Here, we describe a protocol for the preparation of quantum dot nanobeads (QDNB) and the detection of disease biomarkers using QDNB-based lateral flow immunoassay strips. The test results can be qualitatively assessed under UV light illumination and quantitatively measured using a fluorescent strip reader within 15 min.
Quantum dots, also known as semiconductor nanocrystals, are novel fluorescent labels for biological imaging and sensing. However, quantum dot-antibody conjugates with small dimensions (~10 nm), prepared through laborious purification procedures, exhibit limited sensitivity in detecting certain trace disease markers using lateral flow immunoassay strips. Herein, we present a method for the preparation of quantum dot nanobeads (QDNB) using a one-step emulsion evaporation method. Using the as-prepared QDNB, a fluorescent lateral flow immunoassay was fabricated to detect disease biomarkers using C-reactive protein (CRP) as an example. Unlike single quantum dot nanoparticles, quantum dot nanobead-antibody conjugates are more sensitive as immunoassay labels due to signal amplification by encapsulating hundreds of quantum dots in one polymer composite nanobead. Moreover, the larger size of QDNBs facilitates easier centrifugation separation when conjugating QDNBs with antibodies. The fluorescent lateral flow immunoassay based on QDNBs was fabricated, and the CRP concentration in the sample was measured in 15 min. The test results can be qualitatively assessed under UV light illumination and quantitatively measured using a fluorescent reader within 15 min.
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.
The study was approved by the Institutional Review Board of Shanghai Skin Disease Hospital (No. 2020-15). All experimental procedures involving human blood samples were conducted in a Biosafety Level II laboratory. The details of the reagents and equipment used in this study are listed in the Table of Materials.
1. Preparation of QDs nanobeads
NOTE: For QD nanobead synthesis, an emulsion-solvent evaporation technique was used to synthesize QD nanobeads, with a ratio of oil phase to water phase of 1:5. The mini emulsion is generated via ultrasonication, and the nanobeads are solidified through solvent (chloroform) evaporation. Sodium hydroxide was utilized to catalyze the hydrolysis of anhydride groups on the nanobead surface, converting them into carboxyl groups.
2. Preparation of QDNB-antibody conjugates
3. Preparation of lateral flow immunoassay strips
NOTE: The immunochromatographic strip is composed of a nitrocellulose (NC) membrane, a sample pad, a conjugate pad, an absorbent pad, and a polyvinyl chloride (PVC) board (Figure 1). All solutions should be prepared and used immediately. It is recommended that lateral flow immunoassay strips be prepared in a clean room with controlled humidity.
4. Assay operation and qualitative evaluation
NOTE: Operations involving human blood samples are recommended to be conducted in a Biosafety Level II laboratory. The human plasma samples were obtained from a clinical laboratory (from deidentified human subjects). Typically, human blood was collected using an EDTA vacuum tube, and plasma was isolated according to the standard protocol34. Centrifuge at 3,000 × g for 10 min at room temperature for plasma separation. Collect the yellow plasma upper layer using a pipette, and store the plasma in a 1.5 ml tube at -80 °C until use.
The QDNB preparation procedures are schematically illustrated in Figure 1A. The oil phase containing QDs and polymer in chloroform was mixed with the water phase, and a mini-emulsion was obtained by sonication. The emulsion was solidified by gradual evaporation of chloroform. The transmission electron micrograph (TEM) of QDNB is presented in Figure 2A. The QDNBs have a spherical morphology, with average diameters of 96 nm, measured over 50 QDNBs in TEM images. QD nanoparticles are clearly observed inside the nanobeads, indicating successful encapsulation. Figure 2B and Figure 2D show the fluorescence and absorption spectra of QDNB, respectively.
The QDNB and antibody were conjugated using carbodiimide chemistry, as shown in Figure 1B. The QDNB-antibody conjugate was used to prepare QDNB-based LFIA. The structure of LFIA is illustrated in Figure 1C. If the CRP antigen is present in the sample, the QDNB-anti-CRP conjugates accumulate at the test line.
As shown in Figure 3, the measurement is carried out by adding 100 µL of diluted sample to the sample hole. After a reaction time of 15 min, the test results can be qualitatively read under UV light illumination. The quantitative results can be obtained using a fluorescent strip reader, and the concentration of the sample can be calculated using a calibration equation embedded in the strip reader software37,38.
Figure 1: Illustration of the fluorescent lateral flow immunoassay based on quantum dots nanobeads. (A) Procedures for the preparation of QDNB. (B) Principle of QDNB-antibody conjugates. (C) Principle of QDNB-based LFIA for the detection of CRP. Please click here to view a larger version of this figure.
Figure 2: Characterization of QDNBs. (A) TEM image of QDNBs. Scale bar: 100 nm. (B) Fluorescent emission spectra of QDNB. (C) Absorption spectra of QDNB. Please click here to view a larger version of this figure.
Figure 3: Procedures of lateral-flow immunoassay for CRP. (A) Illustration of the detection. (B) Photographs of QDNB-based LFIA strips detecting CRP samples in a range of 0-30 mg/L under UV light. (C) Fluorescent reader for quantification of results. (D) Photograph of the quantitative results. (E) Detection of C-reactive protein (CRP) using logistic transformation followed by linear regression. Please click here to view a larger version of this figure.
Here, we describe a protocol for the preparation of quantum dot nanobeads (QDNB)27 and the use of QDNB for the preparation of fluorescent lateral flow immunoassays (LFIA). The qualitative and quantitative measurement of CRP in samples is demonstrated. This QDNB-based LFIA can also be applied to other disease biomarkers25,32, food toxins29,30, viruses16,27, and more.
The critical steps in this protocol are the preparation of QDNB and QDNB-antibody conjugates. The particle size of QDNB and the QD content in nanobeads should be optimized to obtain highly fluorescent QDNB with low background signals in the LFIA. Capture and detection antibody pairs are critical for the immunoassay and should be screened for the best matches from various vendors. Conjugation of the antibody with QDNB should be optimized, including the antibody/QDNB ratio, buffer pH, and blocking reagents. Specifically, aggregation of QDNBs and antibodies should be avoided by optimizing the pH of the buffer, salt concentration, and reaction time in the conjugation procedures.
Compared with other preparation methods for quantum dot nanobeads, this protocol has the advantages of simplicity, easy operation, and high productivity. Moreover, the polymer composite nanobeads are biocompatible, unlike silica nanobeads, which usually require additional surface modification with polymers. However, the QDNBs prepared using this protocol are not as monodisperse as those prepared using polymer19 or silica21 template nanobeads. The particle size of QDNBs usually ranges from 70 nm to 200 nm.
QDNB-based lateral flow immunoassay (LFIA) strips are prepared similarly to other fluorescent LFIAs. Due to the high quantum yields of QDs and the signal amplification of QDNBs, the QDNB-based LFIA using this protocol is more sensitive than other fluorescent microsphere-based LFIAs, such as time-resolved fluorescent microspheres and fluorescein microspheres. Additionally, the stability of QDNB is superior to traditional fluorescein, providing QDNB-based LFIAs with a longer storage time.
However, cadmium on the surface of quantum dots is prone to chelation with sulfhydryl groups in proteins, especially in aqueous environments. Avoiding the use of high concentrations of protein is critical for the stability of QDNB-based LFIAs. Cadmium is toxic, and its usage is limited in some situations, necessitating the preparation of cadmium-free quantum dots with high quantum yields and stability.
In summary, a versatile method for preparing quantum dot nanobeads in one step is provided, and it is scalable for industrial production. Using the as-prepared QDNBs, fluorescent lateral flow immunoassay strips were created for the qualitative and quantitative detection of disease biomarkers. The ideal QDNB for LFIA is a small-sized nanobead encapsulated with numerous QDs without fluorescence quenching.
The authors have nothing to disclose.
This work was supported by the Project of Shanghai Science and Technology Committee (STCSM) (22S31902000) and the Clinical Research Incubation Program of Shanghai Skin Disease Hospital (NO. lcfy2021-10).
(dimethylamino)propyl)-N’-ethylcarbodiimide hydrochloride | Sigma-Aldrich | 03450 | |
Absorbance paper | Kinbio Biotech | CH37K | |
Bovine serum albumin | Sigma-Aldrich | B2064 | |
Casein | Sigma-Aldrich | C8654 | |
CdSe/ZnS quantum dot | Suzhou Mesolight Inc. | CdSe/ZnS-625 | |
Choloroform | Sino Pharm | 10006818 | |
CRP antibody | Hytest Biotech | 4C28 | |
Fluorescent lateral flow assay reader | Suzhou Helmence Precision Instrument | FIC-H1 | |
Glass fiber pad | Kinbio Biotech | SB06 | |
Goat anti-rabbit IgG | Sangon Biotech | D111018 | |
Nitrocellulose membrane | Satorious | CN140 | |
Poly(styrene-maleic anhydride) copolymer | Sigma-Aldrich | S458066 | |
Rabbit IgG | Sangon Biotech | D110502 | |
Sodium dodecyl sulfate | Sino Pharm | 30166428 | |
Sodium hydroxide | Sino Pharm | 10019718 |