A surface plasmon scattering electrochemical imaging device and imaging method

Abstract

The application belongs to the technical field of electrochemical imaging, and particularly relates to a surface plasmon scattering electrochemical imaging device and an imaging method. The imaging device comprises an electrochemical modulation and microfluidic unit, a surface plasmon resonance excitation and imaging unit, and a surface plasmon scattering imaging unit. The electrochemical modulation and microfluidic unit comprises a microfluidic electrochemical cell. The surface plasmon resonance excitation and imaging unit comprises a light beam forming module, a surface plasmon resonance excitation element, and an imaging module. The light beam forming module comprises an excitation light source and a light beam shaping module, and is used for providing a light source and forming outgoing light. The surface plasmon resonance excitation element receives output light and refracts the output light onto a conductive sensing film of a working electrode. The imaging module comprises a first imaging element and a first detector. The surface plasmon scattering imaging unit comprises a second imaging element and a second detector. The second imaging element is used for collecting scattered light of a sample to be measured on the conductive sensing film, and imaging at the second detector. The application does not need to be labeled, and has high temporal and spatial resolution.

Description

Technical Field

[0001] This invention belongs to the field of electrochemical imaging technology, and particularly relates to a surface plasmon scattering electrochemical imaging device and imaging method. Background Technology

[0002] Electrochemical imaging technology overcomes the limitations of traditional electrochemical techniques, which can only perform overall average measurements of electrochemical processes. When combined with spatial resolution technology, it can visualize and analyze electron transfer, molecular reactions, and mass transport at the electrochemical interface at the micro- and nano-scale, thereby directly revealing the spatial heterogeneity of interfacial reactions. It has demonstrated important roles and broad application prospects in fields such as energy materials, energy storage, and biosensing.

[0003] Current electrochemical imaging mainly includes two types: point-by-point scanning and single-parallel imaging. Point-by-point scanning, such as scanning electrochemical microscopy, has high spatial resolution, but the mechanical scanning at each point limits the temporal resolution. Single-parallel imaging, such as electrochemiluminescence imaging, surface plasmon resonance imaging based on surface plasmon waves, or critical angle reflection electrochemical imaging based on evanescent waves, improves temporal resolution by eliminating the need for scanning. However, electrochemiluminescence imaging requires sample labeling, which may introduce interference or even phototoxicity. Surface plasmon resonance imaging based on surface plasmon waves or critical angle reflection electrochemical imaging based on evanescent waves do not require labeling and can dynamically monitor electrochemical processes in real time. However, their spatial resolution is limited by evanescent waves propagating along the surface, resulting in parabolic trails ranging from several micrometers to tens of micrometers and reducing image contrast.

[0004] Therefore, it would be very important and meaningful to develop a novel label-free electrochemical imaging device and method with high temporal and spatial resolution. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to propose a surface plasmon scattering electrochemical imaging device and method that requires no labeling and has high spatiotemporal resolution.

[0006] In a first aspect, the present invention provides a surface plasmon scattering electrochemical imaging device, comprising an electrochemical modulation and microfluidic unit, a surface plasmon resonance excitation and imaging unit, and a surface plasmon scattering imaging unit. The electrochemical modulation and microfluidic unit includes a microfluidic electrochemical cell, in which a working electrode, a reference electrode and a pair of electrodes are disposed. The working electrode includes an optically transparent substrate and a conductive sensing film covering it. The working electrode also serves as a surface plasmon resonance sensing chip. The surface plasmon resonance excitation and imaging unit is located below the microfluidic electrochemical cell. The surface plasmon resonance excitation and imaging unit includes a beam forming module, a surface plasmon resonance excitation element, and an imaging module. The beam forming module includes an excitation light source and a beam shaping module. The beam shaping module includes a polarizing element, an aperture, and several lenses. The light source is used to provide the excitation light required for detection and to enter the beam shaping module. The beam shaping module is used to adjust the excitation light to form the outgoing light. The surface plasmon resonance excitation element receives the output light from the beam shaping module and refracts the output light onto the conductive sensing film of the working electrode. The imaging module includes a first imaging element and a first detector. The first imaging element collects reflected light from the conductive sensing film of the surface plasmon resonance excitation element and images it with the first detector. The surface plasmon scattering imaging unit is located above the microfluidic electrochemical cell. The surface plasmon scattering imaging unit includes a second imaging element and a second detector. The second imaging element is used to collect the scattered light of the sample to be tested on the conductive sensing film and to image it with the second detector.

[0007] According to an embodiment of the present invention, at least one lens in the beam shaping module is disposed on a two-dimensional beam displacement stage. The position of the lens is adjusted by moving the two-dimensional beam displacement stage to adjust the incident angle of the excitation light, thereby realizing surface plasmon resonance. Furthermore, the working angle used during detection can be further set by moving the two-dimensional beam displacement stage.

[0008] According to an embodiment of the present invention, a refractive index matching liquid is disposed between the surface plasmon resonance excitation element and the working electrode. The output light after passing through the refractive index matching liquid is incident parallel to the conductive sensing film of the working electrode at a certain angle. The refractive index of the refractive index matching liquid is matched with the optically transparent substrate of the surface plasmon resonance excitation element and the working electrode.

[0009] According to an embodiment of the present invention, the first imaging element and the second imaging element are independently selected from the objective lens and / or several lenses.

[0010] According to an embodiment of the present invention, the electrochemical modulation and microfluidic unit further includes an electrochemical workstation, wherein the working electrode, the reference electrode and the counter electrode are electrically connected to the electrochemical workstation.

[0011] According to an embodiment of the present invention, the imaging device further includes a control display unit.

[0012] According to an embodiment of the present invention, the control and display unit includes a control module, a data acquisition module, and a display module. The control module is connected to the electrochemical workstation, the first detector, and the second detector, and is used to set the operating parameters of the electrochemical workstation, the first detector, and the second detector. The data acquisition module is used to acquire data detected by the electrochemical workstation, the first detector, and the second detector in real time. The display module is used to display the data acquired by the data acquisition module.

[0013] According to an embodiment of the present invention, the display module is selected from known displays such as monitors, mobile phones, and iPads.

[0014] According to an embodiment of the present invention, the microfluidic electrochemical cell is provided with an electrolytic buffer inlet and an electrolytic buffer outlet.

[0015] According to an embodiment of the present invention, the surface plasmon resonance excitation element is a high numerical aperture objective lens, an optical prism, or the like.

[0016] According to an embodiment of the present invention, the optically transparent substrate of the working electrode is an optically transparent material such as glass, quartz, silicon, or plastic.

[0017] According to an embodiment of the present invention, the conductive sensing film is a conductive material such as gold or silver that can undergo surface plasmon resonance. The sensing film can be functionally modified as needed, for example, a conductive sensing film formed of 2 nm chromium and 48 nm gold.

[0018] According to an embodiment of the present invention, the counter electrode is a platinum electrode, a gold electrode, a graphite electrode, a carbon rod electrode, etc.

[0019] According to an embodiment of the present invention, the reference electrode is a silver / silver chloride electrode, a saturated calomel electrode, a standard hydrogen electrode, etc. According to an embodiment of the present invention, the light source is a laser diode (LD), a monochromatic light-emitting diode (LED), or a laser light source, etc.

[0020] According to an embodiment of the present invention, the first detector and the second detector are independently selected from array charge-coupled devices (CCD), array complementary metal-oxide-semiconductor (CMOS), or array photodiodes, etc.

[0021] Secondly, the present invention provides a method for surface plasmon scattering electrochemical imaging using the above-mentioned electrochemical imaging device, comprising the following steps: dispersing the analyte on the surface of a conductive sensing membrane, introducing an electrolytic buffer into an electrochemical cell, adjusting the incident angle of the excitation light to achieve surface plasmon resonance, setting the working angle, applying a voltage to the electrochemical cell, recording the surface plasmon scattering imaging signal and the current signal of the electrochemical workstation, and calculating the electrochemical impedance signal or the electrochemical current signal based on the surface plasmon scattering imaging signal.

[0022] According to an embodiment of the present invention, the electrochemical imaging is electrochemical impedance imaging or electrochemical current imaging.

[0023] According to an embodiment of the present invention, when electrochemical imaging is electrochemical impedance imaging, the surface charge density on the electrochemical interface is proportional to the surface plasmon resonance angular displacement, and further proportional to the surface plasmon scattering intensity at a certain incident angle. When an AC modulation voltage is applied to the entire electrochemical cell through the working electrode, reference electrode, and counter electrode, the interface capacitance can be obtained from the change in surface plasmon scattering imaging intensity, and thus the surface impedance imaging can be obtained. The photoelectric quantitative relationship is shown in formula (1): (1) in, ; The frequency is the modulation frequency of the AC voltage. , The amount of electron charge on the conductive sensing film. For electron density, The thickness of the conductive sensing film. , , The dielectric constants are those of the electrolytic buffer, the optically transparent substrate, and the conductive sensing film, respectively. The surface plasmon resonance angle; , denoted as angular displacement due to surface plasmon resonance, and x and y as coordinate positions on the conductive sensing film.

[0024] According to an embodiment of the present invention, when electrochemical imaging is electrochemical current imaging, the current intensity is proportional to the concentration gradient of oxidized or reduced substances on the electrode surface, and the concentration gradient of oxidized or reduced substances near the electrode on the electrode surface corresponds to the refractive index gradient of the solution, and is thus proportional to the surface plasmon resonance angle or the surface plasmon scattering intensity at a certain incident angle, thereby establishing a quantitative relationship between electrochemical current and surface plasmon scattering imaging intensity, as shown in formula (2): (2) in, This is the inverse Laplace transform; On the working electrode Changes in scattering intensity signal at location The Laplace transform of; , This represents the change in refractive index per unit concentration of reducing agent. Let be the diffusion coefficient of the reducing agent. This represents the change in refractive index per unit concentration of oxidant. Let be the diffusion coefficient of the oxidant. This is the sensitivity of the surface plasmon scattering intensity signal to the refractive index change. This represents the number of electrons transferred in a redox reaction. is Faraday's constant.

[0025] Beneficial effects 1) The surface plasmon scattering electrochemical imaging device of this invention, by means of surface plasmon scattering imaging and photoelectric quantitative conversion, can achieve high spatiotemporal resolution and high sensitivity imaging of electrochemical impedance and electrochemical current. Specifically, the light emitted from the light source is excited by the beam shaping module and incident at a certain angle parallel to the working electrode and surface plasmon resonance sensing chip mounted on it by a refractive index matching liquid. Surface plasmon scattering imaging of the sample to be tested is performed from above, and then converted into electrochemical imaging. This invention sets the incident angle to the steepest linear region of the resonance curve to obtain the best detection sensitivity.

[0026] 2) This invention directly observes scattered light, and its spatial resolution is limited only by the Rayleigh criterion. At the same time, it retains the high sensitivity of surface plasmon resonance sensing and has the advantage of low background. It can perform in-situ label-free dynamic imaging and monitoring of electrochemical impedance, electrochemical reaction process, etc. at electrochemical interfaces with high spatiotemporal resolution. Attached Figure Description

[0027] Figure 1 This is a structural block diagram of the surface plasmon scattering electrochemical imaging device of the present invention.

[0028] Figure 2 This is a schematic diagram of the overall structure of the surface plasmon scattering electrochemical imaging device using a high numerical aperture objective lens-type surface plasmon resonance excitation element in Embodiment 1 of the present invention.

[0029] Figure 3 This is a schematic diagram of the overall structure of the surface plasmon scattering electrochemical imaging device using a prism-type surface plasmon resonance excitation element in Embodiment 2 of the present invention.

[0030] Figure 4 The results of single-cell electrochemical impedance imaging and single-ion channel detection in Example 3 of this invention are shown.

[0031] Figure 5 The image shows the cyclic voltammetry curve and current imaging diagram of the redox reaction of a single gold nanowire in Example 4 of this invention. Detailed Implementation

[0032] The structure of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0033] In the description of this invention, it should be noted that the terms "center", "upper", "lower", "left", "right", "vertical", "horizontal", "inner", "outer", etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are only for the convenience of describing this invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on this invention.

[0034] In the description of this invention, it should be noted that, unless otherwise explicitly specified and limited, the terms "installation," "connection," and "linking" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral connection; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; and they can refer to the internal connection of two components. Those skilled in the art can understand the specific meaning of the above terms in this invention based on the specific circumstances.

[0035] Example 1 See Figure 1 , 2 As shown, the surface plasmon scattering electrochemical imaging device of the present invention includes an electrochemical modulation and microfluidic unit 11, a surface plasmon resonance excitation and imaging unit 12, a surface plasmon scattering imaging unit 13, and a control and display unit.

[0036] The electrochemical modulation and microfluidic unit 11 comprises an electrochemical workstation 111 and a microfluidic electrochemical cell 112. The microfluidic electrochemical cell 112 contains a working electrode 113, a reference electrode 114, and a pair of electrodes 115. The microfluidic electrochemical cell 112 also has an electrolytic buffer inlet 116 and an electrolytic buffer outlet 117. The working electrode 113 includes an optically transparent substrate 118 and a conductive sensing film 119 covering it. The working electrode 113 also serves as a surface plasmon resonance sensing chip. The working electrode 113, reference electrode 114, and counter electrode 115 are electrically connected to the electrochemical workstation 111.

[0037] The surface plasmon resonance excitation and imaging unit 12 is located below the microfluidic electrochemical cell 112. The surface plasmon resonance excitation and imaging unit 12 consists of a beam forming module 121, a surface plasmon resonance excitation element 122, and an imaging module 123. The beam forming module 121 includes an excitation light source 1211 and a beam shaping module 1212. The beam shaping module 1212 consists of a polarizing element, an aperture, and several lenses. The light source 1211 provides the excitation light required for detection and directs it into the beam shaping module 1212. The beam shaping module 1212 adjusts the excitation light to the specific p-polarization state required for surface plasmon resonance excitation. Through the combination of the aperture and lenses, it adjusts the spot size and uniformity of the excitation light, adapting the spot to the subsequent control of the surface plasmon resonance excitation element 122. Furthermore, through the lens combination, it optimizes the divergence / convergence state of the beam, ensuring that the light is transmitted to the surface plasmon resonance excitation element 122 via a stable optical path.

[0038] In the beam shaping module 1212, a lens is set on the two-dimensional beam displacement stage 12121. The position of the lens is adjusted by moving the two-dimensional beam displacement stage 12121 to adjust the incident angle of the excitation light, thereby realizing surface plasmon resonance. The working angle used during detection can also be further set by moving the two-dimensional beam displacement stage 12121.

[0039] The surface plasmon resonance excitation element 122 receives the output light from the beam shaping module 121 and refracts the output light onto the conductive sensing film 119 of the working electrode 113. A refractive index matching liquid 14 is disposed between the surface plasmon resonance excitation element 122 and the working electrode 113. The output light after passing through the refractive index matching liquid 14 is incident parallel to the conductive sensing film 119 of the working electrode 113 at a certain angle. The refractive index of the refractive index matching liquid 14 matches the optically transparent substrate 118 of the surface plasmon resonance excitation element 122 and the working electrode 113, which greatly reduces interface reflection, allows light to pass through the interface as much as possible, and allows the light to maintain a parallel transmission state after passing through the interface, so that it is uniformly and stably incident on the surface of the conductive sensing film 119, avoiding non-uniform surface plasmon resonance signal and decreased sensitivity caused by light divergence.

[0040] In this embodiment, the surface plasmon resonance excitation element 122 is a high numerical aperture objective lens.

[0041] In this embodiment, a first reflector is provided between the beam forming module 121 and the surface plasmon resonance excitation element 122. The first reflector is used to reflect the light from the beam forming module 121 and then vertically into the high numerical aperture objective lens, which serves as the surface plasmon resonance excitation element 122, and focus it on the back focal plane of the high numerical aperture objective lens. The surface plasmon resonance excitation element 122 (high numerical aperture objective lens) converts the incident light into parallel light and incidents it at a certain angle onto the working electrode 113 (which also serves as the surface plasmon resonance sensing chip). The light undergoes total internal reflection at the interface between the optically transparent substrate 118 and the electrolytic buffer, and the resulting evanescent wave penetrates into the conductive sensing film 119 to excite the surface plasmons. When the refractive index or charge density of the sample on the conductive sensing film 119 changes, it will cause a change in the surface plasmon resonance excitation conditions.

[0042] The imaging module 123 includes a first imaging element 1231 and a first detector 1232. The imaging module 123 is located below the surface plasmon resonance excitation element 122. The first imaging element 1231 is used to collect the light reflected by the conductive sensing film 119 passing through the surface plasmon resonance excitation element 122 and to image it with the first detector 1232.

[0043] The surface plasmon scattering imaging unit 13 is located above the microfluidic electrochemical cell 112. The surface plasmon scattering imaging unit 13 consists of a second imaging element 131 and a second detector 132. The second imaging element 131 is used to collect the scattered light of the sample to be tested on the conductive sensing film 119 and image it with the second detector 132. The second imaging element 131 can be composed of optical elements such as objective lenses and lenses.

[0044] The electrochemical workstation 111 is connected to the second detector 132 of the surface plasmon scattering imaging unit 13 to achieve synchronization between traditional electrochemical detection and electrochemical imaging detection in this invention. The control and display unit consists of a control module, a data acquisition module, and a display module. The control module is used to set the operating parameters of the electrochemical workstation 111, the first detector 1232, and the second detector 132. The data acquisition module is used to acquire data detected by the electrochemical workstation 111, the first detector 1232, and the second detector 132 in real time. The display module is used to display the data acquired by the data acquisition module. The display module can be a known display such as a monitor, mobile phone, or iPad.

[0045] In this embodiment, the optically transparent substrate 118 of the working electrode and surface plasmon resonance sensing chip 113 is a cover glass, on which a conductive sensing film 119 formed of 2 nm chromium and 48 nm gold is disposed.

[0046] Example 2 In this embodiment, except for the structure of the surface plasmon resonance excitation and imaging unit 12, which is different from that in Embodiment 1, all other structures are the same as in Embodiment 1.

[0047] like Figure 3 The diagram shown is a structural diagram of the prism-type surface plasmon resonance excitation element used in this invention.

[0048] The surface plasmon resonance excitation and imaging unit 12 is located below the microfluidic unit 112 and consists of a beam forming module 121, a surface plasmon resonance excitation element 122, and an imaging module 123. The beam forming module 121 includes a light source 1211 and a beam shaping module 1212. The beam shaping module 1212 consists of a lens, an aperture, and a polarizing element. One lens is mounted on a two-dimensional beam displacement stage 12121. Moving the two-dimensional beam displacement stage 12121 can adjust the position of the lens mounted on it to adjust the incident angle of the excitation light, thereby realizing surface plasmon resonance and further setting the working angle used for detection.

[0049] The surface plasmon resonance excitation element 122 receives the output light from the beam forming module 121 and is incident at a certain angle parallel to the conductive sensing film 119 of the working electrode and surface plasmon resonance sensing chip 113 through the refractive index matching liquid 14. In this embodiment, the surface plasmon resonance excitation element 122 is a prism.

[0050] Example 3 Utilize Figure 2 The imaging device shown performs imaging.

[0051] Among them, the optically transparent substrate 118 of the working electrode 113 (which also serves as a surface plasmon resonance sensing chip) is a cover glass, on which a conductive sensing film 119 formed of 2 nm chromium and 48 nm gold is disposed.

[0052] The bottom of the microfluidic electrochemical cell 112 is the working electrode 113 (which also serves as a surface plasmon resonance sensing chip), and the top is a cover glass with four 1 mm holes. The cover glass is attached to the working electrode 113 (which also serves as a surface plasmon resonance sensing chip) with 400 μm thick adhesive tape. Two of the holes on the cover glass serve as the input port 116 and output port 117 of the electrolytic buffer for the inflow and outflow of the electrolytic buffer. The other two holes are used for the introduction of the reference electrode 114 and the counter electrode 115. In this embodiment, the reference electrode 114 is a silver / silver chloride electrode, and the counter electrode 115 is a platinum electrode.

[0053] The light source 1211 in the surface plasmon resonance excitation and imaging unit 12 is a laser diode (LD) with a center wavelength of 660 nm. The surface plasmon resonance excitation element 122 is a 60X oil immersion objective with a numerical aperture of 1.49. The imaging element 131 in the surface plasmon scattering imaging unit 13 is a combination of a 50X objective with a numerical aperture of 0.42 and a lens. The second detector 132 and the first detector 1232 are arrayed complementary metal-oxide-semiconductor (CMOS) cameras.

[0054] This embodiment describes a method for monitoring the electrochemical impedance spectroscopy (EIS) response of the vanillic acid isoform 1 (TRPV1) ion channel on a single cell stimulated by capsaicin, comprising the following steps: Step 1: Before detection, TRPV1-transfected 293T cells (forming an adherent monolayer) are cultured on the surface of the conductive sensing membrane 119. Live cell imaging solution is introduced into the electrochemical cell. The position of the lens mounted on it is adjusted by the two-dimensional beam shift stage 12121 to adjust the incident angle of the excitation light, thereby realizing surface plasmon resonance. The 50X objective lens in the surface plasmon scattering imaging unit 13 is adjusted to realize surface plasmon scattering imaging of the cells. The two-dimensional beam shift stage 12121 is further adjusted to set the working angle used during detection in the steepest linear region of the resonance curve.

[0055] Step 2: Connect the working electrode, reference electrode, and counter electrode of the electrochemical cell to the electrochemical workstation. Apply a sinusoidal AC voltage with a frequency of 28 Hz and an amplitude of 700 mV to the entire electrochemical cell. Simultaneously record the surface plasmon scattering imaging signal and the current signal of the electrochemical workstation through the control display unit.

[0056] Step 3: Based on the photoelectric quantitative relationship, the monitoring signal of electrochemical impedance imaging is obtained from the real-time recorded surface plasmon scattering imaging signal.

[0057] The change in surface plasmon scattering intensity signal can be obtained using formula (1). ) is converted into an electrochemical impedance signal, (1) in, ; The frequency is the modulation frequency of the alternating current. , The amount of electron charge on the conductive sensing film. For electron density, The thickness of the conductive sensing film. , , The dielectric constants are those of the electrolytic buffer solution, the sensing glass substrate, and the conductive sensing film, respectively. The surface plasmon resonance angle; , denoted as angular displacement due to surface plasmon resonance, and x and y as coordinate positions on the conductive sensing film.

[0058] Figure 4 a is a surface plasmon scattering image of TRPV1-transfected 293T cells. Figure 4 b represents the corresponding surface plasmon scattering electrochemical impedance spectroscopy (SIP). Both SIP and SIP can reveal resolvable single-cell images of single adhesion spots. Figure 4 As shown in c, the electrochemical impedance signal of a single adhesion spot can be used to observe the signal changes from baseline to the opening and closing of a single ion channel before and after capsaicin stimulation of the TRPV1 channel. Figure 4 d shows the switching of single ion channel signals on several typical adhesion plaques over time, with the adhesion plaque of cell 4 also showing the signal accumulation when two ion channels are open simultaneously (around 7s and 8.2s).

[0059] Example 4 Using the surface plasmon resonance imaging device with a high numerical aperture objective lens as described in Example 3, 100 μL of a 5 μg / mL gold nanowire solution was dropped onto the conductive sensing film 119 and dried. The redox reaction of a single gold nanowire was then analyzed. The specific monitoring method steps are as follows: Step 1: Introduce 10 mM sulfuric acid solution into the electrochemical cell (filling the electrochemical cell), and adjust the position of the lens mounted on it using the two-dimensional beam displacement stage 12121 to adjust the incident angle of the excitation light, thereby achieving surface plasmon resonance; adjust the 50X objective lens in the surface plasmon scattering imaging unit 13 to achieve surface plasmon scattering imaging of gold nanowires; further adjust the two-dimensional beam displacement stage 12121 to set the working angle used during detection in the steepest linear region of the resonance curve.

[0060] Step 2: Connect the working electrode, reference electrode, and counter electrode of the electrochemical cell to the electrochemical workstation. Use cyclic voltammetry (0 V, 1.4 V, 100 mV / s) to synchronously record the surface plasmon scattering imaging signal and the current signal of the electrochemical workstation through the control display unit.

[0061] Step 3: Based on the photoelectric quantitative relationship, the monitoring signal for electrochemical current imaging is obtained from the real-time recorded surface plasmon scattering imaging signal.

[0062] During cyclic voltammetric scanning, the surface plasmon scattering imaging signal recorded simultaneously showed that the scattering signal intensity curves of the gold nanowires and the background changed significantly over time.

[0063] The change in surface plasmon scattering intensity signal can be obtained using formula (2). ) is converted into an electrochemical current signal, (2) in, This is the inverse Laplace transform; On the working electrode Changes in scattering intensity signal at location The Laplace transform of; , This represents the change in refractive index per unit concentration of reducing agent. Let be the diffusion coefficient of the reducing agent. This represents the change in refractive index per unit concentration of oxidant. Let be the diffusion coefficient of the oxidant. This is the sensitivity of the surface plasmon scattering intensity signal to the refractive index change. This represents the number of electrons transferred in a redox reaction. is Faraday's constant.

[0064] Figure 5 'a' represents a typical curve showing the change in surface plasmon scattering signal of gold nanowires over time. It can be seen that the intensity of the scattering signal from the gold nanowires exhibits periodic changes (over 5 cycles) with each cyclic voltammetric scan. Figure 5 The intensity of the scattered signal from the background (i.e., the electrode surface without gold nanowires) in step b remains unchanged. Based on the photoelectric quantitative relationship, a cyclic voltammogram of a single gold nanowire can be obtained, such as... Figure 5 In d, the cyclic voltammogram of the four gold nanowires corresponding to the surface plasmon scattering (PSM) image shows obvious oxidation and reduction peaks, indicating heterogeneity. Figure 5 The traditional cyclic voltammograms provided by the electrochemical workstation are overall average results across the entire working electrode, and the signal is weak. Figure 5 e and Figure 5 f gives the pixel-by-pixel anodizing ( ) and cathodic reduction ( Peak current imaging. This demonstrates that this method can not only analyze the redox reaction heterogeneity between gold nanowires, but also detect the redox reaction heterogeneity at different locations on a single gold nanowire.

[0065] The specific embodiments of the present invention have been described above by way of example. However, the scope of protection of the present invention is not limited to the above exemplary embodiments. Any modifications, equivalent substitutions, improvements, etc., made by those skilled in the art within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A surface plasmon scattering electrochemical imaging device, characterized in that, It includes an electrochemical modulation and microfluidic unit, a surface plasmon resonance excitation and imaging unit, and a surface plasmon scattering imaging unit; The electrochemical modulation and microfluidic unit includes a microfluidic electrochemical cell, in which a working electrode, a reference electrode and a pair of electrodes are disposed. The working electrode includes an optically transparent substrate and a conductive sensing film covering it. The working electrode also serves as a surface plasmon resonance sensing chip. The surface plasmon resonance excitation and imaging unit is located below the microfluidic electrochemical cell. The surface plasmon resonance excitation and imaging unit includes a beam forming module, a surface plasmon resonance excitation element, and an imaging module. The beam forming module includes an excitation light source and a beam shaping module. The beam shaping module includes a polarizing element, an aperture, and several lenses. The light source is used to provide the excitation light required for detection and to enter the beam shaping module. The beam shaping module is used to adjust the excitation light to form the outgoing light. The surface plasmon resonance excitation element receives the output light from the beam shaping module and refracts the output light onto the conductive sensing film of the working electrode. The imaging module includes a first imaging element and a first detector. The first imaging element collects reflected light from the conductive sensing film of the surface plasmon resonance excitation element and images it with the first detector. The surface plasmon scattering imaging unit is located above the microfluidic electrochemical cell. The surface plasmon scattering imaging unit includes a second imaging element and a second detector. The second imaging element is used to collect the scattered light of the sample to be tested on the conductive sensing film and to image it with the second detector.

2. The surface plasmon scattering electrochemical imaging device according to claim 1, characterized in that, At least one lens in the beam shaping module is set on a two-dimensional beam displacement stage. The position of the lens is adjusted by moving the two-dimensional beam displacement stage to adjust the incident angle of the excitation light, thereby realizing surface plasma resonance. The working angle used during detection can also be further set by moving the two-dimensional beam displacement stage.

3. The surface plasmon scattering electrochemical imaging device according to claim 1, characterized in that, A refractive index matching liquid is disposed between the surface plasmon resonance excitation element and the working electrode. The output light after passing through the refractive index matching liquid is incident parallel to the conductive sensing film of the working electrode at a certain angle. The refractive index of the refractive index matching liquid is matched with the optically transparent substrate of the surface plasmon resonance excitation element and the working electrode.

4. The surface plasmon scattering electrochemical imaging device according to claim 1, characterized in that, The electrochemical modulation and microfluidic unit also includes an electrochemical workstation, and the working electrode, reference electrode and counter electrode are electrically connected to the electrochemical workstation.

5. The surface plasmon scattering electrochemical imaging device according to any one of claims 1-4, characterized in that, The imaging device further includes a control and display unit, which comprises a control module, a data acquisition module, and a display module. The control module is connected to the electrochemical workstation, the first detector, and the second detector, and is used to set the operating parameters of the electrochemical workstation, the first detector, and the second detector. The data acquisition module is used to acquire data detected by the electrochemical workstation, the first detector, and the second detector in real time. The display module is used to display the data acquired by the data acquisition module.

6. The surface plasmon scattering electrochemical imaging device according to any one of claims 1-4, characterized in that, The microfluidic electrochemical cell is equipped with an electrolytic buffer inlet and an electrolytic buffer outlet.

7. A method for performing surface plasmon scattering electrochemical imaging using the electrochemical imaging device according to any one of claims 1-6, characterized in that, The process includes the following steps: dispersing the analyte on the surface of a conductive sensing membrane, introducing an electrolytic buffer into an electrochemical cell, adjusting the incident angle of the excitation light to achieve surface plasmon resonance, setting the working angle, applying a voltage to the electrochemical cell, recording the surface plasmon scattering imaging signal and the current signal of the electrochemical workstation, and calculating the electrochemical impedance signal or electrochemical current signal based on the surface plasmon scattering imaging signal.

8. The method for surface plasmon scattering electrochemical imaging according to claim 7, characterized in that, The electrochemical imaging is either electrochemical impedance imaging or electrochemical current imaging.

9. The method for surface plasmon scattering electrochemical imaging according to claim 8, characterized in that, When electrochemical imaging is electrochemical impedance imaging, the surface charge density at the electrochemical interface is proportional to the surface plasmon resonance angular displacement, and then, at a certain incident angle, it is proportional to the surface plasmon scattering intensity. When an AC modulation voltage is applied to the entire electrochemical cell through the working electrode, reference electrode, and counter electrode, the interface capacitance can be obtained from the change in surface plasmon scattering imaging intensity, and thus the surface impedance imaging can be obtained. The photoelectric quantitative relationship is shown in formula (1): （1） in, ; The frequency is the modulation frequency of the AC voltage. , The amount of electron charge on the conductive sensing film. For electron density, The thickness of the conductive sensing film. , , The dielectric constants are those of the electrolytic buffer, the optically transparent substrate, and the conductive sensing film, respectively. The surface plasmon resonance angle; , denoted as angular displacement due to surface plasmon resonance, and x and y as coordinate positions on the conductive sensing film.

10. The method for surface plasmon scattering electrochemical imaging according to claim 8, characterized in that, When electrochemical imaging is electrochemical current imaging, the current intensity is proportional to the concentration gradient of oxidized or reduced substances on the electrode surface. The concentration gradient of oxidized or reduced substances near the electrode on the electrode surface corresponds to the refractive index gradient of the solution, and is thus proportional to the surface plasmon resonance angle or the surface plasmon scattering intensity at a certain incident angle. This establishes a quantitative relationship between the electrochemical current and the surface plasmon scattering imaging intensity, as shown in formula (2). （2） in, This is the inverse Laplace transform; On the working electrode Changes in scattering intensity signal at location The Laplace transform of; , This represents the change in refractive index per unit concentration of reducing agent. Let be the diffusion coefficient of the reducing agent. This represents the change in refractive index per unit concentration of oxidant. Let be the diffusion coefficient of the oxidant. This is the sensitivity of the surface plasmon scattering intensity signal to the refractive index change. This represents the number of electrons transferred in a redox reaction. is Faraday's constant.

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