qdnb-antibody conjugates preparation using one-step emulsion evaporation method

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.

QDNB-Antibody Conjugates Preparation Using One-Step Emulsion Evaporation Method

To begin, add 100 microliters of prepared QD nanobeads 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, 523 g 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 nanobead 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, 010 g for five minutes at room temperature, and carefully discard the supernatant.To block the nanobeads, 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.

Research

JoVE Journal

Biochemistry

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