Detection of a Foreign Protein in Milk Using Surface-Enhanced

Feb 9, 2011 - Label-Free Strategy for In-Situ Analysis of Protein Binding ... Byung Ho Lee , Sangjae Seo , Min Hyeok Kim , Youngjin Kim , Soojin Jo ...
0 downloads 0 Views 737KB Size
Download PDF
LETTER pubs.acs.org/ac

Detection of a Foreign Protein in Milk Using Surface-Enhanced Raman Spectroscopy Coupled with Antibody-Modified Silver Dendrites Lili He,† Tom Rodda,† Christy L. Haynes,‡ Timothy Deschaines,§ Todd Strother,§ Francisco Diez-Gonzalez,† and Theodore P. Labuza*,† †

Department of Food Science and Nutrition, University of Minnesota, 1334 Eckles Avenue, St. Paul, Minnesota 55108, United States Department of Chemistry, University of Minnesota, 207 Pleasant Street SE, Minneapolis, Minnesota 55455, United States § Raman Spectroscopy Group, Thermo Fisher Scientific, 5225 Verona Road, Building 4, Madison, Wisconsin 53711, United States ‡

ABSTRACT: Herein we developed a rapid and simple method which used surfaceenhanced Raman spectroscopy (SERS) coupled with antibody-modified silver dendrites to detect ovalbumin (OVA), the egg white protein, introduced into whole milk. OVA was first captured out of milk by use of antibody-modified silver dendrites and then directly measured on the silver dendrites by Raman spectroscopy. Results show that this method is capable of detecting OVA at 0.1 μg/mL in phosphate buffered saline (PBS) and 5 μg/mL in milk within 30 min based on the principal component analysis. This method has the potential for wide use in areas such as allergenic protein detection and bioterrorism agent detection in complex matrixes.

S

ince the accidental discovery of the surface-enhanced Raman scattering (SERS) phenomena in 1974,1,2 SERS, a variant of Raman spectroscopy which exploits nanoscale optical phenomena,3 has been employed for a wide range of analyses. The SERS technique is especially useful when rapid and nondestructive chemical signatures are required, with limits of detection often determined by the properties of the nanoscale-roughened substrates. There are many reports of SERS based immunoassays and most of them used a SERS-active dye as a reporter;4-9 however, few studies have applied this technique in complex matrixes such as foods. One challenge in applying SERS for detection of proteins in a rich protein content matrix such as milk is that the Raman signals of most proteins are very similar and relatively weak. It is almost impossible to discriminate one protein out of a protein matrix without specific extraction of that protein before detection. A second challenge is the fluorescence interference from food components that can overwhelm the Raman signal. In addition, since the SERS phenomenon is dependent on nanosubstrates, a different nanosubstrate may result in different spectral information due to the chemical enhancement mechanism.3 Lack of a uniform pattern of the nanosubstrate may also cause a large data variance due to the uneven “hot-spots” on the nanosubstrate. Therefore, to standardize a reliable and cost-effective substrate is of critical importance for a robust application of the SERS techniques. In this study, we aim to demonstrate the feasibility of SERS detection of a foreign protein that has been introduced into a complex matrix, milk. Current techniques for detecting proteins within a complex matrix are largely limited to immunoassays such as enzyme-linked immunosorbent assay (ELISA),10-12 or chromatography coupled with different detectors.13,14 These methods are time-consuming, labor intensive, and/or very expensive. As of now, there is no method that can rapidly, simply, and r 2011 American Chemical Society

economically detect foreign proteins that have been intentionally or accidentally introduced into a complex matrix. Such a method will have wide applications in the food safety and defense areas. One example is the detection of allergenic proteins, such as tree nut proteins and egg proteins in a complex food or environmental surface swab sample. Cross contamination of foreign allergenic proteins in food products due to improper processing in the factory has caused many incidents of allergic reaction as well as food recalls.15-17 As only a tiny amount of allergenic protein (i.e., as low as 100 μg for peanut protein)18 could trigger an allergic reaction, it is of considerable importance to have a quick, easy, and cost-effective detection method for allergenic proteins in food processing facilities to validate the cleanliness of the equipment. Another application is the detection of potential bioterrorism agents such as botulinum toxin, ricin, abrin, and other protein toxins in complex food matrixes such as milk. The milk processing system is one of the most likely targets for terrorists due to its vulnerability.19 A rapid detection method is critically needed to interrupt an intentionally contaminated load from being introduced into the supply chain. This would eliminate any catastrophic consequences to public health and economic impact. Accordingly, the objective of this study was to develop a simple SERS method coupled with antibody-modified silver dendrites to rapidly detect ovalbumin (OVA), the egg white protein, introduced into whole milk within 30 min. OVA was first captured out of milk by use of antibody-modified silver dendrites and then directly measured on the silver dendrites by Raman spectroscopy. In this case, OVA is a proof-of-concept protein and this method can be generalized to any protein with Received: December 10, 2010 Accepted: February 1, 2011 Published: February 09, 2011 1510

dx.doi.org/10.1021/ac1032353 | Anal. Chem. 2011, 83, 1510–1513

Analytical Chemistry

LETTER

Figure 1. Illustration of SERS detection based on the antibody modified silver dendrites complex.

Figure 3. A PCA plot of spectral data of Ag, Ag-G, Ag-G-antiOVA, and Ag-G-antiOVA-OVA. Ag, silver; G, protein G; OVA, ovalbumin.

Figure 2. Raw SERS spectra of Ag, Ag-G, Ag-G-antiOVA, and Ag-GantiOVA-OVA (a) and the second derivative transformation of spectra of Ag-G-antiOVA, and Ag-G-antiOVA-OVA (b). Ag, silver; G, protein G; OVA, ovalbumin.

an appropriate antibody. The 30 min time frame is critical for the aforementioned applications in milk supply screening. To the best of our knowledge, this is the first report of using SERS coupled with an antibody-modified SERS substrate to rapidly detect a foreign protein in a complex food, milk, within 30 min.

’ EXPERIMENTAL SECTION Preparation of Antibody-Modified Ag Dendrites. Silver (Ag) dendrites were prepared through a simple replacement reaction involving both zinc (Zn) and silver nitrate (AgNO3) according to a previously published method.20 The prepared Ag dendrites were kept in water and stable for at least half a year. The Ag dendrite optical properties make them viable SERS substrates for excitation wavelengths of 500-800 nm.20 Before addition of protein G (Fisher Scientific, Rochester, NY), the Ag dendrite-containing water was adjusted to pH 4.5, the isoelectric point (pI) of protein G, using 0.02 mol/L HCl. Then, 500 μL Ag dendrites (∼2 mg) were mixed with 100 μL of 1 mg/mL protein G under constant rotation for 30 min at room temperature (RT). Bovine serum albumin (BSA) was added to a final concentration of 0.2% (w/v) to stabilize the mixture. After centrifugation at 2000g for 1 min at RT, the pellet (Ag-G) was washed twice using phosphate buffered saline (PBS, pH 7.4) and resuspended in 500 mL of 0.2% BSA. Protein G is an

immunoglobulin-binding protein expressed in Streptococcal bacteria. The protein G-modified Ag dendrites can be used to bind a variety of antibodies with great binding capacity and high stability. Then, the prepared Ag-G substrate was incubated with 500 mL of 1 mg/mL antiOVA (Fisher Scientific, Rochester, NY) under constant rotation for 30 min at RT. After centrifugation at 2000g for 1 min at RT, the pellet (Ag-G-antiOVA) was washed twice with PBS and resuspended in 500 mL of PBS. OVA was added into PBS and whole milk (Roundy’s, Milwaukee, WI) sample at 0, 0.1, 0.5, 1, and 5 μg/mL to act as test samples. Each sample (500 μL) was then incubated with prepared Ag-G-antiOVA colloid (50 μL) under constant rotation for 15 min at RT. After centrifugation at 2000g for 1 min at RT, the pellet (Ag-G-antiOVA-OVA) was washed three times, deposited onto a glass slide, and air-dried at RT ahead of SERS analysis. Raman Instrumentation. A DXR Raman microscope (Thermo Fisher Scientific, Madison, WI) was used in this study. This instrument facilitates 780 nm excitation of SERS spectra through a 10 confocal microscope objective, resulting laser spot diameter of approximately 3 μm, and a 5 cm-1 spectral resolution. Quadruplicate SERS measurements were measured with 4 mW laser power and 25 μm slit aperture for 15 s integration time. Spectra were collected using the Thermo Scientific OMNIC Software with Array Automation. Array Automation allows for automated collection and processing of data from groups of samples that are arranged in an ordered array. Array multiple (n = 25) was used to collect the data by randomly picking 25 spots on the Ag surface for each sample. Spectral Data Analysis. SERS spectral data were analyzed by the TQ Analyst software (Thermo Fisher Scientific, Madison, WI). Principal component analysis (PCA) was applied to analyze the variance of spectral data and build the qualitative predictive model based on the standards. The information provided by 1511

dx.doi.org/10.1021/ac1032353 |Anal. Chem. 2011, 83, 1510–1513

Analytical Chemistry

LETTER

Figure 4. PCA plots of ovalbumin (OVA) spectral data in PBS and milk.

PCA indicates any patterns or trends in the data, which may or may not be significant to a given application. Before PCA analysis, predata processing such as standard normal variant, second derivative transformation, and smoothing were applied when necessary to help PCA capture a more significant variance.21

’ RESULTS AND DISCUSSION As illustrated in Figure 1, the capture procedure consisted of three steps: (1) adsorption of protein G onto Ag dendrites, (2) binding of the antibody to protein G, and (3) immunocapture of antigen. SERS spectra were measured at each step (Figure 2). The spectrum of bare Ag shows a band at around 1070 cm-1, which is assigned to the NO3- stretching from the NO3- residue on the substrate. This band can be used as an internal standard for substrate calibration. Changes in NO3- band intensity can also be used as an indicator of the interaction of the sample with the substrates due to the competition between the NO3- and the sample molecules. Distinct spectra were observed after the adsorption of protein G and the binding of the antiOVA, indicating the successful preparation of the Ag-G-antiOVA complex. After capturing OVA, the spectrum exhibited some differences, although slight as seen in the raw spectra (Figure 2a). After the second derivative transformation, the difference looks much more clear, especially in the range of 1200-1700 cm-1 (Figure 2b), which involves the amide I and III structure,22 indicating the change of the conformation of the three-dimensional structure of the protein complex. The reason for the slight change in the spectrum after capturing the OVA is that most Raman spectra of proteins look similar and the electromagnetic enhancement decreases markedly as the distance between the target and the Ag dendrite surface increases. Nevertheless, the difference can be captured and analyzed by PCA, which is a common statistical tool to separate identical spectral information. As seen in Figure 3, data points of different steps can be separated clearly, showing the success of capture and detection of OVA using this method. SERS spectra from PBS and milk samples with different concentrations of OVA were measured, and PCA was used to reveal spectral differences and build qualitative models. In PBS, the discrimination was observed between the negative control (0 μg/mL) and OVA-spiked samples (0.1, 0.5, and 1 μg/mL). This result indicates that the limit of detection (LOD) in PBS is 0.1 μg/mL or lower. However, the data points of three spiked samples were mixed together, indicating that this method cannot distinguish concentration changes at the tested levels. This may be due to the multilayers of the immuno-complex assembled on

the Ag dendrites that place the OVA binding site outside the ∼10 nm zone of electromagnetic enhancement on the Ag substrate.3 Nevertheless, this method provides a rapid and simple “Yes/No” detection assay. As bioweapons and undeclared allergenic food ingredients should not be present in foods at no matter what level it is, as long as they can be detected, this assay is hence critically important and useful for their detection in food processing lines before the products go into the market. We also validated the method in milk. As seen in Figure 4, data points become distinct between 1 and 5 μg/mL, indicating a limit of detection in milk of approximately 5 μg/mL. The difference between the LODs achieved in PBS and milk may result from nonspecific binding of milk proteins onto the immuno-complex and/or interference from the milk components such as lipid and carbohydrates. In summary, this SERS method provides a rapid and simple “Yes/No” way to detect foreign proteins in a complex food matrix such as milk. The antibody-modified silver dendrite substrate was used for capturing target protein out of complex matrix and enhancing the specific protein signals of interest. The spectral changes after capturing the target protein analyzed by PCA indicate the success of capture and detection of the target. A 780 nm laser excitation wavelength was used in this study to minimize the fluorescence interference from the milk components. If the Ag-G-antibody complex is assembled prior to sensing, the total time for capture and detection of OVA is less than 30 min. This method could be generalized for any protein detection where a binding partner for the molecule of interest is available. The limit of detection for the proof-of-concept OVA was 0.1 μg/mL in PBS and 5 μg/mL in milk. Therefore, future experiments will focus on optimizing substrate performance and decrease nonspecific binding. In addition, DNA aptamers will be employed as target binding partners, in place of the antibody, as the Raman spectra for DNA are quite distinct from protein spectra and should increase the sensitivity.

’ AUTHOR INFORMATION Corresponding Author

*Phone: (612) 624-9701. Fax: (612) 625-5272. E-mail: tplabuza@ umn.edu.

’ ACKNOWLEDGMENT This research was supported by the U.S. Department of Homeland Security (DHS) through the National Center for Food Protection and Defense at the University of Minnesota (Grant Number DHS-3002-11364-00014422). It has not been formally reviewed by DHS. The views and conclusions contained 1512

dx.doi.org/10.1021/ac1032353 |Anal. Chem. 2011, 83, 1510–1513

Analytical Chemistry

LETTER

in this document are those of the authors and should not be interpreted as necessarily representing the official policies, either expressed or implied, of DHS. DHS does not endorse any products or commercial services mentioned in this publication. We appreciate that Thermo Fisher Scientific (Madison, WI) loaned us the DXR Raman microscope for the SERS study.

’ REFERENCES (1) Fleischmann, M.; Hendra, P. J.; McQuillan, A. J. Chem. Phys. Lett. 1974, 26, 163-166. (2) Jeanmaire, D. L.; Van Duyne, R. P. J. Electroanal. Chem. 1977, 84, 1–20. (3) Haynes, C. L.; McFarland, A. D.; Van Duyne, R. P. Anal. Chem. 2005, 77, 338–346. (4) Cui, Y.; Ren, B.; Yao, J.-L.; Gu, R.-A.; Tian, Z.-Q. J. Raman Spectrosc. 2007, 38, 896–902. (5) Yazgan, N. N.; Boyaci, I. H.; Temur, E.; Tamer, U.; Topcu, A. Talanta 2010, 82, 631–639. (6) Song, C.; Wang, Z.; Zhang, R.; Yang, J.; Tan, X.; Cui, Y. Biosens. Bioelectron. 2009, 25, 826–831. (7) Lin, C. C.; Yang, Y. M.; Chen, Y. F.; Yang, T. S.; Chang, H. C. Biosens. Bioelectron. 2008, 24, 178–183. (8) Neng, J.; Harpster, M. H.; Zhang, H.; Mecham, J. O.; Wilson, W. C.; Johnson, P. A. Biosens. Bioelectron. 2010, 26, 1009–1015. (9) Song, C.; Wang, Z.; Yang, J.; Zhang, R.; Cui, Y. Colloids Surf., B: Biointerfaces 2010, 81, 285–288. (10) Garber, E. A.; Eppley, R. M.; Stack, M. E.; McLaughlin, M. A.; Park, D. L. J. Food Prot. 2005, 68, 1294–1301. (11) Duriez, E.; Fenaille, F.; Tabet, J. C.; Lamourette, P.; Hilaire, D.; Becher, F.; Ezan, E. J. Proteome Res. 2008, 7, 4154–4163. (12) Vadas, P.; Wai, Y.; Burks, W.; Perelman, B. JAMA, J. Am. Med. Assoc. 2001, 285, 1746–1748. (13) Kalb, S. R.; Barr, J. R. Anal. Chem. 2009, 81, 2037–2042. (14) Becher, F.; Duriez, E.; Volland, H.; Tabet, J. C.; Ezan, E. Anal. Chem. 2007, 79, 659–665. (15) FDA. http://www.fda.gov/Safety/Recalls/ucm226948.htm 2010. (16) FDA. http://www.fda.gov/Safety/Recalls/ArchiveRecalls/ 2009/ucm192851.htm 2009. (17) FDA. http://www.fda.gov/Safety/Recalls/ArchiveRecalls/ 2009/ucm192368.htm 2009. (18) Hourihane, J. B.; Kilburn, S. A.; Nordlee, J. A.; Hefle, S. L.; Taylor, S. L.; Warner, J. O. J. Allergy Clin. Immunol. 1997, 100, 596–600. (19) Wein, L. M.; Liu, Y. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 9984–9989. (20) He, L.; Lin, M.; Li, H.; Kim, N.-J. J. Raman Sepctrosc. 2009, 41, 739–744. (21) Wu, W.; Walczak, B.; Massarta, D. L.; Prebbleb, K. A.; Last, I. R. Anal. Chim. Acta 1995, 315, 243–255. (22) Li-Chan, E. C. Y. Trends Food Sci. Technol. 1996, 7, 361–370.

1513

dx.doi.org/10.1021/ac1032353 |Anal. Chem. 2011, 83, 1510–1513