Preparation method and application of inverted fluorescence microscope electrolytic tank

Abstract

The invention discloses a preparation method and application of an inverted fluorescence microscope electrolytic tank, and relates to the field of in-situ electrolytic tanks. The method comprises the following steps: 1) cutting and cleaning the bottom of a centrifugal tube, and blow-drying with nitrogen; 2) punching a plurality of holes in the top of the centrifugal tube; (3) the cleaned working electrode and the bottom of the centrifugal tube are bonded through waterproof insulation glue, the bottom of the centrifugal tube is covered with the working electrode, the working electrode is made of a transparent material, the working electrode is thin enough and light-transmitting, and clear focusing can be achieved; 4) adhering the conductor to the working electrode; and 5) after injecting the electrolyte into the electrolytic tank, inserting a counter electrode and a reference electrode into holes in the top of the centrifugal tube. The electrolytic tank is placed on an objective table of an inverted fluorescence microscope and is connected with a conductor through extension of two ends of a working electrode, and the conductor is electrically connected with an external power supply. And a real-time fluorescence picture can be obtained in situ during an electrochemical test, namely a fluorescence signal and an electrochemical signal are obtained at the same time.

Description

Technical Field

[0001] The invention relates to the field of in-situ electrolytic cells, and in particular to a preparation method and application of an electrolytic cell suitable for an inverted fluorescence microscope. Background Art

[0002] Conventional electrochemical techniques study the electron transfer mechanism of microorganisms or chemical substances by measuring electrochemical reaction signals. However, this technology has certain limitations. For example, the measured current, resistance and other signals are the sum of the microorganisms attached to the electrode, ignoring the heterogeneity of individual cells, and it is difficult to intuitively analyze the electron transfer pathway at the protein level. On the contrary, fluorescence microscopy can use fluorescent dyes with specific functions to perform microscopic and intuitive imaging of cell structures and biochemical processes, but it is impossible to quantitatively obtain electron transfer signals. Therefore, in order to evaluate the ability of microorganisms to absorb electrons while monitoring other physiological behaviors of bacteria through fluorescent signals, microbial images, etc., it is necessary to use electrochemical fluorescence microscopy technology. However, the existing in-situ electrolytic cells of electrochemical fluorescence microscopy have the disadvantages of complex processes, high production costs, the electrolytic cell body cannot be protected from light, and assembly and disassembly are troublesome. Summary of the invention

[0003] The purpose of the present invention is to solve the above-mentioned problems in the prior art and to provide a simple electrolytic cell suitable for electrochemical coupled fluorescence microscopy with low cost, simple process and strong universality; the electrolytic cell of the present invention is placed on the stage of the fluorescence microscope, and the bottom of the electrolytic cell is thin enough to achieve clear focus and light transmission. While achieving fluorescence imaging, the three-electrode system in the electrolytic cell can be connected to an electrochemical workstation to perform electrochemical testing, that is, fluorescence signals and electrochemical signals are obtained simultaneously.

[0004] In order to achieve the above object, the present invention adopts the following technical scheme:

[0005] A method for preparing an electrolytic cell suitable for an inverted fluorescence microscope comprises the following steps:

[0006] 1) Cut the bottom of the centrifuge tube, clean it, and blow it dry with nitrogen;

[0007] 2) Punch several holes on the top of the centrifuge tube;

[0008] 3) Using waterproof insulating glue to adhere the cleaned working electrode to the bottom of the centrifuge tube, and the working electrode covers the bottom of the centrifuge tube, and the working electrode is made of a transparent material;

[0009] 4) Glue the conductor to the working electrode;

[0010] 5) After injecting the electrolyte into the electrolytic cell, insert the counter electrode and reference electrode into the hole at the top of the centrifuge tube.

[0011] The holes include two holes for inserting a counter electrode and a reference electrode, and also include holes for introducing gas and / or adding raw materials depending on whether it is necessary to do so during the experiment.

[0012] The centrifuge tube is a transparent or brown centrifuge tube.

[0013] The specifications of the centrifuge tube are 0.2~50 mL.

[0014] The thickness of the working electrode is less than 0.17 mm; the working electrode includes an ITO electrode, a FTO electrode, and an AZO electrode.

[0015] The waterproof insulating glue is epoxy resin AB glue; the conductor is copper conductive glue.

[0016] An electrolytic cell suitable for an inverted fluorescence microscope is prepared by adopting the preparation method.

[0017] The application of the electrolytic cell suitable for an inverted fluorescence microscope is as follows: the assembled electrolytic cell is placed on the stage of the inverted fluorescence microscope, the working electrode is connected to the external power supply through a conductive glue, and according to the electrochemical characteristics of the target object, a suitable electrode potential is applied through an electrochemical workstation to make the substance to be tested react on the electrode. While obtaining the electrochemical reaction signal on the electrode, the fluorescence imaging of the substance to be tested is performed using a fluorescence microscope, so as to obtain other information of the target object at the electrochemical detection interface; the external power supply is an electrochemical workstation.

[0018] The electrolytic cell of the present invention can be used to explore the change of pH during electrochemical reaction, and can also be used to explore the change of membrane potential during extracellular electron transfer of microorganisms.

[0019] The electrolytic cell of the present invention is suitable for an inverted fluorescence microscope. Under an inverted fluorescence microscope, imaging is mainly through the ITO electrode located below the electrolytic cell, and the fluorescence light path is from bottom to top, passing only through the thin and light-transmitting ITO part, so the centrifuge tube material will not affect the data accuracy of the fluorescence imaging, and can provide reliable data support for the experiment.

[0020] Compared with the prior art, the technical solution of the present invention has the following beneficial effects:

[0021] 1. Simple production process: The production process of this electrolytic cell is extremely simple, and batch production can be achieved by manual operation in a laboratory environment. This feature allows researchers to prepare electrolytic cells at any time according to the specific needs of their own experiments, without relying on complex production equipment and professional production processes. For example, when researchers conduct exploratory experiments on the mechanism of microbial electron transfer, they can quickly complete the production of multiple electrolytic cells in the laboratory, greatly improving the efficiency of experimental preparation.

[0022] 2. Excellent cost control: The consumables used to make the electrolytic cell are not only easy to obtain, but also relatively cheap. In addition, due to its low cost, the centrifuge tube can be used as a disposable experimental equipment, and there is no need for tedious cleaning after use, which further reduces the experimental cost.

[0023] 3. Wide application of experiments: The electrolytic cell can be widely used in a variety of experimental conditions such as illumination, light avoidance, aerobic, anaerobic, etc. When light avoidance experiments are required, brown centrifuge tubes can be used to effectively avoid the influence of light on the reaction substances; if the experiment needs to be carried out in an aerobic environment, a simple punching operation can meet the needs; and for anaerobic experiments, an anaerobic atmosphere can be created by sealing and introducing corresponding gases.

[0024] 4. A variety of centrifuge tube specifications: The specifications of the centrifuge tube can be selected in the range of 0.2~50 mL. When the reaction liquid volume requirement is small, the use of small-sized centrifuge tubes can save raw materials; when other tests are required on the liquid after the reaction, and there are specific requirements for the volume of the reaction liquid, such as some mass spectrometry analysis requires 5 mL of reaction liquid, large-sized centrifuge tubes can be used.

[0025] In summary, the simple electrolytic cell suitable for electrochemical coupled fluorescence microscopy provided by the present invention shows significant advantages in many aspects such as production process, cost control, experimental application and experimental operation, which has brought great convenience and promotion to research work in related fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 It is a schematic diagram of the structure of the present invention.

[0027] Figure 2 Schematic diagram of the fluorescent light path of the present invention.

[0028] Figure 3 This is a real picture of the present invention, in which the green tape is used for fixing.

[0029] Figure 4 Cyclic voltammetry curves when Ag and Ag / Cl were used as reference electrodes.

[0030] Figure 5 Fluorescence images of bacteria on the ITO working electrode during a two-step potential sequence (600 s at +300 mV, 600 s at -500 mV) as well as the electrode potential, current and average ThT fluorescence graphs. DETAILED DESCRIPTION

[0031] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention clearer and more understandable, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments.

[0032] Example 1

[0033] 1. See Figure 1 The consumables required for this embodiment are as follows:

[0034] 1) Electrolytic cell body

[0035] Material: Centrifuge tubes commonly used in laboratories, transparent (light-transmissive) and brown (light-proof) are available.

[0036] Specifications: 0.2~50 mL are available according to experimental requirements.

[0037] Others: You can choose cap-type or spiral-shaped centrifuge tubes.

[0038] 2) Working electrode

[0039] Material: transparent ITO, FTO or AZO, etc.

[0040] Thickness: To ensure clear focus at 100x, the ITO thickness should be less than 0.17 mm.

[0041] Size: Both the length and width should be larger than the diameter of the centrifuge tube.

[0042] Others: A whole piece of ITO or patterned ITO is acceptable.

[0043] 3) Counter electrode

[0044] Material: Platinum is commonly used

[0045] Specifications: platinum wire, platinum column, platinum sheet are all acceptable. The electrode diameter / area can be selected according to the experimental requirements, and the length of the electrode must be less than the distance between the stage and the condenser of the fluorescence microscope (make sure the entire experimental device can be placed on the stage, the electrode will get stuck if it is too long).

[0046] Others: In addition to platinum, other materials can also be used.

[0047] 4) Reference electrode

[0048] Material: Conventional saturated calomel electrode, Ag / AgCl electrode, or pseudo reference electrode such as silver wire can be used.

[0049] Specifications: Consistent with the requirements for the counter electrode, the electrode diameter / area can be selected according to the experimental requirements, and the length of the electrode must be less than the distance between the fluorescence microscope stage and the condenser (make sure the entire experimental device can be placed on the stage, the electrode will get stuck if it is too long).

[0050] 5) Lead wire

[0051] Material: Copper conductive glue or other materials with good electrical conductivity.

[0052] 6) Epoxy resin AB glue

[0053] 2. See Figure 1 The preparation of the electrolytic cell of this embodiment comprises the following steps:

[0054] 1) Cleaning: The ITO electrode was ultrasonically cleaned in acetone, ethanol, and ultrapure water for 20 min, then rinsed with ultrapure water and dried with nitrogen. The bottom of the centrifuge tube was cut to make the centrifuge tube at a suitable height, and then the centrifuge tube was ultrasonically cleaned in ethanol and ultrapure water for 20 min, then rinsed with ultrapure water and dried with nitrogen.

[0055] 2) Punching: Punch a hole on the top of the centrifuge tube. (Drill two appropriate holes according to the size of the counter electrode and reference electrode. The number of holes can be increased depending on whether gas or other substances need to be introduced during the experiment.)

[0056] 3) Bonding: Use epoxy resin AB glue to bond the cleaned ITO electrode to the bottom of the centrifuge tube.

[0057] 4) Leads: Glue copper conductive glue onto ITO.

[0058] 5) Assembly: After injecting the electrolyte into the electrolytic cell, insert the counter electrode and reference electrode.

[0059] 3. The working principle and process of the present invention are as follows:

[0060] The assembled electrolytic cell is placed on the stage of an inverted fluorescence microscope. The ITO electrode is connected to the electrochemical workstation via copper conductive glue. According to the electrochemical characteristics of the target object, a suitable electrode potential is applied through the electrochemical workstation to cause a redox reaction of the substance to be tested on the electrode. While obtaining the electrochemical reaction signal on the electrode, a fluorescence microscope is used to perform fluorescence imaging of the substance to be tested, thereby obtaining other information of the target object at the electrochemical detection interface.

[0061] See also Figure 2~3 The electrolytic cell of the present invention is based on an inverted mode, and the fluorescent light path is from bottom to top. The blue light is the excitation light (as shown by the blue arrow), which is irradiated onto the fluorescent dye, and the fluorescent dye emits fluorescence (as shown by the green arrow). The whole process only passes through the ITO part, and ITO is thin and light-transmitting, so it does not affect the fluorescent imaging.

[0062] Fluorescent dyes can be selected according to the purpose of the experiment. For example, pH dyes can be used to explore the changes in pH during electrochemical reactions. Membrane potential fluorescent dyes can be used to explore the changes in membrane potential during extracellular electron transfer in microorganisms.

[0063] The electrolytic cell of the present invention can select centrifuge tubes of different specifications according to experimental requirements. When the volume of the reaction liquid is small, a small centrifuge tube is selected to save raw materials. When other tests are required for the liquid after the reaction, and other tests have requirements on the volume of the reaction liquid, for example, some mass spectrometry analysis requires 5 ml of reaction liquid, then a large centrifuge tube can be selected. Therefore, the present invention can flexibly select the centrifuge tube specifications according to the volume of the reaction liquid.

[0064] The present invention can select brown (light-proof) centrifuge tubes as needed: some reaction substances need to be protected from light, so they need to be light-proofed (such as wrapped in aluminum foil, etc.) during testing, and the present invention can solve this problem well by using brown centrifuge tubes. Moreover, although the light transmittance of brown centrifuge tubes is very poor, it does not affect fluorescence imaging.

[0065] In addition, the present invention can achieve deoxygenation sealing, avoid solution volatilization, and control the atmosphere type inside the cavity. In addition to the advantages of simple process and low cost, it can also meet the aerobic system, and sealing / unsealing can be achieved by whether to punch holes, which is extremely convenient to operate.

[0066] Example 2

[0067] Electrochemical performance characterization

[0068] Silver wire as reference standard potential conversion

[0069] The experimental device of Example 1 was used, with 0.15 mm thick ITO conductive glass as the working electrode, platinum wire as the counter electrode, Ag wire as the quasi-reference electrode, and a nitrogen vent pipe inserted at the top to ensure that the entire experimental process was carried out under anaerobic conditions. The electrolyte was 1 mmol·L -1 [Fe(CN)6)] 4- / [Fe(CN)6)] 3- +0.1 mol·L -1 KCl, scan rate 10 mV·s -1 For detailed results, see Figure 4 Cyclic voltammetry curves.

[0070] Since the standard electrode potential of Ag / AgCl is E ( SHE ) = E ( Ag / AgCl ) + 0.224 V;

[0071] According to the cyclic voltammetry curve, at the oxidation peak potential, E ( Ag / AgCl ) = E ( Ag ) + 0.109 V;

[0072] Reduction peak potential E ( Ag / AgCl ) = E ( Ag ) + 0.118 V;

[0073] It follows that in the case of oxidation, E (SHE) = E (Ag) + 0.333;

[0074] In the reduction case, E (SHE) = E (Ag) + 0.342.

[0075] Example 3

[0076] Electrochemical coupled fluorescence microscopy performance experiment

[0077] The experimental device of Example 1 was used, with 0.15 mm thick ITO conductive glass used as the working electrode, platinum wire as the counter electrode, Ag wire as the quasi-reference electrode, and a nitrogen ventilation tube inserted at the top to ensure that the entire experimental process was carried out under anaerobic conditions.

[0078] Sodium lactate concentration is 10 mmol·L -1 , Thioflavin T concentration was 10 μmol·L -1 .

[0079] See also Figure 5 , a two-step potential sequence was applied, 600 s at +0.3 V (higher than the bacterial oxidation potential) and 600 s at -0.5 V (lower than the bacterial reduction potential). It was found that the fluorescence of ThT at a potential of +0.3 V was stronger than that at a potential of -0.5 V. The fluorescence intensity was quantitatively analyzed using software, and when the potential was switched to -0.5 V, the fluorescence intensity lag time was about 45 s. This shows that the electrolytic cell of the present invention can be used to explore the changes in membrane potential during the extracellular electron transfer of microorganisms.

Claims

1. A method for preparing an electrolytic cell suitable for an inverted fluorescence microscope, characterized in that: The following steps are involved: 1) Cut the bottom of the centrifuge tube, clean it, and blow it dry with nitrogen; 2) Punch several holes on the top of the centrifuge tube; 3) Using waterproof insulating glue to adhere the cleaned working electrode to the bottom of the centrifuge tube, and the working electrode covers the bottom of the centrifuge tube, and the working electrode is made of a transparent material; 4) Glue the conductor to the working electrode; 5) After injecting the electrolyte into the electrolytic cell, insert the counter electrode and reference electrode into the hole at the top of the centrifuge tube.

2. The method for preparing an electrolytic cell suitable for an inverted fluorescence microscope according to claim 1, characterized in that: The holes include two holes for inserting a counter electrode and a reference electrode, and also include holes for introducing gas and / or adding raw materials depending on whether it is necessary to do so during the experiment.

3. The method for preparing an electrolytic cell suitable for an inverted fluorescence microscope according to claim 1, characterized in that: The centrifuge tube is a transparent or brown centrifuge tube.

4. The method for preparing an electrolytic cell suitable for an inverted fluorescence microscope according to claim 1, characterized in that: The specifications of the centrifuge tube are 0.2~50 mL.

5. The method for preparing an electrolytic cell suitable for an inverted fluorescence microscope according to claim 1, characterized in that: The thickness of the working electrode is less than 0.17 mm; the working electrode comprises an ITO electrode, a FTO electrode or an AZO electrode.

6. The method for preparing an electrolytic cell suitable for an inverted fluorescence microscope according to claim 1, characterized in that: The waterproof insulating glue is epoxy resin AB glue; the conductor is copper conductive glue.

7. An electrolytic cell suitable for an inverted fluorescence microscope, characterized in that: Prepared by the preparation method according to any one of claims 1 to 6.

8. The use of an electrolytic cell suitable for an inverted fluorescence microscope as claimed in claim 7, characterized in that: The assembled electrolytic cell is placed on the stage of an inverted fluorescence microscope, and the working electrode is connected to an external power supply through a conductive glue. According to the electrochemical characteristics of the target object, a suitable electrode potential is applied through the electrochemical workstation to make the substance to be tested react on the electrode. While obtaining the electrochemical reaction signal on the electrode, the fluorescence imaging of the substance to be tested is performed using a fluorescence microscope to obtain other information of the target object at the electrochemical detection interface; the external power supply is the electrochemical workstation.

9. The use according to claim 8, characterized in that: Used to investigate pH changes during electrochemical reactions.

10. The use according to claim 8, characterized in that: Used to explore the changes in membrane potential during extracellular electron transfer in microorganisms.

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