The yellow carboxyl fluorescent microspheres are a type of yellow fluorescent material embedded within polystyrene. They have strong and long-lasting fluorescence without quenching, a quantum yield of over 90%, a large Stokes shift, small batch-to-batch differences, good repeatability, and are suitable for the research and large-scale production of lateral chromatography reagents.

Technical parameters: 

Excitation wavelength: 400nm 

2. Emission wavelength: 550nm 

3. Functional group: Carboxyl group (COOH) (Customizable SA) 

3. Particle size range: 200nm - 400nm 

4. Uniformity Index: CV < 5% 

5. Storage conditions: Store at 2-8℃. Do not freeze.

Labeling Process Flow

1. Take 100 µL of carboxyl fluorescent microsphere solution, add 500 µL of MES pH 6.0 buffer to wash once, and centrifuge to remove the supernatant.

2. Add 500 µL of MES pH 6.0 buffer to resuspend the microspheres. Then add 30 µL EDC and 90 µL NHS (both at a concentration of 3 mg/mL, prepared fresh in MES pH 6.0 buffer), rotate and shake in the dark for 30 min, then centrifuge to remove the supernatant.

3. Wash once with 500 µL of MES pH 6.5 buffer. Resuspend the microspheres in 500 µL of MES pH 6.5 buffer, add 20 µg of antibody (antibody amount varies depending on the specific project), rotate and shake in the dark for 2 h, then centrifuge to remove the supernatant.

4. Add 500 µL of blocking solution to resuspend, rotate and shake in the dark for 1 h, then centrifuge to remove the supernatant.

5. Wash once with 500 µL of storage solution and centrifuge to remove the supernatant.

6. Resuspend in 100 µL of storage solution and store at 2–8 °C.

Note: Recommended centrifugation speed is 15,000 rpm (22,000 × g), centrifugation time 15–20 min.

Usage Notes

1. Carboxyl fluorescent microspheres should be vortexed or sonicated to mix thoroughly before use; probe sonication is the most effective way to resuspend microspheres.

2. EDC solution should be prepared fresh before use; NHS should also be prepared fresh.

3. The coupling reaction buffer system must be free of free amino groups; Tris, glycine, and acetate buffers must not be used.

4. Adding trace surfactants (not exceeding 0.01%) to the reaction buffer can effectively prevent microsphere aggregation.

5. The concentration of the microsphere solution can be adjusted according to the signal value of the project; recommended usage for T-line labeling is 3%–8%; for C-line labeling, 1%–2%.

6. Pad material, pad pretreatment buffer, microsphere diluent, activator dosage, and antibody ratio can be optimized based on project performance.