A rapid triple-mode fluorescence switch assay for immunoglobulin detection by using quantum dots-gold nanoparticles nanocomposites
Graphical abstract
The principle of the triple-mode fluorescence switch control system.
Introduction
The detection and quantification of proteins plays an essential role in fundamental research [1]. Establishing a rapid, sensitive and specific technology for protein analysis was of great importance. Advances in nanoscience and nanotechnology have resulted in various novel nanosensors, which have been conditioned for biomolecule detection both in vitro and in vivo. Among the various detection techniques, optical detection has proven to be the most convenient method of all. Quantum dots (QDs) have been favorably adopted in fluorescence switch-based studies because of the large Stokes shift, high quantum yield, good photo stability, and size-dependent maximum emission wavelength tunability [2], [3]. Among fluorescence analyses, fluorescence resonance energy transfer (FRET) systems possess high sensitivity and selectivity. It is a powerful technique for probing small changes in the distance between donor and acceptor fluorophores. With FRET technology, the fluorescence switch can be more sensitive [4], [5]. The emission spectrum of QDs can be adjusted by controlling the synthesis conditions of QDs in the range wavelength of the absorption spectrum of the acceptor. With these good optical characteristics, QDs have been favorably adopted in FRET-based studies for biological analyses. Fluorescence switch combines QDs and Au nanoparticles (AuNPs) perfectly. As an excellent fluorescent quencher, AuNPs open new perspectives in FRET systems owing to their high extinction coefficients and broad absorption spectrum within the visible light range that overlaps with the emission wavelengths of common energy donors [6], [7]. Thus, functionalized QDs and AuNPs could be used to establish fluorescence switch.
The immunoglobulins, most of them are glycoprotein, play an important role on humoral immune. It falls into several classes, such as IgA, IgE, IgM, each with its own functional characteristics. Chicken egg yolk immunoglobulin (IgY), the only antibody existing in chicken egg yolk, has many significant advantages over mammalian immunoglobulin [8]. It has been touted to be a superior alternative to mammalian antibodies for use in various immunological, molecular biology and proteomics applications for several reasons [9]. Presently, IgY has been used extensively for prevention and treatment of various infections in animals and humans with mixed success [10], [11]. Many reports have described its ability to inhibit corresponding antigen bacteria. So it has potential use for food preservation [12]. What is more, IgY can be used as immunological supplements in infant formula and other food. Hence, the determination of IgY is necessary and of great significance.
The current study of IgY mainly concentrated in the purification, and immune titer research [13]. There are also some semiquantitative detection, such as enzyme-linked immunoassay [14], high performance liquid chromatography method [8], electrochemical method [15], lowry assays [16], radial immunodiffusion analysis [17]. However, until now, there are few reports of specific and quantitative detection of IgY. Thus, an efficient and rapid method for the quantitative determination of IgY is still needed. Rabbit anti chicken IgY antibodies can be obtained in rabbit serum through immune rabbits by chicken IgY, which is a subtype of IgG. Staphylococcal protein A (SPA) is a type I membrane protein from the bacterium Staphylococcus aureus. It has found use in biochemical research because of its ability to bind immunoglobulins. In the N-terminal half of the protein is its IgG-binding domains. SPA can bind to IgG in its Fc fragment. However, it does not combine with IgY [18].
In this study, we investigated the applicability of a QDs–AuNPs nanohybrid for an IgY triple-mode sensor utilizing spectroscopic and FRET techniques. Two portions of this immunosensor, namely, QDs-SPA and anti-IgY-AuNPs, are first synthesized. As a light-absorbing material, CdTe QDs provide a “turn on” state in fluorescence measurement. This bright fluorescence is quenched in the presence of anti-IgY-AuNPs due to the immunoreactions between SPA and anti-IgY and FRET (“turn off” state). After the addition of IgY to the QDs-SPA-anti-IgY-AuNPs system, anti-IgY-AuNPs become detached from the QDs-SPA surface and attached to IgY because of competitive immunoreactions, creating a “turn on” state. The whole assay process does not require multiple time-consuming incubation, separation, and washing steps and can be rapidly accomplished within a very short time. These triple-mode fluorescence switch studies will create new opportunities for engineering optically based advance switches for sensing bimolecular.
Section snippets
Reagents and chemicals
All chemicals used were analytical grade. IgY and anti-IgY was purchased from cellwaylab (China). SPA was purchased from BioVision (USA). Avidin was purchased from Sigma-Aldrich (USA). HAuCl4 was purchased from the Shanghai Chemical Reagent Company (Shanghai, China). Salts (Na+, K+), NaOH, glucose, lactose, sodium citrate, Na2HPO4, NaH2PO4, CdCl2, NaBH4, thioglycolic acid (TGA), N-hydroxysuccinimide (NHS), and vitamin C were acquired from the Sinopharm Chemical Reagent Co., Ltd. (Shanghai,
The TEM images of AuNPs and QDs
The TEM images of the obtained AuNPs and QDs is shown in Fig. 1. It showed that the obtained AuNPs (Fig. 1A) were almost spherical morphology and homogeneously distributed in the solution. The relatively mean diameter of AuNPs was 11 nm. The results were similar with the former reference [24]. The shape of QDs (Fig. 1B) was close to spherical, crystalline, sufficiently monodisperse and well separated. The relatively mean diameter of QDs was 2.5 nm. These sizes of AuNPs and QDs we used can
Conclusions
This triple-mode fluorescence nanosensor switch method based on specific interactions made IgY detection using QDs-SPA and anti-IgY-AuNPs more sensitive, rapid and selective. In the established fluorescence switch system, IgY in samples and QDs-SPA would combine with AuNPs-Anti-IgY competitively, thus resulting in the fluorescence intensity recovery from quenching QDs. Under optimal conditions, a linear calibration equation was obtained, with a detection limit of 1.16 ng/mL. The triple-mode
Acknowledgements
This research was supported by the National Natural Science Foundation of China (No.31371810) and the Fundamental Research Funds for the Central Universities (2662015PY080).
Qi Wang received her bachelor in food science and engineering from Huazhong Agriculture University in 2014. She is pursuing her MS in food engineering from Huazhong Agriculture University. She is currently studying in the area of food chemistry.
References (29)
- et al.
Chemical reagents as probes: application to fish protein gels and detection of a cysteine TGase in hake
LWT Food Sci. Technol.
(2011) - et al.
Nitrogen-doped carbon quantum dots: facile synthesis and application as a turn-off fluorescent probe for detection of Hg2+ ions
Biosens. Bioelectron.
(2014) - et al.
Dual optical Hg2+ selective sensing through FRET system of fluorescein and rhodamine B fluorophores
J. Photochem. Photobiol. A
(2014) - et al.
A rapid fluorescence switch-on assay for glutathione detection by using carbon dots-MnO2 nanocomposites
Biosens. Bioelectron.
(2015) - et al.
Fluorescence resonance energy transfer in doubly-quantum dot labeled IgG system
Talanta
(2005) - et al.
A simple method for isolating chicken egg yolk immunoglobulin using effective delipidation solution and ammonium sulfate
Poultry Sci.
(2015) - et al.
A novel, cost-effective and efficient chicken egg IgY purification procedure
J. Immunol. Methods
(2012) - et al.
A new membrane based process to isolate immunoglobulin from chicken egg yolk
Food Chem.
(2010) - et al.
Passive immunity for protection against mucosal infections and vaccination for dental caries
Mucosal Vaccines
(1996) - et al.
Electrochemical immunosensor based on ensemble of nanoelectrodes for immunoglobulin IgY detection: application to identify hen’s egg yolk in tempera paintings
Biosens. Bioelectron.
(2014)
Affinity purification of immunoglobulins from chicken egg yolk using a new synthetic ligand
J. Chromatogr. B
Determination of lysozyme at the nanogram level in food sample using Resonance Rayleigh-scattering method with Au nanoparticles as probe
Spectrochim. Acta A
Water-soluble multicolored fluorescent CdTe quantum dots: synthesis and application for fingerprint developing
J. Colloid Interface Sci.
A simple quantum dot-based fluoroimmunoassay method for selective capturing and rapid detection of Salmonella Enteritidis on eggs
Food Control
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2017, Journal of Pharmaceutical and Biomedical AnalysisCitation Excerpt :As the binding ability of SPA and antibody is very strong, reversible and has no effect on the activity of the antibody, it can be used as chromatographic column filling material for antibody purification [21]. SPA is susceptible to binding with fluorescein, horseradish peroxidase and QDs, which can be applied for antigen detection and disease diagnosis instead of the traditional secondary antibody [22]. In our previous study, B-cell epitopes of Brucella outer membrane protein (OMP) 16, OMP2b, OMP31 and BP26 were predicted and connected, and a multi-epitope fusion protein (rOMP) was expressed.
Qi Wang received her bachelor in food science and engineering from Huazhong Agriculture University in 2014. She is pursuing her MS in food engineering from Huazhong Agriculture University. She is currently studying in the area of food chemistry.
Xuan Fu received his bachelor in food science and engineering from Wu Han Polytechnic University in 2015. He is pursuing his MS in food science from Huazhong Agriculture University. He is currently studying in the area of fluorescence biosensors based on nanomaterial.
Xi Huang received her PhD in college of chemistry from Central China Normal University in 2009. She is currently working at Food Science and Technology College, Huazhong Agriculture University, as an associate professor. She is currently working toward the activity and function of egg proteins.
Fangyi Wu received her bachelor in food science and engineering from Huazhong Agriculture University in 2016. She is currently studying in the area of food chemistry.
Meihu Ma received his PhD from Hunan Agriculture University, ChangSha, in 2005. He is currently working at Food Science and Technology College, Huazhong Agriculture University, as a professor. His research is focused on animal product processing and quality testing.
Zhaoxia Cai received her PhD in analytical chemistry from Nankai University in 2007. She is currently working at Food Science and Technology College, Huazhong Agriculture University, as an associate professor. She studied in the area of light analytical chemistry. She is currently working toward fluorescence biosensors based on nanomaterial.