Development Of Magnetic Enrichment Fluorescence Lateral Flow Immunoassay

Lateral flow immunoassay(LFIA),one of the important means for real-time biomarkers detection,is widely used in the detection of infectious disease pathogens,cardiovascular and tumor markers,due to the advantages of low cost,simple operation and low time consuming.However,the colloidal gold-based colorimetric tests have poor sensitivity and lack the ability of quantitative and multi-channel detection.Quantum dots(QDs)are semiconductor nanomaterials with the size of 2～20nm,which have the following characteristics: photobleaching resistance,stable property,high quantum efficiency.With wide absorption spectrum and narrow emission spectrum,quantum dots of various wavelengths can be excited by a single light source,which has the capability of multi-channel detection.Small-sized QDs exhibit limited signal intensity and difficulty in modification and purification.Thus,this research designed and synthesized the magnetic-quantum dot nanobeads(MQBs)with superparamagnetism and high luminescence to address these above-mentioned application bottlenecks.The antibody-conjugated MQBs can recognize and enrich the biomarks from complex matrix sample,and can be used as the signal probe in LFIA to realize highly sensitive,multi-channel and quantitative real-time detection,which provides a rapid and efficient solution for field detection.In this work,the magnetic fluorescence LFIA,combining nanomaterials,LFIA and signal readout divice,was applied to the detection of tumor markers in serum and respiratory viruses in throat swabs,which improves sensitivity of immunochromatography in handling complex samples.The main research contents are as follows:(1)Synthesis of magnetic-quantum dot nanobeads.Magnetic-quantum dot nanobeads were synthesized by polyetherimide-mediated method,and numerous QDs were densely adsorbed on the magnetic core by electrostatic adsorption.The magnetic core inside the nanostructure had a high saturation magnetization of 83.7 emu/g.Hundreds of quantum dots can be adsorbed on the surface of one magnetic core.Based on theoretical modeling analysis,it was estimated that more than 700 quantum dots can be adsorbed outside a 200-nm-diameter magnetic core,and the experimental fluorescence intensity of MQBs was 114 times that of quantum dots.(2)Magnetic enrichment and lateral flow detection of prostate cancer markers in serum.Prostate-specific antigen(PSA)is a specific biomarker for the diagnosis of prostate cancer.Simultaneous detection of free and complexed prostate-specific antigen(f-PSA and c-PSA)is critical to the prostate cancer diagnostic accuracy.The specific antibodies to c-PSA and f-PSA were conjugated with red and green MQBs,respectively,and the t-PSA antibodies were sprayed on the test line of nitrocellulose membrane.The dual-color antibody-conjugated MQBs can bind with c-PSA and f-PSA to form immunocomplexes,and were captured by the t-PSA antibody.After removing the ambient light by a dual-bandpass filter,their fluorescence signals were recorded by the CMOS image sensor of a smartphone.Then,the concentrations of c-PSA and f-PSA were calculated by in-house algorithm.This assay can simultaneously detect f-PSA and c-PSA with the limits of detection of 0.009 ng/m L and 0.087 ng/m L,respectively.Clinical serum samples of prostate cancer and benign prostatic hyperplasia patients were evaluated to confirm the clinical feasibility.(3)Magnetic enrichment and lateral flow detection influenza A virus in nasopharyngeal swabs.The clinical samples,such as nasopharyngeal or throat swabs,have various components which may lead to the nonspecific adsorption of nanoparticle labels when detecting influenza A viruses in nasopharyngeal using LFIA.Herein,we demonstrated a MQBs based LFIA for magnetic enrichment and fluorescent detection of respiratory virus virions in clinical specimens.The as-synthesized MQBs were conjugated with influenza A virus-specific antibody for efficiently immune capture and magnetic enrichment of respiratory virus virions from complex biological matrix,which also served as highly bright fluorescent probes in lateral flow strips.This assay can achieve quantitative detection of influenza A virus virions with a low limit of detection down to22 pfu/m Lwithin 35 minutes.Furthermore,the presented platform was able to directly detect influenza A virus virions spiked in nasopharyngeal swab dilution,indicating its stability and feasibility in clinical applications.