Multifunctional scanning photoelectrochemical cell microscope and single-individual photoelectric synchronous measurement method

Abstract

The invention relates to a multifunctional scanning photoelectrochemical cell microscope and a single-individual photoelectric synchronous measurement method, which are characterized in that an SECCM system, an optical imaging system and a spectral measurement system are organically integrated by constructing the multifunctional scanning photoelectrochemical cell microscope; synchronous dynamic measurement and imaging of electrochemical information and spectral characteristic information in the reaction process of a single nanoparticle sample to be detected can be realized; the invention further provides a single-individual photoelectric synchronous measurement method, and the complex process of obtaining single-individual sample information through secondary measurement is avoided. According to the method, the timeliness, the accuracy and the multi-dimensional fusion degree of data acquisition are remarkably improved, and a powerful tool platform and a system solution are provided for single-individual physical structure analysis, surface molecular recognition and reaction mechanism research under the micro-nano scale.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of electrochemical test analysis, in particular to a multifunctional scanning photoelectrochemical cell microscope based on spectral imaging and a single-body photoelectric synchronous measurement method. BACKGROUND

[0002] Scanning Electrochemical Cell Microscopy (SECCM) is an electrochemical characterization technology originated in 2010, which was first proposed by the Unwin research group of the University of Warwick in the UK. The core of SECCM technology is to realize high spatial resolution of electrochemical detection by constructing a small electrochemical cell, so as to study the electrochemical process on a microscale. A typical SECCM setup adopts a two-electrode system, in which the research object is used as the working electrode, and the nano probe is used as the quasi-reference electrode, which is filled with solution and inserted into the Ag / AgCl wire to maintain a stable potential. The electrochemical information provided by SECCM mainly reflects the electrochemical processes such as charge transfer and ion transport on the surface of the sample, which cannot be directly and synchronously corresponded to the size and structure of the sample.

[0003] In practical applications, in order to infer the size and structure of the sample, it is usually necessary to combine other characterization techniques such as scanning electron microscopy, transmission electron microscopy, etc. after SECCM characterization to obtain more comprehensive information. For example, the journal Chemistry of Materials (2023, Vol. 35, pp. 4243-4249) published Interfacial Structure and Energy Determine the Heterogeneity in the Electrochemical Metal Dissolution Activity at Grain Boundary. In this paper, by jointly using scanning electrochemical cell microscopy (SECCM) and focused ion beam (FIB), electron microscopy and other means, the structure and energy of the grain boundary how to affect the activity of the metal dissolution reaction were systematically studied. The study pointed out that it is necessary to "first perform SECCM electrochemical imaging, and then combine electron microscopy or other structural characterization means for secondary verification" to infer the structural features of the sample, so this secondary detection method cannot achieve synchronous acquisition of electrochemical information and structural information, which significantly increases the experimental complexity, time cost and error, and may also produce errors due to the resolution difference between different technologies and the change in the secondary detection process of the sample. Therefore, it is necessary to develop a device that can synchronously and dynamically measure and image the electrochemical information and spectral feature information during the reaction process, and there is currently no such integrated device and method. SUMMARY

[0004] In order to overcome the defects of the prior art, the present application aims to provide a multifunctional scanning photoelectrochemical cell microscope and a single-body photoelectric synchronous measurement method, which can realize the synchronous dynamic measurement and imaging of the electrochemical information and spectral characteristic information of a nanoparticle single-body sample to be measured in the reaction process, and also provides a single-body photoelectric synchronous measurement method, which avoids the complex process of obtaining single-body sample information through secondary measurement, and has the advantages of timeliness, accuracy and multi-dimensional fusion.

[0005] To achieve the above object, the present application adopts the following technical solutions: The multifunctional scanning photoelectrochemical cell microscope comprises a SECCM system, an optical imaging system, and a spectral measurement system connected with the optical imaging system; the optical imaging system transmits the collected optical synchronization signals to the spectral measurement system; The optical imaging system is used for positioning and imaging of a single-body sample, and comprises a reverse dark field microscope installed below the body of the reverse dark field microscope, and a dichroic mirror arranged below the reverse dark field microscope, which reflects the optical signals from the reverse dark field microscope in the horizontal direction and guides them to a color CCD camera, forming a reflective dark field microscopic imaging light path; The spectral measurement system is used for measuring the fluorescence spectrum, Raman spectrum, SERS and Rayleigh scattering spectrum of a single body, and comprises a white light or laser light source, a mirror, a spectrometer and a spectral CCD camera, which together constitute a spectral analysis path; wherein the white light or laser light source is focused by the reverse dark field microscope and irradiated to the sample area; after the sample interacts with the light, the spectral signals generated by the sample are collected by the reverse dark field microscope in the reverse direction, and then transmitted to the dichroic mirror, which acts as a partial mirror and reflects part of the optical signals to the color CCD camera, realizing real-time visual positioning and dark field imaging of the sample; the remaining optical signals continue to move in a straight line, are reflected by the mirror to the spectral analysis path, enter the spectrometer for wavelength decomposition, and the multi-band spectral signals after light splitting are received and recorded by the spectral CCD camera, realizing the collection and digital processing of the spectral signals; The SECCM system is used for measuring electrochemical information of a sample, and is composed of a three-dimensional positioning system and a signal acquisition and processing module; the three-dimensional positioning system comprises an electrochemical probe, the electrochemical probe is fixed on a Z-axis piezoelectric ceramic displacement table through a cantilever, the Z-axis piezoelectric ceramic displacement table is connected with an output shaft of a Z-axis stepping motor to realize adjustment and control in the vertical direction; adjustment and control in the horizontal XY-axis direction is realized by cooperation of an XY-axis piezoelectric ceramic and a stepping motor, that is, power outputs of the XY-axis piezoelectric ceramic and the stepping motor are connected with a carrier table; the signal acquisition and processing module comprises a current amplifier, after the electrochemical probe contacts the surface of the sample, an electric signal generated by the electrochemical probe is first amplified by the current amplifier, and then transmitted to an analog-to-digital converter to complete conversion of an analog signal into a digital signal, the converted signal is received and processed by a computer to realize recording, analysis and imaging of the electrochemical signal; meanwhile, the computer also sends control instructions to the XY-axis stepping motor, the XY-axis piezoelectric ceramic, the Z-axis stepping motor and the Z-axis piezoelectric ceramic displacement table to complete path planning and scanning execution of the electrochemical probe in the three-dimensional space, and realize spatial electrochemical measurement.

[0006] The electrochemical probe is composed of an ultrathin gold film attached to the inner wall of a glass tube tip.

[0007] The single-body photoelectric synchronous measurement method based on the multifunctional scanning photoelectrochemical cell microscope comprises the following steps: Step one, noble metal nanoparticles are sprayed on an ITO glass plate to form an electrode surface, the sprayed noble metal nanoparticles are single bodies, the ITO glass plate is placed on the carrier table as a sample, an electrolyte solution is filled into the prepared electrochemical probe, silver / silver chloride is inserted into the electrochemical probe as a quasi-reference electrode, and the tip of the electrochemical probe is in contact with the electrode surface of the ITO glass plate through a liquid bridge to form a three-dimensional micro electrochemical cell; Step two, the three-dimensional positioning system of the SECCM system is controlled by the computer to make the electrochemical probe close to the electrode surface of the ITO glass plate, a weak current is formed between the electrochemical probe and the electrode surface under a set voltage, the feedback current information is amplified by a current amplifier, and the continuous current signal collected by the electrochemical probe is converted into a digital signal that can be processed by the computer through an analog-to-digital converter, so as to obtain position information of the single-body sample and instantaneous electrochemical information of the electrode surface, the instantaneous electrochemical information comprises a cyclic voltammogram, a polarization curve, an impedance spectrum and a linear sweep voltammogram; Step three, white light or laser light is incident in the vertical direction below the carrier table, scattered light on the sample surface is collected by an inverted inverse dark field microscope, a dichroic mirror reflects part of the light signal from the inverse dark field microscope in the horizontal direction and guides the light signal to a color CCD camera, and the color CCD camera obtains positioning and imaging information of the single-body sample; Step four, another part of the light signal from the trans- dark field microscope is further reflected by the mirror into the spectrometer for analysis and conversion, received and recorded by the spectral CCD camera, to obtain the transient fluorescence spectrum, Raman spectrum, transient SERS and transient Rayleigh scattering spectrum of the single sample.

[0008] The single sample position information and the electrode surface transient electrochemical information are obtained by the following method: First, the electrochemical probe is lowered by a set distance Δ z along the Z-axis direction by controlling the Z-axis piezoelectric ceramic displacement table t , and the current value is recorded in real time at a time interval of Δ I , and the speed of the electrochemical probe is controlled to be v vertical_down When the droplet at the tip of the electrochemical probe contacts the electrode surface of the ITO glass plate, the generated current will change obviously, and the feedback current signal can automatically determine whether the electrochemical probe has contacted the sample surface and determine the height information of the electrochemical probe in the Z-axis direction; then, by setting the moving distance Δ x and Δ y of the XY-axis piezoelectric ceramic and the stepping motor, the sample stage is moved with the electrochemical probe fixed, and the spatial position information of the sample in the XY-axis direction is further obtained according to the feedback current signal of the electrochemical probe at each position, so that the synchronous measurement of the sample position and the transient electrochemical response at the specific position is realized.

[0009] The method for obtaining the transient fluorescence spectrum of the single sample is as follows: a white light source with an excitation wavelength in the visible or ultraviolet band is used to locally excite the sample, so that the fluorescent active molecules in the sample transition to the excited state, and release fluorescence in the process of returning to the ground state; the fluorescence signal is collected by the trans- dark field microscope in the reverse direction, and is further transmitted to the dichroic mirror; the dichroic mirror acts as a partial mirror, and reflects a part of the fluorescence signal to the color CCD camera, so as to realize the real-time visual positioning and dark field imaging of the sample; the remaining fluorescence signal continues to travel in a straight line direction, is reflected by the mirror to the spectral analysis path, enters the spectrometer for wavelength decomposition, and the multi-band fluorescence spectrum signal after light splitting is received and recorded by the spectral CCD camera, so as to realize the collection and digital processing of the transient fluorescence spectrum of the single sample.

[0010] The method for acquiring the Raman spectrum of the single sample comprises the following steps: introducing a laser light source as Raman excitation light to irradiate the surface of the sample, so that the molecules of the sample are subjected to Raman scattering with the incident light; the Raman scattering signal is reversely collected by a trans- dark field microscope and continuously transmitted to a dichroic mirror; the dichroic mirror is used as a partial mirror to reflect part of the scattering signal to a color CCD camera, so that the real-time visual positioning and dark field imaging of the sample are realized; the remaining scattering signal continues to move in a straight line and is reflected by a mirror to a spectral analysis path, enters a spectrometer to be subjected to wavelength decomposition, and the Raman spectrum signal after the light splitting is received by a spectral CCD camera and recorded, so that the acquisition and digital processing of the instantaneous Raman spectrum of the single sample are realized.

[0011] The method for acquiring the instantaneous SERS of the single sample comprises the following steps: an electrochemical probe is formed by attaching an ultrathin gold film to the inner wall of the tip of a glass tube, can produce a localized surface plasmon resonance effect, forms a high-intensity localized electromagnetic field under laser irradiation, enhances the molecular signal near the hot spot area of the electrochemical probe, the enhanced molecular signal is reversely collected by a trans-dark field microscope and continuously transmitted to a dichroic mirror; the dichroic mirror is used as a partial mirror to reflect part of the scattering enhanced signal to a color CCD camera, so that the real-time visual positioning and dark field imaging are realized; the remaining enhanced signal continues to move in a straight line and is reflected by a mirror to a spectral analysis path, enters a spectrometer to be subjected to wavelength decomposition, and the SERS spectrum signal after the light splitting is received by a spectral CCD camera and recorded, so that the acquisition and digital processing of the instantaneous SERS signal are realized.

[0012] The method for acquiring the instantaneous Rayleigh scattering spectrum of the single sample comprises the following steps: laser or white light is irradiated to the surface of a sample electrode sprayed with noble metal nanoparticles, so that photons are elastically scattered after interacting with particles smaller than the wavelength; the scattered light signal after the elastic scattering is reversely collected by a trans-dark field microscope and continuously transmitted to a dichroic mirror; the dichroic mirror is used as a partial mirror to reflect part of the scattered light signal to a color CCD camera, so that the real-time visual positioning and dark field imaging of the sample are realized; the remaining scattered light signal continues to move in a straight line and is reflected by a mirror to a spectral analysis path, enters a spectrometer to be subjected to wavelength decomposition, and the Rayleigh scattering spectrum signal after the light splitting is received by a spectral CCD camera and recorded, so that the acquisition and digital processing of the instantaneous Rayleigh scattering spectrum are realized.

[0013] Compared with the prior art, the present application has the following beneficial effects: (1) The present application can realize the photoelectric synchronous dynamic measurement and real-time imaging of electrochemical information and spectral characteristic information on the scale of a single nanoparticle or other single body by constructing a multifunctional scanning photoelectrochemical cell microscope, integrating the SECCM system, the optical imaging system and the spectral measurement system.

[0014] (2) This invention can not only accurately obtain the instantaneous electrochemical response of a single organism during the reaction process (such as CV curve, LSV curve, impedance spectrum, etc.), but also simultaneously record its corresponding fluorescence spectrum, Raman spectrum, SERS and Rayleigh scattering spectrum and other spectroscopic features, thereby revealing the structural, composition and functional changes of a single research object in multiple dimensions, spatially, synchronously, and in multiple dimensions.

[0015] In summary, this invention significantly improves the timeliness, accuracy, and multidimensional fusion of data acquisition, providing a powerful tool platform and system solution for the analysis of individual physical structures, surface molecule recognition, and reaction mechanism research at the micro-nano scale. Attached Figure Description

[0016] Figure 1 This is a schematic diagram of the structure of a multifunctional scanning photoelectrochemical microscope.

[0017] In the figure, 1. Stage; 2. Electrochemical probe; 3. Cantilever; 41. Z-axis piezoelectric ceramic displacement stage; 42. Z-axis stepper motor; 5. Current amplifier; 6. Analog-to-digital converter; 7. Computer; 81. XY-axis stepper motor; 82. XY-axis piezoelectric ceramic; 9. Reverse dark-field microscope body; 10. Reverse dark-field microscope; 11. Dichroic mirror; 12. Color CCD camera; 13. Mirror; 14. Spectrometer; 15. Spectroscopic CCD camera. Detailed Implementation

[0018] The present invention will now be further described with reference to the accompanying drawings.

[0019] like Figure 1 As shown, the multifunctional scanning photoelectrochemical cell microscope includes a SECCM system located above stage 1, an optical imaging system located below stage 1, and a spectral measurement system connected to the optical imaging system; the optical imaging system transmits the collected light synchronization signal to the spectral measurement system.

[0020] The SECCM system is used for measuring electrochemical information of a sample, and is composed of a three-dimensional positioning system and a signal acquisition and processing module; the three-dimensional positioning system comprises an electrochemical probe 2, the electrochemical probe 2 is fixed on a Z-axis piezoelectric ceramic displacement table 41 through a cantilever 3, the Z-axis piezoelectric ceramic displacement table 41 is connected with an output shaft of a Z-axis stepping motor 42 to realize adjustment control in the vertical direction; adjustment control in the horizontal XY-axis direction is realized by cooperation of an XY-axis piezoelectric ceramic 82 and a stepping motor 81, that is, the power outputs of the XY-axis piezoelectric ceramic 82 and the stepping motor 81 are connected with the object table 1, thereby forming a complete three-dimensional positioning system; the signal acquisition and processing module comprises a current amplifier 5, after the electrochemical probe 2 contacts the surface of the sample, the generated electrical signal is first amplified by the current amplifier 5, and then transmitted to an analog-to-digital converter 6 to complete the conversion of the analog signal to the digital signal, the converted signal is received and processed by a computer 7, thereby realizing recording, analysis and imaging of the electrochemical signal; meanwhile, the computer 7 also sends control instructions to the XY-axis stepping motor 81, the XY-axis piezoelectric ceramic 82, the Z-axis stepping motor 42 and the Z-axis piezoelectric ceramic displacement table 41, thereby completing path planning and scanning execution of the electrochemical probe 2 in the three-dimensional space, so as to realize accurate spatial electrochemical measurement, wherein the XY-axis stepping motor 81 and the Z-axis stepping motor 42 realize large displacement adjustment, and the XY-axis piezoelectric ceramic 82 and the Z-axis piezoelectric ceramic displacement table 41 realize fine adjustment.

[0021] The optical imaging system is used for positioning and imaging of a single sample, and comprises a reverse dark field microscope 10 installed below a reverse dark field microscope body 9, which is used for focusing an imaging light path and realizing sample observation, a dichroic mirror 11 is arranged below the reverse dark field microscope 10, the dichroic mirror 11 reflects image light signals from the reverse dark field microscope 10 in the horizontal direction and guides the image light signals to a color CCD camera 12, the color CCD camera 12 is used for receiving the reflected light signals and realizing real-time imaging acquisition, thereby realizing real-time visual positioning and dark field imaging of the sample. The components in the optical imaging system are fixedly connected through standard optical interfaces, thereby forming a stable reflection type dark field microscopic imaging light path.

[0022] The spectral measurement system is used for measuring fluorescence spectrum, Raman spectrum, SERS and Rayleigh scattering spectrum of single bodies, and comprises a white light source or a laser light source, a mirror 13, a spectrometer 14 and a spectral CCD camera 15 which are arranged in a vertical direction below a stage 1 to form a spectral analysis path; wherein the white light source or the laser light source is focused and irradiated to a sample area by the inverted dark field microscope 10; after the sample interacts with the light, the spectral signal is collected reversely by the inverted dark field microscope 10 and then transmitted to a dichroic mirror 11; the dichroic mirror 11 is used as a partial mirror to reflect part of the light signal to a color CCD camera 12 to realize real-time visual positioning and dark field imaging of the sample; the remaining light signal continues to move in a straight line, is reflected by the mirror 13 to the spectral analysis path, is wavelength-decomposed by the spectrometer 14, and then the multi-band spectral signal after the wavelength decomposition is received and recorded by the spectral CCD camera 15 to realize collection and digital processing of the spectral signal. The mirror 13 and the spectrometer 14 are connected by an optical fiber or a standard optical interface, the output end of the spectrometer 14 is connected with the spectral CCD camera 15, and a continuous and stable spectral imaging measurement path is formed.

[0023] The electrochemical probe 2 is composed of an ultrathin gold film attached to the inner wall of the tip of the glass tube, so that a localized surface plasmon resonance effect can be generated.

[0024] The electrochemical probe 2 is prepared according to the following steps: Step one: preparing a nanoliter pipette by laser drawing method: using a glass capillary as a mold, a nanoliter pipette is prepared by using a laser needle drawing instrument of P-2000 type; Step two: preparing an ultrathin gold film attached nanoliter pipette by chemical modification method: mixing C2H5OH and HAuCl4 solution in a volume ratio of 2:3, filling the glass nanoliter pipette prepared in step one, irradiating under a 254 nm 8 W UV light source for 7 hours, washing with C2H5OH and deionized water, and drying and annealing at 100℃ for 1 hour.

[0025] The single body photoelectric synchronous measurement method based on the above multifunctional scanning photoelectrochemical cell microscope comprises the following steps: Step one: spraying noble metal nanoparticles on an ITO glass plate to form an electrode surface, and the sprayed noble metal nanoparticles are single bodies; placing the ITO glass plate as a sample on the stage 1, filling an electrolyte solution into the prepared electrochemical probe 2, inserting silver / silver chloride as a quasi-reference electrode into the electrochemical probe 2, and contacting the tip of the electrochemical probe 2 with the electrode surface of the ITO glass plate through a liquid bridge to form a three-dimensional micro electrochemical cell; Step two, the three-dimensional positioning system of SECCM system is controlled by computer 7 to make electrochemical probe 2 close to the electrode surface of ITO glass plate, and a weak current is formed between electrochemical probe 2 and the electrode surface at a set voltage. The feedback current information is amplified by current amplifier 5, and the continuous current signal collected by electrochemical probe 2 is converted into digital signal which can be processed by computer 7 through analog-to-digital converter 6, so as to obtain single sample position information and instantaneous electrochemical information of electrode surface, including cyclic voltammetry curve (CV curve), polarization curve, impedance spectroscopy and linear scan voltammetry curve (LSV curve); Step three, white light or laser is incident in the vertical direction below the stage 1, and the scattered light of the sample surface is collected by the inverted reverse dark field microscope 10. The dichroic mirror 11 reflects part of the light signal from the reverse dark field microscope 10 in the horizontal direction and guides it to the color CCD camera 12. The color CCD camera 12 obtains the positioning and imaging information of single sample; Step four, another part of the light signal from the reverse dark field microscope 10 is further reflected by the mirror 13 into the spectrometer 14 for analysis and conversion, which is received and recorded by the spectral CCD camera 15, and the instantaneous fluorescence spectrum, Raman spectrum, instantaneous SERS and instantaneous Rayleigh scattering spectrum of single sample are obtained.

[0026] The specific method for obtaining single sample position information and instantaneous electrochemical information of electrode surface is as follows: Firstly, the Z-axis piezoelectric ceramic displacement table 41 is controlled to make the electrochemical probe 2 descend along the Z-axis direction by a set distance Δ z , and the current value I is recorded in real time at a time interval of Δ t during the descending process I , and the speed of the electrochemical probe 2 is controlled to be v vertical_down When the droplet at the tip of the electrochemical probe 2 contacts the conductive electrode surface of the ITO glass plate, the current generated will change obviously (the current mutation threshold value Δ I threshold is determined according to the background current when the droplet at the tip of the electrochemical probe 2 does not contact the sample surface) I I threshold When the droplet contacts the sample surface to form an electrochemical path, the current increases sharply to I ​I threshold ), the feedback current signal automatically determines whether the electrochemical probe 2 has contacted the sample surface and determines the height information of the electrochemical probe 2 in the Z-axis direction; then, the moving distance Δ x and Δ y of the XY-axis piezoelectric ceramic 82 and the stepping motor 81 is set, the sample stage 1 is moved with the electrochemical probe 2 fixed, and the spatial position information of the sample in the XY-axis direction is further obtained according to the feedback current signal of the electrochemical probe 2 at each position, so that the synchronous measurement of the sample position and the instantaneous electrochemical response of the sample at a specific position is realized. Each time the probe moves to a new measurement position and contacts the sample surface again, the system automatically performs current acquisition, and can synchronously trigger laser excitation to realize the synchronous acquisition of the electrochemical signal and the spectral signal.

[0027] The method for acquiring the instantaneous fluorescence spectrum of a single sample specifically comprises: using a white light source with an excitation wavelength in the visible light or ultraviolet band to locally excite the sample, so that the fluorescent active molecules in the sample transition to the excited state and release fluorescence in the process of returning to the ground state. The fluorescence signal is reversely collected by the inverse dark field microscope 10 and continues to be transmitted to the dichroic mirror 11; the dichroic mirror 11 acts as a partial mirror and reflects part of the fluorescence signal to the color CCD camera 12, so as to realize the real-time visual positioning and dark field imaging of the sample; the remaining fluorescence signal continues to travel in a straight line and is reflected by the mirror 13 to the spectral analysis path and enters the spectrometer 14 for wavelength decomposition, and the multi-band fluorescence spectrum signal after being split by light is received and recorded by the spectral CCD camera 15, so as to realize the acquisition and digital processing of the instantaneous fluorescence spectrum of a single sample.

[0028] The method for acquiring the Raman spectrum of a single sample specifically comprises: introducing a laser light source with a specific wavelength as Raman excitation light to irradiate the sample surface, so that the sample molecules and the incident light undergo Raman scattering; the Raman scattering signal is reversely collected by the inverse dark field microscope 10 and continues to be transmitted to the dichroic mirror 11; the dichroic mirror 11 acts as a partial mirror and reflects part of the scattering signal to the color CCD camera 12, so as to realize the real-time visual positioning and dark field imaging of the sample; the remaining scattering signal continues to travel in a straight line and is reflected by the mirror 13 to the spectral analysis path and enters the spectrometer 14 for wavelength decomposition, and the Raman spectrum signal after being split by light is received and recorded by the spectral CCD camera 15, so as to realize the acquisition and digital processing of the instantaneous Raman spectrum of a single sample.

[0029] The method for obtaining the transient SERS of the single sample comprises the following steps: the electrochemical probe 2 is formed by the ultrathin gold film attached to the inner wall of the glass tube tip, which can produce the localized surface plasmon resonance effect, form the high-intensity local electromagnetic field under the laser irradiation, enhance the molecular signal close to the hot spot area of the electrochemical probe 2, collect the enhanced molecular signal by the inverse dark field microscope 10 in the reverse direction, and continue to transmit to the dichroic mirror 11; the dichroic mirror 11 is used as a partial mirror, reflects part of the scattered and enhanced signal to the color CCD camera 12, realizes the real-time visual positioning and dark field imaging of the sample, and the remaining enhanced signal continues to move in the straight line direction, is reflected to the spectral analysis path by the reflector 13, enters the spectrometer 14 to be wavelength resolved, and the SERS spectrum signal after the light splitting is received and recorded by the spectral CCD camera 15, so that the transient SERS signal is obtained, and the detection and reaction process monitoring on the single molecule level are suitable.

[0030] The method for obtaining the transient Rayleigh scattering spectrum of the single sample comprises the following steps: the laser or white light is irradiated to the sample electrode surface sprayed with the noble metal nanoparticles, so that the photon is elastically scattered after the interaction with the particle smaller than the wavelength, the scattered light signal after the elastic scattering is collected by the inverse dark field microscope 10 in the reverse direction, and continues to transmit to the dichroic mirror 11; the dichroic mirror 11 is used as a partial mirror, reflects part of the scattered light signal to the color CCD camera 12, realizes the real-time visual positioning and dark field imaging of the sample, and the remaining scattered light signal continues to move in the straight line direction, is reflected to the spectral analysis path by the reflector 13, enters the spectrometer 14 to be wavelength resolved, and the Rayleigh scattering spectrum signal after the light splitting is received and recorded by the spectral CCD camera 15. The transient Rayleigh scattering spectrum obtained can reflect the particle size, morphology and interaction characteristics of the nanoparticles with the surrounding medium, and can assist the electrochemical measurement to judge the sample type and surface structure.

[0031] The application realizes the synchronous (corresponding to the matrix point-to-point movement of the electrochemical probe) collection and identification of various spectral signals (fluorescence, Raman, SERS, Rayleigh scattering) of a single sample by using the trans- dark field microscopic imaging and spectral measurement system while measuring the single-point kinetics of the SECCM system. The measurement light path propagates from bottom to top, and is transmitted and split along the following path: the laser light source or the white light source emits from the bottom, enters the trans-dark field microscope body 9 in the vertical direction; the light is focused and irradiated to the sample area on the object table 1 by the trans-dark field microscope 10; the interaction between the light and the matter occurs at the sample, including fluorescence emission, Raman scattering, Rayleigh scattering and various signals; after being collected in the reverse direction by the trans-dark field microscope 10, all the uplink scattered light continues to be transmitted to the two-phase color mirror 11; the two-phase color mirror 11 is a partial mirror, which is used for reflecting part of the light signal to the first color CCD camera 12, realizing real-time visual positioning and dark field imaging of the sample; the remaining light signal continues to travel in a straight line and is reflected to the spectral analysis path by the mirror 13; the spectrometer 14 receives the composite light signal introduced by the mirror and performs wavelength decomposition; the multi-band spectral diagram signal after light splitting is received by the spectral CCD 15 and is recorded and digitized.

[0032] The whole measurement light path adopts a two-stage light splitting path design, realizes the parallel operation of “real-time imaging” and “spectrum acquisition”, and considers the acquisition of spatial information and spectral information, which provides key support for photoelectric synchronous measurement. Among them, the two-phase color mirror 11 mainly bears the reflection of the visible light image to the color CCD camera 12, realizing real-time positioning and tracking; and the mirror 13 is specially used for transmitting the sorted spectral signal to the spectrometer 14 and the spectral CCD camera 15 for component analysis and signal analysis.

[0033] The application integrates SECCM with the trans-dark field microscopic imaging system and the spectral measurement system, and constructs a photoelectric cooperative measurement platform with high spatial resolution and high time response capability. The system can synchronously collect the electrochemical signal and the multi-modal spectral signal (including fluorescence spectrum, Raman spectrum, SERS, Rayleigh scattering spectrum, etc.) of the single sample at the moment when the electrochemical probe contacts the single sample, realizes the multi-dimensional and holographic measurement and real-time imaging of the dynamic response process of the single sample such as single nanoparticle, single molecule or micro-area active site.

[0034] The application can accurately reveal the intrinsic characteristics of samples in structure, composition and reaction behavior on micro-nano scale by synergistic collection and fusion analysis of electrochemical information (such as CV curve, polarization curve, charge transfer behavior, etc.) and spectral characteristics (such as Raman peak position, fluorescence intensity, scattering distribution, etc.). The technology breaks the time lag and spatial mismatch limitation brought by traditional multiple asynchronous measurement methods, significantly improves the comprehensive cognitive ability of physical, chemical and biological properties of a single research object, and provides a universal and advanced research tool and methodology system for single molecule reaction mechanism analysis, nanomaterial heterogeneity research and biomolecular recognition.

Claims

1. A multifunctional scanning photoelectrochemical cell microscope, characterized in that, It includes a SECCM system, an optical imaging system, and a spectral measurement system connected to the optical imaging system; the optical imaging system transmits the collected optical synchronization signal to the spectral measurement system; The optical imaging system is used for positioning and imaging of a single sample, including a reverse dark field microscope (10) installed below the reverse dark field microscope body (9). A dichroic mirror (11) is provided below the reverse dark field microscope (10). The dichroic mirror (11) reflects the light signal from the reverse dark field microscope (10) in the horizontal direction and guides it to the color CCD camera (12) to form a reflective dark field microscopy imaging optical path. The spectral measurement system is used to measure the fluorescence spectrum, Raman spectrum, SERS and Rayleigh scattering spectrum of a single object. It includes a white light or laser source incident in the vertical direction, a reflector (13), a spectrometer (14) and a spectral CCD camera (15), which together constitute the spectral analysis path. The white light or laser source is focused onto the sample area by a reverse dark-field microscope (10). After the sample interacts with the light, the spectral signal is collected in reverse by the reverse dark-field microscope (10) and continues to be transmitted to a dichroic mirror (11). The dichroic mirror (11) acts as a partial reflector, reflecting a portion of the light signal to the color CCD camera (12) to realize the real-time visualization and dark-field imaging of the sample. The remaining light signal continues to travel in a straight line and is reflected by the reflector (13) to the spectral analysis path, enters the spectrometer (14) for wavelength decomposition, and the multi-band spectral signal after spectral separation is received and recorded by the spectral CCD camera (15) to realize the acquisition and digital processing of spectral signals.

2. The multifunctional scanning photoelectrochemical cell microscope according to claim 1, characterized in that, The SECCM system is used to measure the electrochemical information of the sample and consists of a three-dimensional positioning system and a signal acquisition and processing module. The three-dimensional positioning system includes an electrochemical probe (2), which is fixed on the Z-axis piezoelectric ceramic displacement stage (41) by a cantilever (3). The Z-axis piezoelectric ceramic displacement stage (41) is connected to the output shaft of the Z-axis stepper motor (42) to achieve vertical adjustment control. The horizontal XY axis adjustment control is completed by the XY axis piezoelectric ceramic (82) and the stepper motor (81) working together, that is, the power output of the XY axis piezoelectric ceramic (82) and the stepper motor (81) are both connected to the stage (1). The signal acquisition and processing module... The module includes a current amplifier (5). After the electrochemical probe (2) contacts the sample surface, the generated electrical signal is first amplified by the current amplifier (5) and then transmitted to the analog-to-digital converter (6) to complete the conversion of analog signal to digital signal. The converted signal is received and processed by the computer (7) to realize the recording, analysis and imaging of electrochemical signal. At the same time, the computer (7) also sends control commands to the XY axis stepper motor (81), XY axis piezoelectric ceramic (82), Z axis stepper motor (42) and Z axis piezoelectric ceramic displacement stage (41) to complete the path planning and scanning execution of the electrochemical probe (2) in three-dimensional space and realize spatial electrochemical measurement.

3. The multifunctional scanning photoelectrochemical cell microscope according to claim 2, characterized in that, The electrochemical probe (2) is composed of an ultrathin gold film attached to the inner wall of the tip of a glass tube.

4. The method for simultaneous photoelectric measurement of a single organism using a multifunctional scanning photoelectrochemical cell microscope according to claim 3, characterized in that, Includes the following steps: Step 1: Spray noble metal nanoparticles onto an ITO glass plate to form an electrode surface. The sprayed noble metal nanoparticles are individual particles. Place the ITO glass plate as a sample on the stage (1). Pour the electrolyte solution into the prepared electrochemical probe (2). Insert silver / silver chloride as a quasi-reference electrode into the electrochemical probe (2). The tip of the electrochemical probe (2) contacts the electrode surface of the ITO glass plate through a liquid bridge to form a three-dimensional micro-electrochemical cell. Step 2: The three-dimensional positioning system of the SECCM system is controlled by the computer (7) to bring the electrochemical probe (2) close to the electrode surface of the ITO glass plate. Under the set voltage, a weak current is formed between the electrochemical probe (2) and the electrode surface. The feedback current information is amplified by the current amplifier (5). The continuous current signal collected by the electrochemical probe (2) is converted into a digital signal that can be processed by the computer (7) through the analog-to-digital converter (6) to obtain the position information of the individual sample and the instantaneous electrochemical information of the electrode surface. The instantaneous electrochemical information includes cyclic voltammetry curves, polarization curves, impedance spectra and linear scanning voltammetry curves. Step 3: White light or laser light is incident vertically below the stage (1), and the scattered light from the sample surface is collected by the inverted reverse dark field microscope (10). The dichroic mirror (11) reflects part of the light signal from the reverse dark field microscope (10) in the horizontal direction and guides it to the color CCD camera (12). The color CCD camera (12) acquires the positioning and imaging information of the individual sample. Step 4: Another portion of the light signal from the reverse dark-field microscope (10) is further reflected by the mirror (13) and enters the spectrometer (14) for analysis and conversion. It is received and recorded by the spectral CCD camera (15) to obtain the instantaneous fluorescence spectrum, Raman spectrum, instantaneous SERS and instantaneous Rayleigh scattering spectrum of the individual sample.

5. The single-entity photoelectric synchronous measurement method according to claim 4, characterized in that, The specific method for obtaining the location information of a single sample and the transient electrochemical information of the electrode surface is as follows: First, the electrochemical probe (2) is lowered by a set distance Δ along the Z-axis by controlling the Z-axis piezoelectric ceramic displacement stage (41). z and during the descent, at Δ t Record current values ​​in real time at time intervals I The speed of the electrochemical probe (2) is controlled as follows: v vertical_down When the droplet at the tip of the electrochemical probe (2) contacts the electrode surface of the ITO glass plate, the generated current will change significantly. The feedback current signal will automatically determine whether the electrochemical probe (2) has contacted the sample surface and determine its height information in the Z-axis direction. Subsequently, the moving distance Δ of the XY-axis piezoelectric ceramic (82) and the stepper motor (81) is set. x and Δ y With the electrochemical probe (2) fixed, the stage (1) is moved, and based on the feedback current signal of the electrochemical probe (2) at each position, the spatial position information of the sample in the XY axis direction of the plane is further obtained, so as to realize the synchronous measurement of the sample position and its instantaneous electrochemical response at a specific position.

6. The single-entity photoelectric synchronous measurement method according to claim 4, characterized in that, The method for obtaining the instantaneous fluorescence spectrum of a single sample is as follows: the sample is locally excited by a white light source with an excitation wavelength in the visible or ultraviolet band, causing the fluorescent active molecules in the sample to transition to the excited state and release fluorescence during the return to the ground state. The fluorescence signal is collected in reverse by a reverse dark-field microscope (10) and then transmitted to a dichroic mirror (11). The dichroic mirror (11) acts as a partial reflector, reflecting a portion of the fluorescence signal to a color CCD camera (12) to achieve real-time visualization and dark-field imaging of the sample. The remaining fluorescence signal continues to travel in a straight line and is reflected by a reflector (13) to the spectral analysis path, entering the spectrometer (14) for wavelength decomposition. The multi-band fluorescence spectrum signal after spectral separation is received and recorded by a spectral CCD camera (15), realizing the acquisition and digital processing of the instantaneous fluorescence spectrum of a single sample.

7. The single-entity photoelectric synchronous measurement method according to claim 4, characterized in that, The method for obtaining the Raman spectrum of a single sample is as follows: a laser light source is introduced as the Raman excitation light to irradiate the sample surface, causing the sample molecules to undergo Raman scattering with the incident light. The Raman scattering signal is collected in reverse by a reverse dark-field microscope (10) and then transmitted to a dichroic mirror (11). The dichroic mirror (11) acts as a partial reflector, reflecting a portion of the scattered signal to a color CCD camera (12) to achieve real-time visualization and dark-field imaging of the sample. The remaining scattered signal continues to travel in a straight line and is reflected by a reflector (13) to the spectral analysis path, entering the spectrometer (14) for wavelength decomposition. The Raman spectral signal after spectral separation is received by a spectral CCD camera (15) and its Raman shift and intensity are recorded, thus realizing the acquisition and digital processing of the instantaneous Raman spectrum of a single sample.

8. The single-entity photoelectric synchronous measurement method according to claim 4, characterized in that, The method for obtaining instantaneous SERS of a single sample is as follows: the electrochemical probe (2) is composed of an ultrathin gold film attached to the inner wall of the glass tube tip, which can generate a local surface plasmon resonance effect and form a high-intensity local electromagnetic field under laser irradiation, which enhances the molecular signal near the hot spot area of ​​the electrochemical probe (2). After the molecular signal is enhanced, it is collected in reverse by the inverted dark field microscope (10) and continues to be transmitted to the dichroic mirror (11). The dichroic mirror (11) acts as a partial reflector, reflecting a portion of the scattered enhanced signal to the color CCD camera (12) to realize real-time visualization positioning and dark field imaging. The remaining enhanced signal continues to move forward in a straight line and is reflected by the reflector (13) to the spectral analysis path, enters the spectrometer (14) for wavelength decomposition, and the SERS spectral signal after spectral separation is received and recorded by the spectral CCD camera (15) to realize the acquisition and digital processing of instantaneous SERS signal.

9. The single-entity photoelectric synchronous measurement method according to claim 4, characterized in that, The method for obtaining the instantaneous Rayleigh scattering spectrum of a single sample is as follows: a laser or white light is irradiated onto the sample electrode surface coated with noble metal nanoparticles, causing photons to generate elastic scattering after interacting with particles smaller than the wavelength. The scattered light signal after elastic scattering is collected in reverse by a reverse dark-field microscope (10) and then transmitted to a dichroic mirror (11). The dichroic mirror (11) acts as a partial reflector, reflecting a portion of the scattered light signal to a color CCD camera (12), thereby realizing real-time visualization and dark-field imaging of the sample. The remaining scattered light signal continues to travel in a straight line and is reflected by a reflector (13) to the spectral analysis path, entering the spectrometer (14) for wavelength decomposition. The Rayleigh scattering spectral signal after spectral separation is received and recorded by a spectral CCD camera (15), thereby realizing the acquisition and digital processing of the instantaneous Rayleigh scattering spectrum.

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