[机械模具数控自动化专业毕业设计外文文献及翻译]【期刊】高度灵敏，高度可重复的激光诱导荧光检测系统-外文文献

10 P PUBLISHING MEASUREMENT SCIENCE AND TECHNOLOGYMeas. Sci. Technol. 19 (2008) 085404 (lOpp) doi: 10.1088/0957-0233/19/8/085404 A highly sensitive, highly reproducible laser-induced fluorescence detection system with optical pickup T Shimomura, C Izawa and T Matsui R&D Engineering Department, Funai Electric Co., Ltd. 1-1 Minami-watarida-chou, Kawasaki-ku. Kawasaki-sfai, Kanagawa 210-0855, Japan E-mail: shimomura.tfunai-atri.co.jp Received 10 February 2008, in final form 23 May 2008 Published 30 June 2008 Online at /MST/19/085404 Abstract In this study, we have developed a novel laser-induced fluorescence detection (LIF) system, which is particularly well suited for measurements in microfluidic processes such as capillary electrophoresis and microchip-based separations and in devices such as microreactors. In order to obtain a high-performance system, we have used a commercially available optical pickup head as a measurement probe along with an objective lens actuator that is vibrated in the vertical and horizontal directions of a light axis according to a simple methodology. Our system is superior to conventional systems because it has high sensitivity and high reproducibility, and it can be implemented without complex and expensive apparatus for high-precision positioning of the component and detection probe. Keywords: laser-induced fluorescence detection, optical pickup, voice coil motor, vibration methodology, without fine-tuning, high sensitivity, high reproducibility, low-cost, compact, microfluidic device (Some figures in this article are in colour only in the electronic version)1. Introduction Recently, the performance of biochemical analyses using small microchips has attracted considerable attention because of its many advantages over conventional systems. The most attractive feature of the microchip is its small size. Its use ensures rapid and efficient separation reactions and reduces the considerable amount of sample and reagent required for analysis. Due to this excellent feature, the microchip has also attracted considerable attention in fields such as environmental monitoring, life sciences and medical care because the quantities of samples used in analyses in these fields are quite small 1,2. However, the extremely small size of these microchips simultaneously requires high detection sensitivity. Among the different types of detection schemes used for measurements in microfluidic devices, the laser-induced fluorescence (LIF) method is considered to be the most sensitive method 3-5 j. Currently, there are various LIF systems available, including an LIF microscope with single- molecule sensitivity in a liquid. However, it is very difficult to develop fast, small and cheap LIF systems without a reduction in the detection sensitivity. For practical implementation, especially in analysis techniques such as immunoassays in medical laboratories, the test chip is required to be a single-use chip. There can be two types of LIF systemsa system that consists of a microchip device and an integrated fluorescence detection system (integrated type) 6 or a system that consists of a disposable microchip and a detection system (separation type). The latter system has technical advantages such as simple handling of single-use test strips and implementation using automated laboratory equipment, which ensures ease of the analysis. However, in the separation-type system, it is difficult to maintain the same relative position between the chip and the detection system for measurements with different chips; this causes substantial experimental error. In the conventional method, a high-precision scanning stage is 1 0957-0233/08/085404+10$30.00 2008 IOP Publishing Ltd Printed in the UK Meas. Sci. Technol. 19 (2008) 085404 T Shimomura et alnecessary in order to adjust the relative position between the chip and detection probe appropriately. This requirement results in very expensive, large and heavy equipment. This situation also makes it difficult to put this system into practical use. and hence it is necessary to develop a cheap and compact system that has a simple mechanism and high sensitivity and high reproducibility. To achieve this purpose, we developed a relatively cheap and compact LIF system with high sensitivity and high reproducibility. Our system utilized a commercially available optical pickup with a high-numerica 1 -aperture lens as a measurement probe. In addition, an objective lens actuator was used, and it was vibrated in the vertical and horizontal directions along the light axis by driving the actuator at an appropriate frequency in both directions using a simple methodology. For example, if we applied our system for measurements in a microfluidic device, the focal spot of the light beam would move in the vertical and horizontal directions and then scan the entire two-dimensional section of the microchannel in the device at high speed. As a result, one can see that measurements can be obtained with high sensitivity and high reproducibility and without fine-tuning of the position between the sample, the microchip and the detection head. Therefore, our microanalytical system has high performance and is both cost and space effective. 2. System description 2.1. Microfluidic test device In this section, we describe the microfluidic device that was used to evaluate the performance of our system. Most microfluidic devices are fabricated in glass or silicon. However, many current studies that are based on polymer-based microfluidic devices are now focusing on p o 1 vdimethyl silox ane (PDMS) due to its low cost and easy handling. Moreover, several studies have reported on PDMS-based microfluidic devices 7-9, PDMS is optically transparent in the wavelength range from 235 run to the near- infrared range, and hence optical detection over the entire visible region is possible. The autofluorescence of PDMS is also low compared to other polymers. We fabricated a test device that included microchannels using PDMS. Figure 1 shows a schematic design of the test device. The chip consisted of a ground plate (50 x 100 x 1.2 mm glass, refractive index: ngiass = 1.52) and a cover plate (30 x 60 x 2.0 mm, npdms = 1.43 10) with five flow channels (width, depth and length of 300 /;- -Chip 5 2 3 ( b ) Channel No. (a) Channel No. Table 1. The RSD values for the measurements in one particular channel (channel 1) and those in all five channels (n = 10). No vibration Vibration RSD value (single-channel ) 3.01% 0.38% RSD value (multi-channel) 6.79% 0.46% Meas. Sci. Technol. 19 (2008) 085404 T Shimomura et al8Figure 8. System response to different concentrations of Resorufin. The least-squares line exhibits a slope of 2.0 x 109 and the detection limit is about 800 pM at a signal-to-noise ratio of 3 (the buffer-only background is 22.23). The excitation light source is a SHG YAG laser (532 nm wavelength, about 9.75 mW laser power) and the Resorufin solution in a 0.1 M phosphate buffer, pH 7.4, flowed through 20 11 m-deep channels at a constant flow rate of 20 /xL min .Figure 9. System response to different concentrations of Resorufin when we use green LED (532 nm wavelength, about 3-1 mW total power) as an excitation light source. The least-squares line exhibits a slope of 3.0 x 108 and the detection limit is about 11 nM at a signal-to-noise ratio of 3 (the buffer-only background is 2.93).is difficult to perform highly sensitive measurements with high reproducibility due to the errors in the position between the chips and the detection probe for different measurements. This implies that our LIF system is well suited to the separation- type system because it can provide cost, time and space- effective measurements and a high performance due to the use of a simplified measurement procedure with an optical pickup. It is also necessary to .consider .the.effect.of.-chromatic. dispersion in our optical system. Our optical pickup lens is designed for 650 nm/780 nm wavelengths. Thus, the pickup head causes axial chromatic aberration in the excitation and fluorescent wavelengths, which results in a shift of focal points (shortened focal lengths). However, this shift does not affect our scanning methodology and our result once the scanning - - bla n k - System response to Kesorufin y = 2E+09X + 37.604 1000 100 10 1.0E-09 1.0E-08 1.0E-07 Resorufin concentration mol/L 10000 1 1.E-1 1.0E-06 V blank System response to Resorufin y = 3E+8x + 4.9739 1000 100 10 1.0E-10 .0E-09 1.0E-09 1.0E-07 Resorufin concentration mol/L 1.0E-06 10000 1 Meas. Sci. Technol. 19 (2008) 085404 T Shimomura et al9range of the object lens is determined for the excitation wavelength. Therefore, chromatic dispersion does not cause severe degradation in the performance of out LIF system and . we can obtain results with high sensitivity and reproducibility. We now consider the results obtained when an LED was adopted as a light source. In our study, we have used a green LED with a maximum .emission myjdengtfa of 525 nm, a half bandwidth of 40 nm, a directivity angle of 15 and an output power of 3.10 mW (NSPG500S; Niehia Corporation, Tokushima. Japan). The beam power after passing through the objective lens was 0.15 mW, which was approximately 4.8% of the total power. Figure 9 shows the calibration curve for Resorufin in this case. One can see that the detection limit was approximately 11 nM at a signal-to-noise ratio of 3, which was approximately 20 dB less than that in the case shown in figure 8. Although the sensitivity is relatively low compared to the case using a laser as a light source, it must be taken into account that this replacement provides an advantage in that the size of the system becomes very compact and hence its price reduces drastically. It might be instructive to compare the sensitivity per unit power of the laser and LED. The detection limit when the laser was used was 800 pM at a signal-to-noise ratio of 3. Therefore, the detection sensitivity per unit power was estimated to be 1.5 nM mW1. On the other hand, the detection sensitivity per unit power when the LED was used was approximately 1.6 nM mW1. Therefore, the detection sensitivity of the LED system is almost comparable to that of LIF. The difference lies in the effectiveness of the light power focused on the microchannel. The effective power of the LED on the channel was only 4.8% of the total power, whereas that of the laser was 77.0% (calculated from output power 2.43 mW after filtering with an ND filter). If a LED with larger output power was available and more effective collimation of that light was accomplished, the sensitivity of the LED system could be improved further. 4. Conclusion A novel LIF system that used a commercially available optical pickup as a measurement probe was developed. The performance of this LIF system was evaluated by using a Resorufin solution and a test microchip with a channel depth of 20 /im: the chip was fabricated by bonding PDMS and glass. It was verified that by using an optical pickup and a simple vibration methodology, the reproducibility of the measurement system can be improved drastically, and it is enough to compensate for the position error arising from the mounting and movement of the chips. We studied the system response for different concentrations of Resorufin in the microchannel chip using the same vibration methodology. The obtained response curve was linear over the measurement range 0.1-100 nM and the detection limit was found to be approximately 800 pM (320 zmol) for a signal-to-noise ratio of 3. which is comparable to that of the conventional system. The reproducibility expressed as RSD values in repeated measurements (n 10) was 1.4% for single-chip measurements and 2.1 % for chip-to-chip measurements. These results are very impressive considering that each chip was fabricated individually and that each chip was mounted by hand in the experimental setup. It shows that the LIF system is particularly well suited for measurements in microfluidic devices in view of its sensitivity and reproducibility. Thus, the system can be effectively used for analysis in microchip chemistries, which require the detection of extremely small amounts of samples accurately. Our system is useful and desirable not only for research and development purposes but also for practical uses such as environmental monitoring, life sciences and medical care because it is relatively cheap, compact and capable of performing fast measurements. We are currently conducting a study on analysis of a type of protein using fluorescent diagnostics. We have applied our system to the qualitative determination of the analysis of the immunochemical assay. In our next paper, we will report on the detailed analysis of the application of our LIF system to such examples. Acknowledgments We thank Professor Uehiyama and Professor Arai for helpful discussions and useful comments. References 1 Krmer P M 1996 Biosensors for measuring pesticide residues in the environment: past, present, and future AOAC Int. 79 1245-54 2 Morgan C L, Newman D J and Price C P 1996 Immunosensors: technology and opportunities in laboratory medicine Clin. Chem. 42 193-209 3 Jacobson S C, Hergenrder R, Moore A W Jr and Ramsey J M 1994 Precolumn reactions with eieetrophoretic analysis integrated on a microchip Anal. Chem. 66 4127-32 4 Liang Z, Chiem N, Ocvirk G, Tang T, Fluri K and Harrison D J 1996 Microfabrieation of a planar absorbanee and fluorescence cell for integrated capillary electrophoresis devices Anal. Chem. 68 1040-6 51 Roddy E S. Price M and Ewing A G 2003 Continuous monitoring of a restriction enzyme digest of DNA on a microchip with automated capillary sample introduction Anal. Chem. 75 3704-11 6 Yang S Y, Hsiung S K. Hung Y C, Chang C M, Liao T L and Lee G B 2006 A cell counting/