CN106896095B - The micro-imaging technique of composite surface plasma resonance and surface-enhanced Raman - Google Patents

The micro-imaging technique of composite surface plasma resonance and surface-enhanced Raman Download PDF

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CN106896095B
CN106896095B CN201710017302.3A CN201710017302A CN106896095B CN 106896095 B CN106896095 B CN 106896095B CN 201710017302 A CN201710017302 A CN 201710017302A CN 106896095 B CN106896095 B CN 106896095B
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CN106896095A (en
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李文雪
时元振
庞霖
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Sichuan University
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    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/65—Raman scattering
    • G01N21/658—Raman scattering enhancement Raman, e.g. surface plasmons
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/55—Specular reflectivity
    • G01N21/552—Attenuated total reflection
    • G01N21/553—Attenuated total reflection and using surface plasmons
    • G01N21/554—Attenuated total reflection and using surface plasmons detecting the surface plasmon resonance of nanostructured metals, e.g. localised surface plasmon resonance

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Abstract

The invention discloses a kind of surface plasma body resonant vibration (Surface Plasmon Resonance,) and surface-enhanced Raman (Surface-enhanced Raman Scattering SPR, SERS compound micro-imaging technique) is related to surface plasma base member and surface-enhanced Raman field.Technical key point: the comprehensive micro imaging system of assembling SPR-SERS is excited using nano slit array grating two mode field and detects SPR and SERS.SPR system excites the variation of angle by the mobile judgement of measurement SPR peak position, to determine the change of the caused surface effective refractive index of chip surface biomolecule reaction；SERS system is by measurement Raman spectrum directly to differentiate biomolecule itself.And efficient, the highly sensitive detection of SPR and Raman is by means of nano slit array grating two mode field: excitation light wave efficiently excites SPR through optical grating construction；SPR generates dipole in the communication process of metal surface with nano gap, and surface field and nano slit dipole collective effect the enhancing surface local electric field that SPR is generated obtain the Raman signal of enhancing.

Description

The micro-imaging technique of composite surface plasma resonance and surface-enhanced Raman
Technical field
The present invention relates to surface plasma base member and surface-enhanced Raman field, large area periodic nanometer gap arrays Structure excites plasma resonance and surface-enhanced Raman and a kind of composite surface plasma resonance and surface-enhanced Raman Micro-imaging technique.
Background technique
Surface plasma resonance (Surface Plasmon Resonance, SPR) is that photon is incident on precious metal surface A kind of quantum photoelectric phenomenon vibrated so as to cause the electronics in metal with electric field.SPR technique passes through measurement metal circle The variation of coupling condition is excited to examine after biological substance interaction occurs on face, caused by the variation of surface effective refractive index Biomolecule is surveyed, is a kind of measurement indirectly；And Raman signal detection is then a kind of complete directly measurement.Raman scattering is to be measured Sample is to the inelastic scattering of incident light, and its essence is when inelastic collision occurs for photon and molecule, photon is by energy transmission After testing molecule, transition and radiation occur for molecular energy state, disclose the vibration of molecule or the spectral technique of rotational energy level.Raman light Spectrum provides the intrinsic vibration of molecule in detected materials and rotary mode, directly reacts the molecular structure of sample to be tested.However it draws Graceful scattering is a kind of weak scattering process, and detecting limitation is in ambient noise and fluorescence background.Raman scattering cross section is about 10-30cm2, And the scattering section of fluorescence process is about 10-15cm2；Relative to Raman scattering, fluorescence signal is significantly larger than Raman scattering, this is also The more commonly used reason of fluorescent technique at present.Raman signal Electromagnetic enhancement is a kind of (such as coarse by local electric field Metal surface can generate the local electric field of enhancing) the Raman enhancement effect that is caused；This so-called surface-enhanced Raman dissipates Penetrate photoelectricity field intensity locating for signal caused by (Surface-enhanced Raman Scattering, SERS) and molecule Biquadratic is directly proportional.Obviously, the enhancing of Raman signal depends on the enhancing of local electric field, and local electric field concentrates on nanometer resonance Near structure, therefore SERS is suitable for directly differentiating and detecting for the protein molecular of surface attached molecules or cell surface.
Direct resolution in view of SPR technique to the indirect detection of biomolecule and Raman spectrum to molecule, in recent years constantly There is researcher to inquire into the enhancing of SPR Raman, or the dual-mode structure that both of which will be combined.It there is now research for nano grain of silver Optical grating construction is embedded in excitating surface plasma, enhances the local fields between nano particle to carry out surface-enhanced Raman inspection It surveys.However, what Argent grain layer used in this method was randomly formed, it is unable to accurately control nanoparticle position and gap is formed Mode, therefore the repeatability of testing result is low, so that practical application can not be carried out.In addition, there is research to make periodicity Gold nano butterfly junction structure forms SPR and SERS substrate；Periodic structure excitating surface plasma, and butterfly junction structure swashs Dipole resonance is sent out, strong local electric field enhancing Raman signal is formed.However nanostructure bowknot manufacture craft is cumbersome, needs electricity consumption The production of the method for beamlet photoetching or ion beam etching, expensive cost of manufacture at all can not applied generalization.Although using nanometer Method for stamping can reduce its cost of manufacture, but its nanostructure, in transfer process, precision not can guarantee.There is research to use 111 crystal orientation carry out wet etching on a silicon substrate and obtain periodic three-legged structure, utilize metal periodic structure excitating surface Plasma local electric field enhances Raman.The method can reach 80% Raman signal detection repetitive rate, basically reach functionization and want It asks.But its enhancement method only enhances Raman signal by surface plasma resonance, lacks nanostructure enhancing local field strength, causes Enhancing rate is not high, can not carry out high-precision biological detection.
The above method can realize surface plasma enhancing Raman detection or surface plasma to a certain extent It is detected while with surface Raman enhancement, but that presently, there are costs of manufacture is high, precision is low, can the disadvantages of the practicability is poor；In addition, SPR-SERS systematic collaboration carries out micro-imaging and has not been reported.
Summary of the invention
The technical problems to be solved by the present invention are: in view of the above problems, a kind of high-precision is provided, can be practical Composite surface plasma resonance and surface-enhanced Raman micro-imaging technique, in same chip realize surface plasma The super enhancing of the efficient excitation and surface local fields of resonance.
The technical solution adopted by the invention is as follows: the comprehensive micro imaging system of assembling SPR-SERS, utilizes nano slit battle array Column grating two mode field excites and detection SPR and SERS simultaneously.
The comprehensive micro imaging system of SPR-SERS is as shown in Figure 1.It introduces and swashs except simple microscope white-light illuminating optical path Light, with the resonance in vitro such as excitating surface on SPR-SERS complex function chip, for incuding biological sample thereon；This laser Local electric field can be excited to enhance simultaneously, excitation biological sample generates Surface enhanced Raman scattering；These two types of signals pass through micro- object Mirror signal collection system, optical path separate and then respectively carry out imaging display and data analysis.
Using large-area nano slit array optical grating construction as SPR-SERS compound chip, as shown in Fig. 2, one week There are the nano gaps of two 10 nanometer scales in phase to generate Local field enhancement.Excitation light wave efficiently excites SPR through optical grating construction: For microscope system, such as Fig. 3, parallel laser is focused through microcobjective, is converged on chip, reaches the incident light packet of chip The angular aperture determined by zero degree to object lens is included, azimuth is zero degree to all light beams in 360 degree of taper；Laser light incident side To in angular aperture and azimuthal variation, variable scanning, i.e. angular aperture and azimuth angle point required for SPR is detected are provided The blanking bar for going out to represent SPR to excite in cloth (see Fig. 3 (b)).SPR generates dipole with nano gap in the communication process of metal surface Oscillation, the Raman that surface field and nano slit dipole collective effect the enhancing surface local electric field that SPR is generated obtain enhancing are believed Number.
Specific detection include: illumination white light (not shown) after the light splitting of Amici prism 3 by 4 focal imaging of lens in receiving screen On 5, general imaging inspection, including sample focusing, regional choice and sample profile observation etc. are carried out.Human eye 1 can be poly- by lens 2 The directly observation imaging of picture of the coke on focal plane.The laser 15 of two kinds of wavelength is introduced except simple microscope white-light illuminating optical path (such as applying same light path system from two ports, introduce 633 nanometers and 785 nanometer lasers respectively), through one group of lens 14 With 12 one-tenth directional lights, wherein position phase diffuser 13 is used to remove the spatial coherence of light, to eliminate laser imaging image patch, (SPR is imaged With), position photo 11, polarizing film 10 are to modulating polarization state.Parallel laser light beam imports microcobjective 8 through Amici prism 7, gathers Nano slit array grating SPR-SERS chip area of the coke on sample stage 9.Focus on the light on SPR-SERS chip area Line imports next Amici prism 6 using Amici prism 7 after microcobjective 8 collects the reflected beams.Collimated light beam is by filtering Piece 16 respectively enters SPR imaging system and SERS detection system: excite a route parallel beams of SPR all pass through filter plate 16 by Lens 17, which focus, imports CCD camera (Charge Coupled Device, charge-coupled device) camera 18, is connect by CCD camera It receives spr signal (Fourier transform plane) computer and shows SPR picture, two blanking bar (such as Fig. 3 of SPR excitation are represented by measuring (b)) movement of characterization excitation angle；Raman scattering light is all reflected by filter plate 16 and is passed through reflecting mirror 19 and change optical path Direction is focused on Raman detection region (including grating 23,24 and CCD camera 25) 26 using after filter plate 20 by lens 21 Slit 22, by Raman detection region 26 receive Raman spectrum signal, the intrinsic peak of analyzing molecules is directly to differentiate molecule.
In conclusion key of the invention are as follows: by surface plasma resonance excitation and surface-enhanced Raman excitation same It realizes on one chip, using microscope imaging, is detected while realizing surface plasma with surface-enhanced Raman.Pass through measurement The variation of the mobile judgement excitation angle of SPR peak position, to determine that the caused surface of chip surface biomolecule reaction is effectively rolled over Penetrate the change of rate.Meanwhile Local field enhancement caused by the coupling in metal surface SPR and nanometer gap, it can be used to measure Raman Spectrum is directly to differentiate biomolecule itself.SPR is measured indirectly and the direct judgement of Raman signal, determines sensitive surface to be accurate On biological respinse increase certainty.To sum up, compound micro-imaging technique proposed by the present invention have it is at low cost, precision is high, Advantage that can be practical, can be widely applied to the fields such as biological detection.
Detailed description of the invention
Examples of the present invention will be described by way of reference to the accompanying drawings, in which:
Fig. 1 is the compound micro-imaging schematic diagram of SPR-SERS dual-mode chip.1 is human eye, and 2,4,12,14,17,21 are Mirror, 3,6,7 be Amici prism, and 5 be receiving screen, and 8 be microcobjective, and 9 be sample stage, and 10 be polarizing film, and 11 be position photo, and 13 are Position phase diffuser, 15 be laser, and 16,20 be filter plate, and 18,25 be CCD camera, and 19 be reflecting mirror, and 22 be slit, and 23,24 are Grating, 26 be Raman detection region.
Fig. 2 (a) is the unilateral type schematic diagram of composite core of atomic force microscope measurement.It shows nanometer gap array structure and receives Rice gap, the elevation information such as Fig. 2 (b) in figure bend region.
Fig. 2 (b) is the slit scan figure of compound chip.Slit scan figure corresponds to oblique in Fig. 2 (a) mesoprosopy schematic diagram The elevation information of line Regional Representative.
Fig. 3 (a) is microscope schematic diagram.For the angle between objective lens optical axis and practical light,For in cone-shaped beam certain With x-axis direction institute at azimuth on one ray cast to disk, azimuth degree variation from 0 degree to 360；Coordinate description In Fig. 3 (b).
Fig. 3 (b) is for incidence angleWhen SPR picture.White circular astragal indicates the Fourier transform plane of SPR in figure, Two black blanking bars represent SPR excitation.X-axis direction is along counterclockwise around a circle, corresponding azimuth in figureDegree change from 0 degree to 360 Change；Angular aperture is radially respectively corresponded outward from the center of circle in x-axis directionFrom 0 degree to from positive and negative maximum aperture angle.
Fig. 4 is SPR excitation-detection platform schematic diagram.15 be laser, and 27 be the microcobjective of 40 times, numerical aperture 0.65, 28 be lens, and 7 be Amici prism, and 8 be 40 times, the microcobjective that numerical aperture is 0.65 or 100 times, numerical aperture is 0.85,9 It is lens for sample stage, 17,18 be CCD camera, and 29 be computer.
Fig. 5 is the SPR picture under different dielectrics.Surrounding's medium on metal grating, by air (air), pure water (H2O), 1%, 10%, 25%, 50%, 75% volumetric ratio ethylene glycol changes to pure ethylene glycol (Ey_Gl), and the corresponding blanking bar for representing SPR excitation is by outer It is mobile at lateral center.
Fig. 6 is that SPR excites angle with the relation curve of compound change in refraction under different dielectrics.SPR surface dielectric from When pure water, 1% ethylene glycol to 100% ethylene glycol change, excite angle with the variation relation of complex refractive index；Provide 7 data points And linear fit curve.
Fig. 7 is that SPR excites angle change curve under various concentration benzenethiol solution.Benzenethiol solution is dense in dielectric solution (unit is mole M) is spent from 0M, 10-2M、10-1M, 1M, 10M variation when, SPR excite angle with benzenethiol solution concentration variation Relationship；Provide 5 data points and linear fit curve.
Fig. 8 is the Surface Enhanced Raman Scattering Spectrum figure under various concentration benzenethiol solution.It is corresponding in turn to benzene from top to bottom Thiophenol concentration is 10-6M、10-5M、10-4M、10-3Four Raman spectrums of M.
Fig. 9 is characterized peak position 1023cm-1Absolute intensity average value with benzenethiol solution concentration variation relation.Dielectric Benzenethiol solution concentration 10 in solution-6M、10-5M、10-4M、10-3M variation, 1023cm-1Peak position absolute intensity average value is with benzene sulphur The variation of phenol concentration；Provide 4 data points and linear fit curve.
In the figure, it is marked as 1 human eye；2 lens；3 Amici prisms；4 lens；5 receiving screens；6 Amici prisms；7 Amici prisms；8 Microcobjective；9 sample stages；10 polarizing films；11 position photos；12 lens；13 phase diffuser laser；14 lens；15 laser；16 filters Wave plate；17 lens；18 CCD cameras；19 reflecting mirrors；20 filter plates；21 lens；22 slits；23 gratings；24 gratings；25 CCD phases Machine；26 Raman detection regions；27 40x/0.65 microcobjectives；28 lens；29 computers；Air refers to that coal quality is air；H2O refers to coal Matter is pure water；It than pure water volumetric ratio is 1 percent that 1% finger coal quality, which is ethylene glycol,；10% refers to that coal quality is ethylene glycol than pure water volumetric ratio It is 10；It than pure water volumetric ratio is 25 percent that 25% finger coal quality, which is ethylene glycol,；50% refers to that coal quality is ethylene glycol than pure Water capacity ratio is 50 percent；It than pure water volumetric ratio is 75 percent that 75% finger coal quality, which is ethylene glycol,；100% refers to that coal quality is Ethylene glycol is 100 percent than pure water volumetric ratio.
Specific embodiment
All features disclosed in this specification or disclosed all methods or in the process the step of, in addition to mutually exclusive Feature and/or step other than, can combine in any way.
Any feature disclosed in this specification unless specifically stated can be equivalent or with similar purpose by other Alternative features are replaced.I.e. unless specifically stated, each feature is an example in a series of equivalent or similar characteristics ?.
Using compound biological chip shown in Fig. 2: common optical grating construction forms new grating, new and old grating after parameter couples Between form nano gap, there are the nano gaps of two 10 nanometer scales in a cycle；Compound chip is that screen periods are big About 400 or 600 nanometers of gap array structure, corresponding SPR excitation wavelength is respectively 633 or 785 nanometers.For microscope system System, directional light are focused through microcobjective, are converged on chip, and the incident light for reaching chip includes determined by zero degree to object lens Angular aperture, all light beams in taper that azimuth is 0 degree to 360 degree.Incident plane wave is focused into conical distribution by microscope, Ranges of incidence angles is,For the maximum aperture angle (being determined by the numerical aperture NA of object lens) of incident light wave,, wherein n is incident medium refractive index.As shown in Fig. 3 (a), incident parallel light be converted into Firing angle is, the plane of incidence (corresponding azimuth) is 0-360 without array light wave.Incident direction is in angular aperture and side The variation of parallactic angle provides variable scanning required for SPR is detected, i.e., goes out to represent in angular aperture and azimuth angle distribution The blanking bar as shown in Figure 3 (b) of SPR excitation.By microscope schematic diagram it is found that Incident angle distribution is symmetrically, if being entered with side Firing angle excites SPR wave, must there is the SPR wave that an opposite incidence angle excites another backpropagation.
The SPR excitation-detection platform of compound dual-mode chip is as shown in Figure 4.633nm or 785nm wavelength laser 15 is through micro- object Mirror 27(40x/0.65) and lens 28 filtering, collimation after become directional light, through Amici prism 7 import microcobjective 8(40x/0.65 Or 100x/0.85) (it is placed in sample stage 9) is focused on print.Light on chip is reflected by object lens 8, is divided rib Change direction after mirror 7, focused by lens 17, receives spr signal, computer with CCD camera 18 on its Fourier transform plane The image-forming information of 29 record CCD cameras 18.The calibration of SPR body refractive index is carried out in this detection platform and face refractive index sensitivity is surveyed Examination.
Carry out SPR body refractive index sensitivity calibration: various concentration percentage ethylene glycol solution respectively according to ethylene glycol with go The volume fraction of ionized water is 0%, 1%, 10%, 25%, 50%, 75%, 100% configuration sample；When carrying out the test of each concentration point, Solution is taken out with pipette, is dripped on chip, surrounding places gasket, covers a piece of coverslip to guarantee liquid sample with a thickness of 100 Micron.It selects 785 nanometer lasers as excitation light source, reflected light is collected by 40x/0.65 microcobjective, changes metal grating On medium, changed by air, pure water, 1% ethylene glycol to 100% ethylene glycol, obtain the micro- SPR picture under various concentration (in Fu Blade face, the focal plane of imaging len), such as Fig. 5；By the relationship of volumetric ratio, according to n complex refractive index=water capacity percentageN water+ Ethylene glycol volume percentageN ethylene glycol can calculate the refractive index of mixed solution under 785 nano wave lengths；The surface linear fit SPR When dielectric changes from pure water, 1% ethylene glycol to 100% ethylene glycol, excitation angle with complex refractive index variation relation such as Fig. 6, It can be calculated 69.8 ° of Shi Jifa angulation change of every variation unit refractive index (Refractive Index Unit, RIU), it is compound SPR body refractive index sensitivity S=69.8 °/RIU of chip.
Selecting, there is the Raman marking materials benzenethiol generallyd use in document to prove to carry out the test of compound chip. SPR biological detection is to measure the variation of the caused surface effective refractive index of metal surface reaction, therefore, reflection SPR detection property The physical quantity of energy is its surface refractive index sensitivity, i.e., superficial molecular adsorbs sensitivity.Sulphur atom and metal in benzenethiol In conjunction with formation monolayer；It is proportional in conjunction with the surface density of monolayer and the concentration of benzenethiol solution on metal.With shifting It is respectively 10 that liquid device, which takes benzenethiol solution to obtain solubility in analysis straight alcohol,-6M、10-5M、10-4M、10-3M、10-2M、10-1M、1M Benzenethiol dilution, be placed in supersonic cleaning machine with 60% power room temperature ultrasound 2 minutes, benzenethiol molecule allowed to be evenly distributed on In solution.
Before carrying out the test of each SPR concentration point, chip is sequentially placed into respectively and above-mentioned matches 10-2M、10-1M、1M、10M In benzenethiol solution, impregnates and allow within 2 hours its adsorption benzenethiol molecule.Print is taken out from benzenethiol solution after immersion, is put Enter and is taken out after being rinsed 2-3 seconds in ethanol solution.Print after rinsing is put into nitrogen drying cupboard, is surveyed after dry in nitrogen stream It tries spare.It takes composite test print on sample stage 9, is focused excitation SPR with 100x/0.85 microcobjective.According to above-mentioned Test process measures spr signal of the compound chip when not depositing benzenethiol solution, the reference point for being 0 M as concentration.
Before carrying out each Raman concentration point test, chip is put into 10-6M、10-5M、10-4M、10-3In M benzenethiol solution It takes out after impregnating 4 hours, is taken out after being rinsed 2-3 seconds in ethanol solution.Print after rinsing is put into nitrogen drying cupboard, It is tested after drying in nitrogen stream spare.Test sample is put into Raman-atomic force combined system Raman detection platform with tweezers On sample stage, using 633 nanometers or 785 nm wavelength lasers as excitation light source, reach sample laser power be 0.1 milliwatt, 2.5 milliwatt.It is focused with 50x/0.5 object lens, excitation spot size is 2, scan 3 times, integrate 3 seconds.
It takes test sample on the sample stage 9 shown in Fig. 4, is focused excitation SPR with 100x/0.85 microcobjective, selects 633 nanometer lasers are as excitation light source.According to aforementioned sample preparation method, the SPR that print to be measured carries out 0M benzenethiol concentration is answered Close chip testing；10 are successively carried out again-2M、10-1M, 1M, 10M benzenethiol strength solution immersion treatment SPR compound chip carry out Test.
When in dielectric solution benzenethiol solution concentration from 0M, 10-2M、10-1M, 1M, 10M variation when, SPR excite angle with The variation relation of benzenethiol solution concentration is as shown in fig. 7, each data point represents on test sample region the flat of 4 different locations Mean value, the standard deviation value in measurement are provided with error bar；Oblique line is the average value fitting that angle is excited with the SPR under each concentration Linearity curve.When benzenethiol concentration is lower, such as 10-2M、10-1M；When benzenethiol concentration is 10M, SPR excites angular deviation It is larger.According to linear fit equation, every change unit concentration swash 1.5 ° of angulation change of hair, the surface induction sensitivity S of compound chip surface=1.5°/M。
Using compound chip, SPR and Raman scattering detection can be obtained simultaneously, only reflection signal need to be passed through Raman spectrum Instrument, and spr signal imports in SPR imager.The Raman of use-atomic force combined system Raman detection platform is examined with Fig. 1 Raman It is identical to survey Some principles.In view of compound chip is very sensitive to high concentration benzenethiol molecule, excitation intensity reaches saturation, uses Low Concentration of Benzene thiophenol solution carries out Raman detection.It is focused with 50x/0.5 object lens, focal beam spot size is 2 microns, scanning 3 It is secondary, it integrates 3 seconds and is illustrated in figure 8 10-6M、10-5M、10-4M、10-3Raman spectrogram under M concentration benzenethiol solution.From it is lower to On be corresponding in turn to 10-6M、10-5M、10-4M、10-3Tetra- Raman spectrums of M, each characteristic peak of benzenethiol are obvious；With benzenethiol concentration It reduces, Raman light intensity decreases.When concentration is reduced to 10-15MWhen, it still can detecte stronger Raman signal.In Raman spectrum Characteristic peak and high signal-to-noise ratio show that the Raman enhancement effect being higher by, the enhancement factor of SERS are 106。
The absolute intensity average value of feature peak position is taken to be Fig. 9 with the variation relation of benzenethiol solution concentration.Each data point Represent test sample region Raman spectrum 1023cm-1The absolute intensity average value of peak position, wherein three times to each concentration spot scan And it is averaged；Oblique line is 1023cm under each concentration-1The linear fit curve of peak position absolute strength value,；Standard deviation is marked with the error line in scheming.When benzenethiol concentration is higher When, such as 10-4M、10-3M；Absolute intensity due to concentration be saturated and deviation is larger.
SPR microscopic imaging device and commercial Raman microscopic system as shown in connection with fig. 4, using nano slit array grating knot Structure realizes surface plasma SPR detection in same chip and detects with Surface enhanced Raman scattering SERS.Using Fig. 1 of the present invention Microscopic system can be in same SPR- in conjunction with large-area nano slit array optical grating construction as SPR-SERS compound chip On SERS chip, while realizing efficient, the highly sensitive detection of SPR and Raman.
The invention is not limited to specific embodiments above-mentioned.The present invention, which expands to, any in the present specification to be disclosed New feature or any new combination, and disclose any new method or process the step of or any new combination.

Claims (4)

1. a kind of composite surface plasma resonance and surface-enhanced Raman micro imaging method, which is characterized in that the method Based on the comprehensive micro imaging system of SPR-SERS, excited simultaneously using nano slit array grating two mode field with detection SPR and SERS is realized；The method based on the comprehensive micro imaging system of SPR-SERS specifically: illumination white light is through Amici prism (3) After light splitting by lens (4) focal imaging on receiving screen (5), the picture that human eye (1) is focused on focal plane by lens (2) is directly seen Imaging is examined, 633 nanometers and 785 nanometers of laser (15) is introduced respectively except simple microscope white-light illuminating optical path, through lens (14) with lens (12) at directional light, wherein using the spatial coherence of position phase diffuser (13) removal light to eliminate laser imaging Image patch, to modulating polarization state, parallel laser light beam imports micro- through Amici prism (7) for position photo (11), polarizing film (10) Object lens (8) focus on the nano slit array grating SPR-SERS chip area on sample stage (9), focus on SPR-SERS core Light in panel region imports next Amici prism using Amici prism (7) after microcobjective (8) collect the reflected beams (6), collimated light beam respectively enters SPR imaging system and SERS detection system by filter plate (16), excites that SPR's is parallel all the way Light beam all passes through filter plate (16) and focuses importing CCD camera (18) by lens (17), receives spr signal by CCD camera (18), Computer shows SPR picture, and the movement of two blanking bars characterization excitation angle of SPR excitation is represented by measuring；Raman scattering light by Filter plate (16) all reflection and pass through reflecting mirror (19) change optical path direction, using after filter plate (20) by lens (21) The slit (22) on Raman detection region (26) is focused on, the signal of Raman spectrum, analysis are received by Raman detection region (26) Directly to differentiate molecule, the Raman detection region (26) includes grating (23), grating (24) and CCD camera at the intrinsic peak of molecule (25)；Screen periods are 400 or 600 nanometers in the nano slit array grating two mode field, and there are two in a cycle The nano gap of 10 nanometer scales.
2. the method according to claim 1, wherein the SPR-SERS integrates micro imaging system for directional light It is focused through microcobjective (8), converges at nano slit array grating SPR-SERS chip area, reach nano slit array grating The incident light of SPR-SERS chip area includes the angular aperture determined by zero degree to microcobjective (8), and azimuth is zero degree All light beams in 360 ° of tapers.
3. the method according to claim 1, wherein nano slit array grating two mode field excites SPR, SPR Intercoupling with the nano gap in nano slit array grating two mode field enhances surface local fields, and the Local field enhancement of enhancing is again Enhance SPR, obtains the excitation of SPR；Micro- SPR picture is received on lens Fourier transform plane by CCD camera (18).
4. the method according to claim 1, wherein nano slit array grating two mode field excitation SPR, SPR generates dipole with the nano gap in nano slit array grating two mode field in the communication process of metal surface；SPR The surface field enhancing of generation obtains strong Raman signal with nano slit local dipole field collective effect enhancing surface local fields.
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