Elsevier

Biosensors and Bioelectronics

Volume 89, Part 2, 15 March 2017, Pages 998-1005
Biosensors and Bioelectronics

Versatility of a localized surface plasmon resonance-based gold nanoparticle-alloyed quantum dot nanobiosensor for immunofluorescence detection of viruses

https://doi.org/10.1016/j.bios.2016.10.045Get rights and content

Highlights

  • An ultrasensitive, rapid and specific immunofluorescence nanobiosensor was developed.

  • This nanobiosensor was based on a gold nanoparticle-induced quantum dot fluorescence signal.

  • Antibody-conjugated gold nanoparticle and QDs induces localized surface plasmon resonance signal.

  • This fluorescence signal was proportional to the concentration of the target virus.

  • The versatility of the biosensor was demonstrated for the detection of norovirus-like particles.

Abstract

Flu infection, caused by the influenza virus, constitutes a serious threat to human lives worldwide. A rapid, sensitive and specific diagnosis is urgently needed for point-of-care treatment and to control the rapid spread of this disease. In this study, an ultrasensitive, rapid and specific localized surface plasmon resonance (LSPR)-induced immunofluorescence nanobiosensor has been developed for the influenza virus based on a gold nanoparticle (AuNP)-induced quantum dot (QD) fluorescence signal. Alloyed quaternary CdSeTeS QDs were synthesized via the hot-injection organometallic route and were subsequently capped with l-cysteine via a ligand exchange reaction. AuNPs were synthesized in HEPES buffer and thiolated with l-cysteine. The concept of the biosensor involves the conjugation of anti-neuraminidase (NA) antibody (anti-NA Ab) to thiolated AuNPs and the conjugation of anti-hemagglutinin (HA) antibody (anti-HA Ab) to alloyed quaternary l-cysteine-capped CdSeTeS QDs. Interaction of the antigens displaying on the surface of the influenza virus target with anti-NA Ab-conjugated AuNPs and anti-HA Ab-conjugated QDs induces an LSPR signal from adjacent AuNPs to trigger fluorescence-enhancement changes in the QDs in proportion to the concentration of the target virus. The detection limit for influenza H1N1 virus was 0.03 pg/mL in deionized water and 0.4 pg/mL in human serum; while, for the clinically isolated H3N2, the detection limit was 10 PFU/mL. The detection of influenza virus H1N1 was accomplished with high sensitivity. The versatility of the biosensor was demonstrated for the detection of clinically isolated influenza virus H3N2 and norovirus-like particles (NoV-LPs).

Introduction

Influenza viruses are classified into three genera types, namely influenza virus A, B and C. Among them, influenza virus A is the most common because it carries the infectious human pathogen and because it is highly diversified and easily mutated (Christman et al., 2011; Hay et al., 2001; Leung et al., 2014). The pandemic influenza virus A H1N1 that occurred in 2009 caused an outbreak of respiratory infection that emanated from Mexico and spread globally to 191 countries (Girarda et al., 2010; Özyer et al., 2011). This outbreak induced a worldwide challenge for health experts and researchers, with the primary aim of developing efficient ways to prevent the rapid spread of this disease (Bimbo et al., 2013; Grabowska et al., 2013).

Conventional diagnostic systems used for the influenza virus have certain limitations that have inspired the continuous development of efficient probes that are capable of meeting the demand for high sensitivity, selectivity and rapidity. Antibody (Ab) detection of the virus based on serological analysis can lead to vague and misguided data interpretation (Centers for Disease Control and Prevention). The commercial rapid influenza detection test (RIDTS) is prone to false-positive and false-negative results (Landry, 2011). Viral culture analysis is time consuming (Treanor, 2005), while existing immunofluorescence assays are cheap but limited in their sensitivity (Lee et al., 2015; Pianciola et al., 2010). Hence, there is a continued demand for the development of rapid, highly sensitive and selective diagnostic probes for the influenza virus.

Fluorescent semiconductor quantum dot (QD) nanocrystals have been widely used as fluorescence reporters in various biosensor designs (Ahmed et al., 2014a, Ahmed et al., 2014b; Lee et al., 2015; Tian et al., 2012) due to their unique optical properties such as their broad absorption and narrow emission spectra, multiplex detection capabilities, size-tunable emission spectra, biocompatibility potential and high photostability (Anderson and Chan, 2008; Bruchez et al., 1998; Chan and Nie, 1998). Recent developments on the applications of QDs in biosensor design have shown that alloyed QD nanocrystals generate higher output efficiencies than conventional binary QD systems (Adegoke et al., 2016a, Adegoke et al., 2016b). It is therefore reasonable to believe that the combination of a metallic nanoparticle with surface plasmon properties could be used to enhance the fluorescence of adjacent QD fluorophores to improve bio-detection sensitivity.

Surface plasmon resonance (SPR) biosensors have been widely used in the fields of biotechnology, biochemistry and bioengineering as diagnostic probes for infectious diseases, ion sensing, protein-DNA interactions, binding events and biological surface modifications (Campbell and Kim, 2007; Eum et al., 2003; Jeong et al., 2008; Lee et al., 2015; Patskovsky et al., 2008; Yeom et al., 2013). Moreover, localized surface plasmon resonance (LSPR) biosensor systems adapted from SPR have drawn active interest in the research community.

In this paper, we report on the rapid and ultrasensitive detection of influenza virus H1N1 using an immunofluorescence biosensor based on a combination of LSPR-induced optical transduction from AuNP-labeled anti-NA Ab and the fluorescence signal generated from adjacent alloyed CdSeTeS QD-labeled anti-HA Ab. The mechanism of the biosensor involves an antigen-antibody interaction in which LSPR induced from AuNPs is used to enhance the fluorescence of adjacent alloyed QDs for the bio-detection of influenza virus. The immunofluorescence biosensor developed in this work is rapid, ultrasensitive and specific. Further analyses demonstrated that the immunofluorescence biosensor developed in this work is also applicable to influenza virus H3N2 and norovirus-like particles (NoV-LPs).

Section snippets

Materials

HEPES buffer, PBS buffer, polyoxyethylene (20) sorbitan monolaurate (Tween 20), sodium acetate, hydrogen peroxide, sulfuric acid, methanol, potassium hydroxide (KOH), chloroform and acetone were purchased from Wako Pure Chemical Ind. Ltd. (Osaka, Japan). HAuCl₄, N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), bovine serum albumin (BSA), 1-octadecene, cadmium oxide (CdO), tellurium (Te), l-cysteine, hexadecylamine (HDA), trioctylphosphine oxide

Characterization of alloyed l-cysteine-capped CdSeTeS QDs

The hydrodynamic particle size distribution of the QDs was determined using DLS. DLS provides three basic pieces of information by (i) probing the hydrodynamic particle size distribution of the nanocrystals in solution, (ii) evaluating the monodispersity of the nanocrystals and (iii) verifying the agglomeration state of the nanocrystals. As shown in Fig. S-3A, Supplementary information, the DLS peak of the QDs shows that they are monodisperse. The hydrodynamic particle size of the QDs was

Conclusion

An LSPR-induced immunofluorescence nanobiosensor for influenza virus has been developed with high sensitivity, rapidity and specificity. In the biosensor, LSPR from adjacent AuNPs can trigger the fluorescence enhancement of QDs based on an antibody-antigen interaction when in direct contact with the target influenza virus. Successful detection of the influenza virus in water and in human serum was accomplished. The versatility of the biosensor was successfully applied for the detection of

Acknowledgements

OA thanks the Japan Society for the Promotion of Science (JSPS) for a postdoctoral fellowship (No. 26-04354). This work was supported by the Suzuken Memorial foundation, Japan, and partly by the Bilateral Joint Research Project of the Japan Society for the Promotion of Science, Japan.

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