Article Journal of Biomedical Nanotechnology

Copyright © 2013 American Scientific Publishers All rights reserved Printed in the United States of America

Vol. 9, 1241–1244, 2013

Simultaneous Detection of SERS and Fluorescence Using a Single Excitation for Microbead-Based Analysis Sa Ram Lee1 , Chang Su Jeon2 , Inseong Hwang2 , Taek Dong Chung2 ∗ , and Hee Chan Kim3 4 ∗ 1

Interdisciplinary Program Biomedical Engineering Major, Seoul National University, 28 Yongon-dong, Chongno-gu, Seoul, Korea Departments of Chemistry, Seoul National University, Seoul 151-742, Korea 3 Departments of Biomedical Engineering, College of Medicine, Seoul National University, Seoul 110-744, Korea 4 Institute of Medical and Biological Engineering, Medical Research Center, Seoul National University, Seoul 110-744, Korea 2

We demonstrate simultaneous detection of surface-enhanced Raman scattering (SERS) and fluorescence signals from a silver microbead. For the dual signal generation, silver microbeads with a diameter of 15 m were functionalized with benzenethiol (BT) as a Raman tag and a cardiac troponin I (cTnI) antibody on their surface. SERS and fluorescence signals were obtained using a single argon laser source with 488 nm wavelength. The SERS signals from Raman tag can be used as identification indices for decoding a particular microbead, while the fluorescence signals provide the information about molecular interactions with a specific biomarker. With simultaneous detection of SERS and fluorescence Delivered by Ingenta to: University of South Carolina signals using single excitation on the functionalized microbeads, we successfully showed the possibility of a simple IP: On:arrays. Sat, 09 Jul 2016 20:02:21 barcoding strategy for multiplex analysis using suspension

Copyright: American Scientific Publishers KEYWORDS: Multiplex Analysis, Surface-Enhanced Raman Spectroscopy (SERS), Fluorescence, Simultaneous Detection, Silver Microbeads.

INTRODUCTION The rapid advance in biochemical studies, clinical diagnostics, and environmental monitoring keeps stimulating the demand in powerful analytical methodology for collecting huge information from smaller sample volume, in shorter reaction time, and at lower cost.1 2 As such, multiplex assay using bead-based suspension array has gathered much attention in that it provides faster binding kinetics, lower costs, higher reproducibility, and superior detection sensitivity.1–3 Together, a number of encoding and decoding methods, such as fluorescence, quantum dots, graphical rods, photobleaching beads, continuousflow lithography based particles, physical method, and electrochemical method, have been suggested.3–11 Although fluorescence is principally employed in multiplex detection methods because of its high sensitivity compared with other signal sources, it has inherent drawbacks, including broad emission band, photobleaching, ∗

Authors to whom correspondence should be addressed. Emails: [email protected], [email protected] Received: 9 November 2011 Accepted: 31 March 2012

J. Biomed. Nanotechnol. 2013, Vol. 9, No. 7

peak overlapping, and thus limited number of barcodes. By contrast, surface-enhanced Raman spectroscopy (SERS) can provide fingerprint spectra of Raman active molecules immobilized on a SERS-active substrate, such as nanostructured gold or silver. Thus, the number of SERS code is virtually unlimited, while providing narrow signal bands and non-destructive detection of analytes even in liquid assay media.12–17 Dual tags on micro- and nanoparticles, which particularly generate fluorescence and SERS signals, have been suggested previously for immunoassay and biomolecular detection for cell biology.18–21 However, for microsized beads, relatively long exposure time (20–30 s) was required to harvest effective SERS signals from the particles, making it difficult to harness them in high-throughput assays.20 21 For nanoparticles, it is difficult to implement suspension array-based assays.19 Furthermore, it is practically difficult to realize the simultaneous detection using a single laser source because the detection wavelength for fluorescence and SERS overlaps in most cases. Herein, we report a simple barcoding strategy using individually functionalized microbeads that can be applied to array-based multiplex analysis. Our strategy exploits




Simultaneous Detection of SERS and Fluorescence Using a Single Excitation for Microbead-Based Analysis

a simultaneous detection of SERS and fluorescence signals using a single excitation source. We have chosen proper bead size and carefully engineered microbeads so that the time required for signal measurement is short enough while holding sensitivity even in much demanding situation, such as high-throughput assay systems. Indeed, we achieved extremely short detection time (100 ms) for SERS and fluorescence, while avoiding signal overlapping between the two by carefully selecting detection ranges.

Lee et al.

room temperature. The functionalized microbeads were then incubated with 0.4 ng/ml of cTnI in 0.5 ml PBS, followed by incubation of 10 ng of mouse anti-cTnI antibody (GeneTex). The fluorescence was generated by using 25 ng/ml of AlexaFluor 610-PE goat anti-mouse IgG (Invitrogen). For control experiments, either mouse anticTnI antibody or cTnI was removed while keeping others same. Between each reaction step, resulting beads were rinsed with PBS for three times to remove unbound antigens or antibodies.

Detection of SERS and Fluorescence The SERS measurements were performed using a cusFunctionalized Microbeads Preparation tomized micro-Raman spectroscopic system equipped with The silver microbeads were fabricated using electroless a microscope. The excitation source was an Argon laser 12 22 In plating method similar to our previous reports. (488 nm, LASOS Lasertechnik GmbH, US) with a maxbrief, colloidal silver nanoparticles (AgNPs, 2–3 nm in imum output power of 20 mW and a beam diamediameter) were attached to amine-terminated poly (methyl ter of 2 m. The laser beam was focused through a methacrylate) beads (PMMA-NH2 , 15 m in diameter, 50 X objective lens. The SERS was detected using a TE Bangs Laboratories, Inc) as seed layers. The silver layer cooled −50  C) charge coupled device (CCD) camera was grown by adding AgNPs electroless plating solu(1,024 × 127 pixels, Andor,iDus DV401). The calibration tion. The Raman tag (1 mM benzenethiol, BT, Sigmaof the spectrometer was achieved using the Raman band Aldrich) was dissolved in 1 ml ethanol, and to this of a silicon wafer at 520 cm−1 to normalize the peak solution were added the silver microbeads for 24 h at intensities of the Raman tags. The fluorescence signals 4  C. BT was immobilized on silver nanoparticles via from functionalized beads were detected by photomultithiol-silver interaction, whereas antibody was chemically plier tube (H10721-20, Hamamatsu Photonics Co. Ltd., conjugated with exposed amine group of Delivered by PMMA Ingenta core. to: University of South Carolina Japan) equipped with a fluorescence filter (624/40 nm The resulting Raman-tagged silver wereOn: cenIP:microbeads Sat, 09 Jul 2016 20:02:21 ® single-band bandpass filter, Semrock, US). BrightLine Copyright: trifuged and washed with DI water several American times to Scientific Publishers For simultaneous detection of SERS and fluorescence sigremove the excess reagents. Next, the modified silver nals from multiple individual silver microbeads, the beads microbeads with Raman tags were added to a solution were spread out on a glass slide and scanned with a laser. containing 100 mM hydrazine hydrate (Sigma-Aldrich) in The SERS spectrum was windowed at 1,020 cm−1 and 50% methanol for 1 h. For the detection of cardiac troprocessed together with fluorescence signals using Matponin I (cTnI) using immunofluorescence assay, 5 ng of lab software (The MathWorks, Inc). The sampling rate of rabbit anti-cTnI antibody was initially incubated with BTFluorescence and SERS signals was 10 kHz and 10 Hz, modified and hydrazine-activated silver microbeads (5 g) respectively. in 0.5 ml of phosphate-buffered saline (PBS) for 1 h at


Scheme 1. Schematic illustrations of experimental setup. (A) The detectors for SERS and fluorescence signals are housed together in a single system. The emission filter (624 nm) is used to select fluorescence signals toward PMT, while SERS is detected through CCD. (B) The microbead has 15 m PMMA bead core where antibody is conjugated with the exposed amine groups and has silver nanoparticles on its surface where Raman tag is chemisorbed (figures not drawn to scale).


J. Biomed. Nanotechnol. 9, 1241–1244, 2013

Lee et al.

Simultaneous Detection of SERS and Fluorescence Using a Single Excitation for Microbead-Based Analysis

RESULTS AND DISCUSSION Scheme 1 shows the overview of experimental setup for SERS and fluorescence signal measurement using functionalized silver microbeads. Scheme 1(b) magnifies the functionalized area of silver microbeads surface. The Raman tag is used as identification indices for decoding particular microbead, while the fluorescence signals provide the information about molecular interactions with specific biomarkers. The detection range of fluorescence signal was designated so that SERS and fluorescence signals are mutually exclusive to prevent signal overlapping. The typical Raman shift wavenumbers in SERS spectra, ranging from 600 to 1,900 cm−1 , correspond to wavelengths from 502.72 to 537.87 nm. Therefore, we chose 624 ± 20 nm filter for fluorescence detection to avoid Figure 2. SERS spectra with various laser exposure time infringement from SERS spectra. Of note, a single laser ranging from 100 ms to 1 s. The Raman tag (BT) chemisorbed source (488 nm) is used for excitation, while two different on the surface of functionalized silver microbead can be detectors, PMT and CCD, are used for the detection of clearly identified from its characteristic peak at 1,020 cm−1 fluorescence and SERS, respectively. with an exposure time of 100 ms. Figure 1 shows the binding of cTnI, a specific biomarker for myocardial-ischemia, with antibody on the bead surIn Figure 2, the intensity of SERS spectra clearly face. The bead was able to detect 0.4 ng/ml cTnI, which increased as the laser exposure time increased from 100 ms falls within a clinically relevant detection cut-off.23 No to 1 s. The BT fingerprint peaks at 998, 1,020, 1,073, and fluorescence could be detected when cTnI or mouse anti1,567 cm−1 clearly stand out of the background. Among cTnI antibody was removed in our immunoassay systhem, the two latters typically originate from benzene tem as a control experiment (data not shown). Thus, the rings. We chose the 1,020 cm−1 peak as a characteristic Delivered by Ingenta to: University of South Carolina fluorescence genuinely originates from antigen-antibody BT09signal for the scanning experiment (vide infra). IP: On: Sat, Jul 2016 20:02:21 interaction. We then spread the silver microbeads on a glass slide Copyright: American Scientific Publishers and scanned with the 488 nm laser, simultaneously monitoring SERS and fluorescence signals. Because detection ranges of both signals are well separated, simultaneous detection was stably achieved with the minimum exposure time of 100 ms (Fig. 3); both signals exactly coincided throughout the scanning, showing that the silver microbead carrying molecular barcode was able to capture cTnI biomarkers effectively. In our system, while the

Figure 1. Representative microscopy image of functionalized silver microbead. (A) A light image. (B) A fluorescence image of sandwich immunoassay using 0.4 ng/ml cTnI cardiac marker proteins. Scale bars are 20 m. J. Biomed. Nanotechnol. 9, 1241–1244, 2013

Figure 3. SERS (upper) and fluorescence (lower) signals from a single silver microbead exactly coincide when simultaneously detected by laser scanning on a glass slide. The peak at 1,020 cm−1 was monitored as a representative SERS peak and fluorescence signals were filtered through 624 ± 20 nm band pass filter.


Simultaneous Detection of SERS and Fluorescence Using a Single Excitation for Microbead-Based Analysis

Lee et al.

6. K. Braeckmans, S. C. De Smedt, C. Roelant, M. Leblans, R. Pauwels, and J. Demeester, Encoding microcarriers by spatial selective photobleaching. Nat. Mater. 2, 169 (2003). 7. D. C. Pregibon, M. Toner, and P. S. Doyle, Multifunctional encoded particles for high-throughput biomolecule analysis. Science 315, 1393 (2007). 8. A. R. Vaino and K. D. Janda, Euclidean shape-encoded combinatorial chemical libraries. Proc. Natl. Acad. Sci. USA. 97, 7692 (2000). 9. K. C. Nicolaou, X. Y. Xiao, Z. Parandoosh, A. Senyei, and M. P. Nova, Radiofrequency encoded combinatorial chemistry. Angew. Chem. Int. Ed. 34, 2289 (1995). 10. X. X. Han, L. J. Cai, J. Guo, C. X. Wang, W. D. Ruan, W. Y. Han, W. Q. Xu, B. Zhao, and Y. Ozaki, Fluorescein isothiocyanate linked CONCLUSIONS immunoabsorbent assay based on surface-enhanced resonance raman The in situ decoding and detection of both SERS and fluscattering. Anal. Chem. 80, 3020 (2008). 11. F. J. Hayes, H. B. Halsall, and W. R. Heineman, Simultaneous orescence are one of the promising strategies that deserve immunoassay using electrochemical detection of metal-ion labels. intensive efforts to practical applications for biological and Anal. Chem. 66, 1860 (1994). chemical analyses. Here, we successfully demonstrated the 12. S. Lee, S. Joo, S. Park, S. Kim, H. C. Kim, and T. D. Chung, facile fabrication of functional silver microbead for simulSers decoding of micro gold shells moving in microfluidic systems. taneous detection of SERS and fluorescence using a single Electrophoresis 31, 1623 (2010). 13. K. Kim, H. B. Lee, J. Y. Choi, and K. S. Shin, Silver-coated dyeexcitation using 488 nm laser source. The SERS signals embedded silica beads: A core material of dual tagging sensors based from a given Raman tag could be detected without interacon fluorescence and Raman scattering. ACS Appl. Mater. Interfaces tion with fluorescence emission signals. Thus, our system 3, 324 (2011). shows the possibility of a simple barcoding strategy and 14. A. Kudelski, Analytical applications of Raman spectroscopy. Talanta multi-functional microbeads for high-throughput multiplex 76, 1 (2008). 15. M. J. Banholzer, J. E. Millstone, L. D. Qin, and C. A. Mirkin, Ratioanalysis. nally designed nanostructures for surface-enhanced Raman spectroscopy. Chem. Soc. Rev. 37, 885 (2008). Acknowledgments: This work was supported by the 16. C. L. Haynes, A. D. McFarland, and R. P. Van Duyne, SurfaceAgency for Defense Development through Chemical and enhanced Raman spectroscopy. Anal. Chem. 77, 338A (2005). by Ingenta of South Carolina Biological Defense Research Delivered Center. And it was to: sup-University 17. Jul G. A. Baker and D. S. Moore, Progress in plasmonic engineering IP: On: Sat, 09 2016 20:02:21 ported by the Public welfare and Safety researchAmerican program Scientific of surface-enhanced Copyright: Publishers Raman-scattering substrates toward ultra-trace analysis. Anal. Bioanal. Chem. 382, 1751 (2005). through the National Research Foundation of Korea (NRF) 18. M. Sanles-Sobrido, W. Exner, L. Rodriguez-Lorenzo, B. Rodriguezfunded by the Ministry of Education, Science and TechGonzalez, M. A. Correa-Duarte, R. A. Alvarez-Puebla, and L. M. nology (No. 2011-0021117). We acknowledge the ConLiz-Marzan, Design of SERS-encoded, submicron, hollow particles verging Research Center Program through the Ministry of through confined growth of encapsulated metal nanoparticles. J. Am. Education, Science and Technology (2011K000895) for its Chem. Soc. 131, 699 (2009). 19. M. A. Woo, S. M. Lee, G. Kim, J. Baek, M. S. Noh, J. E. Kim, S. J. financial support. Park, A. Minai-Tehrani, S. C. Park, Y. T. Seo, Y. K. Kim, Y. S. Lee, D. H. Jeong, and M. H. Cho, Multiplex immunoassay using fluorescentsurface enhanced Raman spectroscopic dots for the detection of bronREFERENCES chioalveolar stem cells in murine lung. Anal. Chem. 81, 1008 (2009). 1. R. Wilson, A. R. Cossins, and D. G. Spiller, Encoded microcarriers 20. B. H. Jun, J. H. Kim, H. Park, J. S. Kim, K. N. Yu, S. M. Lee, for high-throughput multiplexed detection. Angew. Chem. Int. Ed. H. Choi, S. Y. Kwak, Y. K. Kim, D. H. Jeong, M. H. Cho, and 45, 6104 (2006). Y. S. Lee, Surface-enhanced Raman spectroscopic-encoded beads for 2. K. Braeckmans, S. C. De Smedt, M. Leblans, R. Pauwels, and multiplex immunoassay. J. Comb. Chem. 9, 237 (2007). J. Demeester, Encoding microcarriers: Present and future technolo21. B. H. Jun, M. S. Noh, G. Kim, H. Kang, J. H. Kim, W. J. Chung, gies. Nat. Rev. Drug. Discov. 1, 447 (2002). M. S. Kim, Y. K. Kim, M. H. Cho, D. H. Jeong, and Y. S. Lee, 3. Y. Long, Z. L. Zhang, X. M. Yan, J. C. Xing, K. Y. Zhang, J. X. Protein separation and identification using magnetic beads encoded Huang, J. X. Zheng, and W. Li, Multiplex immunodetection of tumor with surface-enhanced Raman spectroscopy. Anal. Biochem. 391, 24 markers with a suspension array built upon core–shell structured (2009). functional fluorescence-encoded microspheres. Anal. Chim. Acta. 22. L. Piao, S. Park, H. B. Lee, K. Kim, J. Kim, and T. D. Chung, 665, 63 (2010). Single gold microshell tailored to sensitive surface enhanced Raman 4. X. H. Gao and S. M. Nie, Quantum dot-encoded mesoporous beads scattering probe. Anal. Chem. 82, 447 (2010). with high brightness and uniformity: Rapid readout using flow 23. R. H. Christenson and H. M. E. Azzazy, Cardiac point of care cytometry. Anal. Chem. 76, 2406 (2004). testing: A focused review of current national academy of clinical 5. S. R. Nicewarner-Pena, R. G. Freeman, B. D. Reiss, L. He, D. J. biochemistry guidelines and measurement platforms. Clin. Biochem. Pena, I. D. Walton, R. Cromer, C. D. Keating, and M. J. Natan, 42, 150 (2009). Submicrometer metallic barcodes. Science 294, 137 (2001).

variation of SERS intensity is not affecting the decoding process, the variation of absolute fluorescence intensity can be minimized by increasing the number of scanned microbeads to ensure reproducibility. Because the size of our silver microbead is well suited for microfluidics and the time required for decoding is extremely short, we envision that the integration of our simple barcoding system with microfluidics will produce high-throughput multiplex assay system.


J. Biomed. Nanotechnol. 9, 1241–1244, 2013

Simultaneous detection of SERS and fluorescence using a single excitation for microbead-based analysis.

We demonstrate simultaneous detection of surface-enhanced Raman scattering (SERS) and fluorescence signals from a silver microbead. For the dual signa...
3MB Sizes 0 Downloads 0 Views