Label-free colorimetric immunoassay for the simple and sensitive detection of neurogenin3 using gold nanoparticles

Biosensors and Bioelectronics 26 (2011) 4245–4248

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Label-free colorimetric immunoassay for the simple and sensitive detection of neurogenin3 using gold nanoparticles Yue Yuan a,b , Jia Zhang a , Hanchang Zhang b,∗ , Xiurong Yang a,∗ a b

State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, PR China Department of Chemistry, University of Science and Technology of China (USTC), Hefei 230026, PR China

a r t i c l e

i n f o

Article history: Received 14 January 2011 Received in revised form 10 April 2011 Accepted 11 April 2011 Available online 28 April 2011 Keywords: Neurogenin3 Peptide Colorimetric immunosensor Au nanoparticles

a b s t r a c t Neurogenin3 (ngn3), as a marker for pancreatic endocrine precursor cells and an essential ingredient in the development of islet cells, was quantitatively detected for the first time. Based on a non-cross-linking specific interaction mechanism, a label-free colorimetric immunoassay for the synthetic peptide fragment of ngn3 (SKQRRSRRKKAND) using glutathione (-Glu-Cys-Gly, GSH) functionalized gold nanoparticles (GNPs) is reported. The anti-ngn3 antibody conjugated GNPs (GNP-Ab) was formed through electrostatic interaction upon the addition of anti-ngn3 antibody to the GSH-modified GNPs solution. Monobinding of the positively charged ngn3 to the negatively charged GNP-Ab will minimize the electrostatic repulsion between nanoparticles by neutralizing the surface charge, and then agglomeration is induced by an increasing salt concentration. Under the optimal conditions, the assay showed a linear response range of 50–300 ng/mL for the peptide with a detection limit being 20 ng/mL. The preliminary study on ngn3 opens up an innovative insight to detect short synthetic peptide fragment of antigen, and may own an opportunity for practical applications in clinical diagnosis and therapeutics. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Neurogenins are a family of basic helix-loop-helix (bHLH) transcription factors that are expressed in neural progenitor cells with the functions of controlling and determining neuronal development (Sommer et al., 1996). Neurogenin3 (ngn3), as a member of neurogenin family, is expressed in endocrine progenitor cells and required for endocrine cell development in the pancreas and intestine (Wang et al., 2006). Some reports suggested that ngn3 played an essential role in determining which progenitor cells will ultimately differentiate into islet cells, and mice in the absence of ngn3 function failed to generate any pancreatic endocrine cells, dying postnatally from diabetes (Schwitzgebel et al., 2000; Gradwohl et al., 2000; Lee et al., 2001; Serafimidis et al., 2007; Johansson et al., 2007). Therefore, ngn3 is an essential ingredient in the development of islet cells and its defect can result in diabetes, which underlines the detection of ngn3 in acquiring new insight into diabetes. Recently, several methods have been developed for the detection of ngn3, such as in situ hybridization (ISH) (Gradwohl et al., 2000; Yoshida et al., 2004; Ma et al., 2009; Yechoor et al., 2009), immunohistochemistry (Schwitzgebel

∗ Corresponding authors. Tel.: +86 431 85262056; fax: +86 431 85689278. E-mail addresses: [email protected] (Y. Yuan), [email protected] (H. Zhang), [email protected] (X. Yang). 0956-5663/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.bios.2011.04.021

et al., 2000; Lee et al., 2001; Serafimidis et al., 2007; Johansson et al., 2007), western blotting (WB) (Sun et al., 2001; Wang et al., 2009), and quantitative real-time PCR (Joglekar et al., 2007). It is possible for these methods to visualize the distribution and localization of ngn3 protein or ngn3 mRNA within a cell or tissue, however, they are insensitive, and the addition of mediator such as dyes, fluorescent substances, isotopes, enzymes, and biotin in the specimen would make the detection procedure complicated, expensive, and time-consuming. To date, no report described the quantitative detection of ngn3, which makes the development of a simple, sensitive and rapid method highly essential and important. Gold nanoparticles (GNPs) (normally 10–50 nm in diameter) have a remarkably high extinction coefficient and strongly distance-dependent optical properties (Storhoff et al., 2000). Different aggregation states of GNPs correspond to distinctive color, which can be appreciably discerned with the naked eye. The last decade has witnessed the expansion of the colorimetric assay using GNPs as the signal reporter, pioneered by the Mirkin group to describe the selective detection of polynucleotides based on the fantastic distance-dependent optical properties of GNPs (Elghanian et al., 1997). Colorimetric immunosensors based on GNPs have been developed rapidly in recent years due to several advantages when compared to traditional immunoassays, such as simple sample preparation, enhanced stability, and a reduction in nonspecific aggregation (Liu et al., 2009; Neely et al., 2009; Wangoo et al., 2010).

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a Hitachi H-800 transmission electron microscope (Hitachi, Japan) operated at 200 kV. Photographs were taken with a Canon digital camera. Zeta potential measurements were performed by a Malvern Zetasizer nano instrument (Malvern, UK). 2.3. Synthesis of GNPs with the diameter of ∼13 nm The synthesis of the citrate-protected GNPs was described in Supporting data. The concentration of the prepared GNPs was ∼14 nM, calculated by the absorption intensity at 520 nm, at which an extinction coefficient of 2.7 × 108 M−1 cm−1 was used (Hill and Mirkin, 2006). 2.4. Preparation of the GNP-Ab conjugate by the electrostatic interaction Scheme 1. Principle of detection of ngn3 using chemically functionalized gold nanoparticles mediated by salt (NaCl). The presence of antigen leads to the aggregation of GNPs resulting in a visible change of color from red to blue. (For interpretation of the references to color in the figure caption, the reader is referred to the web version of the article.)

In this paper, we use a synthetic peptide fragment of ngn3 (SKQRRSRRKKAND) rather than the complete one with full length to test the colorimetric assay, using GNPs as the signal reporter. The concept of the detection procedure is based on a non-crosslinking specific interaction mechanism (Wangoo et al., 2010), where agglomeration of GNPs is caused by an increasing salt concentration, as illustrated in Scheme 1. Monobinding of the positively charged ngn3 to the negatively charged anti-ngn3 antibody conjugated GNPs (GNP-Ab) will minimize the electrostatic repulsion between nanoparticles by neutralizing the surface charge. No cross-linking of GNP-Ab by the antigen is expected, since there is only one binding site on the short peptide fragment for the antibody. In general, no aggregation can be observed in the absence of salt, meanwhile, aggregation of the GNP-immunocomplex is induced by an increasing salt (NaCl) concentration, appreciably revealed by the color change of the solution from red to purple or blue. The concentration of ngn3 can be conveniently accessed by the optical absorption spectra. To the best of our knowledge, this is the first time that a short synthetic peptide fragment of antigen is detected by a label-free colorimetric immunoassay using GNPs as the probe.

Colloidal Au solution (1 mL) was mixed with GSH (7 ␮L, 10 mM) and shaken for 2 h. Excess GSH was removed by two cycles of centrifugation/wash procedure. Then the GSH-modified GNPs solution (1 mL) was incubated with different concentrations of anti-ngn3 antibody for 12 h at 4 ◦ C in PBS media. Excess antibody was removed by three cycles of centrifugation/wash procedure, and the GNP-Ab solutions were finally obtained by each addition of 2 mL of buffer. We prepared six systems by each addition of anti-ngn3 antibody (20, 50, 100, 200, 350, and 500 ␮L, 0.01 g/L), which are denoted as GNP-Ab(J1-6), respectively. 2.5. Preparation of the GNP-Ab conjugate by the glutaraldehyde spacer method Glutaraldehyde (26.5 ␮M) was first added into the GSHmodified GNPs solution which was prepared as described above, and then the mixture was incubated with antibody for 12 h at 4 ◦ C in PBS media. The following process was similar as above. The six systems corresponding to the six concentrations of anti-ngn3 antibody are denoted as GNP-Ab(W1-6), respectively. 2.6. The detection of ngn3 by the GNP-Ab(J1) probe

2. Experimental

Under the optimal conditions (pH 6.5, 30 mM NaCl), different volumes of ngn3 (0.01 g/L) corresponding to certain concentrations (0, 20, 50, 75, 100, 200, 300, 400, and 500 ng/mL) were spiked into the GNP-Ab(J1) solution (50 ␮L GNP-Ab + 50 ␮L PBS). After 10 min of incubation, the absorption spectra were collected. The results were measured three times to assess the standard deviation.

2.1. Materials

2.7. Selectivity test

The synthetic peptide fragment of ngn3 (SKQRRSRRKKAND) and monoclonal anti-ngn3 antibody were obtained from Leagene Co., Ltd. (Beijing, China). The synthetic peptide fragment of ngn1 (AQDDEQERRRRRGRTR) was obtained from Saierbio Co., Ltd. (Tianjin, China). Hydrogen tetrachloroaurate(III) trihydrate (HAuCl4 ·3H2 O) and glutaraldehyde were purchased from Sigma–Aldrich Co. (USA). Glutathione (-Glu-Cys-Gly, GSH) was purchased from Sangon Biotech Co., Ltd. (Shanghai, China). All other chemicals were analytical grade and used without further purification. Phosphate buffer solution (PBS) was prepared by varying the ratio of 0.01 M Na2 HPO4 to 0.01 M NaH2 PO4 . Milli-Q water (18.2 Mcm) was used throughout the experiment, which was conducted at ambient temperature (14 ± 2 ◦ C).

Several interferences were added in the absence and presence of ngn3 to evaluate the selectivity of the immunoassay, which is detailed in the latter section.

2.2. Apparatus UV–vis absorption spectra were recorded by a Cary 50 UV/vis spectrophotometer (Varian, USA). TEM images were obtained from

3. Results and discussion 3.1. GSH-modified GNPs based immunosensor for ngn3 The GSH was covalently connected to the surface of GNPs by the thiol group in the cysteine. The choice of GSH as the modifier seems to be appropriate, in that it can excellently protect the GNPs against precipitation and provide suitable functional groups for the conjugation of the anti-ngn3 antibody. For preparation of the GNP-Ab, two ways were applied, which can be named as the electrostatic interaction method and the glutaraldehyde spacer method. Since the GSH and antibody are zwitterionic molecules, both of which contain the anionic groups (–COO− ) and positively charged amine groups (–NH3 + ) in the experimental pH status, the

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large degree of agglomeration (Fig. 1E), and the color of the solution clearly turned to blue from red (insets of Fig. 1B and E). As control, either addition of ngn3 or NaCl did not cause similar changes in the absorbance or degree of particles aggregation (Fig. 1C and D) or color of the solution (insets of Fig. 1C and D). Moreover, little variation (absorbance or solution color) was detected when we replaced the GNP-Ab with GSH-modified GNPs (Fig. S1, Supporting data). The results unambiguously confirmed that the aggregation of GNPs was caused by the specific binding of ngn3 to the anti-ngn3 antibody.

3.2. Optimization of experimental conditions

Fig. 1. (A) Absorption spectra and (B–E) TEM images plus photos of the GNP-Ab in the presence of none (B), 300 ng/mL ngn3 (C), 80 mM NaCl (D), and 300 ng/mL ngn3 + 80 mM NaCl (E), pH 7.4.

physical adsorption in the former way is supposed to take advantage of ionic/hydrogen bonding between the above two groups (Laczka et al., 2008). The latter way is more understandable, coupling the antibody to the GNPs by covalent interaction of amine groups with glutaraldehyde. Both conjugation methods were effective for the detection of ngn3, and the probes were stable for at least one week when stored in refrigerator (4 ◦ C). To verify the ability of GNP-Ab to behave as colorimetric reporter for ngn3, we initiated on the addition of 300 ng/mL ngn3 to the GNPAb(J4) system. The absorption spectra were collected after 10 min of incubation, as shown in Fig. 1A. The original GNP-Ab conjugate solution exhibited a strong absorption at 520 nm, featuring of the surface plasmon resonance (SPR) of separated GNPs. Little aggregation of particles was observed in the TEM image (Fig. 1B), well agreed with the optical spectrum. Upon the addition of ngn3 and NaCl (80 mM), the intensity of absorption band at 520 nm decreased dramatically and a broad new band corresponding to the absorption of GNP aggregates appeared at 675 nm. The TEM image showed a

Having observed that ngn3 can be visualized by the GNPAb colorimetric probe, we suspected the immunoassay would be dependent on the NaCl concentration, the amount of the antibody on the GNPs, the modification method, and the pH environment. An appropriate salt concentration is crucial for the immunoassay, since less amount of salt will undermine the sensitivity and more salt will greatly reduce its capability against interference. Thus, a critical salt concentration was scrupulously chosen, above which the absorbance spectra became unstable with time. The GNP-Ab(J4) system was chosen to reveal the crucial effect of salt amount on the sensitivity of the colorimetric assay. In order to evaluate the degree of aggregation, the absorbance ratio between 675 nm and 520 nm (A675 /A520 ) was used, in which the A675 and A520 represent the relative quantity of aggregated and dispersed nanoparticles, respectively. As clearly shown in Fig. S2A (Supporting data), the immunoassay was getting more sensitive along with the increase of NaCl concentration till 80 mM. Upon the addition of 100 mM NaCl, the solution turned unstable with time, evidently revealed by the constant change in absorption spectra (Fig. S2B, Supporting data). Similar correlations of sensitivity with salt concentration were also obtained for other assay systems, which we did not detail in this paper. The critical NaCl concentration for GNP-Ab(J1-6) systems were estimated to be 40, 50, 60, 80, 80, and 80 mM respectively, suggesting the increase of the surface charge of gold nanoprobes. This can be evidenced by the zeta potential measurements of the six GNP-Ab solutions, which were averaged to be −35.6, −37.1, −39.4, −44.7, −45.1, and −44.2 mV, respectively. The zeta potential results are in accord well with the critical NaCl concentration data. Such coincidence can be explained by the saturation of antibody on the

Fig. 2. (A) Absorption spectra of the GNP-Ab solution upon the addition of ngn3. The inset shows the solution color change with 1–9 corresponding to 0, 20, 50, 75, 100, 200, 300, 400, and 500 ng/mL ngn3, respectively. (B) The sensitivity of the assay and the calibration curve (red line). (For interpretation of the references to color in the figure caption, the reader is referred to the web version of the article.)

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modified GNPs at 200 ␮L, above which the zeta potential remained stable. The A675 /A520 value correlated with the ngn3 concentration for each GNP-Ab system prepared by both methods, as shown in Fig. S3 (Supporting data). At pH 7.4 and each under the critical salt concentration, it is clear that the immunoassay was nearly unaffected by either the antibody load on the GNPs or the modification method. Therefore, it is reasonable to choose the GNP-Ab(J1) system in the following analysis. pH environment is another important factor for the proposed electrostatic repulsion-based immunoassay, since the charge property of both antibody and the peptide analyte are highly pHdependent. The sensitivities of the GNP-Ab(J1) system under five pH statuses are shown in Fig. S4 (Supporting data). We found that a higher sensitivity was obtained at a lower pH value and meanwhile, some aggregation of nanoparticles was observed at pH 6.0 during sample preparation, suggesting a lower stability. Thus, in spite of the highest sensitivity at a pH of 6.0, we selected a pH of 6.5 as the optimum.

synthetic peptide fragment of antigen. Third, owing to the outstanding biocompatibility and water-solubility of gold nanoprobes, it is highly expected to be further extended to the visualization of ngn3 in living cell samples, just like previous reports did on other analytes (Li et al., 2010; Wang et al., 2010). We thus believe this preliminary immunoassay on ngn3 may have potential opportunity for practical applications in clinical diagnosis and therapeutics. Acknowledgements This work was supported by the National Natural Science Foundation of China (No. 20890022), the National Key Basic Research Development Project of China (No. 2010CB933602, No. 2007CB714500) and the Project of Chinese Academy of Sciences (No. KJCX2-YW-H09). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bios.2011.04.021.

3.3. Quantitative detection for ngn3 References Under the optimal conditions, we performed the quantitation of ngn3, and the absorbance spectra upon the addition of ngn3 is shown in Fig. 2A. The color of the solution was clearly observed to change from red through purple to blue (inset of Fig. 2A). By correlating the A675 /A520 value with the ngn3 concentration, a linear range of 50–300 ng/mL was obtained (R2 = 0.999) (Fig. 2B), with a limit of detection (LOD) being 20 ng/mL. It should be pointed out that the assay output will be some different when the method is applied for the detection of complete ngn3. 3.4. Selectivity test Solutions of cytochrome (Cyt C, 200 ng/mL), l-lysine (Lys, 20 ␮M), l-arginine (Arg, 20 ␮M), lysozyme (200 ng/mL), and a synthetic fragment of neurogenin1 (ngn1, AQDDEQERRRRRGRTR, 200 ng/mL) were each added in the absence and presence of ngn3 (200 ng/mL). Results showed neither of them could elicit a response relative to ngn3 nor interfere with the response of ngn3 (Fig. S5, Supporting data), suggesting an excellent selectivity of the immunosensor. 4. Conclusion In summary, we have developed a label-free colorimetric method for detecting ngn3 by using gold nanoprobes. Besides the excellent sensitivity and selectivity, this methodology has several advantages. First, it is more facile, rapid, and cost-effective than the previous methods, and importantly, it is the first time to quantitatively detect ngn3 that offers a new opportunity for analytical study on neurogenins. Second, it opens up a fresh insight to detect short

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