Microchannel-Based Surface-Enhanced Raman Spectroscopy for Integrated Microﬂuidic Analysis Chun-hong Lai,a,c Li Chen,a Gang Chen,a,* Yi Xu,b Chun-yan Wangb a Key Laboratory of Optoelectronic Technology and Systems (Chongqing University), Ministry of Education, Key Disciplines Lab of Novel Micro-nano Devices and System Technology, and School of Optoelectronics Engineering, Chongqing University, Shapingba, Chongqing 400044, China b School of Chemical Engineering, Chongqing University, Shapingba, Chongqing 400044, China c School of Physics and Electronic Information, China West Normal University, Nanchong 637002, China
We have demonstrated a microchannel-based, surface-enhanced Raman spectroscopy (SERS) sensing approach for integrated microﬂuidic analysis developed using standard micro-fabrication technology. Our approach allows high-sensitivity SERS sensing with a comparatively low-excitation optical power intensity and large cross-sectional microchannel for biological cell analysis. Utilizing a microchannel with a cross section of 100 6 70 lm2, we achieved a detection limit smaller than 10 nM for rhodamine 6G at an excitation power intensity of 132 W/cm2, avoiding any possible heating effects on the sample under investigation. There is great potential for further improvement in the sensitivity of this microchannel-based SERS detection. Index Headings: Surface-enhanced Raman spectroscopy; Integrated microchannel; Micro-ﬂuid.
INTRODUCTION Raman spectroscopy allows ﬁngerprints of molecular characteristics, which can be used for identiﬁcation of material components. One advantage of Raman spectroscopy is that it can be used for samples held in a water solution and on living biological cells; thus it is very important for biochemical analysis. However, the Raman signal is very weak compared with other frequently used analysis methods, such as photoluminescence and infrared spectroscopy. This greatly limits its practical applications, especially for low-concentration material analysis. By using surface-enhanced Raman spectroscopy (SERS), which utilizes a rough metal surface or nanometal particles to amplify a Raman signal, we can detect organic dyestuff in art and in various diseases.1–4 SERS can even reach the level of single-molecule detection.5 Besides its high sensitivity, other advantages include fast measurement, no sample pretreatment, and nondestructive testing. It has great potential in the application of low-concentration biochemistry sample detection and analysis.6 SERS has
been extensively studied since its discovery. Those studies have focused on single-molecule imaging, ultrafast spectroscopic techniques, and other areas.7,8 In recent years, researchers have started combining the advantages of both SERS and micro-ﬂuidics, which might further improve its sensitivity and repeatability.9,10 Utilizing a hollow photonic crystal ﬁber as the SERS platform, Han et al. achieved a detection sensitivity of 1.7 3 10 7 M for thiocyanate anions using a mixture of silver nanoparticles and a target analyte.11,12 Forward-propagating SERS was also demonstrated in a solid-core photonic crystal ﬁber with immobilized discrete silver nano-particles.13 With a 30 cm long solid-core photonic crystal ﬁber, the SERS signal was obtained for a rhodamine 6G (R6G) solution with a concentration of 2 3 10 6 M.14 Although photonic crystals are a promising platform for SERS detection, an integrated on-chip analysis is hard to realize. A liquid-core, anti-resonant reﬂecting optical waveguide was reported for on-chip SERS detection.15 The liquid core was 12 lm in width, 5 lm in height, and 0.7 mm in length. With an incident power intensity of 4 3 104 W/cm2, it was demonstrated to have a detection limit of 30 nM for a R6G solution. Generally a strong intensity is favorable for the enhancement of a SERS signal. However, it might also be harmful for the sample under investigation, especially for biological cells. Additionally, for biochemical analysis, it is plausible for having a wide microchannel for the testing of biological cells, which are on the size of tens of micrometers. In this article, with comparatively lowexcitation power intensity, we demonstrate SERS detection using a microchannel fabricated on an Si substrate. This layout allows for large molecules and biological cell analysis without any sample heating. Because of its simple structure and fabrication steps, it is easy to be integrated with microﬂuidic units for on-chip analysis.
EXPERIMENTAL Received 15 May 2013; accepted 18 September 2013. * Author to whom correspondence should be sent. E-mail: gchen1@ cqu.edu.cn. DOI: 10.1366/13-07146
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Figure 1 provides the diagram of the microchannel cross section, which consists of two major parts: the top is a cover plate, and the bottom is the base with a
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sample cell ﬁlled with rhodamine 6G (R6G) solution. The Raman signal was collected and collimated via a 203 micro-objective lens (O2) with an N.A. of 0.4. The residual laser beam is ﬁltered out by an ultra-steep long-pass edge ﬁlter (F3) with an edge wavelength of 634.5 nm and transition width of 3.2 nm. Finally, through a 103 micro-objective lens (O3) with a N.A. of 0.25, the pure Raman signal was coupled into an optical ﬁber with 600 lm core diameter, which sends the Raman signal into an Ocean Optics QE65000 micro-spectrometer for spectrum analysis.
RESULTS AND DISCUSSION
FIG. 1. The microchannel cross section.
microchannel; both parts used were silicon substrates. To increase the reﬂectivity inside the microchannel, the surface of the cover plate was coated with a 100 nm gold ﬁlm using electron beam evaporation. On the bottom silicon substrate, the microchannel was fabricated using standard wet etching, and then a SiO2 layer was formed on the inner surface of the microchannel with thermal oxidation, on top of which a 5 nm-thick gold ﬁlm was deposited. Finally, Au nanoparticles were formed on the microchannel inner surface by annealing the 5 nm gold ﬁlm in a vacuum oven at a temperature of 300 8C for 30 min.16 The experiment setup for microchannel-based SERS detection is illustrated in Fig. 2. The excitation light source was 632.8 nm He-Ne with an output power of 4 mW and a FWHM bandwidth 2.4 nm. To control the power of the excitation beam, a graded neutral optical ﬁlter (F1) was inserted into the laser beam path right after the laser. The excitation laser beam passes through a laser clean-up ﬁlter (F2) before being coupled into the microchannel. The laser beam is coupled into the microchannel through a 103 micro-objective lens (O1) with a numerical aperture (N.A.) of 0.25. The spot size of the excitation laser beam was measured to be 20 lm at the focal point. The sample used in the experiment was R6G. To avoid the sample concentration variation caused by evaporation, the microchannel was immersed into a sealed
Experimental setup for microchannel–based SERS detection.
The microchannel was originally designed for singlepoint SERS detection on the inner surface of a microchannel with vertical illumination through a transparent glass plate, not speciﬁcally for this experiment, and therefore, its optical loss was very high, especially on the inner surface with gold nanoparticles. The ﬁrst experiment used a 2 mm-long microchannel. The cross section of the microchannel was about 100 lm thick and 70 lm long. The experiment was conducted without silver colloids. No obvious enhancement was observed in the Raman signal of the R6G solution. This might be caused by the fact that the excitation power intensity was low on the inner surface of the microchannel, where the gold nanoparticles are located. In the following experiments, silver nanoparticles were used for the Raman signal enhancement. The silver colloids were prepared following the recipe given by Leopold and Lendl.17 The average particle size was estimated to be about 30 nm.17 The silver colloids and R6G solution were mixed with an ultrasonic mixer, and then the mixture was ﬁlled into the sample cell, in which the microchannel was immersed for the SERS measurements. The microchannel was automatically ﬁlled with the mixture solution using a capillary action. The silver colloids were mixed with R6G solution of different concentrations: 5, 0.5, and 0.05 lM. To avoid any possible contamination caused by the residual R6G in the microchannel, the SERS signal was taken for the R6G from low to high concentration. Figure 3 gives the R6G SERS signal intensity plotted as the function of Raman shift in log scale for the concentration of 5 lM (dotted line), 0.5 lM (dashed line), and 0.05 lM (solid line), respectively. The inset shows the enlarged Raman signal of 0.05 lM R6G. It is clear that the Raman signal is still strong for the 0.05 lM or 50 nM R6G. It should be noted that, in our case, the cross-sectional area of the channel is about 70 3 60 lm2, which is much larger than the reported 12 3 5 lm2 liquid core waveguide.15 Because the biological cell is larger than 10 lm, our larger cross-sectional microchannel allows for biological cell analysis. In addition, in our experiment, the optical power intensity inside the microchannel is only 132 W/cm2, which is much smaller than the reported power intensity of 4 3 104 W/cm2 used by Measor et al.15 The main advantage of low-power intensity is obvious in biochemical analysis, which avoids unnecessary heating induced by the laser beam. As mentioned previously, the inner surface with gold nanoparticles has very poor optical reﬂectivity. It may
FIG. 3. Microchannel-based SERS signal of R6G solution with concentration of 5 lM (dotted line), 0.5 lM (dashed line), and 0.05 lM (solid line), respectively. The inset gives the enlarged SERS signal of the 0.05 lM R6G solution.
greatly reduce the SERS signal generation in two ways. First, the optical loss reduces the excitation laser beam power intensity along the microchannel and, hence, leads to a great reduction in the Raman signal enhancement because the SERS signal is proportional to the square of the excitation beam power intensity. Second, the generated SERS signal decays along the length of the microchannel because of the optical loss. Therefore, it is important to evaluate the optical loss caused by the microchannel inner surface for future improvement of the SERS detection. For this purpose, microchannel with different lengths (2, 3, 5, and 10 mm) were prepared. For the measurement, microchannels were immersed in a R6G solution. The laser beam was coupled into the microchannels from one end, and the output optical power was measured from the other end. The results are given in Fig. 4, where the output optical power is plotted against the microchannel length for a microchannel with cross section of 70 3 60 lm2. An exponential decay function was used to ﬁt the data points. According to the ﬁtting parameters, the optical
FIG. 4. The relation between output optical power and the microchannel length.
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FIG. 5. Microchannel-based SERS signal of R6G solution with concentration of 10 nM.
power coupled with the microchannel is deduced to be 3.5 mW, and we ﬁnd that the decay length L is about 2.48 mm. When the R6G sample solution concentration was 5 lM and a 2 mm microchannel was used, the input light intensity was 3.5 mW and output light intensity was 1.04 mw; thus, the microchannel loss was 2.64 dB/mm. Therefore, we expect to obtain a better SERS signal with less optical loss, which can be simply achieved by replacing the gold nanoparticles on the inner surface of the microchannel with high-reﬂectivity gold ﬁlm. To determine its detection limit, we used a microchannel with a length of 2 mm to obtain the SERS signal of a R6G solution with a concentration of 10 nM. The result is illustrated in Fig. 5. The spectrum clearly shows the typical Raman peaks of R6G. To conﬁrm that other dyes can be detected too, we used a malachite green (BG) solution with a concentration of 10 nM. The corresponding SERS signal is shown in Fig. 6. It indicates that our microchannel-based SERS detection can achieve a detection limit of 10 nM.
FIG. 6. Microchannel-based SERS signal of BG solution with concentration of 10 nM. The inset gives the AFM image of 100 nm gold ﬁlm on a cover plate.
To determine the roughness of the gold ﬁlm on the cover plate, an atomic force microscope (AFM) image was obtained, as given in the inset of Fig. 6. The roughness was not uniform, because the Si substrate was not smooth. The roughness of 100 nm gold ﬁlm was calculated about 21 nm, using an arithmetic average method.
CONCLUSION In conclusion, a microchannel-based SERS detection is introduced. Using standard microfabrication technology, it is easy to be fabricated and integrated with microﬂuidics systems for on-chip SERS analysis. With a comparatively low-excitation optical power intensity and large cross-section microchannel, it achieves a detection limit about 10 nM, although the microchannel used was not optimized for an SERS measurement. The lowintensity and large cross-sectional microchannel are important for biochemical analysis, allowing large living biological cell analysis without any possible harm to the sample. There is much room for further improvement in the performance of this microchannel-based SERS detection. Replacing the gold nanoparticles on the inner surface of the microchannel with high-reﬂectivity gold ﬁlm, we expect a sub-nM detection limit of the SERS signal detection with an optimized length of the microchannel. ACKNOWLEDGMENTS G.C. acknowledges the support of the National Natural Science Foundation of China under Grant Nos. 61077057, 61177093, and 61074177 and the Fundamental Research Funds for the Central Universities, Project No. CDJRC10120014. 1. M. Moskovits. ‘‘Surface Roughness and the Enhanced Intensity of Raman Scattering by Molecules Adsorbed on Metals’’. J. Chem. Phys. 1978. 69(9): 4159-4161. 2. M. Moskovits. ‘‘Surface-Enhanced Spectroscopy’’. Rev. Mod. Phys. 1985. 57(3): 783-826. 3. M. Fleischmann, P.J. Hendra, A.J. McQuillan. ‘‘Raman Spectra of Pyridine Adsorbed at Silver Electrode’’. Chem. Phys. Lett. 1974. 26(2): 163-166. 4. C.L. Brosseau, F. Casadio, R.P. Van Duyne. ‘‘Revealing the Invisible: Using Surface-Enhanced Raman Spectroscopy to Identify
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