Femtogram Level Sensitivity achieved by Surface Engineered Silica Nanoparticles in the Early Detection of HIV Infection

L A Avinash Chunduri,Indira K Hewlett,Aditya Kurdekar,Eswarappa Pradeep Bulagonda,Venkataramaniah Kamisetti,Mohan Kumar Haleyurgirisetty

doi:10.1038/s41598-017-07299-1

Abstract

We have engineered streptavidin labelled Europium doped fluorescent silica nanoparticles which significantly increased sensitivity without compromising the specificity of the immunoassay. As a proof of concept, a time resolved fluorescence based sandwich immunoassay was developed to detect HIV-1 p24 antigen in clinical specimens. The detection range of the silica nanoparticle based immunoassay (SNIA) was found to be between 0.02 to 500 pg/mL in a linear dose dependent manner. SNIA offers 1000 fold enhancement over conventional colorimetric ELISA. Testing of plasma samples that were HIV negative showed no false positive results in the detection of HIV-1 p24 antigen. This highly sensitive p24 assay can help improve blood safety by reducing the antibody negative window period in blood donors in resource limited settings where nucleic acid testing is not practical or feasible. This technology can also be easily transferred to a lab-on-a-chip platform for use in resource limited settings and can also be easily adopted for the detection of other antigens.

Highlights

Nanotechnology could potentially provide a new generation of assays for rapid, highly sensitive and multiplexed detection of pathogens in a miniaturized format
The problem of precipitation of rare earths as hydroxides in a basic pH environment was overcome in the present synthesis of europium doped silica nanoparticles and no sponge like precipitates were observed confirming the optimum concentration of europium for the formation of spherical nanoparticles
SNIA is a linear dose dependent assay. We report this fluorescent silica nanoparticle based immunoassay can achieve an analytical sensitivity of femtogram level, which is approximately thousand times higher in comparison with only picogram level sensitivity achieved with conventional colorimetric ELISA17

Summary

Introduction

Nanotechnology could potentially provide a new generation of assays for rapid, highly sensitive and multiplexed detection of pathogens in a miniaturized format. Well known surface functionalization methods, low cytotoxicity and reliable scale up procedures are the attracting features for various in vitro and in vivo applications[8] Various fluorescent nanoparticles such as lanthanide doped nanoparticles, metal-based dyes, dye impregnated nano and microparticles, luminescent nanoparticles, semiconductor quantum dots, and noble metal nanoclusters have been exploited for various biological applications[9]. Lower detection limits of 1 pg/mL have been achieved for p24 antigen using the boosted ELISA17 Another sensitive method, real-time immuno-Polymerase Chain Reaction (immuno-PCR), can detect 1000 HIV-1 RNA copies, or 40 attogram of HIV-1 p24 antigen, per reaction[18]. Real-time immuno-Polymerase Chain Reaction (immuno-PCR), can detect 1000 HIV-1 RNA copies, or 40 attogram of HIV-1 p24 antigen, per reaction[18] All these improvements of detection sensitivity increase the complexity of testing which makes their deployment in Resource Limited Settings (RLS) infeasible. The development of rapid, portable and accurate Point-of-Care (POC) clinical diagnostics could lower transmission rates, especially in cases of recent HIV infection in resource limited settings by providing timely access to intervention[19]

Methods

Results

Conclusion