allow the DNA to hybridize. Next, flow three percent BSA blocking solution into all 20 channels for an hour. This will remove the non-hybridized DNA.
Now, peel the array slide from the PDMS and dip the slide into three percent BSA once. Then, dip the slide into PBS twice followed by one dip in two percent PBS. Then, as before, dry the slide using a microscope slide spinner.
To proceed, perform a validation step similar to validating one-dimensional slides. Use oligonucleotides one-prime to nine-prime all conjugated to cyanine three. Save the three by three array slide in a 50 milliliter centrifuge tube for subsequent use.
The text protocol further explains how to convert the DNA array into an antibody array. After preparing and purifying the antibody-oligo conjugates, prepare another PDMS mold with micro-chambers. See the text for details.
Mate this mold with the array slide. Now, block the slide with one percent BSA and incubate it for an hour at room temperature. During the incubation, prepare 200 micro-liters of antibody-oligonucleotide conjugates.
After the block, add the antibody-oligo cocktail to the setup. Let the antibodies incubate on the array for an hour at 37 degrees Celsius, and then clean and dry the slide before proceeding with slide dips described in the text protocol. After following the described protocol, using a three by three array, the resulting antibody panel was used in multi-plex detection, mainly through sandwich ELISA platform.
The proteins showed less than 10%variation from one end to the other end of the glass slide. On the three by three array, up to seven proteins can be detected along with a reference labeled by cyanine three and a negative control. In a single-cell experiment performed for a tumor study, the six proteins were all detected simultaneously.
For calibration, recombinant proteins were applied to an array at various concentrations. The sensitivity of detection was quite similar to a conventional sandwich ELISA. While attempting this procedure, its important to remember to keep the PDMS molds free of particulate matter.
Dust in the channels can result in arrays with poor uniformity and low sensitivity. After its development, this technique paved the way for researchers in the field of cancer research to explore cell-cell signaling using models of tumor micro-environments.
