The overall goal of this procedure is to analyze multiple proteins in human saliva samples using microsphere based fluorescent arrays. First, encode the microspheres with different amounts of two fluorescent dyes. Couple the capture antibodies for different proteins onto the encoded microspheres.
Then assemble the multiplexed protein microarray on the fiber optic bundle using the sandwiched immunoassays on the assembled microarrays assay for multiple proteins in the test saliva samples. Ultimately, analysis by fluorescence microscopy can measure the signal responses of different proteins in the saliva samples. The main advantage of this technique over existing methods like Eliza's is that it enables the simultaneous analysis of multiple proteins in complex biofluids.
Although this method can provide insight into human saliva analysis, it can also be applied to other bio flus such as serum urine and sputum. Weigh the opium TTA in an Amber Glass vial and prepare a 200 millimolar stock solution in tetra hydro Furin. Mix gently by pipetting and visually verify that the dye is completely dissolved.
Similarly, prepare a 12 millimolar stock solution of Coumarin 30 mixed gently by pipetting.Visually. Verify that the D is completely dissolved vortex to microsphere suspension, and then transfer 60 microliters to a 1.5 milliliter micro centrifuge tube. To wash the microspheres, add 600 microliters of PBS mixed by pipetting 20 times and centrifuge at 10, 000 RPM for three minutes.
After three washes with tetra hydro furin solution, remove the supernatant resuspend the palate in 600 microliters of working solution, and transfer the mixture to a new 1.5 milliliter micro centrifuge tube. Seal the micro centrifuge tube with para film and place it on a shaker. Protect it from light by covering with aluminum foil.
Add 200 microliters of encoded microsphere suspension into a safe lock, 1.5 milliliter micro centrifuge tube. In a dropwise fashion add 150 microliters, a freshly prepared EDC solution Immediately add 150 microliters of sulfa NHS solution. Mix well by pipetting and verify the suspension is homogenous.
Cap the tube, seal it with paraform and place it on a shaker. Protect it from light by covering with aluminum foil. After three washes of P-B-S-S-D-S buffer reus, suspend the pellet in 200 microliters of P-B-S-S-D-S buffer mix s.
Well then transfer the solution to 300 microliters of P-B-S-S-D-S buffer containing 60 micrograms of capture antibodies. Mix 50 microliters of each type of protein capture microspheres into a new autoclave micro centrifuge tube. Centrifuge the stock microspheres pool at 7, 000 RPM for three minutes and remove all but 100 microliters of supernatant.
Now hand cut fiber optic bundles to approximately five centimeters in length, sequentially polish both ends using diamond lapping films. Then sonicate the polished bundle in deionized water for two minutes. To remove any particles, put a small magnetic stirring bar into a 0.5 milliliter micro centrifuge tube.
Add 400 microliters of protein free PBS buffer and stir on a magnetic stirring plate for 30 minutes. To remove air bubbles etch one end of the polished fiber bundle in a 0.025 normal hydrogen chloride solution for 150 seconds. Immediately submerge and sonicate the etched N in deionized water for one minute.
Then dry the fiber bundles with compressed air mount the fiber bundle on a fiber holder and block the etched end in the bubble free protein free PBS buffer for one hour deposit a one microliter ALI quad of the stored microsphere suspension on the etched end of the fiber optic bundle. Protect the setup from light with a box wrapped with aluminum foil. After 15 minutes, the volume of the suspension will decrease by evaporation and force the microspheres into the etched micro wells.
Use a swab saturated with sample solution to remove microspheres that are not trapped in the micro wells. To make the protein microarray ready for use, add 200 microliters of sample solution into a 0.5 milliliter autoclave micro centrifuge tube. Dip the protein microarray loaded on the fiber into the solution and place on a shaker.
Protect from light by covering with aluminum foil. Next, dip the protein microarray into a new autoclave micro centrifuge tube containing 200 microliters of wash buffer and shake at 600 RPM for two minutes. Now incubate the microarray in a 100 microliter solution of a detection antibody cocktail containing biotinylated detection antibodies and A PBS blocking buffer for 30 minutes.
At room temperature in the dark, clean the distal end of the fiber bundle with a swab saturated with absolute ethanol. Then dry both ends of the fiber with compressed air mount the fiber on an epi fluorescent microscope and image through the distal end of the fiber, acquire the microsphere images corresponding to opium TTAC 30 and SAP fluorescent emissions intensities. The encoding images of seven microsphere types are shown here.
Two intensity levels are observed in the UTTA encoding image, which stand for microspheres encoded with different amounts of UTTA die Similar results are observed in the C 30 encoding image. MATLAB image analysis allows calculation of the fluorescence intensities of different microspheres in the signal image. The decoding results are shown in figure 2D seven.
Different microspheres are successfully decoded. The microspheres show no response when incubated with assay buffer. When incubated with human saliva samples, significant fluorescent responses are observed.
For a better comparison, the contrasts in these two figures have been adjusted to the same level. Once mastered this method enables reproducible analysis of different proteins in the human saliva samples. The results of three independent detections on the same sample are shown.
After watching this video, you should have a good understanding of how to prepare a microsphere based multiplex protein microray and use it for complex bio fluids analysis. Following this procedure. Other protein biomarkers in different complex biofluids can also be analyzed.
