CN103033497A - Microfluidic chip analyzer applying raman spectrum for detection - Google Patents
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Abstract
本发明属于微流控芯片分析仪技术领域，具体涉及一种应用拉曼光谱检测的微流控芯片分析仪。微流控芯片分析仪由与微流控芯片分离的光路模块和固定光路模块的三维自动位移定位台组成，实现了在微流控芯片上精确定位和对样品的拉曼光谱进行检测。具有（1）检测探头与微流控芯片分离，且在微流控芯片上采用倒置检测拉曼光谱的方式，（2）可对微流控芯片显微成像，（3）微流控芯片位置固定，检测探头可在XYZ方向实现精细微调和定位等主要技术特征。该装置各个参数均满足在微流控芯片进行拉曼检测的要求，而且体积小，成本低，是一种新型的可移动式的微流控芯片分析仪，在该领域有着重要的应用潜力和商品市场。
The invention belongs to the technical field of microfluidic chip analyzers, and in particular relates to a microfluidic chip analyzer using Raman spectrum detection. The microfluidic chip analyzer is composed of an optical path module separated from the microfluidic chip and a three-dimensional automatic displacement positioning platform with a fixed optical path module, which realizes precise positioning on the microfluidic chip and detection of the Raman spectrum of the sample. (1) The detection probe is separated from the microfluidic chip, and the Raman spectrum is detected by inversion on the microfluidic chip; (2) Microscopic imaging of the microfluidic chip is possible; (3) The position of the microfluidic chip Fixed, the detection probe can realize the main technical features such as fine fine-tuning and positioning in the XYZ direction. All parameters of the device meet the requirements of Raman detection in microfluidic chips, and it is small in size and low in cost. It is a new type of mobile microfluidic chip analyzer and has important application potential and commodity market.
Description
Technical field
The invention belongs to the micro-fluidic chip analyzer technical field, be specifically related to a kind of micro-fluidic chip analyzer that Raman spectrum detects of using.
Background technology
At present, the micro-fluidic chip technology is fast-developing, it is that the basic operation units such as a kind of handle is biological, chemical, the sample preparation of medical analysis process, reaction, separation, detection are integrated on the chip of a micro-meter scale, automatically finish the technology of analyzing overall process, it has huge application potential in fields such as biology, chemistry, medical science.Chip analyzer mainly is comprised of two parts: the one, and power source.The power source of broad sense comprises electricity, magnetic, sound, light, heat, and present stage, power supply provided the various power that comprise that fluid drives still take power supply as main.The 2nd, detecting device, detecting device then are used for catching the signal that micro-fluidic chip produces.Detecting device is the important component part of Microfluid based Lab on a chip, and the most frequently used is laser induced fluorescence detector, also has in addition ultraviolet, galvanochemistry, mass spectrum, chemiluminescence, Raman spectrum detecting device etc.Wherein Raman spectrum is a kind ofly to exempt from mark, detection mode harmless, high information quantity (Fingerprint), and convenient and other detection means couplings, such as mass spectrum etc.Raman spectrum is a kind of very effective means that sample type is analyzed.If matching surface strengthens technology, even can realize the detection of single molecules level, so it or a kind of high-sensitive detection means.Raman spectrum is very potential new development trend in the microfluidic chip analysis technology.
Micro-fluidic chip analyzer device kind in the market is fewer, and only two kinds that can see on Chinese market are all adopted laser-Induced Fluorescence Detection, significant limitation is arranged.Wherein a kind of is the instrument of the Microfluidic Tool Kit by name of derivative Micralyne company of Canadian Albert university production, and it is focused to hand; Another kind is the Bioanalyzer2100 of Agilent company, and its chip, electrode can not be regulated change so that kit is fixed mode, more are applicable to the application of fixed test project.Raman spectrum can provide than the more information of fluoroscopic examination, a kind of molecular fingerprint spectrum, at aspects such as amalyzing substances kinds obvious advantage is arranged, but do not have at present commercial, as can to detect a Raman signal micro-fluidic chip analyzer, this has just limited the application of Raman spectroscopy on micro-fluidic chip system.The micro-fluidic chip analyzer that detects with Raman spectrum at present also rests on the laboratory study stage, and the universal Raman chip analyzer that is applicable to various micro-fluidic chips detections yet there are no report.The micro-fluidic chip biological analyser that detects with Raman spectrum that develop such as StAndrews university and document (Optics Express187642) report all is that detection part is fixed on the chip, be not easy to regulate and the change chip, be not suitable for universal laboratory.And traditional large-scale micro-Raman spectroscopy is not suitable for directly micro-fluidic chip being detected as universal instrument.Because the detected object of large-scale micro-Raman spectroscopy is generally the liquid in powder or the glass tube, the backreflection detection mode that general employing is just being put, but such structure is not suitable for the detection of micro-fluidic chip and the exploitation of correlation technique.Because the micro-fluidic chip upper surface need to be arranged some pipelines with analysans injection and output, if the detection mode of the backreflection that employing is just being put, microlens and pipeline can collide and the impact detection mutually.Therefore be inverted the structure that detects and be more suitable for carrying out exciting and detecting of Raman spectrum, namely just detect from the bottom surface of micro-fluidic chip and can avoid the problems referred to above.For the spectrometer of fiber Raman sonde-type, although can realize the position of popping one's head in and detection side to flexible control, can not micro-imaging, can't accurately navigate to micron-sized surveyed area on the chip.In addition, traditional Raman spectrometer all is to realize the functions such as location and focusing by the position that the mobile example platform changes sample and object lens, but this is not suitable for micro-fluidic chip.Because may connect some rigidity on the chip with other structures such as unbending pipeline or optical fiber, there is certain difficulty the moving chip position in operation, even some micro-fluidic chip is to be fixed on other modules, can't move at all.Therefore carry out Raman detection at micro-fluidic chip and can guarantee that preferably chip position fixes, the movement by detection probe realizes the location and focuses on.The purpose of micro-fluidic chip development is in order to realize lab-on-a-chip, and a kind of movably chip analyzer is necessary for this technical development, and large-scale micro-Raman spectroscopy is all irremovable, can't Site Detection.Micro-fluidic chip is as a kind of special " sample ", universal Raman spectrometer all can't make things convenient for, effectively it be analyzed at present, and developing a universal micro-fluidic chip analyzer with the Raman spectrum detection will be necessary to the development of micro-fluidic chip technology.
Therefore, except traditional sensitivity, outside the parameters such as resolution and stability need to meet the demands, detect Raman signal for convenient at micro-fluidic chip, Raman detector in the micro-fluidic chip analyzer also needs to satisfy following technical requirement: (1) detection probe is separated with micro-fluidic chip, and adopt the mode that detects Raman spectrum of being inverted at micro-fluidic chip, (2) can be to the micro-fluidic chip micro-imaging, (3) the micro-fluidic chip position is fixed, detection probe can realize fine adjustment and location in the XYZ direction, (4) integrated, be convenient to move, can be used for Site Detection.
The micro-fluidic chip technology is the emphasis of current micro-total analysis system field development, is futures analysis and the direction that detects development.Its target is to realize lab-on-a-chipization and " individual laboratory ".Design a kind of easily, effectively, the Microfluidic chip analyzer device that adopts Raman spectrum to detect is the important tool that promotes this technical development.
Summary of the invention
The micro-fluidic chip analyzer that the object of the present invention is to provide a kind of universal application Raman spectrum to detect, it is the analytical instrument of designing and developing for the Raman spectrum that detects analyte at micro-fluidic chip specially, micro-fluidic chip analyzer is comprised of the light path module of separating with micro-fluidic chip and the three-dimensional automatic displacement positioning table of regulating the light path module, has realized on micro-fluidic chip accurate location and the Raman spectrum of sample is detected.Having (1) detection probe separates with micro-fluidic chip, and adopt the mode that detects Raman spectrum of being inverted at micro-fluidic chip, (2) can be to the micro-fluidic chip micro-imaging, (3) the micro-fluidic chip position is fixed, and detection probe can realize in the XYZ direction technical characteristics such as fine adjustment and location.Light path module and three-dimensional automatic displacement positioning table and control circuit etc. all place in the chlamydate apparatus frame of tool, and micro-fluidic chip to be measured is fixed in the upper surface of apparatus frame, have realized integratedly, are convenient to mobile.This device parameters all satisfies the requirement of carrying out Raman detection at micro-fluidic chip, and volume is little, and cost is low, is a kind of novel portable micro-fluidic chip analyzer, in this field important application potential and commodity market is arranged.
The light path module comprises that inverted micro-imaging light path and inverted Raman spectrum detect light path.Micro-imaging optical routing white light source, convergent lens and CCD form; Raman detection optical routing laser instrument, optical filter, convergent lens and spectrometer form.The exciting light that is sent by laser instrument under the control of three-dimensional automatic displacement positioning table, is focused on the sample in the micro-fluidic chip by the lower surface of micro-fluidic chip behind optical filter, convergent lens, and the Raman signal of excited sample is collected by spectrometer; The white light that is sent by white light source is after convergent lens is assembled, and the lower surface by micro-fluidic chip shines on the micro-fluidic chip equally, and reflected light is by the CCD imaging; Shine the laser facula of micro-fluidic chip lower surface also by the CCD imaging.In conjunction with the micro-imaging light path, can realize laser in accurate location and the focusing of optional position, micro-fluidic chip surface with the movement of outer computer by the three-dimensional automatic displacement positioning table of programmed control, and then realize the co-ordination of spectrometer, CCD.
Compact conformation of the present invention, the indexs such as the resolution of instrument, sensitivity all can satisfy at micro-fluidic chip carries out the needs that Raman spectrum detects, adopt design of the present invention, will conveniently carry out the detection of Raman spectrum at various micro-fluidic chips, thereby the sample in the chip is identified and analyzed.The present invention has following useful feature: (1) Raman detection probe of the present invention separates with micro-fluidic chip, is applicable to various micro-fluidic chips, and chip is changed convenient, to the requirement of chip without special processing and processing, highly versatile.(2) pipeline that runs into of the inversion detection architecture that adopts of the Raman detector of the present invention structure that is conducive to avoid the chip top to detect stops problem, makes things convenient for the detection of micro-fluidic chip Raman spectrum and the design of micro-fluidic chip.(3) but micro-imaging be conducive to realize on the micro-fluidic chip of labyrinth accurate location and measure, make instrument have versatility.The interference of pipeline the etc. when design of (4) moving detection probe with the fixed sample platform can be avoided mobile micro-fluidic chip.(5) realized integratedly, be convenient to mobilely, can be used for Site Detection.
Description of drawings
Below in conjunction with accompanying drawing with describe in conjunction with specific embodiments detection method of the present invention and device in detail, but this several method and the device that are not limited to carry.
Fig. 1: the embodiment of the invention 1 described a kind of one-piece construction schematic diagram of using the micro-fluidic chip analyzer of Raman spectrum detection;
Fig. 2: the described a kind of front elevation of using light path module in the micro-fluidic chip analyzer that Raman spectrum detects of the embodiment of the invention 2;
Fig. 3: the embodiment of the invention 2 described a kind of micro-fluidic chip analyzer Computers of using the Raman spectrum detection are controlled the operational flowchart of three-dimensional automatic displacement positioning table;
Fig. 4: the described a kind of Raman detection sonde configuration schematic diagram in the micro-fluidic chip analyzer that Raman spectrum detects of using of the embodiment of the invention 2;
Fig. 5: the described a kind of front elevation of using light path module in the micro-fluidic chip analyzer that Raman spectrum detects of the embodiment of the invention 3;
Fig. 6: the micro-imaging photo on the micro-fluidic chip surface that the embodiment of the invention 3 described a kind of micro-fluidic chip analyzers of using the Raman spectrum detection detect;
Fig. 7: the Surface enhanced raman spectroscopy of the p-Mercaptoaniline molecule that classical silver sol strengthens in the micro-fluidic chip passage that the embodiment of the invention 3 described a kind of micro-fluidic chip analyzers of using the Raman spectrum detection detect.
Embodiment
Embodiment 1:
Fig. 1 comprises that for conceive the overall schematic of a kind of micro-fluidic chip analyzer of design according to the present invention chip power source 16 and micro-fluidic chip analyzer form.Chip power source 16 is the continuous syringe pump of employing hydrodynamic injection or the high-field electrode of employing voltage extraining sampling.Inverted Raman spectrum detects light path and inverted micro-imaging light path is integrated in the light path module 1, light path module 1 is fixed on the three-dimensional automatic displacement positioning table 2, light path module 1 and three-dimensional automatic displacement positioning table 2 place in the chlamydate apparatus frame 4 of tool, micro-fluidic chip 3 to be measured is fixed in the upper surface of apparatus frame 4, detects, is inverted the technical requirements such as micro-imaging and the accurate location of detection laser thereby realize being inverted Raman spectrum.
Embodiment 2:
Fig. 2 is for conceiving light path module 1 schematic diagram of a kind of concrete structure of design according to the present invention.This optical routing Raman spectrum detects light path and micro-imaging light path two parts form, and is compact conformation, and when carrying out the Raman spectrum detection, neutral semi-transparent semi-reflecting lens 81,82 are switched to outside the light path by converter 12.Can be selected the BRM785 laser instrument of BWTEK company by laser instrument 5() expand behind the laser planoconvex lens 61 that produces or become directional light, mating plate 71 filters the parasitic light except laser after filtration; Again through long logical dichroic filter 72(785nm wavelength reflection, the above wavelength of 785nm sees through) with laser reflection, the laser of reflection changes vertical light path by behind the near-infrared reflection mirror 152 into by horizontal optical path, then on the lower surface of object lens 62 focuses on sample in the micro-fluidic chip 3; The Raman signal (wavelength is greater than 785nm) of institute's excited sample is collected by object lens 62, change vertical light path into horizontal optical path by near-infrared reflection mirror 152, behind long logical dichroic filter 72 and long pass filter 73, filter Rayleigh scattering light again, focus in the modular optical spectrometer 10 finally by lens 63, finish exciting and detecting of Raman spectrum; When carrying out micro-imaging, neutral semi-transparent semi-reflecting lens 81,82 are switched within the light path by converter 12, the illumination light of being launched by white light source 11 through lens 64 focus on, through semi-transparent semi-reflecting lens 81 reflections, through semi-transparent semi-reflecting lens 82, change horizontal optical path into vertical light path post-concentration to the back focal plane of object lens 62 through 152 reflections of near-infrared reflection mirror again, behind object lens 62, become the lower surface that directional light shines micro-fluidic chip 3 uniformly; Reflected light becomes directional light through object lens 62, change vertical light path into horizontal optical path by catoptron 152 after, through 82 reflections of semi-transparent semi-reflecting lens, be imaged onto on the CCD9 through imaging len 65 again.Simultaneously Ear Mucosa Treated by He Ne Laser Irradiation to micro-fluidic chip 3 lower surface imagings also through same light path imaging to CCD9, the image in CCD9 is comprised of micro-image and the laser lighting hot spot of micro-fluidic chip.Above-mentioned whole light path is except object lens 62 and micro-fluidic chip 3, and remaining part all is positioned at the same level plane, and object lens 62 are positioned at the perpendicular vertical with this horizontal plane with micro-fluidic chip 3; Long logical dichroic filter 72, semi-transparent semi-reflecting lens 81,82, catoptron 152 all arrange with incident light angle at 45 °.
Fig. 3 is the process flow diagram of the three-dimensional automatic displacement positioning table 2 of computer control.Main flow process is: the steering order that Labview master routine on computers produces is sent in the Data Detection card through the usb data line, the Data Detection card produces and drives signal controling stepping motor driver generation drive current, drive three-dimensional automatic displacement positioning table 2(VERTEX five phase step motor translation stage) stepper motor motion, thereby realize the control of three-dimensional automatic displacement positioning table.In conjunction with micro imaging system, can realize accurate location and focusing in optional position, micro-fluidic chip surface with outer computer.It is 1.2 microns that displacement platform moves minor increment, satisfies the pinpoint requirement on micro-fluidic chip.
Fig. 4 is the probe segment schematic diagram of micro-fluidic chip analyzer among the present invention.Probe segment separates highly versatile with micro-fluidic chip 3.Testing sample 13 is positioned at the passage of micro-fluidic chip 3 inside, the object lens 62 of long reach with Laser Focusing in the sample 13 of micro-fluidic chip 3 inner passages, thereby effectively excite Raman signal with test sample.
Embodiment 3:
The vertical view of another light path module 1 that Fig. 5 designs according to the present invention, whole light path are integrated into a light path module and are installed on the XYZ three axle automatic displacement positioning tables 2.Be the compact purpose with being convenient to optical path adjusting of implementation structure, the present embodiment has added 3 near-infrared reflection mirrors 151,153,154(reflectivity more than 95% on the basis of Fig. 2).This light path comprises that equally micro-imaging light path and Raman spectrum excite and detect light path two parts.
Carry out Raman spectrum excite and when detecting, semi-transparent semi-reflecting lens 81,82 are switched to outside the light path by converter 12.Be that the laser that produces of the semiconductor laser 5 of 785nm is through 100 microns of optical fiber 14(core diameters by emission wavelength, seeing through wavelength is visible near-infrared wave band, numerical aperture 0.22) transmission, after through lens 61(d=12.7, f=25, d are diameter, and f is focal length, unit is millimeter) become directional light, mating plate 71(sees through wavelength 785nm after filtration) filter the parasitic light except laser; After near-infrared reflection mirror 151, long logical dichroic filter 72,152 triple reflections of near-infrared reflection mirror, change vertical light path into by horizontal optical path again, through 10 times of object lens 62(enlargement factors, numerical aperture 0.22, the long reach apochromatic objective) focus on the sample in the micro-fluidic chip 3 to be measured; The Raman signal of institute's excited sample is collected by object lens 62, change vertical light path into horizontal optical path through near-infrared reflection mirror 152, after long logical dichroic filter 72 by near- infrared reflection mirror 154 and 153 liang of secondary reflections, and then filter Rayleigh scattering light through long pass filter 73, finally by lens 63(d=12.7, f=25) focus on modular optical spectrometer 10(BWTEK112E spectrometer) in, exciting and detecting of Raman spectrum finished.
When carrying out micro-imaging, semi-transparent semi-reflecting lens 81,82 is switched within the light path by converter 12.Micro imaging system comprises illumination path and imaging optical path.The illumination light of illumination path employing Kohler illumination system: by white light source 11(light emitting diode) launching is through lens 64(d=25.4, f=50) focus on, through semi-transparent semi-reflecting lens 81 reflections, through semi-transparent semi-reflecting lens 82, change horizontal optical path into vertical light path post-concentration to the back focal plane of object lens 62 through catoptron 152 reflections again, behind object lens 62, become the lower surface that directional light shines micro-fluidic chip 3 uniformly; Reflected light becomes directional light through object lens 62, change vertical light path into horizontal optical path by near-infrared reflection mirror 152 after, through 82 reflections of semi-transparent semi-reflecting lens, again through imaging len 65(d=25.4, f=150) be imaged onto on the CCD9.Simultaneously Ear Mucosa Treated by He Ne Laser Irradiation to micro-fluidic chip 3 lower surface imagings also through same light path imaging to CCD9, the image in CCD9 is comprised of micro-image and the laser lighting hot spot of micro-fluidic chip.
Above-mentioned whole light path is except object lens 62 and micro-fluidic chip 3, and remaining part all is positioned at the same level plane, and object lens 62 are positioned at the perpendicular vertical with this horizontal plane with micro-fluidic chip 3; Long logical dichroic filter 72, semi-transparent semi-reflecting lens 81,82, near-infrared reflection mirror 151,152 arranges with incident light angle at 45 °, and near-infrared reflection mirror 153,154 becomes 22.5 ° of angles settings with incident light.
Fig. 6 is the microphoto that is detected by CCD9 according to micro-fluidic chip (preparation method's list of references " Modern Scientific Instruments 2001,4, the 10-14 ") surface that the present embodiment detects.Approximately 50 microns of channel diameters in the chip, middle circular speck is laser facula, approximately 60 microns of spot diameters satisfy the requirement of carrying out the spatial resolution of Raman detection at the microcell of micro-fluidic chip.
Fig. 7 is according to the Surface enhanced raman spectroscopy of the present embodiment by the p-Mercaptoaniline molecule of the classical silver sol in the micro-fluidic chip passage of spectrometer 10 detections (J.Phys.Chem.1982,86,3391 described methods obtain) enhancing.The micro-fluidic chip power source is continuous syringe pump, together is installed in the upper surface of apparatus frame 4 with micro-fluidic chip.Be 10 with concentration
-5The p-Mercaptoaniline molecule that mole is every liter pumps in the passage of micro-fluidic chip after the by volume 1:9 mixing of classical silver sol again, and detects the Raman signal of the p-Mercaptoaniline molecule of silver sol enhancing with the designed device of the present invention.The excitation wavelength of laser is 785 nanometers, and light intensity is 30mW, 10 seconds integral time.Stronger peak intensity proves that this device satisfies the requirement that detects low concentration sample at micro-fluidic chip among the figure, i.e. the sensitivity of instrument has reached requirement.Each Raman peaks can be separated clearly among the figure in addition, proves that the wavelength resolution of this device satisfies the requirement that detects Raman spectrum at micro-fluidic chip.The spectral line such as 1585,1443,1385,1072 wave numbers can analyze this to detect thing be p-Mercaptoaniline from figure.Therefore, this embodiment shows that the present invention can be competent at analysis and the identification of low concentration sample kind in the micro-fluidic chip.
Claims (4)
1. use the micro-fluidic chip analyzer that Raman spectrum detects for one kind, it is characterized in that: formed at the accurate mobile three-dimensional automatic displacement positioning table (2) of XYZ direction by the light path module (1) of separating with micro-fluidic chip (3) and adjusting light path module (1); Light path module (1) and three-dimensional automatic displacement positioning table (2) place in the chlamydate apparatus frame of tool (4), and micro-fluidic chip to be measured (3) is fixed in the upper surface of apparatus frame (4); Light path module (1) comprises that inverted micro-imaging light path and inverted Raman spectrum detect light path.
2. a kind of micro-fluidic chip analyzer that Raman spectrum detects of using as claimed in claim 1 is characterized in that: Raman detection optical routing laser instrument (5), optical filter, convergent lens and modular optical spectrometer (10) composition; Micro-imaging optical routing white light source, convergent lens and CCD form; The exciting light that is sent by laser instrument (5) is behind optical filter, convergent lens, under the control of three-dimensional automatic displacement positioning table (2), by the lower surface of micro-fluidic chip to be measured (3) location with focus on the sample in the micro-fluidic chip, the Raman signal of excited sample is collected by modular optical spectrometer (10), thereby realizes that the Raman spectrum for the treatment of sample in the micrometer fluidic chip (3) detects; The white light that is sent by white light source (11) is after convergent lens is assembled, lower surface by micro-fluidic chip to be measured (3) shines on the micro-fluidic chip equally, reflected light is by CCD(9) imaging, shine the laser facula of micro-fluidic chip lower surface also by CCD(9) imaging.
3. a kind of micro-fluidic chip analyzer that Raman spectrum detects of using as claimed in claim 2, it is characterized in that: Raman detection optical routing laser instrument (5), convex lens (61), optical filter (71), long logical dichroic filter (72), near-infrared reflection mirror (152), object lens (62), long pass filter (73), lens (63) and modular optical spectrometer (10) form, expand behind the laser planoconvex lens (61) that is produced by laser instrument (5) or become directional light, mating plate (71) filters the parasitic light except laser after filtration; Again through long logical dichroic filter (72) with laser reflection, the laser of reflection changes vertical light path into by horizontal optical path afterwards by near-infrared reflection mirror (152), then on the lower surface of object lens (62) focuses on sample in the micro-fluidic chip (3); The Raman signal of institute's excited sample is collected by object lens (62), change vertical light path into horizontal optical path by near-infrared reflection mirror (152), behind long logical dichroic filter (72) and long pass filter (73), filter Rayleigh scattering light again, focus in the modular optical spectrometer (10) finally by lens (63), finish exciting and detecting of Raman spectrum; When carrying out micro-imaging, neutral semi-transparent semi-reflecting lens (81,82) are switched within the light path by converter (12), the illumination light of being launched by white light source (11) through lens (64) focus on, through semi-transparent semi-reflecting lens (81) reflection, through semi-transparent semi-reflecting lens (82), change horizontal optical path into vertical light path post-concentration to the back focal plane of object lens (62) through near-infrared reflection mirror (152) reflection again, behind object lens (62), become the lower surface that directional light shines micro-fluidic chip (3) uniformly; Reflected light becomes directional light through object lens (62), change vertical light path into horizontal optical path by catoptron (152) after, through semi-transparent semi-reflecting lens (82) reflections, be imaged onto CCD(9 through imaging len (65) again) on; Simultaneously Ear Mucosa Treated by He Ne Laser Irradiation also arrives CCD(9 through same light path imaging to micro-fluidic chip (3) lower surface imaging) on, at CCD(9) in image formed by micro-image and the laser lighting hot spot of micro-fluidic chip; Above-mentioned whole light path is except object lens (62) and micro-fluidic chip (3), and remaining part all is positioned at the same level plane, and object lens (62) are positioned at the perpendicular vertical with this horizontal plane with micro-fluidic chip (3); Long logical dichroic filter (72), semi-transparent semi-reflecting lens (81,82), catoptron (152) all arrange with incident light angle at 45 °.
4. a kind of micro-fluidic chip analyzer that Raman spectrum detects of using as claimed in claim 3 is characterized in that: increase optical fiber (14) and 3 near-infrared reflection mirrors (151,153,154) in the Raman detection light path; The laser that is produced by laser instrument (5) is through optical fiber (14) transmission, after expand or become directional light through lens (61), mating plate (71) filters the parasitic light except laser after filtration; After near-infrared reflection mirror (151), long logical dichroic filter (72), near-infrared reflection mirror (152) triple reflection, change vertical light path into by horizontal optical path again, on object lens (62) focus on sample in the micro-fluidic chip to be measured (3); The Raman signal of institute's excited sample is collected by object lens (62), change vertical light path into horizontal optical path through near-infrared reflection mirror (152), after long logical dichroic filter (72) by near-infrared reflection mirror (154) and near-infrared reflection mirror (153) two secondary reflections, and then filter Rayleigh scattering light through long pass filter (73), focus in the modular optical spectrometer (10) finally by lens (63), finish exciting and detecting of Raman spectrum; Near-infrared reflection mirror (151,152) arranges with incident light angle at 45 °, and near-infrared reflection mirror (153,154) becomes 22.5 ° of angles to arrange with incident light.
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