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,

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 synth.......

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,.......

Materials

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