Fluorescent Lateral Flow Immunoassay Based on Quantum Dots Nanobeads

Summary

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.

Abstract

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.

Introduction

Lateral flow immunoassay (LFIA) strips serve as crucial rapid detection tools at point-of-care^1,2, 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 latex^4,5, upconversion fluorescent nanoparticles⁶, time-resolved fluorescent microspheres^7,8, and quantum dots^9,10,11. 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-assembly^{14,15,16,17,18}, the swelling method^19,20, and silica microsphere^21,22,23,24 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 detection^28,29,30, infectious disease biomarker detection^31,32, 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.

Protocol

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.

1. Preparation of oil phase and aqueous phase solutions
   1. Prepare 1 mL of oil phase: 0.4 mL of poly (styrene-co-maleic anhydride) (PSMA) solution (50 mg/mL), 0.1 mL of QDs solution (100 mg/mL), fill any volume less than 1 mL with chloroform.
   2. Prepare 5 mL of aqueous phase: SDS aqueous solution (0.5 wt %).
2. Preparation of emulsion and solidification
   1. Add 1 mL of oil phase and 5 mL of aqueous phase to a vial (15 mL), and place the vial on a magnetic stirrer. Turn on the power of the magnetic stirrer.
   2. After the oil and aqueous phases are completely mixed, immediately transfer the flask to the programmed (60 s, a 3 s pulse followed by a 3 s pause, 50% amplitude) ultrasonication and turn on the power.
   3. Observe the solution after ultrasonication. The initially crimson-colored solution transitioned to an opalescent phase that still retains a reddish undertone.
   4. Place the vial back on the magnetic stirrer at room temperature, continuously stirring overnight. The chloroform was gradually evaporated, and the emulsion solidified into composite nanobeads.
3. Surface modification of nanobeads
   1. Add 0.1 mL of 0.1 mM sodium hydroxide to the nanobeads suspension for 1 h. The anhydride groups on the surface of nanobeads get hydrolyzed into carboxyl groups.
4. Purification of nanobeads
   1. Turn off the magnetic stirrer and move the solidified nanobeads to a centrifuge tube. Centrifuge the tube at 13523 x g for 10 min at room temperature, and discard the supernatant.
   2. Add the initial volume of deionized water to the precipitation, cover the tube with its lid, submerge it into the ultrasonic water bath, and maintain immersion until the precipitate is fully dispersed.
   3. Repeat steps 1.4.1-1.4.2 twice, and finally, transfer the nanobeads to the glass bottle. Store it at 4 °C until use.
5. Characterization of nanobeads
   1. Characterize the size and morphology of the QDs nanobeads using transmission electronic microscopy (TEM) imaging²².
      NOTE: Different sizes of nanobeads can be obtained by changing the proportion of QDs solution and PSMA solution. The size of the QDs nanobeads can also be determined through dynamic light scattering (DLS)²⁷.

2. Preparation of QDNB-antibody conjugates

1. Activation of carboxyl groups
   1. Add 100 µL of prepared QDNB (10 mg/mL) in 200 µL of phosphate buffer (PB, 20 mM, pH 6.0), then add 6 µL of EDC (20 mg/mL).
   2. Incubate the mixture for 30 min at room temperature on a rotary mixer.
   3. After the incubation, centrifuge the mixture at 13523 x g for 10 min at room temperature and discard the supernatant.
   4. Add 100 µL of PB (20 mM, pH 6.0) to the precipitation, cover the tube with its lid, and immerse the tube into the operational ultrasonic water bath until the precipitate is fully dispersed.
2. Conjugation of antibody and nanobeads
   1. Disperse 100 µg antibody in 200 µL of PB (20 mM, pH 6.0) and add the activated QDNB suspension obtained from step 2.1 into the antibody solution.
   2. Incubate the mixture at room temperature for 30 min with continuous rotation. Remove excess antibodies by centrifugation at 6010 x g for 5 min (at room temperature), and then carefully discard the supernatant.
3. Blocking of nanobeads
   1. Add 200 µL of 1% casein solution (in 20 mM PB buffer, pH 7.4) to the residue (obtained in step 2.2.2), cover the tube with its lid, and immerse it into the operational ultrasonic water bath until the residue is fully dispersed.
   2. Incubate the mixture at room temperature for 2 h with continuous rotation.
      NOTE: The objective of this step is to block any unreacted sites on the surface of QDNB.
4. Storage of conjugates
   1. Add 100 µL of preservative solution (20 mM PB buffer, pH 7.4, 1% BSA, 5% trehalose, 6% sucrose, 0.1% S9, pH 7.4) to the mixture and store at 4 °C for subsequent use.

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.

1. Preparation of NC membrane
   1. Prepare test line (T-line) and control line (C-line) coating solution: dilute the CRP capture antibody and goat anti-rabbit IgG with PB to a final concentration of 1 mg/mL.
   2. Place the PVC board properly, and remove the protective paper located 25 mm from the edge of the PVC board. Align and adhere the NC membrane along the lower edge of the remaining protective paper to the PVC board.
   3. Dispense T-line and C-line coating solutions onto the NC membrane at a jetting rate of 1 µL/cm, ensuring it is placed 20 mm apart from the upper edge, forming a test line (1 mm wide).
   4. Place the NC membrane in conditions with a humidity of less than 30% and a temperature range of 18-26 °C for drying. The drying time should be at least 24 h.
      NOTE: Ensure the top and bottom edges of the NC membrane are flush with the corresponding edges of the adjacent areas. NC membranes should not be touched with hands. The humidity during this step should be controlled within the range of 45% to 65%. Before proceeding, the T-line coating solution, C-line coating solution, and NC membrane should be equilibrated to the same temperature.
2. Preparation of the conjugate pad
   1. Submerge the conjugate pad into the conjugate pad treatment solution (20 mM PB buffer, pH 7.4, 1%BSA).
   2. Place the soaked conjugate pad into a drying oven for drying treatment (room temperature) overnight.
   3. Gently adhere the modified conjugate pad onto the PVC board.
   4. Dilute the prepared QDNB-antibody conjugate solution with the preservative solution (20 mM PB buffer, pH 7.4, 1% BSA, 5% trehalose, 6% sucrose, 0.1% S9, pH 7.4).
   5. Dispense the QDNB-antibody conjugate solution onto the conjugate pad at a jetting rate of 1 µL/cm and dry the conjugate pad for 24 h.
      NOTE: The specific dilution ratio should be adjusted according to the detection target.
3. Preparation of sample pad
   1. Submerge the sample pad into the sample pad treatment solution (20 mM PB buffer, pH 7.4, 1% BSA, 5% trehalose, 6% sucrose, 0.1% S9, pH 7.4).
   2. Place the soaked sample pad into a drying oven (45 °C) and dry for 24 h.
4. Assembling the card and cutting it into strips
   1. As shown in Figure 1, successively place the absorbent paper (absorbent pad), conjugate pad, and sample pad on the PVC board with NC membrane.
   2. Cut the card into 3.8 mm width strips.
5. Packaging of the strips
   1. Encase the test strips in a plastic cassette and seal an aluminum foil pouch that contains a desiccant.

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.

1. Pipette 10 µL of plasma sample or calibrator solution into 1 mL of a PBS buffer containing 0.1% Tween 20. Then, transfer 100 µL of the prepared diluted sample onto the sample pad of the test strip.
2. After a 15 min run, observe the fluorescence signals of the T line and C line under ultraviolet lightexposure to obtain qualitative results.
3. Insert the strip card into a fluorescent lateral flow assay reader to obtain the fluorescence intensities for quantitative analysis.
   NOTE: In this example, the sandwich immunoassay method is employed^35,36; one red line (control line) indicates a negative result, while the two red lines (test and control lines) indicate a positive result. The absence of the control line indicates that the test conducted is invalid.

Results

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 software^37,38.

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.

Discussion

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 biomarkers^25,32, food toxins^29,30, viruses^16,27, 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.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
(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