Multiplexed Fluorescent Microarray for Human Salivary Protein Analysis Using Polymer Microspheres and Fiber-optic Bundles

Figure 1. Workflow for applying fiber-optic microsphere antibody array to saliva profiling. (1) Microspheres are internally encoded with two fluorescent dyes; (2) the encoded microspheres are externally modified with protein-specific monoclonal antibodies; (3) the multiplexed microspheres are mixed, and (4) randomly deposited in microwells etched at the proximal end of a fiber-optic bundle; (5) salivary proteins are captured by microspheres through sandwich immunoassay, and (6) quantified using fluorescence microscopy.

1. Microspheres Encoding

1. Weigh sodium europium (III) thenoyltrifluoroacetonate trihydrate (Eu-TTA, MW = 869.54 g/mol) in an amber glass vial and prepare a 200 mM stock solution in tetrahydrofuran (THF). Mix gently by pipetting; visually verify the dye is completely dissolved.
2. Weigh coumarin 30 (C30, MW = 347.41 g/mol) in an amber glass vial and prepare a 12 mM stock solution in THF. Mix gently by pipetting; visually verify the dye is completely dissolved.
3. Prepare 700 μl of working solution for each microsphere type using the stock solutions and THF to reach final concentration listed in Table 1.
4. Ensure microspheres (10% w/v) are well suspended by vortexing, and then transfer 60 μl suspension to a 1.5 ml microcentrifuge tube.
5. Add 600 μl PBS to microsphere suspension and mix by pipetting 20x. Centrifuge the tube at 10,000 rpm for 3 min and carefully remove the supernatant. Repeat this process another 2x to wash the microspheres.
6. Wash the microsphere pellet 3x with THF using a similar procedure as step 1.5.
7. Centrifuge the microsphere/THF suspension at 10,000 rpm for 3 min, remove the supernatant, and re-suspend the pellet in 600 μl working solution listed in Table 1. Transfer the mixture to a new 1.5 ml microcentrifuge tube.
8. Seal the microcentrifuge tube with Parafilm and place it on a shaker. Shake at 3,000 rpm and incubate for 24 hr, protect from light by covering the setup using a box covered with aluminum foil.
9. Centrifuge the microsphere suspension at 10,000 rpm for 3 min to form a pellet.
10. Remove the supernatant dye solution, wash the microspheres 6x with 600 μl methanol and then with 600 μl PBS containing 0.01% Tween-20 another 6x, as described in step 1.5.
11. Store the encoded microspheres in 600 μl PBS containing 0.01% Tween-20 at 4 °C until use, protect from light.

Table 1. Concentrations of Eu-TTA and C30 working solutions.

2. Preparation of Protein-capture Microspheres

1. Prepare MES buffer [2-(N-morpholino)ethanesulfonic acid (MES) 0.1 M, NaCl 0.9%, SDS 0.01%, pH 5.7] and PBS/SDS buffer (Sodium Phosphate 0.01 M, NaCl 0.154 M, SDS 0.01%, pH 7.4) in advance at room temperature.
2. Add 200 μl encoded microsphere suspension (containing ~2 mg microspheres) into a Safe-Lock 1.5 ml microcentrifuge tube. It is important to use this type of microcentrifuge tube to avoid spillage during the incubation in step 2.6.
3. Wash the microspheres with 600 μl MES buffer 3x as described in step 1.5. Add 700 μl MES to the microsphere pellet and mix well by pipetting.
4. Prepare 0.105 g/ml 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC, MW = 191.7 g/mol) and 0.163 g/ml sulfo-N-hydroxysulfosuccinimide (sulfo-NHS, MW = 217.13 g/mol) solutions in MES buffer in separate tubes.
5. Add 150 μl freshly prepared EDC solution drop by drop into the microsphere suspension. The EDC hydrolyzes very quickly, thus it is essential to use freshly made solution to ensure the immobilization efficiency. Immediately after the addition of the EDC, add 150 μl sulfo-NHS solution to the microspheres, mix well by pipetting and verify the suspension is homogenous.
6. Cap the tube, seal it with Parafilm, and place it on a shaker. Shake at 1,500 rpm for 4 hr, protect from light using a box covered with aluminum foil.
7. Centrifuge the microsphere suspension at 10,000 rpm for 3 min to form a pellet and remove the supernatant. Wash the microspheres with 500 μl PBS/SDS buffer 3x as described in the step 1.5.
8. Add 200 μl PBS/SDS buffer to the pellet, mix the microspheres well and transfer the solution to 300 μl PBS/SDS buffer containing 60 μg capture antibodies. It is important to suspend the microsphere pellet first and then add the microsphere suspension into the antibody solution.
9. Incubate the antibody-microspheres mixture by shaking at 1,500 rpm for 4 hr on a shaker, protect from light using a box covered with aluminum foil.
10. Centrifuge the microsphere suspension at 10,000 rpm for 3 min to form a pellet and remove the supernatant. Wash the microspheres with 500 μl StartingBlock (TBS) blocking buffer 3x, as described in step 1.5.
11. Mix microspheres with 1 ml of TBS blocking buffer and incubate microspheres at 1,500 rpm for 1 hr on a shaker, protect from light using a box covered with aluminum foil.
12. Centrifuge the microsphere suspension at 7,000 rpm for 3 min to form a pellet and remove the supernatant. Wash the pellet 3x with 500 μl TBS blocking buffer, as described in step 1.5.
13. Centrifuge the microsphere solution at 7,000 rpm for 3 min to form a pellet and remove the supernatant. Add 500 μl TBS blocking buffer and mix by pipetting.
14. Store the modified microsphere suspension at 4 °C until use, protect from light.
15. Repeat this procedure to make other types of microspheres using corresponding antibodies.

3. Fiber-optic Microarray Assembly

1. Mix 50 μl of each type of protein-capture microspheres into a new autoclaved microcentrifuge tube. Centrifuge the stock microspheres pool at 7,000 rpm for 3 min, and remove the supernatant so that the remaining volume is approximately 100 μl.
2. Hand-cut fiber-optic bundles to approximately 5 cm in length and sequentially polish both ends on a polishing machine using 30, 15, 9, 6, 3, 1, 0.5, and 0.05 μm-sized diamond lapping films. Sonicate the polished bundle in deionized water for 2 min to remove any particles.
3. Put a small magnetic stirring bar into a 0.5 ml microcentrifuge tube, add 400 μl of protein-free PBS buffer and stir on a magnetic stirring plate for 30 min to remove air bubbles.
4. Etch one end of the polished fiber bundle in a 0.025 N HCl solution for 150 sec^10,16. Immediately submerge and sonicate the etched end in deionized water for 1 min. Dry the fiber bundles with compressed air.
5. Mount the fiber bundle on a fiber holder and block the etched end in the bubble-free, Protein-free PBS buffer for 1 hr.
6. Deposit 1 μl aliquot of the stored microsphere suspension on the etched end of the fiber-optic bundle. Protect the setup from light with a box with aluminum foil, and wait for 15 min. The volume of the suspension will decrease by evaporation and force the microspheres into the etched microwells. Repeat this loading step one more time.
7. Use a swab saturated with sample solution to remove microspheres that are not trapped in the microwells. The protein microarray is now ready to use. Go immediately to step 4.1 for saliva analysis.

4. Saliva Sample Analysis Using Microspheres Microarray

1. Add 200 μl sample solution (100 μl saliva supernatant diluted with equal volume of StartingBlock T20 (PBS) blocking buffer. The collection protocol for saliva supernatant has been published previously¹⁰). into a 0.5 ml autoclaved microcentrifuge tube. Dip the protein microarray loaded on the fiber into the solution and incubate on the shaker at 600 rpm for 2 hr, protect from light using a box covered with aluminum foil.
2. Prepare 20 ml wash buffer by adding 200 μl 10 % BSA solution and 200 μl 10% Tween-20 into 19.6 ml PBS, mix well by gently shaking.
3. Dip the protein microarray into a new autoclaved microcentrifuge tube containing 200 μl wash buffer and shake at 600 rpm for 2 min. Repeat this wash 3x.
4. Incubate the microarray in 100 μl solution of a detection antibody cocktail containing 5 μg/ml of anti-VEGF, IP-10, IL-8, EGF, MMP-9, IL-1β biotinylated detection antibodies in StartingBlock T20 (PBS) blocking buffer for 30 min at room temperature on the shaker at 600 rpm, protect from light using a box covered with aluminum foil.
5. Wash the microarray 3x with wash buffer as described in step 4.3.
6. Incubate the microarray in 200 μl of 20 μg/ml streptavidin R-phycoerythrin conjugate (SAPE) solution in PBS at 600 rpm on the shaker for 10 min, protect from light using a box covered with aluminum foil.
7. Wash the microarray 5x with wash buffer as described in step 4.3.
8. Clean the distal end of the fiber (i.e., the end away from the microspheres) bundle with a swab saturated with absolute ethanol.
9. Dry both ends of the fiber with compressed air. Mount the fiber on an epifluorescent microscope and image through the distal end of the fiber.
10. Acquire microsphere images corresponding to Eu-TTA, C30, and SAPE fluorescence emission intensities. Optical filters setup and exposure times for fluorescence imaging of Eu-TTA, C30, and SAPE are depicted in Table 2.

Table 2. Parameters for the three fluorescent images of the microarray.