lateral flow immunoassay strips preparation, assay operation and its quantitative evaluation

Quantum Dot Nanobeads for Enhanced Sensitivity in Biomarker Detection

Lateral flow immunoassay (LFIA) strips serve as crucial rapid detection tools at point-of-care1,2, particularly in disease screening during epidemics. However, traditional colloidal gold-based LFIA test strips exhibit low detection sensitivity and only provide qualitative results3. To enhance the detection sensitivity of LFIA, various new nanoparticles have emerged, including colored latex4,5, upconversion fluorescent nanoparticles6, time-resolved fluorescent microspheres7,8, and quantum dots9,10,11. Quantum dots (QDs)12,13, also known as semiconductor nanocrystals, offer tunable emission wavelengths, a wide excitation range, and high luminescence efficiency, making them ideal labels for biological imaging.
However, the fluorescence signal emitted by individual quantum dots remains weak, resulting in relatively low detection sensitivity in immunoassays. Encapsulation of numerous quantum dots within microspheres can amplify signals and improve the sensitivity of quantum dot-based immunoassays. Various methods, such as layer-by-layer self-assembly14,15,16,17,18, the swelling method19,20, and silica microsphere21,22,23,24 encapsulation, have been employed to encapsulate quantum dots inside microspheres. For example, quantum dot-functionalized silica nanosphere labels can be achieved by increasing QD loading per sandwiched immunoreaction25. A spray dryer equipped with an ultrasonic atomizer has also been used to prepare nanoscale QD-BSA nanospheres26. However, the aforementioned methods suffer from complex multi-steps, fluorescence quenching, and low productivity.
In our previous work27, an emulsion-solvent evaporation method for encapsulating quantum dots inside polymer nanobeads was reported. This preparation technique is simple, maintains the fluorescent efficiency of QDs, ensures high encapsulation efficiency, and allows for easy scalable production. Several research groups have successfully developed LFIA strips using QDNBs prepared through this method for applications, including food toxin detection28,29,30, infectious disease biomarker detection31,32, and environmental monitoring33.
This protocol presents specific preparation steps for quantum dot nanobeads (QDNB), QDNB and antibody conjugation, preparation of QDNB-based LFIA, and measurement of C-reactive protein (CRP) in human plasma samples.

Author Spotlight: High-Quality Quantum Dot Nanobeads for Sensitive Fluorescent Lateral Flow Immunoassays

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, upcoercion 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. Common 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.

Lateral Flow Immunoassay Strips Preparation, Assay Operation and its Quantitative Evaluation

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 along 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.

Research

JoVE Journal

Biochemistry

Quantum dots, also known as semiconductor nanocrystals, are novel fluorescent labels for biological imaging and sensing. However, quantum dot-antibody ...