A detection device for multiple neural signal factors

Abstract

The present invention relates to a detection device for multiple neural signal factors, comprising a first substrate and a second substrate stacked in a thickness direction; the first substrate is provided with a microfluidic channel, the microfluidic channel is used to circulate the neural signal factor to be tested and the fluorescent substance that can specifically bind to the neural signal factor to be tested and produce fluorescence in a specific band, providing a platform for the two to react; the second substrate is provided with n photosensitive devices, wherein n≥1; the photosensitive device comprises a first electrode layer, a dielectric layer, and a second electrode layer stacked in sequence in the thickness direction, wherein the current change in the dielectric layer responds to the change in the fluorescence intensity emitted by the microfluidic channel, the first electrode layer is connected to an external current detection unit, and the second electrode layer is connected to a power supply that outputs a constant voltage to the photosensitive device. The present invention can overcome the defect that the electrodes of the traditional electrochemical detection device are easily contaminated, thus affecting the test stability and reproducibility.

Description

Technical Field

[0001] The present invention relates to the field of neural signal factor detection, and in particular to a detection device for multiple neural signal factors. Background Art

[0002] Dopamine, serotonin, calcium ions, sodium ions, etc. are important neural signal factors in the nervous system and play a vital role in many physiological and pathological processes of the human body. Therefore, the accurate measurement of changes in the concentration of neural signal factors is of great significance for studying related physiological and pathological processes in the nervous system. At present, electrochemical sensors or synaptic transistors are usually used to detect the concentration of neural signal factors by utilizing the redox reaction of neural signal factors on these devices. The basic principle is to use the redox reaction between the working electrode of the electrochemical sensor or synaptic transistor and the neural signal factor to generate current or potential signals. These signals can be amplified, detected, processed, and converted into readable physical quantities or digital signals by the sensor chip, thereby realizing quantitative or qualitative analysis of neural signal factors.

[0003] However, the redox process is easily interfered by other electroactive substances, resulting in insufficient selectivity and specificity. In addition, the working electrode is in contact with the neural signal factor, making it easy to be contaminated, which in turn affects the stability and reproducibility of the test. In addition, electrochemical sensors or synaptic transistors usually rely on specific chemical reactions or biorecognition events to detect target molecules. Different neural signal factors have different chemical structures and properties, so different highly specific devices need to be designed to distinguish them, which results in high detection costs. Summary of the invention

[0004] Based on this, the purpose of the present invention is to provide a detection device for multiple neural signal factors, which can overcome the defects of traditional electrochemical detection devices that the electrodes are easily contaminated and affect the test stability and reproducibility, and can be used to detect a variety of different neural signal factors.

[0005] The present invention provides a detection device for multiple neural signal factors, comprising a first substrate and a second substrate stacked along a thickness direction; the first substrate is provided with a microfluidic channel, the microfluidic channel is used to circulate neural signal factors to be detected and fluorescent substances that can specifically bind to the neural signal factors to be detected and generate fluorescence in a specific band, the microfluidic channel comprises a liquid inlet and a liquid outlet, the neural signal factors to be detected and the fluorescent substances flow from the liquid inlet to the liquid outlet along a first direction; the second substrate is provided with n photosensitive devices for responding to the fluorescence in a specific band emitted by the microfluidic channel, wherein n≥1; the photosensitive device comprises a first electrode layer, a dielectric layer and a second electrode layer stacked in sequence along the thickness direction, wherein the first electrode layer and the second electrode layer are used to connect to an external current detection unit, and the current change in the dielectric layer responds to the intensity change of the fluorescence emitted by the microfluidic channel.

[0006] The multi-neural factor detection device of the present invention has the following beneficial technical effects:

[0007] (1) Fluorescence of a specific wavelength band is generated by the specific binding of fluorescent substances to the neural signal factors to be tested, and the light signal is converted into an electrical signal through a photosensitive device that is highly selective for the light of the specific wavelength band, and a photocurrent is output. The concentration of the neural signal factor to be tested is obtained according to the output current value. During the entire photosensitivity period, the photosensitive device does not contact the neural molecules to be tested, so it will not be contaminated, which is conducive to improving the stability of the test. After the test is completed, the microfluidic chip only needs to be pressurized and cleaned to achieve repeated detection, which can overcome the defect that the working electrode of the traditional electrochemical detection device is easily contaminated, affecting the stability and reproducibility of the test;

[0008] (2) By integrating multiple photosensitive devices that are highly selective to specific wavelengths into the detection device, it can be applied to the detection of the concentrations of a variety of neural signal factors, without the need to design highly specific sensors based on the neural signal factors to be detected.

[0009] Furthermore, the photosensitive device is located at a position of the second substrate facing the microfluidic channel.

[0010] Furthermore, when n≥2, the photosensitive devices are distributed in an array along the first direction, and the material of the dielectric layer of each photosensitive device is different from each other, so as to respond to fluorescence of different bands; and the photosensitive devices are connected in parallel.

[0011] Furthermore, the second electrode layer faces the microfluidic channel, and the second electrode layer is made of a transparent conductive material.

[0012] Furthermore, the photosensitive device further includes a photosensitive layer disposed between the second electrode layer and the dielectric layer, and the photosensitive layer is used to improve the photosensitivity characteristics of the photosensitive device.

[0013] Furthermore, the material of the photosensitive layer is any one of MoS2, graphene, germanium, selenium, and silicon carbide.

[0014] Furthermore, the second substrate is also provided with a transimpedance amplifier, and the transimpedance amplifier is connected in series with the photosensitive device.

[0015] Furthermore, the fluorescent substance is a fluorescent dye or a fluorescent probe that can generate fluorescence in a specific band after specifically binding to the neural signal factor.

[0016] Furthermore, the detection device for multiple neural signal factors also includes a third substrate, and the third substrate is provided with an excitation light source for exciting the fluorescent probe in the microfluidic channel to produce fluorescence in a specific band, and the orthographic projection of the excitation light source on the second substrate does not overlap with the photosensitive device.

[0017] Furthermore, the second substrate is also provided with an excitation light source for exciting the fluorescent substance in the microfluidic channel to generate fluorescence in a specific wavelength band.

[0018] For better understanding and implementation, the present invention is described in detail below with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a schematic diagram of the structure of a detection device for multiple neural signal factors according to Embodiment 1 of the present invention;

[0020] Figure 2 It is a top view of the microfluidic chip in Example 1 of the detection device for multiple neural signal factors of the present invention;

[0021] Figure 3 It is a cross-sectional view of the photosensitive device in Embodiment 1 of the detection device for multiple neural signal factors of the present invention;

[0022] Figure 4 It is a cross-sectional view of the photosensitive device in Embodiment 1 of the detection device for multiple neural signal factors of the present invention;

[0023] Figure 5 It is a stereoscopic diagram of the photosensitive device in Embodiment 1 of the detection device for multiple neural signal factors of the present invention;

[0024] Figure 6 This is a schematic diagram of the structure of Embodiment 2 of the detection device for multiple neural signal factors of the present invention;

[0025] Description of reference numerals:

[0026] 10. first substrate; 100. microfluidic chip; 1000. liquid inlet channel; 1001. first liquid inlet channel; 1001a. first liquid inlet port; 1002. second liquid inlet channel; 1002a. second liquid inlet port; 1010. mixing channel; 1010a. mixing liquid inlet port; 1010b. liquid discharge port;

[0027] 20, second substrate; 200, photosensitive device; 201, first photosensitive device; 202, second photosensitive device; 2000, substrate; 2002, first electrode layer; 2004, dielectric layer; 2006, second electrode layer; 2008, photosensitive layer;

[0028] 30. A third substrate; 300. An excitation light source. DETAILED DESCRIPTION

[0029] It should be clear that the described embodiments are only some embodiments of the embodiments of the present application, rather than all embodiments. Based on the embodiments in the embodiments of the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the embodiments of the present application.

[0030] In the description of the present invention, it should be understood that the terms "center", "longitudinal", "lateral", "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inside", "outside", etc., indicating the orientation or positional relationship, are based on the orientation or positional relationship shown in the drawings, and are only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as limiting the present invention. In the description of the present invention, unless otherwise specified, "multiple" means two or more.

[0031] It should be noted that when an element is referred to as being "fixed to" another element, it may be directly on the other element or there may be an intermediate element. When an element is considered to be "connected" or "fixedly connected" to another element, it may be directly connected to the other element or there may be an intermediate element at the same time.

[0032] The present invention provides a detection device for multiple neural signal factors, which can overcome the defect that the working electrodes of traditional detection devices are easily contaminated and affect the test stability and reproducibility, and can be used to detect a variety of different neural signal factors. The specific structure of the detection device for multiple neural signal factors of the present invention is specifically described below through two embodiments.

[0033] Example 1

[0034] Figure 1-5 The specific structure of the embodiment 1 of the detection device of multiple neural signal factors of the present invention is shown. Figure 1 As shown, in this embodiment, the detection device includes a first substrate 10 and a second substrate 20 sequentially stacked from bottom to top along the thickness direction.

[0035] Specifically, the first substrate 10 is provided with a microfluidic chip 100. Figure 2 As shown, the microfluidic chip 100 is rectangular or square, and a microfluidic channel is provided in the microfluidic chip 100. In this embodiment, the microfluidic channel includes a liquid inlet channel 1000 and a mixing channel 1010 connected to the liquid inlet channel 1000. Specifically, the liquid inlet channel 1000 includes a first liquid inlet channel 1001 having a first liquid inlet port 1001a and a second liquid inlet channel 1002 having a second liquid inlet port 1002a. The mixing channel 1010 is a curved serpentine structure as a whole to increase the photosensitive area. It has a mixing liquid inlet port 1010a and a liquid discharge port 1010b. The mixing liquid inlet port 1010a of the mixing channel 1010 is respectively connected to the first liquid inlet channel 1001 and the second liquid inlet channel 1002. A solution containing the nerve signal factor to be tested can be introduced through the first liquid inlet 1001a; at the same time, a fluorescent substance that can specifically bind to the nerve signal factor to be tested can be introduced through the second liquid inlet 1002a. The introduced fluorescent substance and the nerve signal factor to be tested specifically bind in the mixing channel 1010 and can generate fluorescence of a specific wavelength band under light excitation.

[0036] The second substrate 20 is provided with n photosensitive devices 200 for responding to the specific fluorescence emitted by the microfluidic channel at a position facing the mixing channel 1010. Figure 2 As shown, the photosensitive device 200 includes a substrate 2000, a first electrode layer 2002, a dielectric layer 2004 and a second electrode layer 2006 which are stacked in sequence from bottom to top along the thickness direction.

[0037] The substrate 2000 is made of any one of flexible materials such as PDMS (polydimethylsiloxane), PET (polyester resin), PI (polyimide) and PEN (polyethylene naphthalate), and has a thickness of 50-100 μm.

[0038] The first electrode layer 2002 is externally connected to a current detection unit (not shown), and the second electrode layer 2006 is connected to a power source (not shown) that outputs a constant voltage to the photosensitive device 200. The first electrode layer 2002 and the second electrode layer 2006 are made of any conductive material such as ITO, carbon nanotube film, aluminum, silver, gold, and copper. Figure 3As shown, in the detection device of this embodiment, the second electrode layer 2006 is directly opposite to the microfluidic chip 100. The second electrode layer 2006 is deposited by a mask plate with 100-120 μm circular holes uniformly distributed, and is composed of a plurality of cylindrical structures distributed in a matrix, and there are gaps between each cylindrical structure. Such a setting can cause light scattering, increase the propagation path of light in the dielectric layer 2004 to a certain extent, thereby increasing the interaction between light and the dielectric layer 2004, while reducing leakage current, improving electric field distribution, and facilitating more uniform charge injection, and reducing damage caused by excessive local electric field. The second electrode layer 2006 is preferably a transparent conductive material with high visible light transmittance such as ITO or carbon nanotube film, so as to allow the fluorescence generated by the microfluidic chip 100 to irradiate the dielectric layer 2004; the first electrode layer 2002 is made of opaque conductive materials such as aluminum, silver, gold and copper, preferably gold, platinum, aluminum and other materials with high light absorption rate. In this way, by absorbing or totally reflecting the light through the first electrode layer 2002 , the dielectric layer 2004 can be prevented from being affected by the light from one side of the first electrode layer 2002 as much as possible, thereby ensuring the accuracy of detection.

[0039] The material of the dielectric layer 2004 is any one of WO3, Ag-doped TiO2 (i.e., Ag / TiO2), ZnO, IGZO, MoS2, WS2, perfluorosulfonic acid resin, Nafion and graphene. The dielectric layers 2004 of different materials have different absorption rates and corresponding characteristics for fluorescence in different bands, so that the photosensitive device 200 with different dielectric layers 2004 is highly selective for fluorescence in different bands.

[0040] Figure 4 FIG. 2 shows the specific structure of another embodiment of a photosensitive device. Figure 4 As shown, the photosensitive device 200 also includes a photosensitive layer 2008 disposed between the dielectric layer 2004 and the second electrode layer 2006. The photosensitive layer 2008 is a two-dimensional material, specifically MoS2 in this embodiment. The two-dimensional structure of MoS2 provides an extremely high surface / volume ratio, which can improve the photosensitivity of the photosensitive device 200, which helps to improve the light absorption efficiency and photocurrent output of the photosensitive device 200. In addition to MoS2, the material of the photosensitive layer 2008 can also be graphene, or any one of germanium, selenium, silicon carbide and other materials. Germanium, selenium, and silicon carbide have a photoelectric effect, that is, when light is irradiated on the photosensitive layer 2008 material, it can excite electrons to transition from the valence band to the conduction band, generating photogenerated carriers (electron-hole pairs). The carrier mobility of these materials is relatively high, which helps to quickly transmit photogenerated carriers in the photosensitive device, thereby improving photosensitivity.

[0041] In such Figure 1In the detection device shown, the number of photosensitive devices 200 is 2, namely, a first photosensitive device 201 and a second photosensitive device 202. The two photosensitive devices 200 are arranged in a single row array along the first direction D1. The dielectric layer 2004 of the first photosensitive device 201 is made of WO3. WO3 has a high light absorption rate and response characteristics for the red light band (i.e., it is sensitive to red light), and the magnitude of the current generated by it can respond to the magnitude of the fluorescence intensity in the red light band. The dielectric layer 2004 of the second photosensitive device 202 is made of Ag-doped TiO2 (Ag / TiO2). Ag / TiO2 has a high absorption rate and response characteristics for the green light band (i.e., it is sensitive to green light), and the magnitude of the current generated by it responds to the magnitude of the fluorescence intensity in the green light band.

[0042] Since different neural signal factors to be tested will produce fluorescence of different wavelengths when combined with different fluorescent substances, two or more photosensitive devices 200 that can respond to fluorescence of different wavelengths can be integrated on the second substrate 20 according to actual detection needs to detect different types of neural signal factors. By selecting a material with high absorption rate and response characteristics for other wavelengths within the visible light range or even for wavelengths outside the visible light range (such as near-infrared wavelengths or infrared wavelengths) as the dielectric layer 2004 of the photosensitive device 200, it is suitable for the detection of more different types of neural signal factors. If different neural signal factors emit fluorescence of the same or similar wavelengths after combining with corresponding fluorescent probes, the same photosensitive device 200 can be used to detect these different neural signal factors.

[0043] It should be noted that if two or more photosensitive devices 200 are arranged on the second substrate 20, it is necessary to ensure that the orthographic projection of each photosensitive device 200 on the first substrate 10 is located at a position such as Figure 2 The mixing channel 1010 is shown within the outer contour range A. The specific binding of the fluorescent substance and the neural signal factor to be detected occurs in the mixing channel 1010, and the fluorescence generated after the binding mainly comes from the mixing channel 1010, so that the fluorescence generated in the mixing channel 1010 can be irradiated to each photosensitive device 200, and each photosensitive device 200 can also receive the fluorescence irradiation to the greatest extent.

[0044] In order to select a photosensitive device 200 with a narrower spectral response range and ensure that the photosensitive device 200 only responds to fluorescence in a specific color band, after the photosensitive device 200 is formed, the photosensitive device 200 is first placed in a specific color band lighting environment with different light intensities and exposure times for detection to select a photosensitive device 200 with a narrower spectral response range for the specific color band, thereby making the photosensitive device 200 highly selective to light in a specific band (such as red light).

[0045] The inventor of the present application has found through experiments that the selectivity of the photosensitive device 200 to a specific color band is mainly related to the characteristics of the dielectric layer 2004 material itself (including the doping degree). By changing the characteristics of the dielectric layer 2004 material (if the dielectric layer 2004 is made of Ag / TiO2, the doping degree of Ag can be adjusted), and then testing the photosensitive device 200, the photosensitive device 200 finally screened has a high selectivity for fluorescence in a specific band. In the experiment, a spectral response characteristic curve is made, and observing the curve can determine that the photosensitive device 200 has selectivity for light in a specific band.

[0046] Taking the first photosensitive device 201 as an example, a method for preparing a photosensitive device 200 including a photosensitive layer 208 is provided below, comprising the following steps:

[0047] S1: preparing a flexible PDMS substrate 2000, cleaning it, and then irradiating it with a UV lamp in an ozone machine for 20 minutes to enhance the hydrophilicity of the PDMS surface, thereby further improving the quality of the deposited film in the subsequent steps;

[0048] S2: The aluminum target is mounted on the target holder, and magnetron sputtering is performed in an argon environment to align the target position for 45 minutes to form a first electrode layer 2002 with a thickness of 80 nm on the substrate 2000;

[0049] S3: The WO3 target is mounted on the target holder, and magnetron sputtering is performed in an argon environment to align the target position for 45 minutes to form a photosensitive layer 204 with a thickness of 80 nm;

[0050] S4: Using chemical vapor deposition, MoO3 (mass fraction of 99.95%, particle size of 100nm) and S (mass fraction of 99.99%) are used as precursors. The sample formed in the above step S3 is placed in the vacuum chamber of the CVD system. Under the conditions of a carrier gas flow rate of 110 mL / min, a holding time of 10min, a MoO3 mass of 2.5mg, and a S mass of 0.1g, the temperature is raised from room temperature to a reaction temperature of 750℃ at a rate of 40℃ / min. The reaction is carried out for 10 to 30min to form a two-dimensional MoS2 film with a thickness of 0.7 to 3.5nm. After the reaction is completed, the film is cooled to room temperature in the furnace.

[0051] S5: Use high-temperature tape to tightly adhere the mask to the sample surface formed in step S4, and then put it into the electron beam assisted sputtering system. In an argon environment, pre-sputter with the ITO target for 1 minute; formally sputter for 10 minutes to form an ITO second electrode layer 2006 with a thickness of 40nm on the photosensitive layer 2008, and obtain the first photosensitive device 201 after cooling.

[0052] The preparation steps of the second photosensitive device 202 are the same as those of the first photosensitive device 201, and the difference between the two is that when preparing the second photosensitive device 202, step S3 is to use a dual-target magnetron sputtering device, install the silver target and the TiO2 target on the target holder, and align the target position in an argon environment through magnetron sputtering for 60 minutes. The middle time period of the sputtering TiO2 process is 8s for co-sputtering of silver and TiO2 to prepare a dielectric layer 2004 with a thickness of 80nm.

[0053] After the photosensitive device 200 is prepared, a volt-ampere characteristic curve test and a sensitivity test may be performed on the photosensitive device 200 to ensure that the photosensitive device 200 works normally and has a high sensitivity to light in a specific wavelength band.

[0054] (1) Test of volt-ampere characteristic curve under different lighting conditions:

[0055] Apply a forward voltage (e.g. 0→3V→0) to the photosensitive device 200, and observe the current change of the photosensitive device 200 under the condition of no light and under different wavelength conditions (red light band 620~760nm or green light band 495~570nm) under the light stimulation of 50mW. Observe whether the device changes from HRS to LRS during the voltage scanning process, and whether the photosensitive device 200 exhibits memristive characteristics regardless of whether light is added.

[0056] (2) Sensitivity test for a specific wavelength: record the time required for the current to rise from 10% to 90% of the peak value when 50mW light stimulation is applied to the device (under the conditions of 620-760nm in the red light band or 495-570nm in the green light band) and the time required for the current to decay from 90% to 10% of the peak value after the light stimulation is removed (usually in the millisecond level, such as 4 or 5ms). The shorter the time required, the more sensitive the device response.

[0057] The following is a method for preparing the detection device comprising two substrates in Example 1, comprising the following steps:

[0058] S1: preparing a first substrate 10 and a second substrate 20;

[0059] Specifically, a suitable adhesive is used to fix the first photosensitive device 201 and the second photosensitive device 202 on a substrate made of PVA (polyvinyl alcohol), and then the first photosensitive device 201 and the second photosensitive device 202 are connected in parallel using a conductive adhesive, and then they are properly packaged to form a second substrate 20; since MEMS technology and low-temperature pulse radiation irradiation technology are existing technologies, the steps are not described in detail here;

[0060] After the fixing and connection are completed, the first photosensitive device 201 and the second photosensitive device 202 are respectively tested for photoelectric characteristics (including volt-ampere characteristic curve test and sensitivity test) to ensure that they can work normally on the second substrate 20;

[0061] Furthermore, the microfluidic chip 100 is fixed on another PVA substrate by hot pressing or using an adhesive, and then the other parts except the first liquid inlet 1001a, the second liquid inlet 1002a and the liquid discharge port 1010b are packaged to form a first substrate 10;

[0062] S2: The second substrate 20 and the first substrate 10 are stacked and assembled from bottom to top to form a double-layer structure of a multi-neural signal factor detection device.

[0063] Here, the second substrate 20 and the first substrate 10 may be assembled in a fixed connection or a detachable connection.

[0064] The PVA material has certain flexibility, which can make the first substrate 10 and the second substrate 20 have the advantages of being stretchable, not affected by external forces, and highly integrated.

[0065] Furthermore, a transimpedance amplifier 210 is provided on the second substrate 20, and the transimpedance amplifier 210 is connected in series with the first photosensitive device 201 and the second photosensitive device 202. Since the current generated by the photosensitive device 200 after receiving the fluorescent signal is very small, it is necessary to amplify the current signal by setting an amplifier with high input impedance to avoid the loss of the current during the transmission process and to make the current signal change clearly displayed, so as to facilitate the subsequent analysis of the corresponding relationship between the current change and the concentration of the neural signal factor.

[0066] Correspondingly, in order to obtain a detection device having a transimpedance amplifier 210, on the basis of the method for preparing a detection device including two layers of substrates in Example 1, step S2 also includes: using an adhesive to fix the transimpedance amplifier 210 on a second substrate 20 made of PVA, and then using a conductive glue to connect the transimpedance amplifier 210 in series with the first photosensitive device 201 and the second photosensitive device 202 (that is, the first photosensitive device 201 and the second photosensitive device 202 are connected in parallel to form a total resistance, and the transimpedance amplifier 210 is connected in series with the total resistance).

[0067] When using the detection device of the above-mentioned embodiment 1, the solution containing the neural signal factor to be tested is first passed into the first liquid inlet channel 1001 from the first liquid inlet 1001a, and the fluorescent substance is passed into the second liquid inlet channel 1002 from the second liquid inlet 1002a. The neural factor to be tested and the fluorescent substance are gathered in the mixing channel 1010 to specifically bind, and generate fluorescence of a specific band under the irradiation of the light source. The fluorescence intensity is positively correlated with the concentration of the neural signal factor to be tested. The higher the concentration of the neural signal factor to be tested, the greater the fluorescence intensity; the greater the fluorescence intensity, the greater the current output by the photosensitive device 200. After the photosensitive device 200 that is sensitive to the fluorescence in this band is irradiated with fluorescence, its internal carrier concentration changes, thereby generating current inside it. Among them, the fluorescent substance is a fluorescent probe or a fluorescent dye.

[0068] Based on this, the above-mentioned detection device is used to detect the current generated by the target nerve signal factor with different concentration gradients, analyze the relationship between the concentration of the nerve signal factor and the current change, and draw a standard curve of the target nerve signal factor concentration-current; then the same detection device is used to detect the solution containing the target nerve signal factor to be tested, and the corresponding current size is obtained, and then the concentration of the nerve signal factor to be tested is obtained through the drawn standard curve.

[0069] Example 2

[0070] When the fluorescent substance is a fluorescent probe, based on the above detection device embodiment 1, a third substrate 30 is further included. Figure 6 The specific structure of the embodiment 2 of the detection device of multiple neural signal factors of the present invention is shown. Figure 6 As shown, the third substrate 30 is located below the second substrate 20. An excitation light source 300 is provided on the third substrate 30, and the electrode of the excitation light source 300 is connected to an external power supply. When the excitation light source 300 is turned on, the light generated can excite the fluorescent material in the mixing channel 1010 of the microfluidic chip 100 to produce fluorescence. The positive projection of the excitation light source 300 on the third substrate 30 does not overlap with any photosensitive device 200, that is, the excitation light source 300 and any photosensitive device 200 are staggered, so that the light emitted by the excitation light source 300 can irradiate the fluorescent material, but will not directly irradiate the photosensitive device 200, so as to prevent the photosensitive device 200 from receiving crosstalk from irrelevant signals and affecting the detection results. Of course, the third substrate 30 can be arranged above the first substrate 10, as long as it is ensured that the light-emitting chip does not directly irradiate any photosensitive device 200. The color of the excitation light source 300 can be selected according to the selected fluorescent dye or fluorescent probe, as long as it can excite the fluorescent dye or fluorescent probe to produce fluorescence in a specific band. In this embodiment, the excitation light source 300 is a blue light LED chip.

[0071] As an optional method, the third substrate 30 may not be provided, and the excitation light source 300 may be provided on the second substrate 20. In this embodiment, the orthographic projection of the excitation light source 300 on the second substrate 30 does not overlap with any photosensitive device 200, so as to ensure that the light emitted by the excitation light source 300 can irradiate the fluorescent material, but will not directly irradiate the photosensitive device 200, thereby preventing the photosensitive device 200 from receiving crosstalk from irrelevant signals and affecting the detection result.

[0072] Here, a method for preparing a detection device including a three-layer substrate of the above-mentioned embodiment 2 is provided. Compared with the method for preparing the detection device of embodiment 1, the difference is that step S1 also includes preparing a third substrate 30, and step S2 also includes stacking and assembling the third substrate 30 with the second substrate 20 and the first substrate 10 in sequence.

[0073] The specific method for preparing the third substrate 30 is: first, the surface of the PVA substrate is cleaned, roughened and appropriately chemically treated to improve the bonding strength, the excitation light source 300 is fixed on the PVA using conductive glue, and then an electrical connection is established between the electrode of the excitation light source 300 and the flexible circuit on the substrate through the conductive glue, and then the excitation light source 300 is appropriately packaged to prevent the excitation light source 300 from being affected by physical damage and environmental factors, thereby obtaining the third substrate 30.

[0074] After the excitation light source 300 is fixed and connected, electrical and optical tests are performed on the excitation light source 300 to ensure that it can work normally on the PVA substrate.

[0075] Then, the third substrate 30 , the second substrate 20 and the first substrate 10 are stacked and assembled in sequence from bottom to top to form a three-layer structure of a multi-neural signal factor detection device.

[0076] Here, the third substrate 30, the second substrate 20 and the first substrate 10 can be assembled in a fixed connection or a detachable connection. When a detachable assembly method is adopted, the third substrate 30 can be replaced as needed, and a substrate integrated with a light-emitting chip that can generate a specific wavelength can be selected. PVA itself is a colorless and transparent crystalline polymer material with good transparency and glossiness. Therefore, the third substrate 30, the second substrate 20 and the first substrate 10 are prepared using PVA, which allows the light of the LED light-emitting chip to penetrate the three-layer substrate and irradiate to the microfluidic channel to excite the fluorescent substance to produce fluorescence.

[0077] The following provides a method for detecting dopamine concentration and calcium ion concentration based on the detection device of the above-mentioned embodiment 2.

[0078] Application Example 1

[0079] To measure the dopamine concentration, the following steps are included:

[0080] S1: preparing a standard curve of dopamine concentration, fluorescence intensity and electrical characteristic changes of the photosensitive device 200;

[0081] Specifically, first, 1 mL of dopamine test solutions with concentrations of 0 nM, 1 nM, 2.5 nM, 5 nM, 10 nM, 25 nM, 50 nM, 80 nM, 100 nM, 150 nM, 200 nM, 250 nM, 500 nM, 1000 nM, and 2500 nM were prepared using ultrapure water and dopamine solution;

[0082] Then, 100 μL of rGRABDA1m fluorescent probe dispersion was added to 0.5 mL of sodium carbonate-sodium bicarbonate buffer solution (pH=10, 50 mM), and the prepared fluorescent probe dispersion was repeatedly prepared in 15 portions, one of which was introduced into the second liquid inlet 1002a through a syringe pump; and, under the condition of an ambient temperature of 25°C, one of the dopamine test solutions was injected into the first liquid inlet 1001a through a syringe pump; the rGRABDA1m fluorescent probe embeds a fluorescent protein that is sensitive to structural changes into the human dopamine receptor. When dopamine binds to the receptor, it causes a change in the receptor conformation, which can affect the fluorescence intensity of the fluorescent protein to produce detectable red fluorescence;

[0083] The fluorescent probe and dopamine are mixed in the mixing channel 1010 . During this process, the flow rate of the mixed solution in the mixing channel 1010 is controlled to enhance the interaction between the fluorescent probe and dopamine.

[0084] Next, after a period of reaction, the microfluidic chip 100 is irradiated with an LED blue light chip with a wavelength of 480-495 nm and a power of 50 mW to generate a red fluorescence signal, which is measured using confocal line scanning (scanning speed of 1,280 Hz). At the same time, the red fluorescence signal is converted into an electrical signal through the first photosensitive device 201 to output a photocurrent.

[0085] After the measurement is completed, the inlet channel 1000 and the mixing channel 1010 in the microfluidic chip 100 are cleaned by pressurization, and the cleaning effect is monitored and evaluated using an optical microscope, and the above operation is repeated to detect the next concentration solution. After the detection of all concentration solutions is completed, the relationship between the fluorescence intensity and the current change output by the first photosensitive device 201 is analyzed, the corresponding relationship between the dopamine concentration and the current response is established, and a standard curve of dopamine concentration, fluorescence intensity and the change of electrical characteristics of the photosensitive device 200 is drawn.

[0086] S2: Detect the dopamine concentration to be tested;

[0087] Specifically, first, 100 μL of rGRABDA1m fluorescent probe dispersion was added to 0.5 mL of sodium carbonate-sodium bicarbonate buffer solution (pH=10, 50 mM), and the fluorescent probe dispersion was introduced into the second liquid inlet 1002a through a syringe pump; and, under the condition of an ambient temperature of 25°C, a solution containing dopamine to be tested was injected into the first liquid inlet 1001a through a syringe pump;

[0088] Then, the microfluidic chip 100 is irradiated with an LED blue light chip with a wavelength of 480-495nm and a power of 50mW to generate a red fluorescent signal, and the red fluorescent signal is converted into an electrical signal through the first photosensitive device 201 to output a photocurrent;

[0089] Finally, according to the current value output by the first photosensitive device 201 and the standard curve obtained in step S1, the concentration of dopamine to be measured is obtained.

[0090] Application Example 2

[0091] Detection of calcium ions (Ca 2+ ) concentration, comprising the following steps:

[0092] S1: Draw a standard curve of calcium ion concentration, fluorescence intensity and electrical characteristics of the photosensitive device;

[0093] Specifically, first, 1 mL of calcium ion test solutions with concentrations of 0 nM, 1 nM, 2.5 nM, 5 nM, 10 nM, 25 nM, 50 nM, 80 nM, 100 nM, 150 nM, 200 nM, 250 nM, 500 nM, 1000 nM, and 2500 nM were prepared with ultrapure water and calcium ion solution;

[0094] Then, 50 μg of Fluo-4, AM was dissolved in 45.6 μL DMSO to prepare a mother solution, and Pluronic F127 and HBSS were added to dilute the mother solution in turn to make the concentration of Fluo-4, AM 4 μM to prepare a fluorescent probe dispersion, and the prepared fluorescent probe dispersion was repeatedly prepared 15 times, one of which was introduced into the second liquid inlet 1002a through a syringe pump; and, under the condition of an ambient temperature of 37°C, one of the calcium ion test solutions was injected into the first liquid inlet 1001a through a syringe pump; Fluo-4 is a fluorescent probe for monitoring changes in calcium ion concentration in living cells. It is a fluorescent indicator in the form of AM ester, which can penetrate the cell membrane and be hydrolyzed into an active form by esterase in the cell, thereby reacting with Ca 2+ When Fluo-4 binds to Ca 2+ Upon binding, its fluorescence intensity increases dramatically to produce detectable green fluorescence.

[0095] The fluorescent probe and the calcium ions are mixed in the mixing channel 1010 . During this process, the flow rate of the mixed solution in the mixing channel 1010 is controlled to enhance the interaction between the fluorescent probe and the calcium ions.

[0096] Next, after a period of reaction, the microfluidic chip 100 is irradiated with a blue LED chip with a wavelength of 480-495 nm and a power of 50 mW to generate a green fluorescence signal. The fluorescence signal is measured using confocal line scanning (scanning speed of 1,280 Hz), and the green fluorescence signal is converted into an electrical signal through the second photosensitive device 202 to output a photocurrent.

[0097] After the measurement is completed, the inlet channel 1000 and the mixing channel 1010 in the microfluidic chip 100 are cleaned by pressurization, and the cleaning effect is monitored and evaluated using an optical microscope, and the above operation is repeated to detect the next concentration solution. After completing the detection of the calcium ion test solution of each concentration, the relationship between the calcium ion concentration, the green fluorescence intensity and the current change output by the second photosensitive device 202 is analyzed, the corresponding relationship between the dopamine concentration, the green fluorescence intensity and the current response is established, and the standard curve of the calcium ion concentration, the green fluorescence intensity and the change of the electrical characteristics of the second photosensitive device 202 is drawn.

[0098] S2: Detecting the concentration of calcium ions to be tested;

[0099] Specifically, first, 50 μg of Fluo-4, AM was dissolved in 45.6 μL DMSO to prepare a mother solution, and Pluronic F127 and HBSS were added to dilute the mother solution so that the concentration of Fluo-4, AM was 4 μM to prepare a fluorescent probe dispersion, and the fluorescent probe dispersion was introduced into the second liquid inlet 1002a through a syringe pump; and, under the condition of an ambient temperature of 37° C., one portion of the calcium ion test solution was injected into the first liquid inlet 1001a through a syringe pump;

[0100] Next, the microfluidic chip 100 is irradiated with an LED light-emitting chip with a wavelength of 480-495 nm and a power of 50 mW to generate a green fluorescence signal, and the green fluorescence signal is converted into an electrical signal through the second photosensitive device 202 to output a photocurrent;

[0101] Finally, according to the current value output by the second photosensitive device 202 and the standard curve obtained in step S1, the concentration of the calcium ion to be measured is obtained.

[0102] In addition to being used for the detection of dopamine and calcium ion concentrations, the above detection device can also be used for the detection of serotonin and sodium ion concentrations. The fluorescent probes used to detect dopamine, calcium ions, sodium ions and serotonin and the wavelengths for exciting the fluorescent probes are shown in Table 1 below:

[0103] Table 1

[0104]

[0105] As shown in Table 1, the fluorescent probe CoroNa Green or ENG-2 AM can specifically bind to sodium ions, and then under the excitation of the blue light LED chip, the fluorescent probe emits green fluorescence; and after the fluorescent probe GRAB5-HT1.0 specifically binds to serotonin, under the excitation of near-infrared light with a wavelength of 930nm, the fluorescent probe emits green fluorescence. Therefore, the concentration of sodium ions and serotonin can be detected by the detection device of the present invention. The principle and basic steps of the detection method for detecting the concentration of sodium ions and serotonin are basically the same as the principle of the above-mentioned method for detecting the concentration of dopamine and calcium ions, with the difference that: when detecting serotonin, the light source used needs to be replaced with an excitation light source 300 that can emit 930nm near-infrared light; and the fluorescent substance used when detecting serotonin and sodium ions is different from the fluorescent substance used when detecting dopamine and calcium ions; and the reaction temperature in the microfluidic chip 100 may need to be adjusted as needed. Since these are not key parameters of the technical solution of the present invention, they are not described in detail here.

[0106] Compared with the prior art, the multi-neural factor detection device of the present invention has the following beneficial technical effects:

[0107] (1) Fluorescence of a specific wavelength band is generated by the specific binding of fluorescent substances to the neural signal factors to be tested, and the light signal is converted into an electrical signal through a photosensitive device that is highly selective for the light of the specific wavelength band, and a photocurrent is output. The concentration of the neural signal factor to be tested is obtained according to the output current value. During the whole process, the photosensitive device does not contact the neural molecules to be tested and will not be contaminated, which is conducive to improving the stability of the test. After the test, the microfluidic chip only needs to be cleaned under pressure to achieve repeated detection, which can overcome the defect that the working electrode of the traditional electrochemical detection device is easily contaminated, which affects the stability and reproducibility of the test.

[0108] (2) By integrating multiple photosensitive devices that are only sensitive to specific wavelengths in the detection device, it can be applied to the detection of the concentration of multiple neural signal factors, without the need to design highly specific sensors based on the neural signal factors to be detected;

[0109] (3) Based on the high sensitivity and rapid response of fluorescent substances to neural signal factors, and through the fluidity of the solution in the microfluidic channel, real-time monitoring of changes in the concentration of neural signal factors can be achieved.

[0110] The above-mentioned embodiments only express several implementation methods of the present invention, and the description is relatively specific and detailed, but it cannot be understood as limiting the scope of the invention patent. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, and the present invention is also intended to include these modifications and modifications.

Claims

1. A detection device for multiple neural signal factors, Features: It comprises a first substrate (10) and a second substrate (20) stacked in a thickness direction; The first substrate (10) is provided with a microfluidic channel, the microfluidic channel is used to circulate the neural signal factor to be detected and the fluorescent substance that can specifically bind to the neural signal factor to be detected and produce fluorescence in a specific band, the microfluidic channel comprises a liquid inlet and a liquid outlet, the neural signal factor to be detected and the fluorescent substance flow from the liquid inlet to the liquid outlet along a first direction, wherein the fluorescent substance is a fluorescent dye or a fluorescent probe that can specifically bind to the neural signal factor and produce fluorescence in a specific band; The second substrate (20) is provided with n photosensitive devices (200) for responding to fluorescence of a specific wavelength band emitted by the microfluidic channel, wherein n≥1; the photosensitive device (200) comprises a first electrode layer (2002), a dielectric layer (2004) and a second electrode layer (2006) stacked in sequence along the thickness direction, wherein the first electrode layer (2002) is connected to an external current detection unit, the second electrode layer (2006) is connected to a power supply that outputs a constant voltage to the photosensitive device (200), and a change in current in the dielectric layer (2004) responds to a change in the intensity of the fluorescence emitted by the microfluidic channel; The photosensitive device (200) further comprises a photosensitive layer (2008) arranged between the second electrode layer (2006) and the dielectric layer (2004), wherein the photosensitive layer (2008) is used to improve the photosensitive properties of the photosensitive device (200).

2. The detection device for multiple neural signal factors according to claim 1, Features: The photosensitive device (200) is located at a position of the second substrate (20) directly facing the microfluidic channel.

3. The detection device for multiple neural signal factors according to claim 1, Features: When n≥2, the photosensitive devices (200) are distributed in an array along the first direction, and the dielectric layer of each photosensitive device (200) is made of different materials and is used to respond to fluorescence in different wavelength bands. When n=2, the dielectric layer of a photosensitive device (200) is WO 3 The dielectric layer of the other photosensitive device (200) is Ag / TiO 2 ; The photosensitive devices (200) are connected in parallel.

4. The detection device for multiple neural signal factors according to claim 1, Features: The second electrode layer (2006) faces the microfluidic channel, and the material of the second electrode layer (2006) is a transparent conductive material.

5. The device for detecting multiple neural signal factors according to claim 1, Features: The material of the photosensitive layer (2008) is MoS 2 , graphene, germanium, selenium, and silicon carbide.

6. The device for detecting multiple neural signal factors according to claim 1, Features: The second substrate (20) is further provided with a transimpedance amplifier (210), and the transimpedance amplifier (210) is connected in series with the photosensitive device (200).

7. The device for detecting multiple neural signal factors according to claim 1, Features: The invention also comprises a third substrate (30), wherein the third substrate (30) is provided with an excitation light source (300) for exciting the fluorescent substance in the microfluidic channel to produce fluorescence of a specific wavelength band, and the orthographic projection of the excitation light source (300) on the second substrate (20) does not overlap with the photosensitive device (200).

8. The device for detecting multiple neural signal factors according to claim 1, Features: The second substrate (20) is also provided with an excitation light source (300) for exciting the fluorescent substance in the microfluidic channel to produce fluorescence in a specific wavelength band.

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