A rapid triple-mode fluorescence switch assay for immunoglobulin detection by using quantum dots-gold nanoparticles nanocomposites

Accepted Manuscript Title: A rapid triple-mode fluorescence switch assay for immunoglobulin detection by using quantum dots-gold nanoparticles nanocomposites Author: Qi Wang Xuan Fu Xi Huang Fangyi Wu Meihu Ma Zhaoxia Cai PII: DOI: Reference:

S0925-4005(16)30367-7 http://dx.doi.org/doi:10.1016/j.snb.2016.03.073 SNB 19878

To appear in:

Sensors and Actuators B

Received date: Revised date: Accepted date:

3-2-2016 15-3-2016 17-3-2016

Please cite this article as: Qi Wang, Xuan Fu, Xi Huang, Fangyi Wu, Meihu Ma, Zhaoxia Cai, A rapid triple-mode fluorescence switch assay for immunoglobulin detection by using quantum dots-gold nanoparticles nanocomposites, Sensors and Actuators B: Chemical http://dx.doi.org/10.1016/j.snb.2016.03.073 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

A rapid triple-mode fluorescence switch assay for immunoglobulin detection by using quantum dots-gold nanoparticles nanocomposites Qi Wang, Xuan Fu, Xi Huang, Fangyi Wu, Meihu Ma, Zhaoxia Cai＊

National Research and Development Center for Egg Processing, College of Food Science and Technology, Huazhong Agricultural University, Wuhan, Hubei 430070, PR China

＊

Corresponding author.Tel.: +86 027 87283177.

E-mail address: [email protected] (Z.Cai).

Graphical Abstract

The principle of the triple-mode fluorescence switch control system.

Highlights

We herein report a triple-modenano sensor with a fluorometric readout based on CdTe quantum dots (QDs) and gold nanoparticles (AuNPs) for discriminative detection of immunoglobulin.

We used anti-IgY and staphylococcal protein A (SPA) to make the detection discriminatively to IgY. The immune interaction between QDs-SPA and anti-IgY-AuNPs leads to drastic quenching of the donor by a fluorescence resonance energy transfer (FRET) process.

After the addition of IgY, anti-IgY-AuNPs peel off from the QDs-SPA surface and bind to IgY due to competitive immunoreactions, resulting in the restoration of the fluorescence intensity of QDs.

The fluorescence intensity of the system increased linearly with increasing concentrations of IgY from 5 to 200 ng/mL with a detection limit of 1.16 ng/mL.

Abstract The immunoglobulins, most of them are glycoprotein, play an important role on humoral immune. We herein report a triple-modenano sensor with a fluorometric readout based on CdTe quantum dots (QDs) and gold nanoparticles (AuNPs) for discriminative detection of immunoglobulin. Immunoglobulins fall into several classes, each with its own functional characteristics. In this research, we use chicken egg yolk immunoglobulin (IgY) as an example to establish the specific detection methods. IgY is the only

antibody existing in chicken egg yolk, which has many significant advantages. We used anti-IgY and staphylococcal protein A (SPA) to make the detection discriminatively to IgY. The immune interaction between QDs-SPA and anti-IgY-AuNPs leads to drastic quenching (turning off) of the donor by a fluorescence resonance energy transfer (FRET) process. After the addition of IgY, anti-IgY-AuNPs peel off from the QDs-SPA surface and bind to IgY due to competitive immunoreactions, resulting in the restoration of the fluorescence intensity of QDs (turning on). Consequently, this process can be utilized for the selective detection of IgY via optical responses. This proposed assay demonstrated a favorable anti interference ability and highly selectivity toward IgY with a detection limit of 1.16 ng/mL. More importantly, the nanosensor could not only function in aqueous solution for IgY detection with high sensitivity but also exhibit sensitive responses toward IgY in complicated biological environments, demonstrating its potential in bioanalysis, which might be significant in biochemical field in the future.

Keywords: Immunoglobulin; IgY; Triple-mode nanosensor; Fluorescence resonance energy transfer; Anti-IgY; Staphylococcal Protein A

1. 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 fall 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 becomes 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. 2. Experiment 2.1 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, China). Glycine, cysteine, glutathione, aspartic acid, L-serine, tyrosine, lysozyme, and BSA were purchased from the Biosharp Company (China). PEG20000 was acquired from Beijing Solarbio Science and Technology Company (China). Te powder was

acquired

from

Delan

Fine

Chemical

Reagent

Company

(Tianjin,

China).

1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC) and 1-Thioglycerol (TG) were acquired from Aladdin Chemical Reagent Company Ltd. (China). All solutions were prepared using ultrapure water generated by an ELGA Purelab Option (UK) with an electric resistance ≥ 18.2 MΩ. IgY was prepared in a 1 mg/mL stock solution and diluted to 1 μg/mL as the working solution. Anti-IgY was prepared at 2 μg/mL. SPA was prepared at 2 μg/mL. All the solutions were stored at 4 °C. The 0.1 mg/mL HAuCl4 and 10 mg/mL sodium citrate solutions were prepared with ultrapure water. 7.65 g CdCl2 was added to 200 mL ultrapure water and take 5 mL from it diluted to 160 mL as the standby liquid. Phosphate buffered solutions (PBS) with different pH values were prepared by mixing 0.01 mol/L Na2HPO4 and 0.01 mol/L NaH2PO4, according to certain proportions. Egg products (yolk powder and whole egg powder) were purchased from the Kangde Biological Products Co. Ltd. (China). 2.2 Preparation of the AuNP solution The glass apparatus used for preparing AuNPs was immersed in aqua regia (HNO 3: HCl = 1:3, v/v) for 48 h, washed by ultrapure water several times, and then dried before use. AuNPs were prepared according to previously published methods [19, 20]. In a beaker, 100 mL of 0.1 mg/mL HAuCl4 was heated until boiling. Next, 5 mL of 10 mg/mL sodium citrate solution with the same temperature was quickly added to the beaker and the solution was stirred on a magnetic heater stirrer (SZCL-2A

magnetic stirring apparatus from Shanghai Dongxi Refrigeration Equipment CO.,

Ltd, China). The solution was boiled with stirring for 10 min. The color of the solution turned from blue-black to bright red. The cooled solution was stored at 4 °C, sealed and in the dark. The concentration of the above AuNP solution was 47.8 μg/mL. A JEM-2100 transmission electron microscope (TEM) (JEOL Ltd, Japan) was used to observe the formation of AuNPs and to measure their diameters. AuNPs were diluted to suitable concentrations and ultrasonically dispersed for 10 min. One to two drops were placed on a copper grid. After water volatilization, the copper grid was placed on the rod sample for TEM detection. The acceleration voltage was 200 KV.

2.3 Conjugation of AuNPs with anti-IgY To prepare the anti-IgY-AuNPs nanoprobe, 1 mL of 2 μg/mL anti-IgY was added to 10 mL of 47.8 μg/mL AuNPs with magnetic stirring at pH 7.5 (PHS-3C pH meter from Shanghai Precision & Scientific Instrument CO., Ltd, China). After stirring for 10 min, 40 μL of 3% PEG-20000 was added as a stabilizer. Next, the mixture was stirred for 15 min and stored at 4 °C. 2.4 Synthesis of CdTe QDs and conjugation of CdTe QDs with SPA Referring to the existing method [21, 22], highly fluorescent CdTe QDs were synthesized in aqueous solution. The oxygen-free NaHTe solutions were prepared by stirring a mixture of 36.4 mg of NaBH4, 26.7 mg of Te powder, and 1.5 mL of ultrapure water at 37 °C until the black Te powder disappeared (AR-2140 analytical balance from Mettler Toledo instrument CO., Ltd, Switzerland). The freshly prepared oxygen-free NaHTe solution reacted with a mixture of 160 mL CdCl2 standby liquid, 140 μL of TG, and 27.5 μL of TGA (pH 11.2 adjusted with 1 mol/L NaOH) at 97 °C for 1 h. TEM images were obtained with an accelerating voltage of 200 kV. TEM samples were prepared by dropping the aqueous solution of CdTe QDs on 800-mesh carbon-coated copper grids. The water-soluble CdTe QDs were activated by using carbodiimide chemistry for conjugation with SPA. In particular, 3 mL of QDs solution was put into the mixed solutions containing 1 mL of PBS (0.01 mol/L, pH 7.4) and 200 μL of 4 mg/mL EDC. The mixture was placed under gentle stirring at room temperature for 5 min. Approximately 20 min were required to activate free carboxylic acid groups on the QDs fully [23]. Next, 200 μL of 0.15 mg/mL sulfo-NHS was added to the mixed solutions, and the mixture was stirred for another 20 min. Thereafter, 0.5 mL of 0.002 mg/mL SPA were added into the system and reacted at 37 °C for 2 h in the dark. In this step, CdTe QDs and SPA were conjugated through strong covalent bonds. The final bioconjugated CdTe QDs were collected and kept at 4 °C. 2.5 Detection of IgY In a 10 mL tube, 0.5 mL of QDs-SPA solution, 2 mL of AuNPs-anti-IgY solution, 0.5 mL of 0.01 mol/L PBS (pH 7.4), and various amounts of IgY (5 to 200 ng/mL) or real samples were added. The mixture was diluted to 5 mL with ultrapure water, mixed thoroughly, and reacted for 80 min at 37 °C.

The concentration of IgY in the food sample solutions was diluted until it reached the appropriate the linear range of the calibration curve established in this study. The absorption spectra of AuNPs were obtained using a Nanodrop 2000c spectrometer (Thermo, USA). An RF-5301 spectrofluorometer (Shimadzu, Japan) was used to record fluorescence spectra and to measure the fluorescence intensity. The excitation wavelength was 330 nm. The excitation and emission slit width were 5.0 nm. The fluorescence intensity was recorded as F 0 and F respectively in the absence and in the presence of IgY. The change in fluorescence intensity (ΔF) was obtained by subtracting the fluorescence intensity of the binding product from that of the blank. ΔF and the concentration of IgY were used for quantitative detection of IgY. 2.6 Preparation of real sample We selected yolk powder and whole egg powder as real samples to detect the IgY concentration because they are frequently used as raw materials in food processing. The egg products were pretreated by a separation process. The powder samples (178.5 mg) were added to 10 mL of ultrapure water. These mixtures were centrifuged at 5000 rpm for 15 min at 4 °C to deplete the high-abundance proteins, using a 3-30N bench type cryogenic centrifuge (sigma, Germany). The supernatant was collected and diluted as food samples for future detection. 3. Results and Discussion 3.1 The TEM image of AuNPs and QDs The TEM image 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 establish the FRET system appropriately. 3.2 The mechanism of the triple-mode nanosensor fluorescence switching The principle of the fluorescence switch control system is shown in scheme 1. QDs can

conjugate with SPA under the coupling effect of EDC and NHS. QDs-SPA showed triple-mode fluorescent responses from monomer emission to aggregation-induced emission over the IgY concentration within the scope of the appropriate changes. QDs-SPA indeed behaved as a strong fluorescence emitter, with the monomer emission peak at 525 nm under excitation wavelength at 330 nm. When there is QDs-SPA monomer, it provides a “turn on” state. Under suitable pH and temperature conditions, QDs-SPA and anti-IgY-AuNPs assembled a QDs-SPA-anti-IgY-AuNPs immunocomplex, with the help of immune recognition between SPA and anti-IgY. Thus, AuNPs and QDs were close to each other, and the distance between them is less than 10 nm. One of the necessary conditions for FRET is that the distance between donor and acceptor is less than 10 nm. So the electronic excitation energy of QDs is transferred to AuNPs nearby via a through-space dipole-dipole interaction between the QDs-AuNPs pair. Then FRET occurred. As shown in Fig. 2, there was a necessary overlap between the emission spectrum of QDs (donor) and the absorption spectrum of AuNPs (acceptor) [25]. The broad absorption of AuNPs and narrow emission spectra of QDs used in this FRET laid a good foundation for energy transfer. In this system, QDs conjugated with SPA were used as energy donors, whereas AuNPs conjugated with anti-IgY were used as energy acceptors to form the FRET system. The fluorescence of QDs can be quenched by AuNPs, providing the “turn off” state. With the measured samples containing IgY, through antigen-antibody interaction, IgY and QDs-SPA combined with anti-IgY-AuNPs competitively. With an increased concentration of IgY in the samples, the probability of the combination of QDs-SPA and anti-IgY-AuNPs decreased. Next, the combination of QDs and AuNPs via the binding site on anti-IgY was blocked and FRET between them was prohibited. Thus, the fluorescence quenching of QDs by AuNPs was reduced. The fluorescence intensity of QDs recovered. It provides the third mode, a “turn on” state. In other words, the concentration of IgY was successfully converted into optical fluorescence signals. The selectivity experiment showed that the triple-mode probe exhibited high selectivity under complex conditions. Moreover, the fluorescence intensity was enhancing with increasing concentrations of IgY. In this way, we can not only qualitative detection IgY selectively, and also establishing a sensitive accurate quantity method of IgY detection.

3.3 Fluorescence quenching of QDs-SPA by Anti-IgY-AuNPs Different concentrations of the energy receptor affect the triple-mode nanosensor system. We investigated the concentration of the energy receptor, anti-IgY-AuNPs, in this study. As shown in Fig. 3, after adding anti-IgY-AuNPs to QDs-SPA, QDs-SPA and anti-IgY-AuNPs were closed to each other, assembled a QDs-SPA-anti-IgY-AuNPs immunocomplex, with the help of immune recognition between SPA and anti-IgY. The energy of QDs-SPA transferred to anti-IgY-AuNPs. Thus, the fluorescence intensity of QDs-SPA decreased and provided a “turn off” state. With the increasing concentration of anti-IgY-AuNPs from 0 to 29 μg/mL, the fluorescence intensity of QDs-SPA decreased continuously. To obtain a good transfer effect and a high sensitivity at the same time, we chose 19 μg/mL anti-IgY-AuNPs in this study. 3.4 The anti-interference ability of system The influence of coexisting substances such as Na+, K+, glucose, lactose, glycine, cysteine, glutathione, aspartic acid, L-serine, tyrosine, lysozyme, and BSA was investigated. The experimental results are listed in table 1. IgY detection concentration was significantly lower than the concentrations of all the coexisting substances. Many of the coexisting substances, such as ions, amino acids, and proteins, had little influence on the determination of IgY within the permission of 5.0% relative error. Therefore, this method was resistant to disturbance. 3.5 The quantitative detection of IgY based on triple-mode nanosensor fluorescence switch

3.5.1 Effect of pH on the conjugation between QDs-SPA and anti-IgY-AuNPs Fig. 4 shows the influence of pH in the conjugation between QDs-SPA and anti-IgY-AuNPs. When considering the stability of the native structure of the protein, the pH value varied from 4.6 to 9.0, around neutral. The fluorescence intensity declined remarkably with increasing pH. When the pH was in the acidity range, the fluorescence intensity was high because IgY can better bind to anti-IgY-AuNPs. Thus, the relieve of FRET lead to QD’s fluorescence recovery. When the pH was in the neutral and alkaline range, the fluorescence intensity remained relatively steady as the pH value moved up because the QDs were stable under alkaline conditions. Moreover, the neutral pH value

offered the advantage of helping to maintain the native conformation and biological activity and ensure later immune recognition of IgY and anti-IgY. Overall, the pH 6 was chosen as the preferred reaction condition for the conjugation of QDs-SPA and anti-IgY-AuNPs. 3.5.2 Effect of PBS concentration on the detection system The effect of concentrations of PBS buffered saline during the detection was shown in Fig. 5. It is found that with the increasing concentration of PBS buffered saline, the change of fluorescence intensity between binding product and the blank showed a trend of rise first then fall. The maximum change in fluorescence intensity occured at 2 mmol/L. This may be because the reaction between antigen and antibody needs certain ionic strength. When the concentration of PBS is low, the IgY can’ t react well with anti-IgY-AuNPs. However, when the PBS concentration is too high, it may promote the immune complex dissociation. Thus, the concentration of 2 mmol/L was selected in this study. 3.5.3 Effect of detection temperature on the conjugation system Fig. 6 shows the influence of temperature in the detection. The temperatures tested were 4, 25 and 37 °C. The maximum change of fluorescence intensity occurred at 4 °C. This may be caused by the higher fluorescence intensity and better sensitivity of QDs at lower temperature. When the temperature was higher than 4 °C, the change in fluorescence intensity may have declined because QDs had lower fluorescence intensity and the ability of fluorescence recovery declined at high temperature. Therefore, the detection temperature chosen for further study was 4 °C. 3.5.4 Effect of reaction time on the conjugation system Fig. 7 shows the influence of time in the detection. The fluorescence intensity of the system increased gradually with an increase in the reaction time. IgY and QDs-SPA combined with anti-IgY-AuNPs competitively through the antigen-antibody interaction. When the incubation time was over 45 min, there was no obvious increase in fluorescence intensity, which means the reaction reached a state of equilibrium [26]. When the incubation time was from 45 min to 120 min, the fluorescence intensity kept stable. Therefore, 45 min to 120 min was chosen as the experimental condition in this study.

3.5.5 The detection of IgY using QDs-SPA-anti-IgY-AuNPs system

According to the above procedures, the calibration curve for IgY determination was constructed under the optimal conditions. The fluorescence spectra of the QDs-SPA-anti-IgY-AuNPs system with different concentrations of IgY are presented in Fig. 8. With increasing concentrations of IgY (from 5 to 200 ng/mL), the fluorescence intensity of the system was enhanced proportionately. The more IgY there was in the sample, the more conjugated anti-IgY-AuNPs could bind. The probability of the combination of QDs-SPA and anti-IgY-AuNPs decreased with increased concentrations of IgY in the samples. Thus, the fluorescence quenching of QDs by AuNPs was reduced. The fluorescence intensity of the system was enhanced as macro behavior. The concentration of IgY was successfully converted into optical fluorescent signals. In this way, we established the immunofluorescence analysis method for IgY detection.

3.5.6 The detection range and limit of detection Under optimal conditions, the fluorescence intensity of the system with different concentrations of IgY was measured, and the calibration curve was constructed according to the relationship between the IgY concentration (C) and the corresponding fluorescence intensity change (ΔF). Fig. 9 illustrates the linear increase in the fluorescence intensity with increased concentrations of IgY. The linear range for IgY was from 5 to 200 ng/mL and the standard regression equation was ΔF = 0.5255C + 2.8505 (C: ng/mL), with the correlation coefficient R2 = 0.996. The limit of detection (LOD) refers to three times the value of the instruments background signal produced by the matrix blank. It indicates the sensitivity of the methods and instruments. It is defined by the equation LOD =3S0/K, where S0 is the standard deviation of blank measurements (n=11) and K is the slope of the calibration graph. Here, the LOD was 1.16 ng/mL. 3.5.7 Analytical performance Table 2 displays the analysis of five synthetic samples. In real egg products, salts, amino acids, proteins, saccharides, vitamins, and others are present. Therefore, in each synthetic sample, we selected salts, amino acids, proteins, saccharides, and vitamins to simulate a real sample and assess the anti-interference of our method. Table 2 showed that a certain concentration of the synthetic samples

had little influence on the detection system. The strong interaction between IgY and anti-IgY might contribute to the results. Thus, the detection of IgY had a strong anti-interference ability.

To examine the applicability of this method, we applied this method in real products. We chose egg powder because there is a relatively high concentration of IgY in egg yolk. Two egg powder samples were pretreated to remove the high-abundance proteins by the separating procedures mentioned in section 2.6. Following the experimental procedures, the concentration of IgY in food samples was determined (Table 3). The content of IgY in these egg powder samples as shown in table 3 was lower compared with the standard value of IgY concentration in eggs, namely, 5 mg IgY per mL plain yolk [27]. This may be due to the IgY loss or activity decreases during the process. Thus, the method established in this study was suitable for the determination of IgY concentration in egg products.

The analytical performance of our method was compared with other reports. Chicken IgG ELISA quantitation kit is a relatively common method to detect IgY. Kitaguchi et al [28] reported an ELISA assay to detect IgY in egg yolk with the detection range of 5.7 to 12.2 mg per g egg yolk. This method was not inexact and a little expensive. Recently, some methods, such as electrochemical immunosensor, to detect immunoglobulin were reported. Bottari et al [29] used electrochemical immunosensor based on ensemble of nanoelectrodes for IgY detection. This is a semi-quantitative method. The minimum response value of this method was 0.02 mg/mL. The detection range was from 0.02 to 0.1 mg/mL. And it needs a relatively complex electrode processing. Spindel et al [30] reported low volume planar surface fluorescent immunoassays to detect IgY with QDs. The limit of detection was 25 ng/mL. From the above, compared with these methods, the method we established can quantitative determinate immunoglobulin, and had a relatively wide linear range and a lower LOD. It was because spectroscopic detection has it’s own inherent advantage. In the fluorescence detection, fluorescence intensity is measured in the orthogonal direction of incident light. That is to say, the detection is under the background of black. We can change the incident light intensity or increase the magnification of the fluorescence signals to improve the sensitivity. And the energy transfer from the donor to acceptor

could amplify the signal change. In summary, this method had a strong specificity without a long analysis time, complex operations and use of an expensive apparatus. 4. 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 combined 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 fluorescence switch method established in this work was applied to the quantitative determination of IgY in egg products. We believe that this novel triple-mode switch study will open new opportunities for engineering optically based advance switches for sensing bimolecular. 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).

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Biography 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.

Captions 1 Fig. 1. The TEM of AuNPs (A) and QDs (B). Fig. 2. Fluorescence spectra of CdTe QDs (a) and absorption spectra of AuNPs (b). Fig. 3. Fluorescence spectra of QDs-PA-anti IgY-AuNPs complex with different concentration of anti IgY-AuNPs. The concentration of anti IgY-AuNPs from a to g are 0, 5, 10, 14, 19, 24, and 29 μg/mL, respectively. The calculation is based on the concentration of AuNPs. Fig. 4. Effect of pH on the conjugation system. IgY concentration: 200 ng/mL. Fig. 5. Effect of concentration of PBS on the conjugation system. The changed concentration of IgY: 90 ng/mL. Fig. 6. Effect of the detection temperatures on conjugation system. The changed concentration of IgY: 120 ng/mL. Fig. 7. Effect of the incubation time on fluorescence intensity. The incubation time is 0, 10, 20, 30, 45, 60, 80, 100, 120, 140, 160, and 180 min. IgY concentration: 200 ng/mL. Fig. 8. Changes in fluorescence spectra of the binding system in the presence of different concentrations of IgY. Concentration of IgY from (a) to (i) (ng/mL): 0, 5, 20, 40, 60, 90, 120, 160, and 200. λex =330 nm. Fig. 9. Linear relationships between concentrations of IgY and the changes in fluorescence intensity. IgY concentration is 5, 20, 40, 60, 90, 120, 160, and 200 ng/mL.

Scheme. 1. The principle of the triple-mode fluorescence switch control system.

Fig. 1.

Fig. 2.

Fig. 3.

Fig. 4.

Fig. 5.

Fig. 6.

Fig. 7.

Fig. 8.

Fig. 9.

Scheme. 1.

Table 1 Interference of coexisting substances Coexisting Substances

Concentration

Change

of

(10-6 mol/L)

intensity (%)

Na+

200

-4.16

K+

200

+3.13

Glucose

100

+3.72

Lactose

100

+2.39

Glycine

10

+4.41

Cysteine

2

+4.98

Glutathione

10

+3.72

L-serine

2

+3.91

Tyrosine

2

+3.13

Aspartic acid

10

+4.41

Lysozyme

0.1

+4.78

BSA

0.01

+3.13

fluorescence

Table 2 Results for IgY determination in synthetic samples. Synthetic

Main interferences

Change of fluorescence

sample

(10-8 mol/L)

intensity (%)

Na+ 10000, Tyrosine 20, Glucose 100,

1

-4.91

BSA 0.1, Vitamin C 10 Na+ 10000, L-serine 10, Lactose 1000,

2

+5.17

Albumin 300, Glutathione 10 K+ 20000, Glycine 200, Glucose 200,

3

+8.46

Avidin 300, Vitamin C 20 K+ 20000, Cysteine 4, Glucose 200,

4

+5.33

BSA 0.2, Vitamin C 20 K+ 10000, L-serine 10, Lactose 1000,

5

+5.82

Avidin 150, Lysozyme 0.1

Table 3 Results for the determination of IgY in egg products. Concentration of IgY Samples (mg IgY per g egg powder) Yolk powder

2.010

Whole egg powder

2.650