CN112834476B - Method for realizing metal nanoparticle aggregation and SERS detection based on photophoresis technology - Google Patents
Method for realizing metal nanoparticle aggregation and SERS detection based on photophoresis technology Download PDFInfo
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- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- 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
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Abstract
The invention discloses a method for realizing metal nanoparticle aggregation and SERS detection based on a photophoresis technology. The method is realized based on a light control-micro-Raman detection system, the capture, SERS detection and micro-Raman imaging of the metal nanoparticles by the aid of the photophoretic force can be realized simultaneously, metal nanoparticle aggregates are formed on the surface of the auxiliary substrate by the metal nanoparticles, and the high-sensitivity SERS substrate is obtained. The generation of the photophoresis phenomenon of the invention is caused by the uneven surface heat distribution of the metal nano particles irradiated by the Gaussian beam to generate reverse photophoresis close to the center of a laser spot; the light beam applying the photophoretic force to the particles and the SERS detection light beam are the same light beam, the defects of limited number of captured metal nanoparticles, complex experimental device and long time consumption are overcome, and compared with the traditional metal nano sol substrate, the technology can greatly improve the SERS signal of the molecules to be detected.
Description
Technical Field
The invention relates to a method for controlling metal nanoparticles by utilizing a photophoresis technology, in particular to a method for realizing the aggregation of the metal nanoparticles and SERS detection based on the photophoresis technology, which can realize the aggregation of the metal nanoparticles on an auxiliary substrate, can greatly improve the detection sensitivity of molecules to be detected and has wide application prospect.
Background
Surface Enhanced Raman Spectroscopy (SERS) is a nondestructive detection technique using photons as probes, and is directly linked with a vibration spectrum of a molecular structure, so that fingerprint authentication can be performed on a substance, any small change of the structure of the substance can be very sensitively reacted in the Raman spectrum, and a carrier of the molecule is very important because the Surface morphology of a substrate adsorbed by the molecule is an important influence factor on whether SERS effect occurs and the strength of an SERS signal. The research on SERS-active substrates has been one of the research hotspots in this field. The metal nano particles have optical properties completely different from bulk metal, so that the scattering and absorption of incident light with specific wavelength can be enhanced, the light field energy is localized on the surface of the metal nano particles, the electric field intensity around the metal nano particles is enhanced, the spectral signal intensity of detection molecules around the metal nano particles can be obviously improved, and the metal nano particles have wide application in the SERS field due to the special optical properties of the metal nano particles. Research shows that a plurality of gold particles are gathered together to form a polymer of hundreds of nanometers, under the action of exciting light (usually near infrared) with proper frequency, SERS enhancement factors can reach 14 orders of magnitude even after the influence of resonance Raman is eliminated, nanoparticles are mutually gathered and fused to generate a large number of particle connection points, the site regions generating large surface plasma enhancement effects are called hot points, plasma coupling of the gold nanoparticles at the hot points generates strong local electromagnetic fields, and SERS signal intensity of molecules in the hot point regions can be greatly improved. Thus, the metal nanoparticle aggregates can cause the rough surface to generate electromagnetic enhanced "hot spots" and thus a stronger electromagnetic enhancement effect, so that the probe molecules generate stronger Raman signals.
At present, the preparation of metal nanoparticle aggregates mostly depends on adding an aggregating agent such as inorganic salt ions, inorganic acid or organic amine into a solution to promote the aggregation of nanoparticles, reduce the inter-particle distance and generate effective coupling, so that obvious SERS enhancement is obtained, but the excessive aggregation of metal nano-sol can cause the sedimentation of nanoparticles, and the difficulty in controlling the aggregation leads to poor SERS effect reproducibility.
Research shows that the interaction of light and substances can be used for controlling the metal nanoparticles, wherein gradient force and scattering force are proved to be used for controlling the metal nanoparticles, but the magnitude of the gradient force and the scattering force of the light is only from a few flying cows to a leather cow, and the action range is only a few micrometers, so that a large number of objects are difficult to capture and control in a large range, the working efficiency is low, the number of captured particles is limited, the duration is long, and the two beams of laser are often used for respectively controlling and exciting, and the experimental device is complex. The photophoretic force is also a tool for manipulating tiny objects, and the photophoretic phenomenon is caused by the uneven distribution of heat on the surface of tiny objects irradiated by a light beam. When the energy distribution of the light facing surface of the object is more concentrated, the temperature of liquid in contact with the light facing surface is higher, and the frequency of water molecules impacting the object is higher than the impacting frequency of the back light surface of the object, so that the object is caused to move from a high-temperature part (close to a light source) of the liquid to a low-temperature part (far away from the light source), namely forward photophoresis, otherwise, if the energy distribution of the back light surface of the object is more concentrated, the object can generate reverse photophoresis movement. At present, no research work is available for capturing metal nanoparticles by utilizing a photophoretic force and performing SERS spectral detection.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a set of micro-Raman device capable of capturing metal nanoparticles by utilizing a photophoresis force and a method for detecting the to-be-detected molecule SERS by utilizing the device, and the defects that the number of the captured metal nanoparticles is limited, an experimental device is complex and the time consumption is long in the prior art are overcome. The functions of imaging, photophoretic force capture and SERS signal detection can be realized simultaneously. Compared with the traditional metal nano sol substrate, the technology can greatly improve the SERS signal of the molecule to be detected.
In a first aspect, a device for realizing metal nanoparticle aggregation and SERS detection based on a photophoresis technology is characterized by comprising a light control-micro Raman system and an SERS enhanced substrate module, wherein the same light path is used for realizing the photophoresis capture and SERS detection through a Raman detection light path, the light path comprises a Raman detection module and a micro imaging module, the Raman detection module irradiates light emitted by a laser to the SERS enhanced substrate module, namely a sample pool, scattered light collected by a micro objective lens is guided into the Raman detection module by using a beam splitter, light of an image of an object to be detected collected by the micro objective lens is guided into the micro imaging module by using the beam splitter, the Raman spectrum measurement and the micro imaging of the object to be detected are realized, the micro imaging module is used for recording a motion state generated by the action of the photophoresis of metal nanoparticles, the SERS enhanced substrate module is composed of an auxiliary substrate, a first substrate, a second substrate, a third substrate, a fourth substrate, the laser comprises a sample pool and a mixed solution of metal nano sol and an object to be detected, wherein the power of the laser is 50-120mw, the auxiliary substrate is positioned at the bottom of the sample pool, and the thickness of the mixed solution of the metal nano sol and the object to be detected is 0.8-1.2 mm.
Further, the raman detection module comprises: the device comprises a laser, an incident optical fiber, a first collimating convex lens, a band-pass filter, a first plane reflector, a first high-pass dichroic sheet, a beam splitter, a microscope objective, a SERS enhanced substrate enhancing module, a second high-pass dichroic sheet, a second plane reflector, a high-pass filter, a first focusing convex lens, a collecting optical fiber, a spectrometer and a computer, wherein a laser beam emitted by the laser reaches the collimating convex lens through the incident optical fiber, is filtered by the band-pass filter to reach the first plane reflector, is reflected to the beam splitter and the microscope objective by the first high-pass dichroic sheet to be vertically focused to the SERS enhanced substrate module, the capture and SERS detection of gold nanoparticles are realized by photophoresis in a mixed solution of an object to be detected and gold nanoparticle sol in the SERS enhanced substrate module, and a scattering light beam generated by excitation reaches the first high-pass dichroic sheet and the second high-pass dichroic sheet after passing through the microscope objective and the beam splitter for filtering, and then the SERS signals reach the second plane reflecting mirror, reach the high-pass filter after being reflected, are collected by the collecting optical fiber after passing through the focusing convex lens and are transmitted to the spectrometer, are converted into electric signals through spectrometer light splitting and a CCD (charge coupled device) and are transmitted to a computer for displaying and storing, and the detection of the SERS signals is realized.
Further, the microscopic imaging module comprises: the light emitted by the LED array white light source sequentially passes through the second collimating convex lens, the first diaphragm, the second high-reflection mirror, the SERS enhanced substrate module, the microscope objective, the beam splitter, the low-pass filter, the first plano-convex lens, the diaphragm and the second plano-convex lens and then reaches the imaging CCD.
Further, the numerical aperture of the collection fiber is matched with the numerical aperture of the spectrometer slit.
Further, the ratio of the transmittance to the reflectance of the first high-pass dichroic sheet is 1: 9.
Furthermore, the first plane mirror and the second plane mirror are installed in the cage-type right-angle adjustable reflection type installation seat.
Furthermore, the microscopic imaging light path is transmission type illumination, and the diaphragm 23 can effectively adjust the size of the illumination light spot.
Further, the auxiliary substrate is made of a transparent quartz plate.
Further, the average particle size of the metal nanosol is 60nm, and the ratio of the gold nanosol to the object to be measured is 1: 3.
the device for realizing the metal nanoparticle aggregation and SERS detection based on the photophoresis technology realizes SERS signal detection and the movement state of the metal nanoparticles under the photophoresis acting force.
Compared with the background technology, the technical scheme has the following advantages:
the metal nanoparticles in the metal nano sol can be controlled in a large range through the photophoresis force, a large number of metal nanoparticles can be gathered in a short time, and compared with the traditional metal nano sol, the metal nano sol has a large enhancement effect on an SERS signal of an object to be detected.
The system can realize the capture of metal nanoparticles and the excitation of SERS signals only by using one laser beam, and the microscopic imaging module can monitor and record the motion state of the metal nanoparticles in the solution in real time.
Drawings
Fig. 1 is a light path diagram of a light manipulation-micro raman spectroscopy system.
Fig. 2 is a temporal diagram of the movement of gold nanoparticles captured by an imaging CCD, wherein (a) and (b) are temporal diagrams of the gold nanoparticles gathering to the center of a light spot in a field of view, and the time interval between the two diagrams is 1 s.
FIG. 3 is a contrast micrograph of the gold nanoparticles after aggregation without and with the action of the photophoresis.
FIG. 4 shows pyrene solution (5.0X 10) on two different substrates-7 mol/L), a gold nano sol substrate (a) and a gold nano particle aggregate SERS substrate generated by combining an auxiliary substrate with a photophoretic force.
FIG. 5 pyrene solution (5.0X 10)-7mol/L) of a three-dimensional waterfall plot with SERS enhancement effect varying with aggregation time, t<10min。
FIG. 6 laser focusing time and on/off state vs. pyrene solution (5.0X 10)-7mol/L) strong influence of SERS Peak at characteristic Peak (t)<10 min: continuously turning on the laser; t is t>10 min: turn off the laser for 10 minutes, and detect SERS signal of pyrene solution every one minute).
Fig. 7 field of view the extent of the annular region of the tracking gold nanoparticles.
FIG. 8 is a graph of the velocity and power of a particle fitted to an annular region
In the figure, 1 is a 785nm semiconductor laser, 2 is an incident optical fiber, 3 is a collimating convex lens, 4 is a band-pass filter, 5 is a first plane mirror, 6 is a first high-pass dichroic plate, 7 is a beam splitter, 8 is a microscope objective, 9 is a sample cell, 10 is a second high-pass dichroic plate, 11 is a second plane mirror, 12 is a high-pass filter, 13 is a focusing convex lens, 14 is a collecting optical fiber, 15 is a spectrometer, and 16 is a computer; 21 is an LED array white light source, 22 is a collimating convex lens, 23 is a diaphragm, 24 is a high-reflection mirror, 25 is a low-pass filter, 26 is a first plano-convex lens, 27 is a diaphragm, 28 is a second plano-convex lens, and 29 is an imaging CCD; the device comprises a reference numeral 31, an auxiliary substrate 32, a mixed solution of gold nano sol and an object to be detected 33, a gold nano particle aggregate 34 and a sample cell for containing the mixed solution, wherein the reference numeral 31 is 785nm control laser, the auxiliary substrate is 32, the sample cell is composed of a glass slide, a gasket and a cover glass.
Detailed description of the preferred embodiments
Example one
The technical scheme adopted by the invention comprises the following steps:
(1) constructing a light control-micro Raman detection system;
the system mainly comprises a Raman detection light path and a microscopic imaging light path which are coupled together through a beam splitter, wherein the microscopic imaging light path is a vertical light path, the microscopic imaging light path plays a vital role in the invention for the gold nanoparticles captured by the photocatalysis, and the verticality of the imaging light path is adjusted through the image of a light spot formed by irradiating laser on the surface of a sample pool in the imaging light path, so that the light beam can be strictly and vertically irradiated on the surface of the sample. The phenomenon of photophoresis is caused by the nonuniformity of light absorption of gold nanoparticles irradiated by Gaussian beams at different r positions in the liquid surface, wherein the r refers to the distance from the optical axis of the Gaussian beams. The light path is vertical, so that the uniformity of light absorption of the particles at the same r distance can be ensured.
Wherein the ratio of the transmittance to the reflectivity of the beam splitter is 1: 9.
Wherein the microscopic imaging light path is transmission type illumination.
Wherein the power of the laser is 50 mw-120 mw.
Wherein the focal length of the microscope objective is 6 mm-8 mm of long focus.
Wherein the size of the beam waist after the laser passes through the microscope objective is 10-20 μm.
(2) Preparing gold nano sol and a solution to be detected with a certain concentration;
(3) the preparation of a sample cell in the SERS enhanced substrate module in the process of realizing the photo-electrophoresis capture is realized, wherein the sample cell (from bottom to top) comprises:
the container is made of glass and has a cuboid space and is used for containing the gold nano sol and the solution to be detected.
And the auxiliary substrate is a quartz plate, and exciting light is focused into the auxiliary substrate through a cover glass during SERS detection.
And the cover glass plays a role in sealing, reduces the flow of the gold nano sol and improves the stability in the detection process.
Before Raman spectrum collection, the auxiliary substrate is placed in a vessel formed by the first step, gold nano sol is dripped into the rest space, and finally a cover glass is covered above the liquid level, so that bubbles generated by manual operation are avoided.
Wherein the solution to be detected is polycyclic aromatic hydrocarbon or pesticide.
Wherein the volume ratio of the gold nano sol to the mixed solution of the object to be detected is 1: 3.
The distance from the upper surface of the auxiliary substrate to the cover glass, namely the thickness of the liquid level, is 0.8-1.2mm, and adverse effects can be generated on the aggregation process of the gold nanoparticles due to over-thickness and over-thinness, so that the strength of an SERS detection signal is influenced.
(4) The detection sample is placed on a three-dimensional electric adjustable object stage, the distance between a laser focal plane and the auxiliary substrate is adjusted, and the laser focal point is focused into the auxiliary substrate, which is another key point for realizing the capture of gold nanoparticles by the photophoresis.
The following detailed description of embodiments of the invention is provided in connection with the accompanying drawings and examples. The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
The technical scheme of the invention is realized by the following modes: a method for realizing gold nanoparticle aggregation and SERS detection based on a photophoresis technology comprises the following steps:
(1) construction of light control-micro Raman system
As shown in fig. 1, 1 to 16 are raman detection modules, excitation light reaches a collimating convex lens 3 from an incident optical fiber 2, is filtered by a band-pass filter 4, then reaches a first plane mirror 5, light beams reach a first high-pass dichroic sheet 6 after being reflected by the first plane mirror, wherein laser with a wavelength of more than 785nm penetrates through the dichroic sheet, laser with a wavelength of less than 785nm is reflected and reaches a beam splitter 7, the laser reflected by the beam splitter reaches a microscope objective 8, the laser is focused by the microscope objective and reaches a sample cell 9, the focused light beams realize capture and SERS detection of gold nanoparticles by means of photochemistry in a mixed solution inside 1 to 9, raman signals and rayleigh scattered light generated by excitation return according to an original path, pass through the microscope objective 8 and the beam splitter 7 and reach the first high-pass dichroic sheet 6 and a second high-pass dichroic sheet 10 for filtering, and rayleigh light and stray light are reflected, raman scattered light with the wavelength larger than 785nm passes through the second plane mirror 11, then reaches the high-pass filter 12 after being reflected, finally the Raman scattered light passes through the focusing convex lens 13, is collected by the collecting optical fiber 14 and is transmitted to the spectrometer 15, and is converted into an electric signal through spectrometer light splitting and a CCD (charge coupled device) and then is transmitted to the computer 16 for displaying and storing, so that SERS signal detection is realized.
Fig. 1 shows that 21 to 29 are microscopic imaging modules, the microscopic imaging light path is transmission illumination, and the microscopic imaging modules include: the light emitted by the LED array white light source sequentially passes through the second collimating convex lens, the first diaphragm, the second high-reflection mirror, the SERS enhanced substrate module, the microscope objective, the beam splitter, the low-pass filter, the first plano-convex lens, the diaphragm and the second plano-convex lens and then reaches the imaging CCD. The light emitted by the LED white light array light source 21 passes through the collimating convex lens 22 and then passes through the diaphragm 23 to illuminate a field of view, 25 is a low-pass filter, preferably a 750nm low-pass filter, the collimating convex lens can be flexibly disassembled, 26 plano-convex lens, 27 diaphragm and 28 plano-convex lens form an aberration eliminating combination, the size of the field of view imaged by the CCD can be adjusted, the aberration is eliminated, and 29 is an imaging CCD.
The invention realizes the photophoresis capture and SERS detection through the same optical path, the optical path comprises a Raman detection module and a microscopic imaging module, the Raman detection module irradiates light emitted by a laser to an SERS enhanced substrate module, namely a sample pool, then scattered light (scattered light generated by irradiating the SERS enhanced substrate module by the laser) collected by a microscopic objective lens is guided into the Raman detection module by a beam splitter, light (images generated by particles due to illumination of an LED array white light source) of an image of an object to be detected collected by the microscopic objective lens is guided into the microscopic imaging module by the beam splitter, the Raman spectrum measurement and the microscopic imaging of the object to be detected are realized, and the microscopic imaging module is used for recording the motion state of the nano particles generated by the action of the photophoresis force.
Aiming at the response degree of the gold nanoparticle aggregate to 785nm laser, a laser light source is selected to be a 785nm semiconductor laser;
wherein, in order to guarantee the axiality of light beam, the plane mirror is installed in the adjustable reflection formula mount pad in cage right angle, can adjust the every single move and the slope of plane mirror through adjusting the knob on the mount pad to avoid the light beam off-axis.
Wherein the ratio of the transmittance to the reflectance of the beam splitter is preferably 1:9,
among them, it is preferable that the focal length of the objective lens is 6 mm.
The second plane mirror 11 is used for adjusting the reflection angle of the collected light beam by the adjusting knob so that the collected light beam can strictly vertically pass through the high pass filter 12, and when the collected light beam vertically passes through the high pass filter, more rayleigh scattered light can be filtered.
The first and second plano- convex lenses 26 and 28 have the same focal length, and preferably have a focal length of 3 cm.
Wherein, low pass filter 25 is that can dismantle in a flexible way, when adjusting the relative distance between laser focal plane and the auxiliary substrate, can unload it, when the motion process of record gold nanoparticle, can install to the motion state of clear observation particle.
Wherein, first plane mirror and second plane mirror install in the adjustable reflective mount pad of cage right angle, its purpose is in order to guarantee the axiality of light beam, the contained angle that can adjust laser and vertical direction simultaneously makes its vertical incidence auxiliary substrate, and if and only when laser beam from the top down vertical incidence when the determinand, just form obvious SERS reinforcing effect, when taking place the skew, gold nanoparticle of same r distance department produces inhomogeneous photophoresis power because of receiving inhomogeneous light beam irradiation, do not have a large amount of effects production of assembling to the facula center, consequently, the SERS signal of determinand can not have apparent reinforcing.
Wherein the numerical aperture of the collection fiber 14 matches the numerical aperture of the spectrometer slit.
Wherein, the diaphragm 23 can effectively adjust the size of the illumination light spot.
(2) Preparation of gold nano sol and solution to be measured with certain concentration
Preparing gold nano sol: the preparation of gold sol refers to the traditional Frens method, and 5.8 multiplied by 10 are prepared-3And (3) taking a mol/L trisodium citrate solution as a reducing agent, slowly adding the reducing agent into a boiling chloroauric acid solution with the volume fraction of 1%, and continuously stirring and heating for reacting for 1 h. The particle size of the gold nanoparticles obtained by the reaction can be controlled by adjusting the temperature and adding different amounts of trisodium citrate. Among them, the average particle diameter of the gold nanoparticles is preferably 60 nm.
Preparation of a solution to be tested: and selecting the solution to be detected as a pyrene solution with the concentration of 500 nM.
(3) Preparation of sample cell in SERS enhanced substrate module
A large number of researches show that the thickness of the liquid surface is closely related to the detection result of SERS, the aggregation process of the gold nanoparticles can be adversely affected by over-thickness and over-thickness, and the intensity of an SERS detection signal is sharply reduced outside a certain thickness range, so that the detection of the SERS detection signal is affected.
Therefore, in order to realize the aggregation of gold nanoparticles and SERS detection based on a photophoresis technology, the invention provides a brand-new sample pool aiming at the problem that the existing sample pool can not accurately realize the liquid level thickness, and specifically as shown in the right diagram in FIG. 1, 32 is an auxiliary substrate, 33 is a mixed solution of gold nanoparticle sol and an object to be detected, 34 is a gold nanoparticle aggregate generated under the action of a photophoresis force, 35 is a sample pool for containing the mixed solution, a vessel made of glass and provided with a cuboid space for containing the mixed solution of the gold nanoparticle sol and solution pyrene to be detected, and the sample pool comprises a glass slide, a gasket and a cover glass.
The sample cell is made of glass, the transparent auxiliary substrate is made of quartz, the volume of the quartz plate is preferably length × width × height =20mm × 10mm × 2mm, and the distance from the auxiliary substrate to the cover glass, that is, the liquid surface thickness of the mixed solution is 0.8-1.2mm, preferably 1 mm.
The volume ratio of the gold nano sol to the object to be detected in the sample cell can be 2:1, 1:1, 3:1, 1:2, 1:3 and the like, and preferably, the volume ratio of the gold nano sol to the object to be detected is 1: 3.
(4) SERS detection of solutions to be detected
The detection process of the probe molecule pyrene: placing the auxiliary substrate in a sample cell, taking 500nM of probe pyrene and gold nano sol, and mixing the probe pyrene and the gold nano sol in a volume ratio of 3:1, injecting the laser beam into a sample cell, keeping the propagation direction of the laser beam vertical to the surface of the auxiliary substrate, focusing the focal point of the laser beam inside the auxiliary substrate, preferably, adjusting the focal plane of the laser to make the distance between the focal plane of the laser and the upper surface of the quartz plate be 0.6mm, and when the focal plane of the laser is positioned at the position, the particles in the solution are converged toward the center of a light spot fastest. Research shows that when the laser beam is vertically incident to the object to be measured from top to bottom, an obvious SERS enhancement effect is formed, when deviation occurs, gold nanoparticles at the same r distance are irradiated by uneven light beams to generate uneven photophoresis force, and the effect of gathering towards the center of a light spot is not generated in a large amount, so that the SERS signal of the object to be measured cannot be enhanced obviously.
In order to explore the capturing effect of the invention on gold nanoparticlesAt 5X 10-7 Taking a pyrene solution in mol/L as a detection molecule, preferably, the volume ratio of the gold nano sol to the detection molecule is 1: and 3, after the laser is turned on, the gold nanoparticles covered by the laser facula are converged towards the center of the facula, because of the action of reverse photophoresis generated by uneven heat distribution on the surfaces of the gold nanoparticles irradiated by the light beam, the light intensity at the central position of the Gaussian light beam is strongest, and the particles distributed around the Gaussian light beam are lower than the particles at the central position to absorb light, so that the temperature of the particles at the peripheral position is lower than that of the particles at the central position, and the particles are estimated to generate reverse photophoresis by observing that the movement direction of the particles shot by the CCD is close to the central strongest position of the light beam. FIG. 2 is a transient diagram of the movement of gold nanoparticles photographed by an imaging CCD, the time interval between (a) and (b) is 1s, because the particles are still subjected to longitudinal binding force, some particles will deviate from a white light focal plane in 1s, so the particles are not easy to observe in a view field, in order to more clearly show the process that the gold nanoparticles converge towards an optical axis, more obvious 10 particles are circled to observe the movement condition of the particles, in order to compare the relative positions of the particle movement, a nick is etched on a quartz plate as a mark, and the process that the particles converge towards the center can be obviously observed through comparison. FIG. 3 is a comparative micrograph of gold nanoparticles in a field of view after aggregation and initial process of aggregation of gold nanoparticles under a photodynamic action, and the gold nanoparticle aggregates generated on the surface of the gold nanoparticle auxiliary substrate can be clearly seen from FIG. 3.
In order to explore the influence of gold nanoparticle aggregates generated based on the photophoretic force on the SERS signal intensity of the object to be detected, the SERS signal intensity was 5 × 10-7 Taking a pyrene solution in mol/L as a detection molecule, preferably, the volume ratio of the gold nano sol to the detection molecule is 1:3, a gold nano sol substrate and a gold nano particle aggregate SERS substrate generated by combining an auxiliary substrate with a photophoresis force are compared and researched in the figure 4, the integration time of a spectrometer is 1s, the laser power is 90mw, and as can be seen from the figure 4, compared with a gold nano sol system, the method for generating the efficient SERS substrate by combining the auxiliary substrate with a light control technology enables an SERS signal to be obviously enhanced, and pyrene is in a 403cm range-1、586 cm-1、1056 cm-1、1233 cm-1、1396 cm-1、1609 cm-1The SERS signal intensity of characteristic peaks at equal positions is obviously enhanced, and the generation of the gold nanoparticle aggregate is proved to be directly related to the detected SERS signal intensity. Wherein at 586cm-1The peak value is enhanced by more than 20 times compared with the peak value in the gold nanometer sol substrate, because the auxiliary substrate plays a role of bearing the gold nanometer particle aggregate, when the gold nanometer particles are converged to the center of a light beam, the ultrahigh electromagnetic field enhancement can be generated at the gap of the metal nanometer particle aggregate, more Raman signal 'hot spots' can be generated in a unit solid angle, and the Raman signal of a sample in the gap can be greatly enhanced.
To investigate the influence of the gold nanoparticle aggregation process on the SERS signal intensity of the analyte, the value is 5 × 10-7 Taking a pyrene solution in mol/L as a detection molecule, preferably, the volume ratio of the gold nano sol to the detection molecule is 1: 3. during the detection, the intensity of SERS signal increases with the increase of the aggregation time, as shown in FIG. 5, which is a pyrene solution (5.0X 10)-7 mol/L) of a three-dimensional waterfall plot with SERS enhancement effect varying with aggregation time, t<For 10 min. In order to more intuitively see the enhancement effect of the intensity of the SERS signal of pyrene under the action of electrophoresis, the peak intensity of pyrene at the characteristic peak is changed along with the aggregation time, as shown in FIG. 6, the pyrene solution is (5.0X 10)-7 mol/L) 586cm at the characteristic peak-1、1233 cm-1The SERS signal intensity is along with the change trend generated by the laser gathering time and the opening and closing state, and as can be seen from the graph, the signal reaches the maximum value within 7 and 8 minutes, then the signal tends to the stable state, and within 8 minutes, the SERS signal almost linearly increases because the distance between the gold nanoparticles is reduced along with the increase of the number of the particles and the number of the hot points generated between the particles is gradually increased when the gold nanoparticles in the sol are gathered to the beam waist of the light spot under the action of the optophoretic force. The SERS signal does not increase any more between 8 and 10 minutes because the size of the gold nanoparticle aggregate exceeds the coverage range of the laser spot, so that the particles which are not irradiated by the laser have no SERS enhancement effect on the molecules to be detected. After 10 minutes, the laser was turned off,and opening the laser to detect the SERS signal at an interval of one minute, and continuously keeping the intensity of the SERS signal of pyrene at an interval of 10 minutes, which shows that the gold nanoparticle aggregate is not dispersed immediately due to the disappearance of the optical pressure, and keeps a stable aggregation state within a period of time.
The forming process of the gold nanoparticle aggregate is dynamic, the gold nanoparticles are converged towards the center of a light spot under the action of reverse photophoresis, the flow velocity of the gold nanoparticles is related to the laser power, in order to explore the relationship between the particle flow velocity and the laser power, the laser powers of 60mw, 70mw, 80mw, 90mw, 100mw, 110mw and 120mw of different parameters are set, the motion states of the particles under different powers are recorded by using a CCD (charge coupled device), the same annular region range in a field of view is selected in the experimental process, as shown in figure 7, 5 particles are randomly tracked, the motion speed of the particles is calculated, figure 8 is a variance fitting curve of instantaneous motion speeds of 5 particles randomly selected under different powers, the judgment coefficient is 0.95852, the result shows that the motion speed of the particles and the laser power have a better linear relationship, therefore, the forming time of the gold nanoparticle aggregate can be accelerated by properly increasing the laser power, and effectively shorten the time for the SERS signal to reach saturation. In the detection of the SERS on the object to be detected, the method has the advantages of rapidness, real-time performance, in-situ performance, high sensitivity and the like, and the time for effectively shortening the saturation time of the SERS signal has obvious significance for the practical application of the SERS.
Example two
The difference between the second embodiment and the first embodiment is that the gold nano sol in the first embodiment is replaced by silver nano sol, the wavelength of the laser in the raman detection light path of the laser is replaced by 532nm, and parameters of other optical structures are changed correspondingly, so that the device and the method for realizing silver nano particle aggregation and SERS detection based on the optophoresis technology are obtained.
Compared with the prior art, the device and the method have the advantages that an experimental device is simple, the metal nanoparticles can be captured and the SERS signal can be excited only by one laser beam, the number of the captured particles is large, and the time for forming the nanoparticle aggregate is short.
Claims (9)
1. A device for realizing metal nanoparticle aggregation and SERS detection based on a photophoresis technology is characterized by mainly comprising a light control-micro Raman system, wherein the light control-micro Raman system comprises a Raman detection module and a micro imaging module; the Raman detection module comprises: the device comprises a laser, an incident optical fiber, a first collimating convex lens, a band-pass filter, a first plane reflector, a first high-pass dichroic sheet, a beam splitter, a microscope objective, a SERS enhanced substrate module, a second high-pass dichroic sheet, a second plane reflector, a high-pass filter, a focusing convex lens, a collecting optical fiber, a spectrometer and a computer, wherein the SERS enhanced substrate module consists of an auxiliary substrate, a mixed solution of metal nano sol and an object to be measured and a sample pool for containing the mixed solution, a laser beam emitted by the laser reaches the first collimating convex lens through the incident optical fiber, is filtered by the band-pass filter to reach the first plane reflector, reaches the first high-pass dichroic sheet after being reflected by the first plane mirror, is vertically focused to the SERS enhanced substrate module after being reflected to the beam splitter and the microscope objective, and realizes the capture of nanoparticles by the light in the mixed solution of the metal nano sol and the object to be measured in the sample pool for containing the mixed solution, the Raman scattering light beam generated by excitation reaches a first high-pass dichroic sheet and a second high-pass dichroic sheet for filtering after passing through a microscope objective and a beam splitter, then reaches a second plane reflector, reaches a high-pass filter after being reflected, is collected by a collecting optical fiber and transmitted to a spectrometer after passing through a focusing convex lens, is converted into an electric signal by the spectrometer and a CCD (charge coupled device), and is transmitted to a computer for displaying and storing, so that the detection of an SERS signal is realized;
the microscopic imaging module includes: the light emitted by the LED array white light source sequentially passes through the second collimating convex lens, the first diaphragm, the second high-reflection lens, the SERS enhanced substrate module, the microscope objective, the beam splitter, the low-pass filter, the first plano-convex lens, the second diaphragm and the second plano-convex lens and then reaches the imaging CCD;
the Raman detection module irradiates light emitted by the laser to the SERS enhanced substrate module, scattered light collected by the microscope objective is guided into the Raman detection module by the beam splitter, light of an image of an object to be detected collected by the microscope objective is guided into the microscopic imaging module by the beam splitter, the microscopic imaging module is used for recording the motion state of the metal nano particles generated by the action of optoelectrophoresis, so that Raman spectrum measurement and microscopic imaging of the object to be detected are realized, the power of the laser is 50-120mw, the sample pool for containing the mixed solution consists of a glass slide, a gasket and a cover glass, the auxiliary substrate is positioned at the bottom of the sample pool, so that the laser focus is focused inside the auxiliary substrate, the distance from the upper surface of the auxiliary substrate to the cover glass is the thickness of the mixed solution of the metal nano sol and the object to be detected, and the thickness of the mixed solution of the metal nano sol and the object to be detected is 0.8-1.2 mm.
2. The apparatus of claim 1, wherein the collection fiber has a numerical aperture that matches the numerical aperture of the spectrometer slit.
3. The apparatus of claim 2, wherein the first high-pass dichroic plate has a ratio of transmittance to reflectance of 1: 9.
4. The apparatus of claim 1, wherein the first and second planar mirrors are mounted in a caged right angle adjustable reflective mount.
5. The apparatus of claim 1, wherein the microscopic imaging optical path is transmissive illumination and the aperture is configured to adjust the size of the illumination spot.
6. The apparatus of claim 1, wherein the auxiliary substrate is made of a transparent quartz plate.
7. The device according to claim 1, wherein the metal nanosol is a gold nanosol or a silver nanosol.
8. The device as claimed in claim 7, wherein the metal nanosol is a gold nanosol, the average particle diameter of the gold nanosol is 60nm, and the volume ratio of the gold nanosol to the analyte is 1: 3.
9. a method for realizing metal nanoparticle aggregation and SERS detection based on a photophoresis technology, which is based on the device for realizing metal nanoparticle aggregation and SERS detection based on the photophoresis technology of any one of claims 1-8, and realizes SERS signal detection and recording of the motion state of nanoparticles generated by the action of the photophoresis force.
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