TW200841010A - Multi-wavelength fluorescence detecting method of a microchip system - Google Patents

Abstract

A multi-wavelength fluorescence detecting method of a microchip system comprising following steps: providing a microchip comprising a board, an injection channel, a separation channel and a reference channel, and injecting a sample into the injection channel connecting with the separation channel; providing a potential in the microchip to drive the sample to move to a detecting position on the separation channel; providing an optical apparatus with a continuous light source and a receiving unit to excite the sample to emit a fluorescence via an exciting light emitted by the continuous light source and receiving the fluorescence by the receiving unit, wherein the exciting light doesn't beam the receiving unit; and transmitting the fluorescent signal received by the receiving unit to an optical analyzing unit to analyze; wherein the reference channel is provided to measure the data of the background to correct the data of the sample.

Description

200841010 IX. INSTRUCTIONS: [Technical Leadership of the Invention] The present invention relates to a multi-wavelength fluorescence detection method for a microfluidic wafer system, and more particularly to exciting a microfluidic wafer system using a continuous light source of an optical instrument. Fluorescent detection and detection of the fluorescence to improve detection efficiency, accuracy, simplification of equipment and cost reduction of multi-wavelength fluorescence detection methods for microfluidic wafer systems. [Prior Art] A fluorescent detection method for a conventional microfluidic wafer system, such as the Republic of China Bulletin No. 490558 "Sample analysis system with a wafer type electrophoresis device" ^

The patent includes an automatic sample introduction device, a wafer, a mine, a shield, a X 曰曰々 package source supply, a detection unit, a signal acquisition unit and a signal processing unit. The autosampler is used to fill or introduce a sample into the crystal.曰 s 1 Green Moon is equipped with several channels, which are used by the autosampler to sample and separate the sample. The power supply is configured to provide a voltage in the wafer, and the voltage is used to separate the sample in the wafer. The unit is configured to select a fluorescent detection unit, which is used to measure the wood Xtm. The nickname produced. The signal operation unit is configured to capture the signal of the sample measured by the test unit. The signal processing unit rotates the signal. The glory detecting unit comprises a light source, a lens, an excitation ferrite, a beam splitter, a radiation filter, a pinhole and a photomultiplier tube. When utilizing the conventional microfluidic wafer system, the sample is introduced into the wafer via the autosampler, and then the voltage is applied to the crystal.

Linda\PKHat\PU03ll.doc f* 200841010 An electroosmotic flow [electr〇〇sm〇sis n〇w] is generated on-chip to influence the flow rate of each component in the channel in each port, thereby generating separation. A detection position is then preset in the channel. After the sample flows to the detecting position, the light source generates excitation-excitation light through the excitation filter to filter and obtain excitation light of a special wavelength, and then reflects the excitation light by the beam splitter, and the lens is reflected through the lens The excitation light is focused to the _ position, and the focused excitation light is used to illuminate the sample at the position. The sample irradiated with the excitation light will generate fluorescence emission. Then, the fluorescent light generated by the feminine product is collected by the lens, and then sequentially passed through the spectroscope, the radiation filter and the “pinhole” to the photomultiplier tube to enlarge the glory signal generated by the # product by using the photomultiplier tube. After the domain information acquisition unit compares the analog signal, the signal processing unit outputs the sample analysis data. This completes the speculation of the sample. In general, the fluorescent detection method of the conventional microfluidic wafer system has the following disadvantages: for example, the excitation light is directly incident on the signal receiving unit I', and the intensity of the excitation light is greater than that emitted by the sample. The glory of the glory makes the fluorescent signal relatively insignificant, which will reduce the measurement accuracy. Therefore, the conventional method needs to set the excitation wave to filter out the excitation light of a specific wavelength, which causes it to be expensive and has disadvantages: in addition, the conventional debt measurement method is limited by the excitation and debt of a single wavelength. The 'thermal method simultaneously produces multi-wavelength fluorescence emission, so that the fluorescence excitation wavelength of the ς sample adjusts the wavelength of the excitation light and repeats the two systems. In addition to increasing the sample loss, it also increases the disadvantage of low efficiency on the detection time side. At the same time, due to the long-term excitation and detection limitation of the _'33 P:\01-l LindaNPK Pat\PKI03ll.doc one 7-200841010, the detected data range is small, and the quantified data obtained subsequently is accurate. Insufficient. For the above reasons, it is necessary to further improve the multi-wavelength fluorescence fingerprinting method of the conventional microfluidic wafer system. In view of the above, the present invention improves the above-mentioned disadvantages by exciting a microfluidic wafer using a continuous light source of an optical instrument. Fluorescence is emitted and the fluorescence is detected using an optical analysis unit. Thereby, the present invention can indeed improve the detection efficiency, accuracy, cost reduction and simplification of the multi-wavelength fluorescence detection method of the microfluidic wafer system of the device. SUMMARY OF THE INVENTION The present invention is directed to a multi-wavelength fluorescence detection method for a microfluidic wafer system that utilizes a continuous light source of an optical instrument to excite a microfluidic wafer system to emit fluorescence and utilize an optical analysis unit. The detection makes the invention have the effect of improving the detection efficiency.

---- I * W i A secondary object of the present invention is to provide a multi-wavelength fluorescence detection method for a microfluidic wafer system, wherein the optical instrument is a dark field optical group, so that the invention has the advantages of reducing cost and simplifying equipment The effect. Another object of the present invention is to provide a multi-wavelength fluorescence detecting method for a microfluidic wafer system, wherein a microfluidic wafer on the microfluidic wafer system is further provided with a reference channel to obtain a background value, thereby The invention has the effect of improving the detection accuracy. VIII. According to the multi-wavelength fluorescence detection method of the Maoyue microfluidic wafer system, the method comprises the steps of: providing - the microfluidic wafer has a substrate, a two-injection W, a separation channel, and a reference channel, and - sample introduction and the division D: \ ni-1 LindaNPl; Pat \ PKI03H.doc η7 / η « ! / η3 / ιι: ηι aji — 8 — 200841010 in the injection channel of Shangyutong; provide a voltage in the micro The fluid wafer is moved to the separation channel, and the sample is moved to the separation channel, and the continuous light-receiving unit is provided by the broken light, and the connection is used, and the original light is activated. __Light, where 'the excitation light is not directly illuminated: 4, and the fluorescent signal received by the optical instrument $ is transmitted to the optical sub

- and performing "analysis, the towel, the reference channel is used to measure a background value to further correct the data measured by the sample. ', [Embodiment] The purpose, features, advantages can be more obvious embodiments, BRIEF DESCRIPTION OF THE DRAWINGS In order to make the above and other aspects of the present invention readily apparent, the following detailed description of the present invention will be described as follows: Cup-like photographs 1 and 2 show a microfluid of the preferred embodiment of the present invention. The first step S1 of the method of making crystals of the crystals and the method of the method of the invention is provided. The microfluidic wafer 1 has a substrate η, a sample injection channel 12, a separation channel, and a test slot. 14 ' and a sample (not shown) is introduced into the injection channel 12 which is in communication with the separation tank 13. In more detail, the sample is selected from the mixture of the analyte and the background solution. The test object (not shown) is pre-calibrated with a fluorescent dye (not shown) to facilitate subsequent fluorescence excitation. The substrate u is selected as A - glass substrate. The sample channel 12 is for the sample to be introduced, and the length is selected to be 0.8 cm. 13 is used to separate the sample, and the system is connected to and intersects to form a cross shape. The separation channel u is preset with a detection position a, and the length of the separation channel 13 Choose ^ as

Linda\PK Pat\PKI03H.doc —9 — 200841010 5cm. The reference channel M is disposed on one side of the separation channel u. 'Reading the 2 scene solution guide and _ its work light signal as a background value of subsequent data analysis' further reduces detection error and improves detection accuracy . The distance between the test core U and the separation channel 13 is selected to be five sides. The micro-cylinder blade 1 is preferably provided with a scale line 15 and the scale line 15 is placed on one side of the separation channel 13 to facilitate the determination of the moving position of the sample. Wherein, the width of each channel is selected to be _, and the depth system is selected to be 36 // m 明. As shown in Figures 1 and 2, in the preferred embodiment of the present invention, the input is; The opposite ends of the 12 are respectively formed with a first through hole I], ι21, and the sample channel 12 is in communication with the sample channel 12, the first through hole 121 is for introducing the sample, and the other first through hole 121 is used for To exclude excess samples. The separation channel 13 ^ is divided into two ends - a second through hole 131, 131, which communicates with the separation groove 13. The opposite ends of the reference channel 14 respectively form a third through hole (4), (4), and communicate with the reference channel 14, the third through hole i4i is used for introducing the background solution to determine the background value, and another A three-way aperture 141 is provided to remove excess background solution. The second step S2 of the multi-wavelength fluorescence detecting method of the microfluidic wafer system of the preferred embodiment of the present invention is to provide a voltage on the microfluidic wafer 1 as shown in FIGS. 1 and 2, To detect the movement of the sample to the detection channel sa on the separation channel 13. More specifically, the electric dust is applied to both ends of the separation channel 13 to form an electric field (not shown) and to generate an electroosmosis flow. The components in/from the sample are affected by the electroosmotic flow. Thus, the B:\0I-I Linda\PK Pat\PK103ll.doc ——10——200841010 can be driven via the electroosmotic flow. The components of the ncr are such that the sample can be separated along the separation channel and moved to the detection position a. Referring again to FIGS. 1 to 3, a third step S3 of the multi-wavelength fluorescence detecting method of the microfluidic chip system of the preferred embodiment of the present invention provides a continuous light source 21 via an optical instrument 2. And a receiving unit 22, wherein the excitation light 211 is emitted by the continuous light source 21 to excite the sample to emit a fluorescent light, and the fluorescent light is received through the receiving unit 22, wherein the excitation light 211 does not directly illuminate the receiving unit twenty two. More specifically, the optical instrument 2 of the present embodiment is selected but not limited to a dark field optical group. The optical instrument 2 comprises a continuous light source 21, a receiving unit 22, a baffle, a concentrating unit 24 and a pinhole 25. Preferably, the sample is between the continuous light source 21 and the receiving unit 22. The continuous light source 21 emits a plurality of wavelengths of excitation light 211 to excite the sample at the detection position a to emit fluorescence (not shown). The continuous light source 21 can simultaneously excite a plurality of different fluorescent dyes, thereby improving detection efficiency. The continuous light source 21 is selected but not limited to a tungsten lamp. The receiving unit 22 is configured to receive the fluorescent light emitted by the sample. The intensity of the excitation light 211 is greater than the amount of fluorescence emitted by the sample. If the receiving unit 22 simultaneously receives the signal of the excitation light 211 and the fluorescent light, the signal of the fluorescent light is relatively insignificant. Therefore, the excitation light 2 emitted by the continuous light source 21 is not directly irradiated to the receiving unit 22, so as to prevent the receiving unit 22 from receiving the excessively intense excitation light 211, and affecting the relative signal of the fluorescent light emitted by the sample port. strength. The receiving unit 22 is selected as an objective lens. 4, in the preferred embodiment of the present invention, the 200841010 continuous light source 21 is selectively disposed below the receiving unit 22. In order to prevent the continuous light source 21 from directly illuminating the receiving unit 22, the baffle 23 is disposed in the horizontal direction between the continuous light source 21 and the receiving unit 22 to block the excitation light 211 emitted by the continuous light source 21. After the baffle 23 is blocked, the light emitted by the continuous light source 21 forms a hollow tubular excitation light 211. The baffle 23 is selected to be circular. The baffle 23 and the receiving unit u are provided with a concentrating unit 24 for focusing the intermediate-second excitation light 211 by the concentrating unit %, thereby exciting the sample to emit fluorescence. The poly unit 24 is selected as a dark field concentrating mirror. The sample is located between the receiving unit 22 and the concentrating unit 24. The pinhole 25 is disposed above the receiving unit 22, and after receiving the glory signal of the sample, the receiving unit 22 transmits the signal to the subsequent optical analysis unit 3 via the pinhole 25 for analysis of the JJ signal. In this way, the excitation light 2 ιι emitted by the continuous light source 21 is blocked by the baffle 23 to form a hollow tubular excitation light 2 ιι. Then, the illuminating unit 211 is configured to form a hollow cone-shaped luminaire 1 211 ′ to focus on the detection position of the microfluidic chip i via the excitation light 211 to be located at the detection position & The sample emits fluorescence 'and the fluorescence is received by the receiving unit 22. Since the excitation light 2 ιι forms the hollow cone excitation light 211, and the cone apex is located on the microfluidic wafer 1 'the excitation light 211 can be prevented from directly entering the receiving unit ^ ^ ^ and only a few corrections The excitation light 211 of the Na-shaped shot can enter the connection. The acquirer: 22. The hollow cone-shaped excitation light 211 is preferably focused on the detection. Referring again to Figures 1 to 3, the microflow i»:\0l-l LindaNPK Pat\Pi;l〇3l of the preferred embodiment of the present invention 1. doc η7/η4/ίχνπ:〇ί —12 — 200841010 The fourth step of the multi-wavelength fluorescence detection method of the makeup system is S4: the photon is transmitted to the received fluorescent signal The optical analysis unit 3 performs knife analysis. More specifically, the fluorescent signal received by the optical device 2 is selected, and the fluorescent signal is transmitted from the optical fiber to the optical analysis, t13 for analysis. The fluorescent signal can also be selected to be amplified by a photomultiplier (not shown) and then transmitted to the optical analysis unit 3. The light: the knife unit 3 is preferably selected as a UV-visible_near-infrared light spectrum analyzer to simultaneously illuminate the fluorescent light of various wavelengths. After the optical analysis of the camping light signal, the relative change of time [second], wave, [nm] and transmittance [%] can be obtained, and more accurate quantitative data can be further obtained. In addition, please refer to the figure, in order to make the measured data more accurate, preferably after the above steps are completed, another step is taken to obtain the background value: the background solution is guided to the reference slot : In the third through hole 141, a voltage is applied to drive the background solution to move. The tea test channel U is on the reference position b; the background light of the position b is measured by the excitation light 2, and the W light is received via the receiving unit 22, and the light of the Jing Qianguang is transmitted to the optical analysis unit 3 to obtain the f view value. Wherein the reference position W is located on the reference palpe 14 and corresponds to the position a. Finally, the sample obtained from the sample is deducted from the background value of the lining, so that the fluorescent data measured by the fresh product can be corrected, and the fluorescence data measured by the fresh product can be further improved to obtain a more accurate quantified wife = please refer to the 1 to 3 figure. As shown, when implementing the preferred embodiment of the present invention, the first step is to enter the dragon [pour S1: to be calibrated via the fluorescent axis ^ D:\ni-* Lirwln\PK Pat\PKI03H.doc η7/η·ι/η3 / ιι; 〇,,,, 13 200841010 [not depicted] injected into the injection channel 12 of the microfluidic wafer i. After the product is gradually flowing to the intersection of the sampling channel 12 and the separation channel 13, the second (four) S2 is supplied: voltage is supplied to the microfluidic wafer i; the sample is driven along the axis of the channel 13 And performing the third step S3: using the continuous light source 21 to emit the multi-wavelength light-emitting 211, and sequentially forming the bamboo shoot light 211 into a hollow cone via the baffle 23 and the concentrating unit %. The excitation light 211 is shaped and focused on the detector a to excite the sample located at the detection position a to emit fluorescence. The fluorescent light emitted by the sample 1 will be received by the receiving unit 22, and the excitation light 2lj": is not directly irradiated to the receiving unit 22 to avoid affecting the relative intensity of the glory signal; finally, the fourth step is performed: The _fluorescent signal received by the optical device 2 is transmitted to the optical analysis unit 3 via the optical fiber and analyzed to obtain a relative change in time, wavelength, and transmittance. Thus, the multi-wavelength detection side of the microfluidic wafer system of the preferred embodiment of the invention is completed. Thus, the present invention does improve the efficiency, accuracy, cost, and simplification of equipment. Referring to Fig. 4, in the examples of the present invention, fitc, RhB and Atto610 were selected as the fluorescent dyes of the sample. Figure 4 is a graph of the absorption and emission spectra of each of the phosphor dyes measured by the multi-wavelength fluorescence detection method of the present invention. In the figure, the dotted line means the wavelength of the absorption light of each of the fluorescent dyes, and the solid line means the wavelength of the emitted light of each of the fluorescent dyes. It can be seen that the maximum absorption wavelengths of FITC, RhB and Atto610 are 49 〇 nm [nano], 553 nm and 614 nm, respectively, and the maximum emission wavelengths are 514 nm, 578 nm and 633 nm, respectively. The maximum emission light of each of the fluorescent dyes D:\01-I LindaSPK Pat\PKI0311.doc - - ητ/ηΊ/η-νιι;^ ^ 200841010 The long-term is an important indicator for the subsequent analysis of fluorescence. 5 f 5 (5) Analysis of relative transmittance & ^ When the wavelength intensity is obtained, the background data is subtracted from the background data of the product. 'The 3D electrophoresis pattern can be used to compare the time, wavelength and transmittance. It is used to judge the intensity of the glory. Figures 5 and 6 =; _ system 1 stomach fluorescence detection method needs to correspond to the long intensity of each of the above-mentioned syllabary = second brother 5 maps to conduct multiple debt tests, and the beneficial method simultaneously pairs the large poor light radiation & Long fluorescent dyes are detected. In the sixth picture, the different radiations are not directly entered into the reception due to the excitation light 211: the preferred embodiment must be installed with any = ^ this unsubscribed debt test, which can be simultaneously entered; , please refer to the 5th ==::_ test: ! When the light is quantitatively analyzed, it will be in the 5th picture: the peak == the obtained quantitative data, but because of the measured data only, to = The detection result under the radiation wavelength intensity is such that !1=product=, the electrophoresis pattern can use (4) plane 1 as the volumetric knife, so that the obtained quantization is low:::=* has _ measurement efficiency, accuracy = as described above Compared with the conventional multi-fluidic wafer system, multi-wavelength fluorescence D:\0I-1 LindaNPJ: Pal\PXl〇3U. dix 15 200841010 == There is a specific wavelength excitation for the multi-wavelength of the image-bearing wafer n 7 test the detective, when the thermal method is detected at the same time, because of the shortcomings of low efficiency. The same is true - the wave is miscellaneous _ limit, so that the accuracy of the data is insufficient. In contrast, the first wafer i $_ learns the continuous light source 21 of the instrument 2 to excite the microfluid

</ RTI> </ RTI> and using the optical analysis unit 3, === the reference channel 14 to determine the background value to correct the sample of the fire:: and = set although the invention has been disclosed using the preferred embodiment described above, The present invention is not intended to be used in the present invention, and it is still within the spirit and scope of the present invention. This is subject to the definition of the scope of the patent application. 16 — D:\OI-i Linda\PK Pat\PK103n.doc 07/0^1/03/11: 200841010 [Simple description of the diagram] The microfluidic chip system of the first embodiment And the flow block diagram of the multi-wavelength detection method. Figure 3 is a perspective view of a microfluidic wafer in accordance with a preferred embodiment of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS A schematic diagram of a multi-wavelength gold first detection method for a microfluidic wafer according to a preferred embodiment of the present invention. Shoot: Hey. Absorbing and Discharging of Each of the Fluorescent Dyeing Agents in a Preferred Embodiment of the Invention FIG. 5 is a 3D electropherogram of a microfluidic wafer system fluorescent detection method according to a preferred embodiment of the present invention. 4 ' 第 Figure 6 · The glory of the conventional microfluidic wafer system method The analysis of the time relative transmittance [%]. ‘,亍之

[Main component symbol description] 1 microfluidic wafer 12 injection channel 121 'first through hole 131 second through hole 14 reference channel 14 Γ third through hole 2 optical instrument 211 excitation light 23 baffle 25 pinhole 11 substrate 121 First through hole 13 separation channel 131 'second through hole 141 third through hole 15 tick mark 21 'continuous light source 22 receiving unit 24 concentrating unit 3 optical analysis unit D: \OM LindaXPK Pat\PKin3H.doc — 17 200841010 a Detection position b Reference position SI First step S2 Second step S3 Third step S4 Fourth step

Claims (1)

200841010 X. Patent Application Range: 1. A multi-wavelength fluorescence detection method for a microfluidic wafer system, comprising the steps of: providing a microfluidic wafer having a substrate, a sampling channel, a separation channel and a reference slot a channel and introducing a sample into the injection channel in communication with the separation channel; Providing a voltage on the microfluidic wafer to drive the sample to move to a detection position on the separation channel; providing a continuous light source and a receiving unit via an optical instrument to emit excitation light by using the continuous light source The sample emits a fluorescent light, and receives the fluorescent light through the receiving unit, wherein the excitation light does not directly illuminate the receiving unit; and transmits the fluorescent signal received by the optical instrument to the optical analysis unit for analysis Wherein the reference channel is used to determine a background value to further correct the data measured by the sample. 2. A multi-wavelength fluorescence detection method for a microfluidic wafer system according to claim 1, wherein the optical instrument is a dark field optical group. 3. The multi-wavelength fluorescence detecting method of the microfluidic wafer system according to claim 1, wherein the sample is prepared from a sample to be tested and a background solution. 4. The multi-wavelength fluorescence detection method of the microfluidic wafer system according to claim 3, wherein the background value is obtained by: introducing the background solution into the tea-chamber channel and applying a voltage to drive The background D:\0i-! LindaXPK Pat\PKi03H.doc — 19 — 200841010 Loose liquid moves to a reference value on the reference channel; with the excitation light, the background solution at the reference position emits glory, and The $7 (four) background is transmitted to the optical analysis unit via the interface to analyze and obtain the background value. 5. The multi-wavelength edge light detecting method of the microfluidic chip system according to claim 1, wherein the fluorescence value of the sample is subtracted from the background value to correct the fluorescence analysis of the sample. data. 6. A multi-wavelength a-light detection method for a microfluidic wafer system as described in claim 4, wherein the reference position corresponds to a position to be detected. 7. The multi-wavelength 1 method of the microfluidic wafer system according to the scope of the invention, wherein the optical device comprises a shaft continuous light source, a receiving unit, a baffle, a concentrating unit and a pinhole. . 8. The multi-wavelength gm:fluorescent method of the microfluidic wafer system according to item 7 of the patent application scope, wherein the continuous light-emitting light system is blocked by the gastric raft and forms a hollow tubular excitation light. . 9. According to t, please use the multi-wavelength camp side method of Wei Wei 8 韵 微 微 微 微 微 微 ' 其 其 其 其 其 其 其 其 其 其 其 其 其 其 其 其 其 其 其 其 其 其 其 其 其 其 其 其 其 其 其 其 其 其 其 其 其The receiving unit is not directly illuminated. 10. The multi-wavelength fluorescent method of the microfluidic wafer (4) according to claim 9 of the patent application, wherein the bee-shaped hair reading of the towel is focused on a detection position on the brittle fluid crystal moon. 11. According to the patent application, the microfluidic wafer system and the multi-wavelength fluorescence measurement method described in the seventh paragraph of the patent application, wherein the fluorescent light emitted by the sample enters the reception ^ D:\0M Linda\PJ; Pat\PKI03n (foc — 20 — 07/04/03/11:0.} AV 200841010 yuan, then passed through the pinhole and passed to the optical analysis unit. 12. Microfluidic wafer according to claim 1 The multi-wavelength fluorescence detecting method of the system, wherein the continuous light source is a tungsten lamp. 13. The multi-wavelength fluorescence detecting method of the microfluidic chip system according to claim 1, wherein the optical instrument The fluorescent signal is transmitted to the optical analysis unit via an optical fiber. D:\0t-l LindaXPK Pat\PKl03H.doc —21 —