Fluorescent Lateral Flow Immunoassay Based on Quantum Dots Nanobeads

Our research interests currently focus on constructing high-quality multifunctional nanomaterials and their application in the development of ultrasensitive point-of-care diagnostics. Lateral flow immunoassay strips serve as critical rapid detection tools at the point-of-care, particularly in disease screening during epidemics. To enhance the detection sensitivity to lateral flow immunoassay, various new nanoparticle has emerged, including colored latex, upconversion fluorescent nanoparticles, timed-result fluorescent microspheres, and quantum dots.

We presented a one-step emulsion evaporation method for preparing quantum dot nanobeads and other applications in sensitivity, for instance, lateral fluoro-immunoassays. Compared to other methods of preparing quantum dot nanobeads, this protocol offers the advantage of simplicities of operation and high productivity. Additionally, the polymer composite nanobeads are biocompatible, unlike silica nanobeads, which generally need additional surface modification with polymers.

Sensitive fluorescent lateral flow immunoassay strips have been developed using prepared quantum dot nanobeads. These strips can be used to qualitatively and quantitatively detect disease biomarkers at point-of-care. This new assay could help medical professionals diagnose diseases quickly and accurately.

To begin, mix 0.4 milliliters of polystyrene-co-maleic anhydride solution and 0.1 milliliters of QD solution to prepare one milliliter of the oil phase. Then fill the rest with chloroform until the volume reaches one milliliter. Next, prepare the aqueous phase using sodium dodecyl sulfate solution.

Combine one milliliter of the oil phase and five milliliters of the aqueous phase in a vial. Place the vial on a magnetic stirrer and turn it on. Once the oil and aqueous phases are thoroughly mixed, immediately transfer the vial to the programmed ultrasonication device and turn on the power.

Then place the vial back on the magnetic stirrer at room temperature, and stir continuously overnight. After solvent evaporation, add 0.1 milliliters of 0.1 millimolar sodium hydroxide to the nano bead suspension, and allow it to react for one hour. To begin, add 100 microliters of prepared QD nano beads to 200 microliters of phosphate buffer.

Then add six microliters of EDC. After that, incubate the mixture for 30 minutes at room temperature on a rotary mixer. Centrifuge the mixture at 13, 523G for 10 minutes at room temperature, and discard the supernatant.

Next, add 100 microliters of phosphate buffer to the precipitate. Cover the tube with its lid and immerse it in the operational ultrasonic water bath until the precipitate is fully dispersed. Disperse 100 micrograms of antibody in 200 microliters of phosphate buffer, and add the activated QD nano bead suspension to the antibody solution.

Incubate the mixture at room temperature for 30 minutes with continuous rotation. Then, remove excess antibodies by centrifugation at 6, 010G for five minutes at room temperature, and carefully discard the supernatant. To block the nano beads, add 200 microliters of 1%casein solution to the residue.

Cover the tube with its lid and immerse it into the operational ultrasonic water bath until the residue is fully dispersed. Incubate the mixture at room temperature for two hours with continuous rotation. Add 100 microliters of preservative solution to the mixture and store at four degrees Celsius for subsequent use.

To begin, prepare the test line and control line coating solution. Dilute the C-reactive protein capture antibody and goat anti-rabbit immunoglobulin G with phosphate buffer. Next, place the polyvinyl chloride board properly and remove the protective paper located 25 millimeters from the edge of the board.

Align and adhere the nitrocellulose membrane a long the lower edge of the remaining protective paper to the polyvinyl chloride board. Dispense the test line and control line coating solutions onto the nitrocellulose membrane, ensuring it is placed 20 millimeters apart from the upper edge, forming a test line. For drying, place the nitrocellulose membrane in conditions with a humidity of less than 30%and a temperature range of 18 to 26 degrees Celsius.

To prepare the conjugate pad, submerge the conjugate pad into the conjugate pad treatment solution. Then place the soaked conjugate pad in a drying oven for overnight drying treatment. Dilute the prepared QD nano bead antibody conjugate solution with the preservative solution and dispense it onto the conjugate pad.

For sample pad preparation, submerge the sample pad into the sample pad treatment solution. Then place the soaked sample pad into a drying oven set at 45 degrees Celsius and dry for 24 hours. Successively, place the absorbent paper, conjugate pad, and sample pad on the polyvinyl chloride board with the nitrocellulose membrane.

Pipette 10 microliters of plasma sample or calibrator solution into one milliliter of a PBS buffer containing 0.1%Tween 20. Then transfer 100 microliters of the prepared diluted sample onto the sample pad of the test strip. After a 15 minute run, observe the fluorescent signals of the test line and control line under ultraviolet light exposure to obtain qualitative results.

Insert the strip card into a fluorescent lateral flow assay reader to obtain the fluorescence intensities for quantitative analysis.