Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 131 (2014) 243–248

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Highly sensitive homogenous chemiluminescence immunoassay using gold nanoparticles as label Jing Luo, Xiang Cui, Wei Liu, Baoxin Li ⇑ Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi’an 710062, China

h i g h l i g h t s

g r a p h i c a l a b s t r a c t

 A universal platform for

A novel and sensitive strategy to convert the antibody–antigen recognition event into chemiluminescence signal by employing AuNPs as signaling probes is proposed.

homogeneous immunoassay was proposed using gold nanoparticles (AuNPs) as label.  This simple protocol consisted of just a one-step incubation followed by injection and reading.  The assay exhibited excellent sensitivity with a detection limit as low as 3 pg/mL.

a r t i c l e

i n f o

Article history: Received 12 March 2014 Received in revised form 9 April 2014 Accepted 17 April 2014 Available online 26 April 2014 Keywords: Chemiluminescence Homogeneous immunoassay Gold nanoparticles

a b s t r a c t Homogeneous immunoassay is becoming more and more attractive for modern medical diagnosis because it is superior to heterogeneous immunoassay in sample and reagent consumption, analysis time, portability and disposability. Herein, a universal platform for homogeneous immunoassay, using human immunoglobulin G (IgG) as a model analyte, has been developed. This assay relies upon the catalytic activity of gold nanoparticles (AuNPs) on luminol–AgNO3 chemiluminescence (CL) reaction. The immunoreaction of antigen and antibody can induce the aggregation of antibody-functionalized AuNPs, and after aggregation the catalytic activity of AuNPs on luminol–AgNO3 CL reaction is greatly enhanced. Without any separation steps, a CL signal is generated upon addition of a trigger solution, and the CL intensity is directly correlated to the quantity of IgG. The detection limit of IgG was estimated to be as low as 3 pg/ mL, and the sensitivity was better than that of the reported AuNPs-based CL immunoassay for IgG. Ó 2014 Elsevier B.V. All rights reserved.

Introduction Chemiluminescence (CL) immunoassay combines the high sensitivity of CL detection with the high specificity of immunoassay, and has been widely utilized to detect various biomolecules in ⇑ Corresponding author. Tel.: +86 2981530779; fax: +86 29 81530727. E-mail addresses: [email protected], [email protected] (B. Li). http://dx.doi.org/10.1016/j.saa.2014.04.076 1386-1425/Ó 2014 Elsevier B.V. All rights reserved.

clinical, pharmaceutical and environmental and biochemical fields [1,2]. The established CL immunoassay methods were mostly based on using luminor (such as luminol, isoluminol and acridinium ester) or enzyme (such as horseradish peroxidase and alkaline phosphatase) as label. Luminol is the best known and one of the most efficient CL reagents. In general, luminol is coupled to antibody or antigen via reactions involving the amino group. However, the resulting conjugates have much lower CL efficiency than that of

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luminol itself [1,3]. Meantime, labeled directly with the CL tags, the biomolecules activity could decrease more or less. Enzyme label is easily denaturalized, short lifetime and low stability [4], and the labeling procedure are complex and require low temperature [3]. Therefore, searching for the stable, easy-to-prepare, low-cost and highly biocompatible labels is of critical importance for the CL immunoassay. In recent years, nanomaterials have been widely employed as biological labels for immunoassays and other bioanalyses to overcome the safety problems, poor sensitivity, and poor stability associated with the radioisotopic, fluorescent, and enzyme labels [5]. Among these nanomaterial labels, gold nanoparticles (AuNPs) have drawn much attention because of several advantages, such as easy preparation of different nanostructures with special properties, small size and correspondingly large surface-to-volume ratio, chemically tailorable physical properties and good biocompatibility [6,7]. AuNPs have already been used as label in CL immunoassay [4,8–10]. In 2005, Lu group [8] and Li group [9] have separately developed AuNPs-based CL immunoassay using Au3+-catalyzed luminol CL reaction. Recently, Qi et al. [4] proposed a competitive flow injection CL immunoassay for IgG using AuNPs as label. After immunoreaction, the AuNPs, CL label, were dissolved through chemical oxidation to Au3+, and then the CL system was employed to measure Au3+ [4,8,9]. Because about 2.3  105 gold atoms are theoretically contained in one 20-nm spherical gold particle, the sensitivity of the CL immunoassay could be improved. However, the dissolution of AuNPs was conducted under extremely strict conditions (high concentration HNO3–HCl or poisonous HCl–Br2), and it needed a long time to ensure that the dissolution was completed. To avoid the dissolution of AuNPs, Li et al. [11] directly utilized the catalytic activity of irregular AuNPs on luminol–H2O2 CL reaction to establish a non-stripping CL immunoassay. Although this protocol avoided the strict stripping procedure, the synthesis of irregular nanoparticles was hard to control, requiring stirring for long time with temperature control (40 °C for 24 h), purging of oxygen and relatively low monodispersity, which may influence the repeatability among different batches, limiting the practical application of this method. Cui group found the catalytic activity of the normal spherical AuNPs on luminol system [12,13], and used the normal spherical AuNPs to label the second antibody for developing a non-stripping CL immunoassay [14]. The reported AuNPs-based CL immunoassays [4,8–11,14] were performed in heterogeneous format. A typical heterogeneous immunoassay involves many steps, such as antibody immobilization, incubation, multiple separation and washing cycles, signal amplification and measurement. From the initial antibody immobilization to the final reading of the assay results, the entire immunoassay can usually take several hours to complete. The heterogeneous immunoassay is rather time-consuming and labor-intensive. To overcome these problems, there is an

increasing need for homogeneous immunoassays without any separation steps, especially in the field of modern diagnosis because of their simplicity, ease of automation, and high throughput [15–17]. Recently, it is reported that the normal spherical AuNPs or silver nanoparticles can catalyze luminol–AgNO3 reaction to produce a CL [13,18], and this novel CL reaction has the merits of low background, good stability and good reproducibility. In this work, an interesting phenomenon is observed that the catalytic activity of the aggregated AuNPs on luminol–AgNO3 CL reaction is much higher than that of dispersed AuNPs (ca. 13 nm). By taking advantage of this phenomenon, a homogenous CL immunoassay is proposed. A diagram of this method is shown in Scheme 1. Human immunoglobulin G (IgG) was taken as the model analyte to provide the ‘‘proof-of-principle’’ verification of the concept. Aqueous AuNPs were prepared and modified with antibody (goat-anti-human IgG). In the presence of antigen (IgG), the functionalized AuNPs can bind antigen to form dimers (or oligomers) [16,19], and the aggregated AuNPs induce a strong CL signal of luminol system. In the absence of antigen, the dispersed AuNPs induce weak CL emission. The increased CL emission intensity will be positively related to the concentration of the antigen added in the assay solution. Homogeneous assays offer some unique advantages compared to heterogeneous assays, especially in the simplicity of the assay. However, the sensitivity of homogeneous assays is often not as high as heterogeneous amplification assays [20]. In this work, the low background of luminol–AgNO3–AuNPs CL system made the homogenous CL immunoassay exhibit high sensitivity. The detection limit of IgG was estimated to be as low as 3 pg/mL, and the sensitivity was better than that of the reported AuNPs-based heterogeneous CL immunoassay for IgG [4,8–11,14]. Experimental Reagents and materials Chloroauric acid (HAuCl4) and silver nitrate was purchased from Sinopharm Chemical Reagent Company (Shanghai, China). Luminol was obtained from Sigma. Sodium citrate was purchased from Xi’an Chemical Reagent Company (Xi’an, China). Human IgG, goat-anti-human IgG, bovine serum albumin (BSA) and HRP-labeled goat-anti-human IgG were purchased from Beijing Dingguo Biotechnology Company (Beijing, China). All the measurements of IgG have been performed in 0.01 M sodium phosphate buffer (pH 7.4). The human serum, provided by Shaanxi Normal University Hospital, was used as the sample to evaluate the reliability of the proposed immunoassay. Polystyrene 96-well microtiter plates were used to perform the immunoreactions. The luminol stock solution (50 mM) was prepared by dissolving luminol (0.4428 g) in 0.1 M NaOH and then diluting to 50 mL with 0.1 M NaOH. The luminol solution was stored in the dark for one

Scheme 1. Schematic illustration of CL immunoassays based on the catalytic effect of aggregated AuNPs to luminol–AgNO3 CL system.

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Apparatus The CL intensity was measured and recorded with a model IFFSA Chemiluminescence Analyzer (Xi’an Remex Electronic Sci. Tech. Co. Ltd., Xi’an, China). CL data acquisition and treatment were performed using RFL-1 software (Xi’an Remax Electronic Sci. Tech. Co. Ltd., Xi’an, China). A HH-4 thermostatic water bath (Changzhou Guohua Instrumental Factory, Changzhou, China) was used to control the temperature of the immunoreactions at 0.1 °C intervals. The results of UV were obtained using SHIMADZU UV-1800 recording spectrophotometer (Shimadzu, Japan). The transmission electron microscopy (TEM) images of GNPs were taken using a Hitachi H-600 TEM (Tokyo, Japan). Dynamic Light Scattering (DLS) was used to see the size distributions of the particles on a photon correlation spectrometer (Brookhaven BI-90Plus, US). Preparation and characterization of AuNPs All glassware and stirrer used in the following procedure were thoroughly cleaned in aqua regia (HNO3–HCl 1:3, v/v), rinsed in distilled water, and then oven-dried prior to use, to avoid unwanted nucleation during the synthesis. The AuNPs (ca. 13 nm diameter) were prepared by the citrate reduction method according to the published protocol [21]. Briefly, a sodium citrate solution (0.1 mol/L, 1.94 mL) was rapidly added to a boiled HAuCl4 solution (50 mL H2O, 0.167 mL 10% HAuCl4) under vigorous stirring. The mixed solution was maintained for 8 min at boiling and then removed from the heating mantle. Stirring was continued for another 13 min, and after cooling to room temperature, the prepared AuNPs solution was stored in the refrigerator (4 °C). The synthesized AuNPs were characterized by using UV–visible spectroscopy and TEM in order to measure the particle size. The concentration of the AuNPs solution was 17 nM, which was estimated by using UV–visible spectroscopy, based on an extinction coefficient of 2.7  108 M 1 cm 1 at k = 520 nm for 13 nm particles [22]. Preparation of AuNPs-labeled goat-anti-human IgG The preparation of AuNPs-labeled goat-anti-human IgG was performed according to the literature procedure [9]. Briefly, after AuNPs solution was adjusted to pH value 9 with 0.1 M K2CO3 solution, 1.0 mL of 0.5 mg/mL goat-anti-human IgG was added to 5 mL of pH-adjusted AuNPs solution. The mixed solution was stirred and incubated at room temperature for 1 h. The resulting bio-conjugate was centrifuged at 13,000 rpm at 4 °C for 30 min to get rid of the unbound goat anti-human IgG, and the supernatant was removed. The oily ruby sediment was re-dispersed by 6 mL of 0.01 M sodium phosphate buffer (PBS, pH 7.4) containing 1% BSA. The bio-conjugate solution was stored at 4 °C. The synthesized AuNPs-labeled goat-anti-human IgG were characterized by using UV–visible spectroscopy, TEM and DLS. The stability of AuNPs-labeled goatanti-human IgG solution solution was estimated by measurement of the absorbance at 520 nm every day. The results showed that the absorbance of AuNPs-labeled goat-anti-human IgG solution significantly changed after one week because the AuNPs themselves aggregate during the stored time. So, we used the fresh AuNPs every week in this experiment.

Procedure of chemiluminescence-based immunoassay A typical CL-based immunoassay was realized by the following steps. First, 200 lL of the AuNPs modified with goat anti-human IgG (0.5 mg/mL) was added to 200 lL of human IgG with different concentrations or diluted serum samples. Afterward, 100 lL PBS (pH 7.4, containing 3.0 mM NaCl) was added. The mixtures were blended by the vortex mixer and incubated for 40 min at 37 °C water bath. Second, 20 lL of the resulting solution was decanted to the quartz cuvette (used as CL reactor), and the CL reaction was triggered by injecting 100 lL of the mixed solution containing AgNO3 (10 lM) and luminol (0.3 mM). The CL signal was measured and recorded by using the RFL-1 ultraweak Chemiluminescence Analyzer. The concentration of human IgG was quantified by the CL peak intensity. The CL experiments in this work were not consecutive measurements.

Results and discussion We first evaluate the feasibility of our new design. Fig. 1 shows the CL emission of the luminol–AgNO3 sensing system under different conditions. In the absence of antigen, the antibodyfunctionalized AuNPs show the rather weak catalytic activity, and the catalytic activity changed slightly compared to the bare AuNPs. In contrast, in the presence of 0.1 lg/mL IgG, the CL intensity is significantly increased. The control experiment showed that IgG did not affect the CL intensity of luminol–AgNO3–bare AuNPs. It is clear that the antibody-antigen recognition would be easily distinguishable by a CL analysis of the luminol–AgNO3–AuNPs CL system. This suggests that the enhanced CL intensity may be attributed to the immunoreaction between antigen and antibody on the surface of AuNPs. The AuNPs (ca. 13 nm) solution has a surface plasma resonance absorption peak at about 520 nm and appears pink [23]. It is well known that the surface plasmon absorption of AuNPs is very sensitive to their interparticle distance and surface state [16]. In this work, we used AuNPs to label the antibody. After the introduction of antibody to AuNPs, a slight red-shift of spectra appeared, and the absorption intensity at about 520 nm was increased (Fig. 2), which was considered as the confirmation of the protein absorption onto the particle surface [24]. In the presence of IgG, the absorption intensity of antibody-functionalized AuNPs at ca. 520 nm increased, which could suggest the occurrence of the immunoreaction [16]. TEM images were used to explore the change of AuNPs induced by the immunoreaction in this system (Fig. 3). Compared

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week prior to use to ensure that the reagent had stabilized. Working solution H2O2 were prepared fresh daily from 30% (w/w) H2O2. Unless other indicated, all reagents and solvents were purchased in their highest available purity and used without further purification. Millipore Milli-Q water (18 MX cm 1) was used in all experiments.

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Time (s) Fig. 1. CL emission of luminol–AgNO3 system under different conditions. Experimental condition: 0.5 mM luminol, 10 lM AgNO3 and 0.1 lg/mL IgG.

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with the bare AuNPs, the antibody-functionalized AuNPs became larger. Before the immunoreaction with human IgG, AuNPs were highly dispersed, and after goat-anti-human IgG functionalized AuNPs solution mixed with human IgG, the immunoreaction between human IgG and the goat-anti-human IgG resulted in crosslinking of the functionalized AuNPs and led to form dimers. In order to further verify this phenomenon, DLS was also used to explore the change of AuNPs induced by the immunoreaction. Analysis of the hydrated size distributions reveals the average size of 60.3 ± 1.5 nm (Fig. 3B) for the goat-anti-human IgG functionalized AuNPs, which was larger than 32.9 ± 0.8 nm (Fig. 3A) for the bare AuNPs, suggesting the successful absorption of the protein absorption onto the particle surface. What is more persuasive, analysis of size distributions reveals the average size of 130.2 ± 6.2 nm (Fig. 3C) for the antibody-functionalized AuNPs in the presence of antigen, which is just about twice of the size of the antibody-functionalized AuNPs themselves. These results suggest that the immunoreaction causes AuNPs to mainly form dimers. It is worthwhile to note that the particle size obtained with DLS measurement is different from the size obtained with TEM measurement [25,26]. The obtained size with DLS measurement is the hydrodynamic diameter, whereas the obtained size with TEM measurement is primary particle. When nanoparticles are dispersed in liquids, the hydrodynamic size is often larger than the primary particle size in dry state [25,26]. In our recent work [27–29], we found that the catalytic activity of the aggregated AuNPs on luminol–H2O2 CL reaction was much higher than it of dispersed AuNPs, and discussed the mechanism on enhancement of activity of AuNPs after aggregation. Based on the previous studies [27–30], we reasoned that the enhancement effect of the aggregated AuNPs on luminol–AgNO3 reaction may be attributed to the change of the AuNPs surface-charge density. Analysis revealed that the zeta potential of the goat-anti-human IgG functionalized AuNPs (dispersed AuNPs) was 30.3 mV; after incubation with 0.1 lg/mL IgG, the zeta potential was 22.2 mV. It is clear that the AuNPs surface negative charge density is greatly decreased after aggregation. Luminol anion is the main molecular form of luminol in strong basic media. The anionic luminol does not easily interact with the anionic AuNPs because of electrostatic repulsion. The dispersed AuNPs, which have a high surface negative charge density, have a rather low catalytical activity on this CL reaction. After immunoreaction, the linked AuNPs (dimers or oligomers) with low surface negative charge density can more easily interact with anionic luminol, resulting in a higher catalytic effect on the luminol CL reaction. Thus, we reasoned that the decrease of the surface negative charge density of AuNPs resulted in the enhanced catalysis of the aggregated AuNPs.

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Diamiter (nm) Fig. 3. DLS results of AuNPs (A), AuNPs-antibody conjugates (B) and AuNPsantibody + antigen (C). Inset shows the corresponding TEM images.

Luminol–H2O2 CL system could also be applied in the AuNPsbased CL immunoassays. So, we compared the sensitivity of luminol–H2O2 CL system with luminol–AgNO3 CL system in this AuNPsbased immunoassay. The experimental results showed that the S/N (signal/noise ratio) of 0.1 lg/mL IgG reached 8.6 in luminol–AgNO3 CL system, (Fig. 1), and the S/N was only 1.5 in luminol–H2O2 CL system (Fig. 4), which indicates that luminol–AgNO3 CL system is more suitable for the CL immunoassay. The high sensitivity of luminol–AgNO3 CL system is attributed to low background and good stability of this system [18]. To improve the performance of

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Time (s) Fig. 4. CL emission of luminol–H2O2 system under different condition. Experimental condition: 0.5 mM luminol, 0.1 mM H2O2, and 0.1 lg/mL IgG.

the CL immunoassay, we optimized the immunoreaction condition. Both the immunoreaction and the aggregation of the AuNPs involve charged species and electrostatic interaction [16]. The media pH would affect the charge distribution of antigen, antibody and AuNPs surface, which consequently influence the aggregation of the AuNPs. The experiments showed that the CL intensity reached a maximum at pH 7.4. So, we chose 0.01 M phosphate buffered saline (PBS, pH 7.4) as the immune reaction media. Ionic strength (meaning the concentration of NaCl added to the buffer) also influenced the CL response of the system. When the added NaCl concentration was greater than 3 mM, the CL intensity decreased, which might be attributed to the denaturation of protein at high ionic intension. In this system, 100 mL of 3 mM NaCl was used to control the ionic strength for the immunoassay. A long incubation time of IgG and the antibody-functionalized AuNPs is expected to yield a high CL signal. The response was recorded for increasing incubation time. When the assay was conducted at 37 °C, the CL signal reached a maximum at 40 min, and the CL response remained almost constant over 40 min. The amount of antibody-functionalized AuNPs influenced the sensitivity. When antibody-functionalized AuNPs concentration was too high, the system is not sensitive enough to detect small amounts of IgG; when antibody-functionalized AuNPs concentration was too low, the aggregation of AuNPs was very slow. Therefore, 200 lL 17 nM antibody-functionalized AuNPs were used in all experiments. In addition, we also optimized the CL reaction condition. The experimental results showed that the optimal concentrations of luminol and AgNO3 were 0.3 mM and 10 lM, respectively. The optimal pH for the CL reaction was 12. Under the optimized conditions, experiments were carried out by adding increasing amounts of IgG to the CL system to examine whether the CL change could be used for IgG quantification. The CL intensity increases with the increasing IgG concentration, to reveal a linear in the IgG concentration 0.01–1.0 ng/mL (Fig. 5). Fig. 5A and B shows the linear regression equation (I is the CL intensity, C is the concentration of IgG, and R is the correlation coefficient) in 0.01–0.08 ng/mL range and 0.1–1.0 ng/mL range, respectively. The relative standard deviation (RSD) was less than 6% for 0.1 ng/ mL IgG (n = 7). The detection limit (taken to be 3 times the standard deviation in the blank solution) was 3 pg/mL. The detection limit is lower than that obtained in the reported AuNPs-based CL immunoassay for IgG [4,8,9,11,14,29]. The present limit of detection for this present system is superior to most of the reported immunoassay for IgG so far [31].

Fig. 5. Representative recorder outputs of the CL system in the presence of IgG in 0.01–0.08 ng/mL range (A) and 0.1–1.0 ng/mL range (B). Inset shows a linear relationship between the CL intensity (I) and target antigen concentration (C). Error bars represent the standard deviations of three independent measurements.

Table 1 Detection results of IgG in human sera using the proposed CL immunoassay.

a

Sample

Proposed methoda (mg/mL)

Colorimetric ELISAa (mg/mL)

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9.40 14.53 12.01

9.97 13.66 11.24

Relative deviation (%) 5.7 6.4 6.8

Average of three replicates.

In order to evaluate the reliability and application of the proposed immunoassay to clinical diagnostics, human serum samples were analyzed simultaneously with the proposed method and the ELISA. The fresh human blood samples from some volunteers were collected from Shaanxi Normal University Hospital and used as testing samples. Before the test, the samples were diluted with 1000 million times step by step to be in the linear range of the proposed method. At the same time, the conventional ELISA method was used as the standard method, and the ELISA test procedures were according to the operational manuals of the ELISA kits. The experimental results (Table 1) show that the values obtained by the proposed method were comparable with those obtained by the ELISA method and the relative deviation between the two methods was over the range ±7%. Therefore, the developed method is applicable for the determination of IgG in real sample.

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Conclusion In conclusion, a novel and sensitive strategy to convert the antibody-antigen recognition event into CL signal by employing AuNPs as signaling probes is proposed. Without any separation and washing steps, the assay is homogeneous and occurs in the liquid phase. This simple protocol consisted of just a one-step incubation followed by injection and reading, and the assay exhibited excellent sensitivity with a detection limit as low as 3 pg/mL. Therefore, given the simplicity and sensitivity, the proposed immunoassay has great potential for protein assay in the fields of molecular biology and clinical diagnostics. Acknowledgements This work was supported financially by the National Natural Science Foundation of China (No. 21275096) and the Program for Key Science and Technology Innovation Team of Shaanxi Province. References [1] [2] [3] [4]

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Highly sensitive homogenous chemiluminescence immunoassay using gold nanoparticles as label.

Homogeneous immunoassay is becoming more and more attractive for modern medical diagnosis because it is superior to heterogeneous immunoassay in sampl...
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