Article pubs.acs.org/JAFC
Ultrasensitive and Quantitative Detection of a New β‑Agonist Phenylethanolamine A by a Novel Immunochromatographic Assay Based on Surface-Enhanced Raman Scattering (SERS) Mingxin Li,† Hong Yang,‡ Shuqun Li,§ Kang Zhao,† Jianguo Li,*,† Danni Jiang,† Lulu Sun,† and Anping Deng*,† †
The Key Laboratory of Health Chemistry and Molecular Diagnosis of Suzhou, College of Chemistry, Chemical Engineering and Materials Science, and ‡College of Pharmacy Sciences, Soochow University, Suzhou 215123, China § China State Institute of Pharmaceutical Industry, Zhangjinag Institute, Shanghai 201203, China S Supporting Information *
ABSTRACT: Phenylethanolamine A (PA) is a new kind of β-agonist, which was illegally used as a feed additive for growth promotion in China. In this study, a novel immunochromatographic assay (ICA) based on surface-enhanced Raman scattering (SERS) for the ultrasensitive and quantitative detection of phenylethanolamine A is presented. The principle of this new ICA is similar to that based on colloidal gold particles, but using AuMBA@Ag-Ab [e.g., polyclonal antibody of PA labeled Au@Ag core− shell nanoparticles (NPs) sandwiched with a Raman reporter (4-mercaptobenzoic acid, MBA)] as a probe. After ICA procedures, the specific Raman scattering intensity of MBA on the test line was measured for quantitative detection of PA. This assay was completed within 15 min. The IC50 and limit of detection (LOD) values of the ICA for PA detection were 0.06 ng mL−1 and 0.32 pg mL−1, respectively, which were 1−3 orders of magnitude lower than those obtained by other immunoassays, indicating the ultrasensitivity of this ICA. There was no cross-reactivity (CR) of the assay with another three β-agonists (ractopamine, clenbuterol, and salbutamol), suggesting high specificity of the SERS-based ICA. A spiking experiment revealed that the recoveries of PA from pig urine samples were in range of 99.9− 101.2% with relative standard deviations (RSDs) of 3.6−5.8%. The results demonstrated that this SERS-based ICA was able to quantitatively detect PA in urine samples with high sensitivity, specificity, precision, and accuracy and might be a powerful method for the analysis of other target analytes in the food area. KEYWORDS: phenylethanolamine A, surface-enhanced Raman scattering (SERS), immunochromatographic assay (ICA), core−shell nanoparticles, Raman reporter, antibody
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INTRODUCTION β-Adrenergic agonists are synthetic phenethanolamine compounds originally used in the therapeutic treatment of asthma and preterm labor in humans.1 They also have the function to improve the production of lean meat and were often illicitly abused as growth promoters in livestock by the promotion of repartitioning of fat into muscles and as doping drugs to enhance the performance of athletes.2,3 However, the illegal use of β-agonists in livestock production has led to toxic effects after human consumption of meat products. Long-term or high-dose use has been shown to illicit deleterious physiological side effects, and a large enough single dosage may initiate an acute toxic response such as cardiac palpitation, tachycardia, nervousness, muscle tremors, and confusion.4 Therefore, βagonists are banned as feed additives for growth promotion in animals in many countries. Unfortunately, although great efforts have made to reinforce the supervision and monitor the use of β-agonists, some other new β-agonists have emerged, such as phenylethanolamine A (PA, Figure 1), which is a byproduct during the ractopamine synthesis process. PA has the same effect as the well-known β-adrenergic agonists clenbuterol, ractopamine, and salbutamol (Figure 1). As a newly found βadrenergic agonist in China, PA has been banned from being used in feeds and animal drinking water in 2010 in the Bulletin 1519 issued by the Ministry of Agriculture of China.5 © XXXX American Chemical Society
Therefore, it is very urgent to establish a simple, low-cost, rapid, sensitive, and specific analytical method for the ultrasensitive determination of PA to ensure food safety and public health. Currently, instrumental analysis is the main analytical method for the determination of β-adrenergic agonists in biological and food samples including liquid chromatography (LC),6 liquid chromatography−mass spectrometry (LC-MS),7 liquid chromatography−tandem mass spectrometry (LC-MS/ MS),8,9 and gas chromatography−mass spectrometry (GCMS).10 In 2010, the Ministry of Agriculture of China issued a standard for the detection of PA in feed using highperformance liquid chromatography−tandem mass spectrometry (HPLC-MS/MS).11 Up to now, seldom have analytical methods been reported to monitor PA except the studies of LC-MS/MS, 1 2 enzyme-linked immunosorbent assay (ELISA),13,14 and electrochemical method.15 Chromatographic methods have high precision, but the instruments are expensive and the pretreatment procedure is complicated and timeconsuming. ELISA is highly selective and sensitive, but a Received: July 27, 2014 Revised: October 21, 2014 Accepted: October 24, 2014
A
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Figure 1. Molecular structures of phenylethanolamine A, clenbuterol, ractopamine, and salbutamol.
Figure 2. (a) Schematic diagrams of the assembly of the ICA strip and the principle of SERS-based ICA for PA detection; (b) preparation of AuMBA@Ag-Ab probe.
detection of some analytes (e.g., pregnancy testing), it is not suitable in some cases when the quantitative level of an analyte is important.25 In past years, some attempts of ICA using fluorescence or quantum dots for quantitative analysis were made.26,27 Nevertheless, a significant limitation of these modified ICA methods is that the results may suffer from optical interference (e.g., photobleaching). Fluorescence ICAs are often complicated by the requirement of an elaborate excitation and detection scheme and by broad emission bands. Surface-enhanced Raman scattering (SERS) is one of the most sensitive analytical techniques available for the scientist today.28 Colloidal Au and Ag in solution can render SERSactive by adsorbate-induced particle aggregation.29,30 Au
primary drawback is its time-consuming and laborious incubation washing and rinsing procedures. The electrochemical method is disturbed by many interferents. Immunochromatography assay (ICA) represents a popular and widely used test principle for point-of-care applications. The main features of the normal Au colloidal particle-based ICA are the user-friendly operation, quickly obtained results, less interference due to chromatographic separation, relatively low cost, and fairly good shelf life.16 At present, ICA has been widely used for the rapid detection of toxic or harmful substances in many fields such as food safety monitoring17−20 and point-of-care diagnostics.21−24 However, although the conventional qualitative analysis can meet the needs of B
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into the above mixture. With the continuous addition of AgNO3 solution in amounts of 0.08, 0.12, 0.16, 0.20, 0.25, 0.30, and 0.40 μmol to 1 mL of AuMBA solution, silver nitrate was reduced by ascorbic acid and the resultant Ag shells continuously grew on the surface of Au seeds. When the purple-red of solution changed to orange-yellow, the solution was further stirred for 30 min and centrifuged at 9000 rpm for 10 min. After removal of the supernatant, the AuMBA@Ag NPs were obtained. The pH value of AuMBA@Ag solution was adjusted to 9.0 with 0.1 mol L−1 K2CO3, and then 1 mL of the solution was mixed with Ab against PA for 3.5 h. Finally, 100 μL of 5% BSA was added to the mixtures and incubated overnight to block the immune complex probes. The final AuMBA@Ag-Ab probes were separated from solutions by centrifugation at 8500 rpm for 10 min. Sediment was diluted with an equal volume of phosphate (PB) buffer and stored at 4 °C until use. Preparation of ICA Strip. The ICA strip consisting of an absorbent pad, a nitrocellulose membrane with a test line and a control line, a sample pad, and a PVC bottom plate in a length of 6 cm was assembled (Figure 2a). The test and control lines were formed by spreading 10 μL of PA−BSA (5.0 mg mL−1) and 10 μL of goat antirabbit IgG (1:100 dilution), respectively, and then dried for 20 min at room temperature. The NC membrane was pasted on the center of the PVC bottom plate. The absorbent pad and the sample pad were pasted to both ends of the NC membrane, overlapping it about 1.5 mm. The assembled plate was cut to 4 mm in width. Strips were sealed into glass bottles in the presence of desiccant gel and nitrogen gas and stored at 4 °C until use. Procedure of SERS-ICA and SERS Signal Acquisition. Six microliters of AuMBA@Ag-Ab was added onto the sample pad 1.0 cm near the NC membrane, and then 150 μL of PA standard/sample solution was pipetted onto the sample pad at 0.5 cm from the end of the strip. The standard/sample solutions together with the AuMBA@ Ag-Ab probes were flowing toward the absorbent pad by capillary action. The color was developed on the test and control zones visually. All ICA procedures were completed within 15 min. The immune complex probes captured by the coating antigen on the test line could be measured with the portable laser Raman Analyzer coupled with a microscope (Eplan, 40 × 0.6). Excitation source was tuned at 785 nm with a laser power of 20 mW at the test line area. The typical integration time used in this assay was 10 s, average with 2 and boxcar with 1. The peak intensity at 1074 cm−1 arose from MBA measured from an average of spectra collected at 10 different spots along the middle part of the test line used for quantification.
colloids have advantages of easy preparation, homogeneity, and biocompatibility with biomolecules such as proteins, antibodies, and DNA, in contrast to Ag NPs, which are not biocompatible and less stable under biological conditions.31 However, the extinction coefficient of the surface plasmon band of Ag NP is approximately 4 times as large as that for an Au NP of the same size.32 Currently, the SERS activity of the Ag@Au or Au@Ag core−shell bimetallic nanoparticles were investigated and compared.33−35 The Au@Ag core−shell nanoparticles were found to have a higher SERS activity mainly due to electronic ligand effect and localized electric field enhancement in core− shell naoparticles.35 Au@Ag core−shell nanoparticles have been explored for biological applications such as oligonucleotide conjugation and SERS-based immunoassay.36 Herein, we describe a novel ICA based on SERS for the detection of PA in urine samples for the first time. The principle and procedures of this approach (Figure 2a) were similar to those based on normal Au colloidal particles. To develop this ICA, a new probe (e.g., AuMBA@Ag-Ab) was synthesized (Figure 2b) by immobilizing a polyclonal antibody against PA on the surface of Au/Ag core−shell nanoparticles that have been sandwiched with a Raman reporter (MBA, which is the most common Raman reporter with specific Raman peaks at 1074 and 1583 cm−1 applied to the development of a SERS-based immunoassay). After ICA procedures, the specific Raman scattering intensity of MBA on the test line was measured for the quantitative detection of PA. The proposed ICA displayed the properties of high sensitivity, specificity, accuracy, and precision for PA detection.
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MATERIALS AND METHODS
Materials and Apparatus. Bovine serum albumin (BSA) and Tween-20 were purchased from Sigma (St. Louis, MO, USA). 4Mercaptobenzoic acid (MBA) was purchased from Aladdin China Ltd. (Shanghai, China). Chloroauric acid (HAuCl4), silver nitrate (AgNO3, 99%), ascorbic acid (99%), and trisodium citrate were obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Phenylethanolamine A (PA) was bought from Toronto Research Chemicals Inc. (Toronto, ON, Canada). Clenbuterol, ractopamine, and salbutamol were obtained from Zhongmu Pharmaceutical Co., Ltd. (Hubei, China). All other chemicals were of analytical grade. The polyclonal Ab against PA and coating antigen (PA−BSA conjugate) were prepared by our group.14 Nitrocellulose (NC) membranes were purchased from Whatman (Shanghai, China). PVC sheets, adhesive tape, and filter paper were purchased from Jieyi Biotechnology Co. Ltd. (Shanghai, China). A deionized-RO water machine (Dura 12FV) was purchased from THE LAB Co. (USA). The digital photographs of the samples were taken with a Cannon IXUS 125 HS digital camera (Cannon Inc., Japan). Transmission electron microscopy (TEM) photographs were taken on a Tecnai G220 from USA FEI Co. The portable Raman Analyzer RamTracer-200-HS was obtained from OptoTrace Technologies, Inc. (Suzhou, China). Preparation of AuMBA@Ag-Ab Probe. The preparation of the AuMBA@Ag-Ab probe is illustrated in Figure 2b. The colloidal gold particles were first prepared according to ref37. Briefly, 1.5 mL of 1% trisodium citrate solution was added rapidly to the boiling aqueous solution of 100 mL of 0.01% HAuCl4 with vigorous stirring. The mixture was kept boiling for 15 min until the color did not change. After the solution cooled down, 10 μL of 1 mmol L−1 MBA solution was added to 10 mL of colloidal gold solution and stirred for 3 h. The MBA modified Au colloids (e.g., AuMBA) were purified by centrifugation and resuspension with the same volume of ultrapure water. After pH adjustment with 0.01 mol L−1 HCl, 10 mL of the above AuMBA colloid was mixed with 2 mL of 0.1 mol L−1 ascorbic acid in a beaker under stirring. Then 1 mmol L−1 AgNO3 was dropwise added
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RESULTS AND DISCUSSION Principle of the SERS-Based ICA. As illustrated in Figure 2a, the principle of SERS-based ICA is similar to that based on conventional gold colloidal particles. When 150 μL of PA standard/sample solution was pipetted onto the sample pad at 0.5 cm from the end of the strip, the standard/sample solutions would move toward the absorbent pad and AuMBA@Ag-Ab would migrate together with liquid solution. If there was no PA in the standard/sample solution (e.g., negative result), the AuMBA@Ag-Ab would be captured by PA−BSA coated on the membrane (test line). Excess AuMBA@Ag-Ab would be moved continuously to get the control line and be captured by goat anti-rabbit IgG. Two orange-yellow bands on the test line and control line would appear due to the accumulation of orangeyellow AuMBA@Ag-Ab. In contrast, for standard/sample solution containing a large amount of PA (e.g., positive result), the specific antibody binding sites on the probe would be occupied first by PA, leaving no binding sites for PA−BSA. Consequently, no AuMBA@Ag-Ab would remain at the PA− BSA location on the NC membrane, but AuMBA@Ag-Ab would move continuously to get to the control line and be captured by goat anti-rabbit IgG. In this case, only the orange-yellow band on the control line appeared. Thus, the degree of intensity of C
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Figure 3. (a, c) TEM images and DLS of Au NPs; (b, d) TEM images and DLS of AuMBA@Ag NPs with Ag shell thickness of 2.5 nm.
AuMBA@Ag-Ab color on the test line was the reverse of the concentration of PA. With the portable Raman analyzer, the SERS scattering intensity arising from the MBA captured on the test line was measured for the quantitative analysis of PA. Preparation and Characterization of AuMBA@Ag NPs. The colloidal gold NPs were first prepared and characterized. The TEM images and dynamic light scattering (DLS) of 100 individual Au NPs are shown in Figure 3a,c. The TEM images revealed that the colloidal gold NPs were homogeneous with a diameter size about 30 nm, whereas the DLS results showed that the Au NPs size distribution was in the range of 30 ± 5 nm. To increase the sensitivity of the SERS-based ICA for PA, it will be desirable to have more MBA sandwiched in the probe. MBA could be easily attached to Au NPs via the thiol group in the MBA molecule. When 10.0 mL of the Au colloids was mixed with different amounts of MBA, more MBA addition to Au colloid solution would make the Au NPs aggregate and even precipitate. We found that 10.0 μL of MBA solution at the concentration of 1 × 10−3 mol L−1 was considered the appropriate amount of MBA required to mix with 10.0 mL of the Au colloids to form the AuMBA NPs. The effect of the amount of AgNO3 in the preparation of AuMBA@Ag NPs on the SERS enhancement of MBA was investigated. With the amount of AgNO3 increasing in the range of 0.08, 0.12, 0.16, 0.20, 0.25, 0.30, and 0.40 μmol to 1 mL of AuMBA solution, silver nitrate was reduced by ascorbic acid and the Ag shells continuously grew on the surface of AuMBA seeds. Accordingly, the color of the resultant AuMBA@Ag NPs was gradually changed from purple-red to orange-yellow to yellow (Supporting Information, Figure S1a). The Raman intensity of the AuMBA@Ag solutions was tested by the portable
Raman Analyzer. As illustrated in Figure S1b, there is a strong dependence of the Raman intensity on the amount of AgNO3 for NPs preparation, for example, on the Ag shell thickness. The maximal Raman intensity was obtained for the AuMBA@Ag solution by employing 0.25 μmol of AgNO3 for NPs preparation. Apparently, the Ag shell of AuMBA@Ag NPs plays an important role in enhancing the MBA signals in the SERS detection. The TEM image and DLS of 100 individual AuMBA@Ag NPs using 0.25 μmol of AgNO3 for NPs preparation are presented in Figure 3, panels b and d, respectively. The TEM image indicated the size of AuMBA@Ag NPs was about 35 nm. The DLS results showed that the AuMBA@Ag NPs size distribution was in the range of 35 ± 5 nm. Compared with the size of gold NPs, the thickness of the Ag shell in AuMBA@Ag NP was estimated to be 2.5 nm. Test of Nonspecific Adsorption. Before PA detection, whether there was nonspecific adsorption was tested. The ICA procedures at zero concentration of analyte in three situations were performed: (a) AuMBA@Ag-Ab as a probe, PA−BSA coated on the test line; (b) AuMBA@Ag-BSA as a probe, PA− BSA coated on the test line; (c) AuMBA@Ag-Ab as a probe, Na2CO3−NaHCO3 solution spread on the test line. The Raman spectra in the above three situations are illustrated in Figure S2. From Raman spectrum a, it was seen that both specific Raman scattering peaks of the MBA at 1074 and 1583 cm−1 with the signal values of 9200 (AU) and 8100 (AU) appeared, which clearly demonstrated that the probe of AuMBA@Ag-Ab was specifically bound with PA−BSA at the test line on the membrane. From Raman spectra b and c, there were almost no SERS signals appearing either at 1074 cm−1 or D
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at 1583 cm−1, indicating that there was no nonspecific adsorption. Optimization of Assay Conditions. The sensitivity of the SERS-based ICA was improved by optimizing a series of conditions such as the amount of the antibody used for preparing AuMBA@Ag-Ab probes, the coating antigen, and the probe utilized on the strip. Here we used the inhibition ratio (B0/B0.1) to evaluate the optimization. B0 and B0.1 are the SERS intensities at 1074 cm−1 when PA concentrations were at 0 and 0.1 ng mL−1, respectively. The bigger the inhibition ratio value, the higher the sensitivity of the assay. In the preparation of AuMBA@Ag-Ab probe, different amounts of antibody were consecutively coupled to 1.0 mL of AuMBA@Ag NPs. The effects of the amount of Ab on the inhibition ratio were investigated (Figure S3a). It was found that when different volumes of antibody (at concentration of 2.0 mg mL−1) from 0.5 to 3.0 μL were applied, the highest value of B0/B0.1 was achieved at 2.0 μL. Thus, the optimal amount of Ab for AuMBA@Ag probe preparation was 2.0 μL. The amount of coating antigen (PA−BSA) immobilized on the test line and the AuMBA@Ag-Ab probe applied in the ICA strip significantly influence the assay sensitivity. As shown in Figure S3b,c, when 10 μL of PA−BSA at concentrations from 3.0 to 7.0 mg mL−1 was applied to the ICA procedure, the highest value of B0/B0.1 was achieved at 5.0 mg mL−1, whereas when volumes of AuMBA@Ag-Ab from 2.0 to 10 μL were tested, the highest value of B0/B0.1 was obtained at 6.0 μL. Therefore, the optimal conditions applied to one ICA strip were 10 μL of PA-BSA at a concentration of 5.0 mg mL−1 and 6.0 μL of AuMBA@Ag-Ab. Sensitivity of the Assay. Under optimal assay conditions, the SERS-based ICA for PA detection was developed. The PA standard solutions were in a range of 0−100 ng mL −1. After the addition of 150 μL of PA standard solution to the sample pad, 15 min later, the color arising from AuMBA@Ag-Ab on the test line was visualized by the naked eye (Figure 4a). With PA concentration increasing, the intensity of the color on test lines was gradually decreased. The SERS intensity at 1074 cm−1 generated from MBA on the test lines was measured by SERS analyzer. As illustrated in Figure 4b, with PA concentration increasing, the SERS intensity at 1074 cm−1 gradually decreased. The standard curve of the SERS-based ICA for PA was constructed in the form of B/B0 × 100% versus log C (Figure 4c), where B and B0 were the SERS intensities when PA solutions were at the standard points and zero concentration. It was seen from Figure 4c that values of IC50 and LOD at 3 × SD were 0.064 ng mL−1 and 0.32 pg mL−1, respectively. The comparison of the IC50 and LOD values achieved in this study with those obtained in other immunoassays for PA detection is given in Table S1. It can be seen from Table S1 that IC50 and LOD values in this work are 1−3 orders of magnitude lower than those obtained by other immunoassays, indicating the ultrasensitivity of the SERS-based ICA. Reproducibility of the SERS Signals. To investigate the reproducibility of the SERS signals, three strips set up with PA standard solutions at 0, 0.1, and 1.0 ng mL−1 after ICA procedures were tested. The SERS intensities at 1074 cm−1 from 10 different spots along the middle parts of the test line in the strips were measured and are illustrated in Figure S4a. It was seen that among the three strips, the higher the PA concentration, the lower the SERS intensities; in each strip, the RSD value of the SERS intensities at 10 measurements were
Figure 4. (a) Digital photograph of the strips after ICA procedure. The numbers on the test lines are the standard concentration of analyte (ng mL−1). (b) Raman spectra arising from MBA on test lines. (c) Standard curve of the SERS-based ICA for PA. The bars represent the standard deviation of the 10 measurements of the intensities of MBA at 1074 cm−1 on test lines.
5.17, 7.93, and 5.64%, respectively, indicating high precision of the SERS signal. Specificity of the Assay. The specificity of a competitive immunoassay is often expressed by cross-reactivity (CR) value. Here we chose another three β-adrenergic agonists (ractopamine, salbutamol, and clenbuterol hydrochloride) to test the specificity of this assay. All compounds including PA were prepared at concentrations of 0, 0.01, 0.1, and 1 ng mL−1 and E
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ment of Jiangsu Higher Education Institutions for financial support of this study.
applied to ICA procedures. The CR values were calculated according to CR (%) = [IC50 of PA)]/[IC50 of tested compound] × 100%. As shown in Figure S4b, for all three βagonists, with the concentration increasing, the SERS intensities were almost constant, suggesting that there was almost no cross-reactivity of this SERS-based ICA with these three βagonists, which demonstrated the high specificity of the proposed assay. Test of Recovery. We chose pig urine samples for the spiking experiment. After dilution of the urine to 1:50 with PVP−PBST (polyvinylpyrrolidone−phosphate buffer saline with 0.1% Tween-20, v/v = 1:1, 1%), the urine sample was spiked with PA to concentrations of 0.01, 0.1, and 1.0 ng mL−1, then 150 μL of the diluted urine sample was applied to ICA procedures. The contents of PA in spiked samples were quantified according to the standard curve run in the same day. As shown in Table 1, the recoveries of the PA from spiked
Notes
The authors declare no competing financial interest.
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(1) Vestergaard, M.; Sejrsen, K.; Klastrup, S. G. Composition and eating quality of longissimus dorsi from young bulls fed the β-agonist cimaterol at consecutive developmental stages. Meat Sci. 1994, 38, 55− 66. (2) Prezelj, A.; Obreza, A.; Pecar, S. Abuse of clenbuterol and its detection. Curr. Med. Chem. 2003, 10, 281−290. (3) Mitchell, G. A.; Dunnavan, G. Illegal use of adrenergic agonists in the United States. J. Anim. Sci. 1998, 76, 208−211. (4) Chan, T. Y. K. Food-borne clenbuterol may have potential for cardiovascular effects with chronic exposure (commentary). J. Toxicol. Clin. Toxicol. 2001, 39, 345−348. (5) Bulletin of the Ministry of Agriculture of the People’s Republic of China, No. 1519, 2010. (6) Zhang, Y. T.; Zhang, Z. J.; Sun, Y. H.; Wei, Y. Development of an analytical method for the determination of β2-agonist residues in animal tissues by high-performance liquid chromatography with online electrogenerated [Cu(HIO6)2]5−-luminol chemiluminescence detection. J. Agric. Food Chem. 2007, 55, 4949−4956. (7) Courant, F.; Pinel, G.; Bichon, E.; Monteau, F.; Antignac, J. P.; LeBizec, B. Development of a metabolomic approach based on liquid chromatography-high resolution mass spectrometry to screen for clenbuterol abuse in calves. Analyst 2009, 134, 1637−1646. (8) Mazzarino, M.; Fiacco, I.; delaTorre, X.; Botrèa, F. Screening and confirmation analysis of stimulants, narcotics and beta-adrenergic agents in human urine by hydrophilic interaction liquid chromatography coupled to mass spectrometry. J. Chromatogr., A 2011, 1218, 8156−8167. (9) Blanca, J.; Muñz, P.; Morgado, M.; Méndez, N.; Aranda, A.; Reuvers, T.; Hooghuis, H. Determination of clenbuterol, ractopamine and zilpaterol in liver and urine by liquid chromatography tandem mass spectrometry. Anal. Chim. Acta 2005, 529, 199−205. (10) Caban, M.; Stepnowski, P.; Kwiatkowski, M.; Migowska, N.; Kumirska, J. Determination of β-blockers and β-agonists using gas chromatography and gas chromatography−mass spectrometry − a comparative study of the derivatization step. J. Chromatogr., A 2011, 1218, 8110−8122. (11) Bulletin of the Ministry of Agriculture of the People’s Republic of China, No. 1486-1-2010, Beijing, 2010. (12) Zhang, M. X.; Li, C.; Wu, Y. L. Determination of phenylethanolamine a in animal hair, tissues and feeds by reversed phase liquid chromatography tandem mass spectrometry with QuEChERS. J. Chromatogr., B 2012, 900, 94−99. (13) Bai, Y. H.; Liu, Z. H.; Bi, Y. F.; Wang, X.; Jin, Y. Z.; Sun, L.; Wang, H. J.; Zhang, C. M.; Xu, S. X. Preparation of polyclonal antibodies and development of a direct competitive enzyme-linked immunosorbent assay to detect residues of phenylethanolamine a in urine samples. J. Agric. Food Chem. 2012, 60, 11618−11624. (14) Cao, B. Y.; He, G. Z.; Yang, H.; Chang, H. F.; Li, S. Q.; Deng, A. P. Development of a highly sensitive and specific enzyme-linked immunosorbent assay (ELISA) for the detection of phenylethanolamine a in tissue and feed samples and confirmed by liquid chromatography tandem mass spectrometry (LC−MS/MS). Talanta 2013, 115, 624−630. (15) Lai, Y. J.; Bai, J.; Zhu, W.; Xian, Y. Z.; Jin, L. T. Electrochemical determination of phenylethanolamine a based on Nafion/MWCNTs/ AuNPs modified carbon electrode. Chin. J. Chem. 2013, 31, 221−229. (16) Posthuma-Trumpie, G. A.; Korf, J.; Amerongen, A. Lateral flow(immuno)assay: its strengths, weaknesses, opportunities, and threats − a literature survey. Anal. Bioanal. Chem. 2009, 393, 569−582. (17) Liu, B. H.; Tsao, Z. J.; Wang, J. J.; Yu, F. Y. Development of a monoclonal antibody against ochratoxin A and its application in
Table 1. Recoveries of PA from Spiked Urine Samples Measured by SERS-Based ICA concn spiked (ng mL−1) 0.01 0.1 1
recovery measured (100%) 101.9 97.5 96.36
94.7 101 103.6
106.3 105 99.7
average
RSD (100%)
100.9 101.2 99.9
5.8 3.7 3.6
samples were 99.9−101.2% with RSD values in range of 3.6− 5.8% (n = 3), indicating that this new approach was an applicable method to effectively detect the target analyte in urine samples. In conclusion, a novel ICA based on SERS using AuMBA@AgAb as a probe for the sensitive and quantitative detection of PA was developed in this study. Under optimal conditions, the IC50 and LOD values of this new approach for PA were 0.06 ng mL−1 and 0.32 pg mL−1, respectively, indicating the ultrasensitivity of the assay. This ICA also displayed high specificity and reproducibility. Furthermore, by sandwich format, the proposed SERS-based ICA might be applied for the detection of macromolecular compounds; and by immobilizing more than two different kinds of coating antigens on the NC membrane as test lines and utilizing corresponding AuMBA@AgAb probes, it might be used for multiplex assays. The proposed SERS-based ICA was proven to be a rapid, simple, and ultrasensitive analytical method for PA and might be also applied for the determination of various target analytes in clinical, biological, food, and environmental analyses.
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ASSOCIATED CONTENT
S Supporting Information *
Figures S1−S4 and Table S1. This material is available free of charge via the Internet at http://pubs.acs.org.
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REFERENCES
AUTHOR INFORMATION
Corresponding Authors
*(A.P.) Phone: +86 512 65882362. Fax: +86 512 65882362. Email: [email protected]. *(J.L.) Phone: +86 512 65882362. Fax: +86 512 65882362. Email: [email protected]. Funding
We thank the National Natural Science Foundation of China (NSFC, Contracts 20835003, 21075087, and 21175097) and a Project Funded by the Priority Academic Program DevelopF
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enzyme-linked immunosorbent assay and gold nanoparticle immunochromatographic strip. Anal. Chem. 2008, 80, 7029−7035. (18) Molinelli, A.; Grossalber, K.; Fuhrer, M.; Baumgartner, S.; Sulyok, M.; Krska, R. Development of qualitative and semiquantitative immunoassay-based rapid strip tests for the detection of T-2 toxin in wheat and oat. J. Agric. Food Chem. 2008, 56, 2589−2594. (19) Sheibani, A.; Tabrizchi, M.; Ghaziaskar, H. S. Determination of aflatoxins B1 and B2 using ion mobility spectrometry. Talanta 2008, 75, 233−238. (20) Xu, Y.; Huang, Z. B.; He, Q. H.; Deng, S. Z.; Li, L. S.; Li, Y. P. Development of an immunochromatographic strip test for the rapid detection of deoxynivalenol in wheat and maize. Food Chem. 2010, 119, 834−839. (21) Krajaejun, T.; Imkhieo, S.; Intaramat, A.; Ratanabanangkoon, K. Development of an immunochromatographic test for rapid serodiagnosis of human pythiosis. Clin. Vaccine Immunol. 2009, 16, 506−509. (22) Sithigorngul, P.; Rukpratanporn, S.; Pecharaburanin, N.; Suksawat, P.; Longyant, S.; Chaivisuthangkura, P.; Sithigorngul, W. A simple and rapid immunochromatographic test strip for detection of pathogenic isolates of Vibrio harveyi. J. Microbiol. Methods 2007, 71, 256−264. (23) Sithigorngul, W.; Rukpratanporn, S.; Sittidilokratna, N.; Pecharaburanin, N.; Longyant, S.; Chaivisuthangkura, P.; Sithigorngul, P. A convenient immunochromatographic test strip for rapid diagnosis of yellow head virus infection in shrimp. J. Virol. Methods 2007, 140, 193−199. (24) Sundar, S.; Maurya, R.; Singh, R. K.; Bharti, K.; Chakravarty, J.; Parekh, A.; Rai, M.; Kumar, K.; Murray, H. W. Rapid, noninvasive diagnosis of visceral Leishmaniasis in India: comparison of two immunochromatographic strip tests for detection of anti-K39 antibody. J. Clin. Microbiol. 2006, 44, 251−253. (25) Liu, G.; Lin, Y. Y.; Wang, J.; Wu, H.; Wai, C. M.; Lin, Y. Disposable electrochemical immunosensor diagnosis device based on nanoparticle probe and immunochromatographic strip. Anal. Chem. 2007, 79, 7644−7653. (26) Zou, Z. X.; Du, D.; Wang, J.; Smith, J. N.; Timchalk, C.; Li, Y. Q.; Lin, Y. H. Quantum dot-based immunochromatographic fluorescent biosensor for biomonitoring trichloropyridinol, a biomarker of exposure to chlorpyrifos. Anal. Chem. 2010, 82, 5125−5133. (27) Kim, Y. M.; Oh, S. W.; Jeong, S. Y.; Pyo, D. J.; Choi, E. Y. Development of an ultrarapid one-step fluorescence immunochromatographic assay system for the quantification of microcystins. Envrion. Sci. Technol. 2003, 37, 1899−1904. (28) Petry, R.; Schmitt, M. J. Raman spectroscopy − a prospective tool in the life sciences. ChemPhysChem 2003, 4, 14−30. (29) Senapati, D.; Dasary, S. S. R.; Singh, A. K.; Senapati, T.; Yu, H. T.; Ray, P. C. A label-free gold-nanoparticle-based SERS assay for direct cyanide detection at the parts-per-trillion level. Chem.−Eur. J. 2011, 17, 8445−8451. (30) Lee, P. C.; Meisel, D. Adsorption and surface-enhanced Raman of dyes on silver and gold sols. J. Phys. Chem. 1982, 86, 3391−3395. (31) Chandran, S. P.; Ghatak, J.; Satyam, P. V.; Sastry, M. Interfacial deposition of Ag on Au seeds leading to Au core Ag shell in organic media. J. Colloid Interface Sci. 2007, 312, 498−505. (32) Cao, Y. W.; Jin, R.; Mirkin, C. A. DNA-modified core-shell Ag/ Au nanoparticles. J. Am. Chem. Soc. 2001, 123, 7961−7962. (33) Rivas, L.; Sanchez-Cortes, S.; Garca-Ramos, J. V.; Morcillo, G. Mixed silver/gold colloids: a study of their formation, morphology, and surface-enhanced Raman activity. Langmuir 2000, 16, 9722−9728. (34) Yang, Y.; Shi, J.; Kawamura, G.; Nogami, M. Preparation of Au− Ag, Ag−Au core−shell bimetallic nanoparticles for surface enhanced Raman scattering. Scr. Mater. 2008, 58, 862−865. (35) Huang, Y.; Yang, Y.; Chen, Z.; Li, X.; Nogami, M. Fabricating Au−Ag core-shell composite films for surface-enhanced Raman scattering. J. Mater. Sci. 2008, 43, 5390−5393. (36) Cui, Y.; Ren, B.; Yao, J. L.; Gu, R. A.; Tian, Z. Q. Synthesis of AgcoreAushell bimetallic nanoparticles for immunoassay based on surface-enhanced Raman spectroscopy. J. Phys. Chem. B 2006, 110, 4002−4006.
(37) Frens, G. Controlled nucleation for the regulation of the particle size in monodisperse gold suspensions. Nat. Phys. Sci. 1973, 241, 20− 22.
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Ultrasensitive and Quantitative Detection of a New β‑Agonist Phenylethanolamine A by a Novel Immunochromatographic Assay Based on Surface-Enhanced Raman Scattering (SERS) Mingxin Li,† Hong Yang,‡ Shuqun Li,§ Kang Zhao,† Jianguo Li,*,† Danni Jiang,† Lulu Sun,† and Anping Deng*,† †
The Key Laboratory of Health Chemistry and Molecular Diagnosis of Suzhou, College of Chemistry, Chemical Engineering and Materials Science, and ‡College of Pharmacy Sciences, Soochow University, Suzhou 215123, China § China State Institute of Pharmaceutical Industry, Zhangjinag Institute, Shanghai 201203, China S Supporting Information *
ABSTRACT: Phenylethanolamine A (PA) is a new kind of β-agonist, which was illegally used as a feed additive for growth promotion in China. In this study, a novel immunochromatographic assay (ICA) based on surface-enhanced Raman scattering (SERS) for the ultrasensitive and quantitative detection of phenylethanolamine A is presented. The principle of this new ICA is similar to that based on colloidal gold particles, but using AuMBA@Ag-Ab [e.g., polyclonal antibody of PA labeled Au@Ag core− shell nanoparticles (NPs) sandwiched with a Raman reporter (4-mercaptobenzoic acid, MBA)] as a probe. After ICA procedures, the specific Raman scattering intensity of MBA on the test line was measured for quantitative detection of PA. This assay was completed within 15 min. The IC50 and limit of detection (LOD) values of the ICA for PA detection were 0.06 ng mL−1 and 0.32 pg mL−1, respectively, which were 1−3 orders of magnitude lower than those obtained by other immunoassays, indicating the ultrasensitivity of this ICA. There was no cross-reactivity (CR) of the assay with another three β-agonists (ractopamine, clenbuterol, and salbutamol), suggesting high specificity of the SERS-based ICA. A spiking experiment revealed that the recoveries of PA from pig urine samples were in range of 99.9− 101.2% with relative standard deviations (RSDs) of 3.6−5.8%. The results demonstrated that this SERS-based ICA was able to quantitatively detect PA in urine samples with high sensitivity, specificity, precision, and accuracy and might be a powerful method for the analysis of other target analytes in the food area. KEYWORDS: phenylethanolamine A, surface-enhanced Raman scattering (SERS), immunochromatographic assay (ICA), core−shell nanoparticles, Raman reporter, antibody
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INTRODUCTION β-Adrenergic agonists are synthetic phenethanolamine compounds originally used in the therapeutic treatment of asthma and preterm labor in humans.1 They also have the function to improve the production of lean meat and were often illicitly abused as growth promoters in livestock by the promotion of repartitioning of fat into muscles and as doping drugs to enhance the performance of athletes.2,3 However, the illegal use of β-agonists in livestock production has led to toxic effects after human consumption of meat products. Long-term or high-dose use has been shown to illicit deleterious physiological side effects, and a large enough single dosage may initiate an acute toxic response such as cardiac palpitation, tachycardia, nervousness, muscle tremors, and confusion.4 Therefore, βagonists are banned as feed additives for growth promotion in animals in many countries. Unfortunately, although great efforts have made to reinforce the supervision and monitor the use of β-agonists, some other new β-agonists have emerged, such as phenylethanolamine A (PA, Figure 1), which is a byproduct during the ractopamine synthesis process. PA has the same effect as the well-known β-adrenergic agonists clenbuterol, ractopamine, and salbutamol (Figure 1). As a newly found βadrenergic agonist in China, PA has been banned from being used in feeds and animal drinking water in 2010 in the Bulletin 1519 issued by the Ministry of Agriculture of China.5 © XXXX American Chemical Society
Therefore, it is very urgent to establish a simple, low-cost, rapid, sensitive, and specific analytical method for the ultrasensitive determination of PA to ensure food safety and public health. Currently, instrumental analysis is the main analytical method for the determination of β-adrenergic agonists in biological and food samples including liquid chromatography (LC),6 liquid chromatography−mass spectrometry (LC-MS),7 liquid chromatography−tandem mass spectrometry (LC-MS/ MS),8,9 and gas chromatography−mass spectrometry (GCMS).10 In 2010, the Ministry of Agriculture of China issued a standard for the detection of PA in feed using highperformance liquid chromatography−tandem mass spectrometry (HPLC-MS/MS).11 Up to now, seldom have analytical methods been reported to monitor PA except the studies of LC-MS/MS, 1 2 enzyme-linked immunosorbent assay (ELISA),13,14 and electrochemical method.15 Chromatographic methods have high precision, but the instruments are expensive and the pretreatment procedure is complicated and timeconsuming. ELISA is highly selective and sensitive, but a Received: July 27, 2014 Revised: October 21, 2014 Accepted: October 24, 2014
A
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Figure 1. Molecular structures of phenylethanolamine A, clenbuterol, ractopamine, and salbutamol.
Figure 2. (a) Schematic diagrams of the assembly of the ICA strip and the principle of SERS-based ICA for PA detection; (b) preparation of AuMBA@Ag-Ab probe.
detection of some analytes (e.g., pregnancy testing), it is not suitable in some cases when the quantitative level of an analyte is important.25 In past years, some attempts of ICA using fluorescence or quantum dots for quantitative analysis were made.26,27 Nevertheless, a significant limitation of these modified ICA methods is that the results may suffer from optical interference (e.g., photobleaching). Fluorescence ICAs are often complicated by the requirement of an elaborate excitation and detection scheme and by broad emission bands. Surface-enhanced Raman scattering (SERS) is one of the most sensitive analytical techniques available for the scientist today.28 Colloidal Au and Ag in solution can render SERSactive by adsorbate-induced particle aggregation.29,30 Au
primary drawback is its time-consuming and laborious incubation washing and rinsing procedures. The electrochemical method is disturbed by many interferents. Immunochromatography assay (ICA) represents a popular and widely used test principle for point-of-care applications. The main features of the normal Au colloidal particle-based ICA are the user-friendly operation, quickly obtained results, less interference due to chromatographic separation, relatively low cost, and fairly good shelf life.16 At present, ICA has been widely used for the rapid detection of toxic or harmful substances in many fields such as food safety monitoring17−20 and point-of-care diagnostics.21−24 However, although the conventional qualitative analysis can meet the needs of B
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into the above mixture. With the continuous addition of AgNO3 solution in amounts of 0.08, 0.12, 0.16, 0.20, 0.25, 0.30, and 0.40 μmol to 1 mL of AuMBA solution, silver nitrate was reduced by ascorbic acid and the resultant Ag shells continuously grew on the surface of Au seeds. When the purple-red of solution changed to orange-yellow, the solution was further stirred for 30 min and centrifuged at 9000 rpm for 10 min. After removal of the supernatant, the AuMBA@Ag NPs were obtained. The pH value of AuMBA@Ag solution was adjusted to 9.0 with 0.1 mol L−1 K2CO3, and then 1 mL of the solution was mixed with Ab against PA for 3.5 h. Finally, 100 μL of 5% BSA was added to the mixtures and incubated overnight to block the immune complex probes. The final AuMBA@Ag-Ab probes were separated from solutions by centrifugation at 8500 rpm for 10 min. Sediment was diluted with an equal volume of phosphate (PB) buffer and stored at 4 °C until use. Preparation of ICA Strip. The ICA strip consisting of an absorbent pad, a nitrocellulose membrane with a test line and a control line, a sample pad, and a PVC bottom plate in a length of 6 cm was assembled (Figure 2a). The test and control lines were formed by spreading 10 μL of PA−BSA (5.0 mg mL−1) and 10 μL of goat antirabbit IgG (1:100 dilution), respectively, and then dried for 20 min at room temperature. The NC membrane was pasted on the center of the PVC bottom plate. The absorbent pad and the sample pad were pasted to both ends of the NC membrane, overlapping it about 1.5 mm. The assembled plate was cut to 4 mm in width. Strips were sealed into glass bottles in the presence of desiccant gel and nitrogen gas and stored at 4 °C until use. Procedure of SERS-ICA and SERS Signal Acquisition. Six microliters of AuMBA@Ag-Ab was added onto the sample pad 1.0 cm near the NC membrane, and then 150 μL of PA standard/sample solution was pipetted onto the sample pad at 0.5 cm from the end of the strip. The standard/sample solutions together with the AuMBA@ Ag-Ab probes were flowing toward the absorbent pad by capillary action. The color was developed on the test and control zones visually. All ICA procedures were completed within 15 min. The immune complex probes captured by the coating antigen on the test line could be measured with the portable laser Raman Analyzer coupled with a microscope (Eplan, 40 × 0.6). Excitation source was tuned at 785 nm with a laser power of 20 mW at the test line area. The typical integration time used in this assay was 10 s, average with 2 and boxcar with 1. The peak intensity at 1074 cm−1 arose from MBA measured from an average of spectra collected at 10 different spots along the middle part of the test line used for quantification.
colloids have advantages of easy preparation, homogeneity, and biocompatibility with biomolecules such as proteins, antibodies, and DNA, in contrast to Ag NPs, which are not biocompatible and less stable under biological conditions.31 However, the extinction coefficient of the surface plasmon band of Ag NP is approximately 4 times as large as that for an Au NP of the same size.32 Currently, the SERS activity of the Ag@Au or Au@Ag core−shell bimetallic nanoparticles were investigated and compared.33−35 The Au@Ag core−shell nanoparticles were found to have a higher SERS activity mainly due to electronic ligand effect and localized electric field enhancement in core− shell naoparticles.35 Au@Ag core−shell nanoparticles have been explored for biological applications such as oligonucleotide conjugation and SERS-based immunoassay.36 Herein, we describe a novel ICA based on SERS for the detection of PA in urine samples for the first time. The principle and procedures of this approach (Figure 2a) were similar to those based on normal Au colloidal particles. To develop this ICA, a new probe (e.g., AuMBA@Ag-Ab) was synthesized (Figure 2b) by immobilizing a polyclonal antibody against PA on the surface of Au/Ag core−shell nanoparticles that have been sandwiched with a Raman reporter (MBA, which is the most common Raman reporter with specific Raman peaks at 1074 and 1583 cm−1 applied to the development of a SERS-based immunoassay). After ICA procedures, the specific Raman scattering intensity of MBA on the test line was measured for the quantitative detection of PA. The proposed ICA displayed the properties of high sensitivity, specificity, accuracy, and precision for PA detection.
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MATERIALS AND METHODS
Materials and Apparatus. Bovine serum albumin (BSA) and Tween-20 were purchased from Sigma (St. Louis, MO, USA). 4Mercaptobenzoic acid (MBA) was purchased from Aladdin China Ltd. (Shanghai, China). Chloroauric acid (HAuCl4), silver nitrate (AgNO3, 99%), ascorbic acid (99%), and trisodium citrate were obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Phenylethanolamine A (PA) was bought from Toronto Research Chemicals Inc. (Toronto, ON, Canada). Clenbuterol, ractopamine, and salbutamol were obtained from Zhongmu Pharmaceutical Co., Ltd. (Hubei, China). All other chemicals were of analytical grade. The polyclonal Ab against PA and coating antigen (PA−BSA conjugate) were prepared by our group.14 Nitrocellulose (NC) membranes were purchased from Whatman (Shanghai, China). PVC sheets, adhesive tape, and filter paper were purchased from Jieyi Biotechnology Co. Ltd. (Shanghai, China). A deionized-RO water machine (Dura 12FV) was purchased from THE LAB Co. (USA). The digital photographs of the samples were taken with a Cannon IXUS 125 HS digital camera (Cannon Inc., Japan). Transmission electron microscopy (TEM) photographs were taken on a Tecnai G220 from USA FEI Co. The portable Raman Analyzer RamTracer-200-HS was obtained from OptoTrace Technologies, Inc. (Suzhou, China). Preparation of AuMBA@Ag-Ab Probe. The preparation of the AuMBA@Ag-Ab probe is illustrated in Figure 2b. The colloidal gold particles were first prepared according to ref37. Briefly, 1.5 mL of 1% trisodium citrate solution was added rapidly to the boiling aqueous solution of 100 mL of 0.01% HAuCl4 with vigorous stirring. The mixture was kept boiling for 15 min until the color did not change. After the solution cooled down, 10 μL of 1 mmol L−1 MBA solution was added to 10 mL of colloidal gold solution and stirred for 3 h. The MBA modified Au colloids (e.g., AuMBA) were purified by centrifugation and resuspension with the same volume of ultrapure water. After pH adjustment with 0.01 mol L−1 HCl, 10 mL of the above AuMBA colloid was mixed with 2 mL of 0.1 mol L−1 ascorbic acid in a beaker under stirring. Then 1 mmol L−1 AgNO3 was dropwise added
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RESULTS AND DISCUSSION Principle of the SERS-Based ICA. As illustrated in Figure 2a, the principle of SERS-based ICA is similar to that based on conventional gold colloidal particles. When 150 μL of PA standard/sample solution was pipetted onto the sample pad at 0.5 cm from the end of the strip, the standard/sample solutions would move toward the absorbent pad and AuMBA@Ag-Ab would migrate together with liquid solution. If there was no PA in the standard/sample solution (e.g., negative result), the AuMBA@Ag-Ab would be captured by PA−BSA coated on the membrane (test line). Excess AuMBA@Ag-Ab would be moved continuously to get the control line and be captured by goat anti-rabbit IgG. Two orange-yellow bands on the test line and control line would appear due to the accumulation of orangeyellow AuMBA@Ag-Ab. In contrast, for standard/sample solution containing a large amount of PA (e.g., positive result), the specific antibody binding sites on the probe would be occupied first by PA, leaving no binding sites for PA−BSA. Consequently, no AuMBA@Ag-Ab would remain at the PA− BSA location on the NC membrane, but AuMBA@Ag-Ab would move continuously to get to the control line and be captured by goat anti-rabbit IgG. In this case, only the orange-yellow band on the control line appeared. Thus, the degree of intensity of C
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Figure 3. (a, c) TEM images and DLS of Au NPs; (b, d) TEM images and DLS of AuMBA@Ag NPs with Ag shell thickness of 2.5 nm.
AuMBA@Ag-Ab color on the test line was the reverse of the concentration of PA. With the portable Raman analyzer, the SERS scattering intensity arising from the MBA captured on the test line was measured for the quantitative analysis of PA. Preparation and Characterization of AuMBA@Ag NPs. The colloidal gold NPs were first prepared and characterized. The TEM images and dynamic light scattering (DLS) of 100 individual Au NPs are shown in Figure 3a,c. The TEM images revealed that the colloidal gold NPs were homogeneous with a diameter size about 30 nm, whereas the DLS results showed that the Au NPs size distribution was in the range of 30 ± 5 nm. To increase the sensitivity of the SERS-based ICA for PA, it will be desirable to have more MBA sandwiched in the probe. MBA could be easily attached to Au NPs via the thiol group in the MBA molecule. When 10.0 mL of the Au colloids was mixed with different amounts of MBA, more MBA addition to Au colloid solution would make the Au NPs aggregate and even precipitate. We found that 10.0 μL of MBA solution at the concentration of 1 × 10−3 mol L−1 was considered the appropriate amount of MBA required to mix with 10.0 mL of the Au colloids to form the AuMBA NPs. The effect of the amount of AgNO3 in the preparation of AuMBA@Ag NPs on the SERS enhancement of MBA was investigated. With the amount of AgNO3 increasing in the range of 0.08, 0.12, 0.16, 0.20, 0.25, 0.30, and 0.40 μmol to 1 mL of AuMBA solution, silver nitrate was reduced by ascorbic acid and the Ag shells continuously grew on the surface of AuMBA seeds. Accordingly, the color of the resultant AuMBA@Ag NPs was gradually changed from purple-red to orange-yellow to yellow (Supporting Information, Figure S1a). The Raman intensity of the AuMBA@Ag solutions was tested by the portable
Raman Analyzer. As illustrated in Figure S1b, there is a strong dependence of the Raman intensity on the amount of AgNO3 for NPs preparation, for example, on the Ag shell thickness. The maximal Raman intensity was obtained for the AuMBA@Ag solution by employing 0.25 μmol of AgNO3 for NPs preparation. Apparently, the Ag shell of AuMBA@Ag NPs plays an important role in enhancing the MBA signals in the SERS detection. The TEM image and DLS of 100 individual AuMBA@Ag NPs using 0.25 μmol of AgNO3 for NPs preparation are presented in Figure 3, panels b and d, respectively. The TEM image indicated the size of AuMBA@Ag NPs was about 35 nm. The DLS results showed that the AuMBA@Ag NPs size distribution was in the range of 35 ± 5 nm. Compared with the size of gold NPs, the thickness of the Ag shell in AuMBA@Ag NP was estimated to be 2.5 nm. Test of Nonspecific Adsorption. Before PA detection, whether there was nonspecific adsorption was tested. The ICA procedures at zero concentration of analyte in three situations were performed: (a) AuMBA@Ag-Ab as a probe, PA−BSA coated on the test line; (b) AuMBA@Ag-BSA as a probe, PA− BSA coated on the test line; (c) AuMBA@Ag-Ab as a probe, Na2CO3−NaHCO3 solution spread on the test line. The Raman spectra in the above three situations are illustrated in Figure S2. From Raman spectrum a, it was seen that both specific Raman scattering peaks of the MBA at 1074 and 1583 cm−1 with the signal values of 9200 (AU) and 8100 (AU) appeared, which clearly demonstrated that the probe of AuMBA@Ag-Ab was specifically bound with PA−BSA at the test line on the membrane. From Raman spectra b and c, there were almost no SERS signals appearing either at 1074 cm−1 or D
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at 1583 cm−1, indicating that there was no nonspecific adsorption. Optimization of Assay Conditions. The sensitivity of the SERS-based ICA was improved by optimizing a series of conditions such as the amount of the antibody used for preparing AuMBA@Ag-Ab probes, the coating antigen, and the probe utilized on the strip. Here we used the inhibition ratio (B0/B0.1) to evaluate the optimization. B0 and B0.1 are the SERS intensities at 1074 cm−1 when PA concentrations were at 0 and 0.1 ng mL−1, respectively. The bigger the inhibition ratio value, the higher the sensitivity of the assay. In the preparation of AuMBA@Ag-Ab probe, different amounts of antibody were consecutively coupled to 1.0 mL of AuMBA@Ag NPs. The effects of the amount of Ab on the inhibition ratio were investigated (Figure S3a). It was found that when different volumes of antibody (at concentration of 2.0 mg mL−1) from 0.5 to 3.0 μL were applied, the highest value of B0/B0.1 was achieved at 2.0 μL. Thus, the optimal amount of Ab for AuMBA@Ag probe preparation was 2.0 μL. The amount of coating antigen (PA−BSA) immobilized on the test line and the AuMBA@Ag-Ab probe applied in the ICA strip significantly influence the assay sensitivity. As shown in Figure S3b,c, when 10 μL of PA−BSA at concentrations from 3.0 to 7.0 mg mL−1 was applied to the ICA procedure, the highest value of B0/B0.1 was achieved at 5.0 mg mL−1, whereas when volumes of AuMBA@Ag-Ab from 2.0 to 10 μL were tested, the highest value of B0/B0.1 was obtained at 6.0 μL. Therefore, the optimal conditions applied to one ICA strip were 10 μL of PA-BSA at a concentration of 5.0 mg mL−1 and 6.0 μL of AuMBA@Ag-Ab. Sensitivity of the Assay. Under optimal assay conditions, the SERS-based ICA for PA detection was developed. The PA standard solutions were in a range of 0−100 ng mL −1. After the addition of 150 μL of PA standard solution to the sample pad, 15 min later, the color arising from AuMBA@Ag-Ab on the test line was visualized by the naked eye (Figure 4a). With PA concentration increasing, the intensity of the color on test lines was gradually decreased. The SERS intensity at 1074 cm−1 generated from MBA on the test lines was measured by SERS analyzer. As illustrated in Figure 4b, with PA concentration increasing, the SERS intensity at 1074 cm−1 gradually decreased. The standard curve of the SERS-based ICA for PA was constructed in the form of B/B0 × 100% versus log C (Figure 4c), where B and B0 were the SERS intensities when PA solutions were at the standard points and zero concentration. It was seen from Figure 4c that values of IC50 and LOD at 3 × SD were 0.064 ng mL−1 and 0.32 pg mL−1, respectively. The comparison of the IC50 and LOD values achieved in this study with those obtained in other immunoassays for PA detection is given in Table S1. It can be seen from Table S1 that IC50 and LOD values in this work are 1−3 orders of magnitude lower than those obtained by other immunoassays, indicating the ultrasensitivity of the SERS-based ICA. Reproducibility of the SERS Signals. To investigate the reproducibility of the SERS signals, three strips set up with PA standard solutions at 0, 0.1, and 1.0 ng mL−1 after ICA procedures were tested. The SERS intensities at 1074 cm−1 from 10 different spots along the middle parts of the test line in the strips were measured and are illustrated in Figure S4a. It was seen that among the three strips, the higher the PA concentration, the lower the SERS intensities; in each strip, the RSD value of the SERS intensities at 10 measurements were
Figure 4. (a) Digital photograph of the strips after ICA procedure. The numbers on the test lines are the standard concentration of analyte (ng mL−1). (b) Raman spectra arising from MBA on test lines. (c) Standard curve of the SERS-based ICA for PA. The bars represent the standard deviation of the 10 measurements of the intensities of MBA at 1074 cm−1 on test lines.
5.17, 7.93, and 5.64%, respectively, indicating high precision of the SERS signal. Specificity of the Assay. The specificity of a competitive immunoassay is often expressed by cross-reactivity (CR) value. Here we chose another three β-adrenergic agonists (ractopamine, salbutamol, and clenbuterol hydrochloride) to test the specificity of this assay. All compounds including PA were prepared at concentrations of 0, 0.01, 0.1, and 1 ng mL−1 and E
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ment of Jiangsu Higher Education Institutions for financial support of this study.
applied to ICA procedures. The CR values were calculated according to CR (%) = [IC50 of PA)]/[IC50 of tested compound] × 100%. As shown in Figure S4b, for all three βagonists, with the concentration increasing, the SERS intensities were almost constant, suggesting that there was almost no cross-reactivity of this SERS-based ICA with these three βagonists, which demonstrated the high specificity of the proposed assay. Test of Recovery. We chose pig urine samples for the spiking experiment. After dilution of the urine to 1:50 with PVP−PBST (polyvinylpyrrolidone−phosphate buffer saline with 0.1% Tween-20, v/v = 1:1, 1%), the urine sample was spiked with PA to concentrations of 0.01, 0.1, and 1.0 ng mL−1, then 150 μL of the diluted urine sample was applied to ICA procedures. The contents of PA in spiked samples were quantified according to the standard curve run in the same day. As shown in Table 1, the recoveries of the PA from spiked
Notes
The authors declare no competing financial interest.
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(1) Vestergaard, M.; Sejrsen, K.; Klastrup, S. G. Composition and eating quality of longissimus dorsi from young bulls fed the β-agonist cimaterol at consecutive developmental stages. Meat Sci. 1994, 38, 55− 66. (2) Prezelj, A.; Obreza, A.; Pecar, S. Abuse of clenbuterol and its detection. Curr. Med. Chem. 2003, 10, 281−290. (3) Mitchell, G. A.; Dunnavan, G. Illegal use of adrenergic agonists in the United States. J. Anim. Sci. 1998, 76, 208−211. (4) Chan, T. Y. K. Food-borne clenbuterol may have potential for cardiovascular effects with chronic exposure (commentary). J. Toxicol. Clin. Toxicol. 2001, 39, 345−348. (5) Bulletin of the Ministry of Agriculture of the People’s Republic of China, No. 1519, 2010. (6) Zhang, Y. T.; Zhang, Z. J.; Sun, Y. H.; Wei, Y. Development of an analytical method for the determination of β2-agonist residues in animal tissues by high-performance liquid chromatography with online electrogenerated [Cu(HIO6)2]5−-luminol chemiluminescence detection. J. Agric. Food Chem. 2007, 55, 4949−4956. (7) Courant, F.; Pinel, G.; Bichon, E.; Monteau, F.; Antignac, J. P.; LeBizec, B. Development of a metabolomic approach based on liquid chromatography-high resolution mass spectrometry to screen for clenbuterol abuse in calves. Analyst 2009, 134, 1637−1646. (8) Mazzarino, M.; Fiacco, I.; delaTorre, X.; Botrèa, F. Screening and confirmation analysis of stimulants, narcotics and beta-adrenergic agents in human urine by hydrophilic interaction liquid chromatography coupled to mass spectrometry. J. Chromatogr., A 2011, 1218, 8156−8167. (9) Blanca, J.; Muñz, P.; Morgado, M.; Méndez, N.; Aranda, A.; Reuvers, T.; Hooghuis, H. Determination of clenbuterol, ractopamine and zilpaterol in liver and urine by liquid chromatography tandem mass spectrometry. Anal. Chim. Acta 2005, 529, 199−205. (10) Caban, M.; Stepnowski, P.; Kwiatkowski, M.; Migowska, N.; Kumirska, J. Determination of β-blockers and β-agonists using gas chromatography and gas chromatography−mass spectrometry − a comparative study of the derivatization step. J. Chromatogr., A 2011, 1218, 8110−8122. (11) Bulletin of the Ministry of Agriculture of the People’s Republic of China, No. 1486-1-2010, Beijing, 2010. (12) Zhang, M. X.; Li, C.; Wu, Y. L. Determination of phenylethanolamine a in animal hair, tissues and feeds by reversed phase liquid chromatography tandem mass spectrometry with QuEChERS. J. Chromatogr., B 2012, 900, 94−99. (13) Bai, Y. H.; Liu, Z. H.; Bi, Y. F.; Wang, X.; Jin, Y. Z.; Sun, L.; Wang, H. J.; Zhang, C. M.; Xu, S. X. Preparation of polyclonal antibodies and development of a direct competitive enzyme-linked immunosorbent assay to detect residues of phenylethanolamine a in urine samples. J. Agric. Food Chem. 2012, 60, 11618−11624. (14) Cao, B. Y.; He, G. Z.; Yang, H.; Chang, H. F.; Li, S. Q.; Deng, A. P. Development of a highly sensitive and specific enzyme-linked immunosorbent assay (ELISA) for the detection of phenylethanolamine a in tissue and feed samples and confirmed by liquid chromatography tandem mass spectrometry (LC−MS/MS). Talanta 2013, 115, 624−630. (15) Lai, Y. J.; Bai, J.; Zhu, W.; Xian, Y. Z.; Jin, L. T. Electrochemical determination of phenylethanolamine a based on Nafion/MWCNTs/ AuNPs modified carbon electrode. Chin. J. Chem. 2013, 31, 221−229. (16) Posthuma-Trumpie, G. A.; Korf, J.; Amerongen, A. Lateral flow(immuno)assay: its strengths, weaknesses, opportunities, and threats − a literature survey. Anal. Bioanal. Chem. 2009, 393, 569−582. (17) Liu, B. H.; Tsao, Z. J.; Wang, J. J.; Yu, F. Y. Development of a monoclonal antibody against ochratoxin A and its application in
Table 1. Recoveries of PA from Spiked Urine Samples Measured by SERS-Based ICA concn spiked (ng mL−1) 0.01 0.1 1
recovery measured (100%) 101.9 97.5 96.36
94.7 101 103.6
106.3 105 99.7
average
RSD (100%)
100.9 101.2 99.9
5.8 3.7 3.6
samples were 99.9−101.2% with RSD values in range of 3.6− 5.8% (n = 3), indicating that this new approach was an applicable method to effectively detect the target analyte in urine samples. In conclusion, a novel ICA based on SERS using AuMBA@AgAb as a probe for the sensitive and quantitative detection of PA was developed in this study. Under optimal conditions, the IC50 and LOD values of this new approach for PA were 0.06 ng mL−1 and 0.32 pg mL−1, respectively, indicating the ultrasensitivity of the assay. This ICA also displayed high specificity and reproducibility. Furthermore, by sandwich format, the proposed SERS-based ICA might be applied for the detection of macromolecular compounds; and by immobilizing more than two different kinds of coating antigens on the NC membrane as test lines and utilizing corresponding AuMBA@AgAb probes, it might be used for multiplex assays. The proposed SERS-based ICA was proven to be a rapid, simple, and ultrasensitive analytical method for PA and might be also applied for the determination of various target analytes in clinical, biological, food, and environmental analyses.
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ASSOCIATED CONTENT
S Supporting Information *
Figures S1−S4 and Table S1. This material is available free of charge via the Internet at http://pubs.acs.org.
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REFERENCES
AUTHOR INFORMATION
Corresponding Authors
*(A.P.) Phone: +86 512 65882362. Fax: +86 512 65882362. Email: [email protected]. *(J.L.) Phone: +86 512 65882362. Fax: +86 512 65882362. Email: [email protected]. Funding
We thank the National Natural Science Foundation of China (NSFC, Contracts 20835003, 21075087, and 21175097) and a Project Funded by the Priority Academic Program DevelopF
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enzyme-linked immunosorbent assay and gold nanoparticle immunochromatographic strip. Anal. Chem. 2008, 80, 7029−7035. (18) Molinelli, A.; Grossalber, K.; Fuhrer, M.; Baumgartner, S.; Sulyok, M.; Krska, R. Development of qualitative and semiquantitative immunoassay-based rapid strip tests for the detection of T-2 toxin in wheat and oat. J. Agric. Food Chem. 2008, 56, 2589−2594. (19) Sheibani, A.; Tabrizchi, M.; Ghaziaskar, H. S. Determination of aflatoxins B1 and B2 using ion mobility spectrometry. Talanta 2008, 75, 233−238. (20) Xu, Y.; Huang, Z. B.; He, Q. H.; Deng, S. Z.; Li, L. S.; Li, Y. P. Development of an immunochromatographic strip test for the rapid detection of deoxynivalenol in wheat and maize. Food Chem. 2010, 119, 834−839. (21) Krajaejun, T.; Imkhieo, S.; Intaramat, A.; Ratanabanangkoon, K. Development of an immunochromatographic test for rapid serodiagnosis of human pythiosis. Clin. Vaccine Immunol. 2009, 16, 506−509. (22) Sithigorngul, P.; Rukpratanporn, S.; Pecharaburanin, N.; Suksawat, P.; Longyant, S.; Chaivisuthangkura, P.; Sithigorngul, W. A simple and rapid immunochromatographic test strip for detection of pathogenic isolates of Vibrio harveyi. J. Microbiol. Methods 2007, 71, 256−264. (23) Sithigorngul, W.; Rukpratanporn, S.; Sittidilokratna, N.; Pecharaburanin, N.; Longyant, S.; Chaivisuthangkura, P.; Sithigorngul, P. A convenient immunochromatographic test strip for rapid diagnosis of yellow head virus infection in shrimp. J. Virol. Methods 2007, 140, 193−199. (24) Sundar, S.; Maurya, R.; Singh, R. K.; Bharti, K.; Chakravarty, J.; Parekh, A.; Rai, M.; Kumar, K.; Murray, H. W. Rapid, noninvasive diagnosis of visceral Leishmaniasis in India: comparison of two immunochromatographic strip tests for detection of anti-K39 antibody. J. Clin. Microbiol. 2006, 44, 251−253. (25) Liu, G.; Lin, Y. Y.; Wang, J.; Wu, H.; Wai, C. M.; Lin, Y. Disposable electrochemical immunosensor diagnosis device based on nanoparticle probe and immunochromatographic strip. Anal. Chem. 2007, 79, 7644−7653. (26) Zou, Z. X.; Du, D.; Wang, J.; Smith, J. N.; Timchalk, C.; Li, Y. Q.; Lin, Y. H. Quantum dot-based immunochromatographic fluorescent biosensor for biomonitoring trichloropyridinol, a biomarker of exposure to chlorpyrifos. Anal. Chem. 2010, 82, 5125−5133. (27) Kim, Y. M.; Oh, S. W.; Jeong, S. Y.; Pyo, D. J.; Choi, E. Y. Development of an ultrarapid one-step fluorescence immunochromatographic assay system for the quantification of microcystins. Envrion. Sci. Technol. 2003, 37, 1899−1904. (28) Petry, R.; Schmitt, M. J. Raman spectroscopy − a prospective tool in the life sciences. ChemPhysChem 2003, 4, 14−30. (29) Senapati, D.; Dasary, S. S. R.; Singh, A. K.; Senapati, T.; Yu, H. T.; Ray, P. C. A label-free gold-nanoparticle-based SERS assay for direct cyanide detection at the parts-per-trillion level. Chem.−Eur. J. 2011, 17, 8445−8451. (30) Lee, P. C.; Meisel, D. Adsorption and surface-enhanced Raman of dyes on silver and gold sols. J. Phys. Chem. 1982, 86, 3391−3395. (31) Chandran, S. P.; Ghatak, J.; Satyam, P. V.; Sastry, M. Interfacial deposition of Ag on Au seeds leading to Au core Ag shell in organic media. J. Colloid Interface Sci. 2007, 312, 498−505. (32) Cao, Y. W.; Jin, R.; Mirkin, C. A. DNA-modified core-shell Ag/ Au nanoparticles. J. Am. Chem. Soc. 2001, 123, 7961−7962. (33) Rivas, L.; Sanchez-Cortes, S.; Garca-Ramos, J. V.; Morcillo, G. Mixed silver/gold colloids: a study of their formation, morphology, and surface-enhanced Raman activity. Langmuir 2000, 16, 9722−9728. (34) Yang, Y.; Shi, J.; Kawamura, G.; Nogami, M. Preparation of Au− Ag, Ag−Au core−shell bimetallic nanoparticles for surface enhanced Raman scattering. Scr. Mater. 2008, 58, 862−865. (35) Huang, Y.; Yang, Y.; Chen, Z.; Li, X.; Nogami, M. Fabricating Au−Ag core-shell composite films for surface-enhanced Raman scattering. J. Mater. Sci. 2008, 43, 5390−5393. (36) Cui, Y.; Ren, B.; Yao, J. L.; Gu, R. A.; Tian, Z. Q. Synthesis of AgcoreAushell bimetallic nanoparticles for immunoassay based on surface-enhanced Raman spectroscopy. J. Phys. Chem. B 2006, 110, 4002−4006.
(37) Frens, G. Controlled nucleation for the regulation of the particle size in monodisperse gold suspensions. Nat. Phys. Sci. 1973, 241, 20− 22.
G
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