CN111855766A - Cell multi-parameter detection micro-nano sensor and manufacturing method thereof - Google Patents
Cell multi-parameter detection micro-nano sensor and manufacturing method thereof Download PDFInfo
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/27—Association of two or more measuring systems or cells, each measuring a different parameter, where the measurement results may be either used independently, the systems or cells being physically associated, or combined to produce a value for a further parameter
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- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
- B01L3/502707—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by the manufacture of the container or its components
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- G01K—MEASURING TEMPERATURE; MEASURING QUANTITY OF HEAT; THERMALLY-SENSITIVE ELEMENTS NOT OTHERWISE PROVIDED FOR
- G01K7/00—Measuring temperature based on the use of electric or magnetic elements directly sensitive to heat ; Power supply therefor, e.g. using thermoelectric elements
- G01K7/16—Measuring temperature based on the use of electric or magnetic elements directly sensitive to heat ; Power supply therefor, e.g. using thermoelectric elements using resistive elements
- G01K7/18—Measuring temperature based on the use of electric or magnetic elements directly sensitive to heat ; Power supply therefor, e.g. using thermoelectric elements using resistive elements the element being a linear resistance, e.g. platinum resistance thermometer
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/02—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating impedance
- G01N27/04—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating impedance by investigating resistance
- G01N27/06—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating impedance by investigating resistance of a liquid
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/02—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating impedance
- G01N27/04—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating impedance by investigating resistance
- G01N27/06—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating impedance by investigating resistance of a liquid
- G01N27/07—Construction of measuring vessels; Electrodes therefor
Abstract
The invention provides a cell multi-parameter detection micro-nano sensor and a manufacturing method thereof, the sensor comprises a glass substrate (1) and a PDMS sheet (2) which are mutually attached, a flow groove (7) is arranged on the attachment surface of the PDMS sheet (2), a flow channel is formed on the glass substrate (1) after attachment, a sample inlet hole (5) and a sample outlet hole (6) are arranged at two ends of the flow groove (7), an opening dam for allowing cells to flow in and be reserved is arranged in the flow groove (7), the opening of the opening dam faces the sample injection direction, a plurality of micro electrode blocks are arranged on the attachment surface of the glass substrate (1) at the corresponding position of the opening dam and are positioned below the opening dam, and a height gap smaller than the individual outer diameter of the cells to be detected is reserved between the opening dam and the glass substrate (1); each micro electrode block is provided with a lead-out wire (4) made of Pt material; the invention can detect a plurality of parameters of cells and can realize on-line continuous monitoring.
Description
Technical Field
The invention relates to the technical field of cell detection and micro-nano sensors, in particular to a cell multi-parameter detection micro-nano sensor and a manufacturing method thereof.
Background
In the field of biology, parameters such as temperature, conductivity and pH value are important indexes indispensable for researching the life process of organisms in the field of life science. In life science research, various physiological and chemical reactions are continuously performed in living cells, and the expressions closely related to the reactions are changes in temperature, conductivity and pH. On the other hand, when the cells are stimulated by external factors, the cells rapidly adjust their metabolic activities to cope with environmental changes, and the changes are accompanied by significant changes in parameters such as temperature, conductivity, and pH. Therefore, the real-time detection of parameters such as cell temperature, conductivity, pH value and the like is realized, and the method has important significance for the research of cell physiological functions and heterogeneity, and from the aspects of adaptability and stress response of cells to external environments. The cell parameters change slightly and are easily influenced by the extracellular environment, so the cell parameter detection method needs high precision and high response speed to accurately detect the parameter fluctuation of the cell. At present, no mature effective detection means exists, and especially no mature effective detection means for parameters such as cell temperature, conductivity, pH value and the like exist.
With the continuous improvement and development of Micro-fluidic and Micro-machining technologies, the advantages of Micro total analysis systems (μ -TAS) in the fields of biological research and clinical experimental diagnosis at the cellular level are becoming more and more significant. The micro-fluidic chip has the advantages of equivalent size of functional units and cells, high precision, rapid and convenient detection and the like, and has obvious advantages in cell analysis. The technology can integrate all the existing cell analysis steps and processes (such as cell manipulation, cell capture/screening, cell culture, online real-time dynamic monitoring analysis and the like) on one microchip, realize the integration of analysis and operation, can reduce the damage and pollution to cell samples in the operation process, and is very suitable for the rapid and high-sensitivity detection of a small amount of cell characteristic parameters. The cell multi-parameter detection micro-nano sensor is the micro-fluidic sensor and is also called a micro-fluidic chip.
At present, a series of novel microfluidic chips and devices are developed for single cell detection, and the microfluidic chips and the devices can be mainly divided into the following types: (1) and an emulsion drop unit is utilized to wrap a single cell, so that an independent space is provided for cell detection. And then the microstructure is used for controlling liquid drop fusion to realize the lysis of single cells and nucleic acid amplification, such as patent document CN 109988821A. (2) The microcavity structure unit is used for capturing single cells, the opening and closing of a specific channel are controlled through a micro valve structure, lysis solution is introduced to lyse the cells, and lysate is driven to flow into a micro reaction cavity for amplification, such as patent document CN 106065391A. (3) By using the principle of dielectrophoresis, electrodes are integrated in a microchip, single cells are captured, and then high voltage is applied to lyse the cells, as in patent document CN 107267382A. The method for detecting the cell gene in the microfluidic chip mainly comprises the following steps: (1) adding a specific Taqman probe into the nucleic acid amplification reagent, and analyzing gene expression according to a fluorescence signal. (2) Recovering the nucleic acid amplification product, and detecting the target nucleic acid by adopting a second-generation sequencing technology. (3) By using in situ nucleic acid hybridization techniques, genes can be analyzed without lysing the cells.
The above microfluidic devices applied to single cell parameter detection still have the following problems: 1) the method can only realize the detection of single parameter, but cannot realize the integrated simultaneous detection and real-time online continuous monitoring of a plurality of parameters. 2) The chip capable of realizing cell detection integration has a complex structure and needs external sample introduction or control equipment. 3) The chip with simple structure only realizes single cell capture and lysis, and does not integrate all detection steps.
Polydimethylsiloxane, abbreviated as PDMS. Is a high molecular organic polymer, has optical transparency and internal fine structure porosity, and has good biocompatibility. Applications of PDMS include micro-fluidic channel systems in bio-microelectromechanical systems, caulks, lubricants, contact lenses. The liquid dimethyl siloxane is a viscous liquid called silicone oil, and is an organic siloxane mixture with chain structures with different polymerization degrees, and the end group and the side group of the organic siloxane mixture are all hydrocarbon groups (such as methyl, ethyl, phenyl and the like).
Disclosure of Invention
The invention aims to solve the technical problem of providing a cell multi-parameter detection micro-nano sensor, which integrates the functions of cell capture and multi-parameter detection and also provides a manufacturing method of the cell multi-parameter detection micro-nano sensor.
In order to solve the technical problems, the cell multi-parameter detection micro-nano sensor adopts the following technical scheme:
a cell multi-parameter detection micro-nano sensor comprises a glass substrate (1) and a PDMS (polydimethylsiloxane) sheet (2) which are mutually attached, wherein the attaching surface of the PDMS sheet (2) is a slotted structure surface, a runner (7) is arranged on the attaching surface of the PDMS sheet (2), a runner for cell solution to flow through during detection is formed on the glass substrate (1) after the PDMS sheet is attached to the glass substrate (1), through holes penetrating through the PDMS sheet (2) are respectively arranged at two ends of the runner (7), and a sample inlet hole (5) and a sample outlet hole (6) are formed; the cell flow groove is characterized in that an opening box dam for flowing and retaining a cell solution is arranged in the flow groove (7), and an opening of the opening box dam faces to the direction of sample injection; more than 2 micro electrode blocks which are respectively used for detecting and sensing different parameters are arranged on the binding surface of the glass substrate (1) at the corresponding position of the opening dam, so that each micro electrode block is positioned below the area occupied by the opening dam, a height gap for sample fluid and air to flow through during detection is reserved between the opening dam and the glass substrate (1), and the height gap is smaller than the outer diameter of the cell to be detected; lead wires (4) made of Pt are respectively arranged on the micro electrode blocks.
The following is a further scheme of the cell multi-parameter detection micro-nano sensor of the invention:
the height of the opening box dam in the runner (7) is lower than the binding surface of the PDMS sheet (2), and the height difference (h) is used as the height gap between the opening box dam and the glass substrate (1) after the PDMS sheet (2) is bound with the glass substrate (1).
The opening box dam is a C-shaped dam (8) provided with an opening and in a C-shaped circular ring shape, and the opening width of the C-shaped dam (8) is larger than the inner radius of the C-shaped dam (8); or the opening box dam is a square dam with an opening at one side and a square ring in the shape of a square, and the opening width of the square dam is larger than half of the side length of the inner side of the opening side of the square dam; the runner (7) is provided with an arc-shaped notch (16) at the position of the opening dam.
The dam height of the opening box dam is 50um, and the height gap between the opening box dam and the glass substrate (1) is 5 um; the internal diameter of the C-shaped dam (8) is 150um, or the side length of the inner side of the square dam is 150 um.
The flow grooves (7) are arranged on the PDMS sheet (2) in a rectangular ring shape with openings, and the sample inlet holes (5) and the sample outlet holes (6) are 2 ports with rectangular ring openings; the open dams are respectively arranged in the launders (7) on the other 3 sides of the rectangular ring.
The miniature electrode block comprises a temperature detection electrode block (3), an electric conductivity detection electrode block (13) and a pH value detection electrode block (14), and the pH value detection electrode block (14) is also provided with a reference electrode block (15) corresponding to the pH value detection electrode block.
The temperature detection electrode block (3) and the conductivity detection electrode block (13) comprise a Ti layer with the thickness of 500a sputtered on the glass substrate (1) as an adhesion layer, a Pt layer with the thickness of 1500a sputtered on the adhesion layer to form a Ti/Pt layer (17) with the thickness of 500/1500a, and a Si layer with the thickness of 5000a deposited on the Ti/Pt layer (17)3N4A layer (12) as an insulating layer; the pH value detection electrode block (14) comprises a Ti layer (18) with the thickness of 2500a sputtered on the glass substrate (1); the reference electrode block (15) comprises a layer (20) of Ag/AgCl having a thickness of 2500 a.
Each micro electrode block also comprises an ion detection electrode and an ORP detection electrode.
The bottom end of the leading-out wire (4) of each micro electrode block is positioned at the side edge of the glass substrate (1); the temperature detection electrode block (3) is in a shape that a Pt wire roundly and repeatedly turns back, two ends of the temperature detection electrode block (3) are respectively connected with a 2-side outgoing line (4), and the temperature detection electrode block (3) is located at the top end of the 2-side outgoing line (4).
In order to solve the technical problems, the manufacturing method of the cell multi-parameter detection micro-nano sensor adopts the following technical scheme:
cell multi-parameter detection micro-nano sensorThe manufacturing method of the sensor comprises the steps of manufacturing the PDMS sheet (2) and the glass substrate (1) and bonding the two, wherein the manufacturing of the PDMS sheet (2) comprises the preparation of the silicon substrate (9) as a mould and the manufacturing of the PDMS sheet (2), and the manufacturing method is characterized in that the manufacturing of the PDMS sheet (2) specifically comprises the following steps: step 1, pretreatment of a silicon substrate (9): from 98% H 2SO4、30%H2O2Mixing the components in a ratio of 7:3 to prepare piranha solution, continuously soaking the silicon substrate (9) in the piranha solution at 120 ℃ for 30min, then taking out the silicon substrate (9), repeatedly washing the silicon substrate with deionized water, putting the silicon substrate (9) in a spin dryer, and spin-drying the surface water for later use; step 2, gluing: uniformly coating the LC100A photoresist (10) on the surface of a silicon substrate (9) by using a gumming machine; step 3, photoetching: developing after exposure for 15s to obtain a required pattern; step 4, etching: etching the silicon substrate (9) downwards to a depth of 5um by using a deep reactive ion etching method; step 5, removing the photoresist: removing the LC100A photoresist (10) by using piranha solution; step 6, gluing: throwing SU8-3050 photoresist (11) on a cleaned silicon substrate (9) to form a layer with the thickness of 50um, and horizontally standing for 2 hours; and 7, photoetching: after exposure for 10s, developing and hard baking to obtain a required silicon substrate (9); and 8: pouring vacuumized PDMS on a silicon substrate (9), standing for half an hour, and then drying in a 90 ℃ oven for 1 h; step 9, demolding: cut into individual PDMS pieces (2) with a cutter for use.
The manufacturing method of the glass substrate (1) specifically comprises the following steps: step 10, sputtering: sputtering a Ti/Pt layer (17) with the thickness of 500/1500a on the whole glass substrate; step 11, gluing: uniformly coating LC100A photoresist (10) on the metal surface by using a gumming machine; step 12, photoetching: developing after exposure for 15s to obtain a required pattern; step 13, etching: etching 2000a downwards on the glass substrate by using a deep reactive ion etching method, removing the redundant Ti/Pt layer (17), and only keeping the temperature detection electrode block (3), the conductivity detection electrode block (13) and the Ti/Pt layer (17) of the electrode lead; step 14, removing the photoresist: removing the photoresist (10) of the LC100A on the surface of each electrode by using acetone; step 15, sputtering: sputtering a Ti layer (18) with a thickness of 2500a on the basis of the above pattern; Step 16, gluing: uniformly coating LC100A photoresist (10) on the metal surface by using a gumming machine; step 17, photoetching: developing after exposure for 15s to obtain a required pattern; step 18, etching: etching 2500a downwards on the glass substrate by using a deep reactive ion etching method, only keeping the Ti layer (18) on the pH value detection electrode block (14), and removing the Ti layer (18) at other positions; step 19, removing the photoresist: removing the photoresist (10) of the LC100A on the surface of the electrode by using acetone; step 20, gluing: uniformly coating LC100A photoresist (10) on the above pattern by using a gumming machine; step 21, photoetching: developing after exposure for 15s to obtain a required pattern; step 22, vacuum plating: plating an Ag layer (19) with the thickness of 2500a on the reference electrode block (15) by using a vacuum plating method, and then performing electrolytic chlorination by using an electrochemical workstation to form an Ag/AgCl layer (20); step 23, removing the photoresist: removing the photoresist by using dry etching, and removing the LC100A photoresist (10); step 24, PDCVD: depositing a layer of Si with the thickness of 5000a by a vapor deposition method3N4A layer (12) as an insulating layer; step 25, gluing: the LC100A photoresist (10) is uniformly coated on Si by using a coater3N4A surface of the layer (12); step 26, photoetching: developing after exposure for 15s to obtain a required pattern; step 27, etching: etching down to 5000a by deep reactive ion etching to remove excessive Si 3N4Layer (12), i.e. Si on top of the pH value detection electrode block (14) and the reference electrode block (15) with the lead wires removed3N4A layer (12), the remainder remaining; step 28, removing the photoresist: dry stripping is used to remove the excess LC100A photoresist (10); step 29, preparing a layer of Ag/AgCl film on the Ag layer (19) of the reference electrode block (15) by using an electrochemical constant potential deposition method to form an Ag/AgCl reference electrode; obtaining the processed glass substrate (1) for standby.
Step 30, bonding: and cleaning the processed PDMS sheet and the glass substrate together, taking out, quickly aligning and bonding under a microscope in a structure-to-structure manner, and heating on a hot plate at 105 ℃ for 2h to ensure that the PDMS sheet and the glass substrate are firmly bonded.
The cell multi-parameter detection micro-nano sensor adopts a glass substrate as a substrate material, various sensors are prepared on the surface of a glass sheet and are bonded with a PDMS sheet, and real-time online continuous monitoring of parameters such as temperature, conductivity and pH value in a micro environment is realized. The novel micro-system for monitoring the change of the components of the microenvironment is provided for cell research, and real-time data is provided for the influence of the change of the parameters of the microenvironment on the growth state of the cells. The multi-parameter detection micro-nano sensor can observe the changes of the conductivity, the temperature, the PH value and the like of cells in the states of division, death and the like in real time, and can well control the growth distribution of the cells in the micro-nano sensor through the opening box dam, thereby effectively solving the problem of disordered cell distribution of an array chip. Wherein the detection accuracy of the temperature sensor is +/-0.01 ℃, and the response time is less than or equal to 0.1 s; the detection accuracy of the conductivity sensor is +/-0.0005S/m, and the response time is less than or equal to 0.1S; the detection range of the pH sensor is 1-14, and the detection accuracy is +/-0.005 pH.
The cell multi-parameter detection micro-nano sensor can continuously monitor the change of parameters such as cell temperature, conductivity, pH value and the like on line in real time on the premise of not influencing the normal physiological function of cells. The device is mainly formed by bonding two parts, namely an electrode layer on a glass sheet and a PDMS sheet with a flow groove. The former uses photolithography, sputtering, and wet stripping processes, and the latter uses photolithography, Deep Reactive Ion Etching (DRIE), SU8 photoresist lithography processes. Although the photolithography process is the same, the two processes are obviously different. To facilitate observation of cell morphology under a microscope, the electrode was partially sputtered onto a glass slide and a layer of Si was applied to the Pt electrode3N4And the electrode is prevented from conducting electricity after being contacted with water. And scribing the glass sheet after the above steps are finished. After a needed structure is photoetched on a silicon chip, a pattern structure on a silicon chip mould is transferred to PDMS through an epoxy glue mould-reversing process, a needed micro-fluidic chip channel appears on a PDMS film at the moment, and the solidified PDMS film is cut and punched by a cutter. And finally, carrying out plasma cleaning on the processed PDMS chip and the scribed electrode chip, quickly aligning and bonding under a microscope after cleaning, and then placing on a hot plate at 105 ℃ for heating for 2h to ensure that the PDMS chip and the scribed electrode chip are firmly bonded.
The diameter of the cell is larger than the height of the channel under the structure of the C-shaped dam or the square dam, so the cell can be retained in the opening dam, and each micro-electrode block is positioned below the area occupied by the opening dam, so the micro-fluidic chip can accurately detect the parameter change conditions of temperature, conductivity, pH value and the like in the growth and proliferation processes of the adhered cell, and can detect the parameter change conditions of temperature, conductivity, pH value and the like of the cell in normal physiological activities (metabolism, division, apoptosis and the like) and under the stimulation of drugs in real time. In addition, the cell multi-parameter detection micro-nano sensor can be manufactured in batch, has the characteristics of low cost, high detection sensitivity and the like, and has important practical application value.
Drawings
FIG. 1 is a three-dimensional schematic diagram of the appearance of a cell multi-parameter detection micro-nano sensor.
FIG. 2 is a schematic perspective view of a cell multiparameter detection micro-nano sensor PDMS sheet and a glass substrate in a separated state.
FIG. 3 is a schematic perspective view of a PDMS sheet.
Fig. 4 is a perspective view of a glass substrate.
Fig. 5 is an enlarged view of a portion a of fig. 3.
Fig. 6 is an enlarged view of a portion B of fig. 4.
FIG. 7 is a schematic top view of a C-dam.
FIG. 8 is a schematic top view of a square dam.
FIG. 9 is a schematic diagram showing the cross-sectional shape change of each process in the process of preparing PDMS sheet.
FIG. 10 is a schematic view showing changes in the cross-sectional shapes of the respective processes in the process of manufacturing a glass substrate.
Note: fig. 9 and 10 are schematic and do not correspond to the actual cross-sectional shape of the PDMS sheet or glass substrate.
Detailed Description
The invention is described in further detail below with reference to the accompanying examples.
The cell multi-parameter detection micro-nano sensor disclosed by the invention comprises a glass substrate 1 and a PDMS sheet 2 which are mutually attached as shown in figure 1. As shown in fig. 2, the bonding surface of the PDMS sheet 2 is a grooved structure surface, the bonding surface of the PDMS sheet 2 is provided with a flow channel 7, a flow channel for flowing through of cell solutions during detection is formed on the glass substrate 1 after bonding with the glass substrate 1, and two ends of the flow channel 7 are respectively provided with through holes penetrating through the PDMS sheet 2 to form a sample inlet hole 5 and a sample outlet hole 6. As shown in fig. 3, an opening dam for flowing and retaining the cell solution is disposed in the flow cell 7, and an opening of the opening dam faces the injection direction. As shown in fig. 2, more than 2 micro electrode blocks for detecting and sensing different parameters are arranged on the binding surface of the glass substrate 1 at the corresponding positions of the opening dam, so that each micro electrode block is located below the area occupied by the opening dam, a height gap for sample fluid and air to flow through during detection is reserved between the opening dam and the glass substrate 1, and the height gap is smaller than the outer diameter of the cell to be detected. As shown in fig. 2 and 4, a lead wire 4 made of Pt is provided for each micro-electrode block. Since the vast majority of cells are 10-20um in diameter, this height gap can be set to 5 um.
As shown in fig. 5 and 7, the open box dam is a C-shaped annular C-shaped dam 8 provided with an opening, and the opening width of the C-shaped dam 8 is greater than the inner radius of the C-shaped dam 8. Alternatively, as shown in fig. 8, the opening box dam is a square dam with an opening at one side and a square ring, and the opening width of the square dam is greater than half of the side length of the inner side of the opening side of the square dam. As shown in fig. 3 and 5, the runner 7 may be provided with a circular arc-shaped recess 16 at the position of the opening dam. Because most cells have the diameter of 10-20um, the detected cells cannot be limited in the C-shaped ring through the gap, so that the special C-shaped structure can fix the cells on the microelectrode, and the existence of the gap with the height of 5um between the opening dam and the glass substrate 1 does not influence the circulation of air and liquid, so that the survival of the detected single cells is not influenced. The same is true of the square dam. As shown in fig. 3, the width of the flow cell 7 should be at least slightly larger than the outer diameter of the opening box dam, and a circular arc-shaped notch 16 may be formed at the position of the opening box dam of the flow cell 7 to facilitate the cell solution to be measured to flow forward.
The height gap between the above-mentioned opening dam and the glass substrate 1 can be realized by: as shown in fig. 3 and 5, the height of the opening dam in the flow channel 7 is lower than the bonding surface 5um of the PDMS sheet 2, and the height difference h is used as the height gap between the opening dam and the glass substrate 1 after the PDMS sheet 2 is bonded to the glass substrate 1. The dam height of the opening box dam is 50um, and the height gap between the opening box dam and the glass substrate 1 is 5 um; the internal diameter of the C-shaped dam 8 is 150um, or the side length of the inner side of the square dam is 150 um.
As shown in fig. 3, the flow cell 7 is arranged on the PDMS sheet 2 in a rectangular ring shape with openings, and the sample inlet hole 5 and the sample outlet hole 6 are 2 ports with rectangular ring openings; open dams are respectively arranged in the launders 7 on the other 3 sides of the rectangular ring, namely 3 open dams are arranged along the launders 7. And the glass substrate 1 below the 3 opening box dams is respectively provided with each micro electrode block. Cell solution flows in the flow groove 7, when the cell solution flows through the 3 opening dams, the cells to be detected are respectively captured by the 3 opening dams, and the cells to be detected are received in the respective opening dams for detection and sensing of the micro electrode blocks below.
As shown in fig. 4 and 6, in the embodiment shown in the figures, the micro electrode block includes a temperature detection electrode block 3, a conductivity detection electrode block 13, a pH value detection electrode block 14, and the pH value detection electrode block 14 is further provided with a reference electrode block 15 corresponding to the pH value detection electrode block 14. The temperature detection electrode block 3 and the conductivity detection electrode block 13 comprise a Ti layer with the thickness of 500a sputtered on the glass substrate 1 as an adhesion layer, a Pt layer with the thickness of 1500a sputtered on the adhesion layer to form a Ti/Pt layer 17 with the thickness of 500/1500a, and a Si layer with the thickness of 5000a deposited on the Ti/Pt layer 173N4A layer 12 as an insulating layer; the pH value detection electrode block 14 comprises a Ti layer 18 with the thickness of 2500a sputtered on the glass substrate 1; the reference electrode block 15 comprises a layer 20 of Ag/AgCl with a thickness of 2500 a.
The micro electrode blocks are provided with an ion detection electrode and an ORP detection electrode which can be added or converted according to requirements, in addition to the temperature detection electrode block 3, the conductivity detection electrode block 13 and the pH value detection electrode block 14 in the embodiment shown in the figure.
As shown in fig. 4 and 6, the bottom end of the lead-out wire 4 of each micro electrode block is positioned at the side edge of the glass substrate 1; the temperature detection electrode block 3 is in a shape that a Pt wire roundly turns back and forth, two ends of the temperature detection electrode block 3 are respectively connected with 2 side outgoing lines 4 of the temperature detection electrode block, and the temperature detection electrode block 3 is located at the top end of the 2 side outgoing lines 4.
The method for manufacturing the cell multi-parameter detection micro-nano sensor comprises the steps of manufacturing the PDMS sheet 2 and the glass substrate 1 in sequence and bonding the two, wherein the manufacturing of the PDMS sheet 2 comprises the steps of preparing the silicon substrate 9 serving as a mould of the PDMS sheet and manufacturing the PDMS sheet 2.
As shown in fig. 9, the preparation of the PDMS slab 2 specifically includes the following steps:
And 7, photoetching: after exposure for 10s, developing and hard-baking to obtain the required silicon substrate 9; see fig. 9-7.
And 8: pouring vacuumized PDMS on the silicon substrate 9, see FIGS. 9-8; standing for half an hour, and oven drying at 90 deg.C for 1 hr.
Step 9, demolding: cutting the PDMS into single PDMS pieces 2 for later use by a cutter; see fig. 9-9.
As shown in fig. 10, the manufacturing of the glass substrate 1 specifically includes the following steps:
Step 11, gluing: uniformly coating LC100A photoresist 10 on the metal surface by using a gumming machine; see fig. 10-3.
Step 21, photoetching: developing after exposure for 15s to obtain a required pattern; see fig. 10-13.
Step 22, vacuum plating: plating an Ag layer 19 with the thickness of 2500a on the reference electrode block 15 by using a vacuum plating method, and then performing electrolytic chlorination by using an electrochemical workstation to form an Ag/AgCl layer 20; see fig. 10-14.
Step 23, removing the photoresist: removing the photoresist by using dry etching, and removing the LC100A photoresist 10; see fig. 10-15.
Step 24, PDCVD: depositing a layer of Si with the thickness of 5000a by a vapor deposition method3N4A layer 12 as an insulating layer; see fig. 10-16.
Step 25, gluing: the LC100A photoresist 10 was uniformly coated on Si using a coater3N4The surface of layer 12; see fig. 10-17.
Step 26, photoetching: developing after exposure for 15s to obtain a required pattern; see fig. 10-18.
Step 27, etching: etching down to 5000a by deep reactive ion etching to remove excessive Si3N4Layer 12, i.e. Si on top of the lead-out wire, pH value detection electrode block 14, reference electrode block 153N4Layer 12, the remainder remaining; see fig. 10-19.
Step 28, removing the photoresist: removing the redundant LC100A photoresist 10 by using dry photoresist removal to obtain a glass substrate 1 for later use; see fig. 10-20.
Step 29, preparing a layer of Ag/AgCl film on the Ag layer 19 of the reference electrode block 15 by using an electrochemical constant potential deposition method to form an Ag/AgCl reference electrode; see fig. 10-21. The processed glass substrate 1 is obtained for use.
Step 30, bonding: and cleaning the processed PDMS sheet and the glass substrate together, taking out, quickly aligning and bonding under a microscope in a structure-to-structure manner, and heating on a hot plate at 105 ℃ for 2h to ensure that the PDMS sheet and the glass substrate are firmly bonded.
Step 31, surface mounting: and adhering the bonded chip on a PCB (printed Circuit Board), namely, pasting the chip on the PCB, and adopting gold wire ball bonding for routing.
Step 32, encapsulation: and manually welding the rear end lead, and sealing all electrode lead interfaces by using AB glue to finish the preparation of the whole device.
The manufacturing method of the cell multi-parameter detection micro-nano sensor aims at the temperature, conductivity and pH value 3 parameter detection micro-nano sensor provided with the temperature detection electrode block 3, the conductivity detection electrode block 13 and the pH value detection electrode block 14. The multi-parameter cell detection micro-nano sensor comprising other parameters can be adjusted according to the manufacturing method.
Claims (10)
1. A cell multi-parameter detection micro-nano sensor comprises a glass substrate (1) and a PDMS (polydimethylsiloxane) sheet (2) which are mutually attached, wherein the attaching surface of the PDMS sheet (2) is a slotted structure surface, a runner (7) is arranged on the attaching surface of the PDMS sheet (2), a runner for cell solution to flow through during detection is formed on the glass substrate (1) after the PDMS sheet is attached to the glass substrate (1), through holes penetrating through the PDMS sheet (2) are respectively arranged at two ends of the runner (7), and a sample inlet hole (5) and a sample outlet hole (6) are formed; the cell flow groove is characterized in that an opening box dam for flowing and retaining a cell solution is arranged in the flow groove (7), and an opening of the opening box dam faces to the direction of sample injection; more than 2 micro electrode blocks which are respectively used for detecting and sensing different parameters are arranged on the binding surface of the glass substrate (1) at the corresponding position of the opening dam, so that each micro electrode block is positioned below the area occupied by the opening dam, a height gap for sample fluid and air to flow through during detection is reserved between the opening dam and the glass substrate (1), and the height gap is smaller than the outer diameter of the cell to be detected; lead wires (4) made of Pt are respectively arranged on the micro electrode blocks.
2. The cell multiparameter detection micro-nano sensor according to claim 1, wherein the height of the opening box dam in the flow cell (7) is lower than the bonding surface of the PDMS sheet (2), and the height difference (h) is used as the height gap between the opening box dam after the PDMS sheet (2) is bonded to the glass substrate (1) and the glass substrate (1).
3. The cell multiparameter detection micro-nano sensor according to claim 1, wherein the open box dam is a C-shaped dam (8) having a C-shaped circular ring and provided with an opening; or the opening box dam is a square dam with an opening at one side and a square ring in the shape of a square.
4. The cell multiparameter detection micro-nano sensor according to claim 1, wherein the flow cell (7) is provided with a circular arc-shaped notch (16) at the position of the opening box dam.
5. The cell multiparameter detection micro-nano sensor according to claim 1, wherein the flow cell (7) is arranged in a rectangular ring shape with openings on the PDMS sheet (2), and the sample inlet hole (5) and the sample outlet hole (6) are 2 ports with rectangular ring openings; the open dams are respectively arranged in the launders (7) on the other 3 sides of the rectangular ring.
6. The cell multiparameter detection micro-nano sensor according to claim 1, wherein the micro electrode block comprises a temperature detection electrode block (3), an electric conductivity detection electrode block (13), and a pH value detection electrode block (14), and the pH value detection electrode block (14) is further provided with a reference electrode block (15) corresponding thereto.
7. The micro-nano sensor for cell multiparameter detection according to claim 6, wherein the temperature detection electrode block (3) and the conductivity detection electrode block (13) comprise a Ti layer with a thickness of 500a sputtered on the glass substrate (1) as an adhesion layer, a Ti/Pt layer (17) with a thickness of 500/1500a sputtered on the adhesion layer, and a Si layer with a thickness of 5000a deposited on the Ti/Pt layer (17)3N4A layer (12) as an insulating layer; the pH value detection electrode block (14) comprises a Ti layer (18) with the thickness of 2500a sputtered on the glass substrate (1); the reference electrode block (15) comprises a layer (20) of Ag/AgCl having a thickness of 2500 a.
8. The cell multiparameter detection micro-nano sensor according to claim 6, wherein each micro electrode block further comprises an ion detection electrode and an ORP detection electrode.
9. The cell multiparameter detection micro-nano sensor according to claim 1, wherein the bottom end of the outgoing line (4) of each micro electrode block is positioned at the side edge of the glass substrate (1); the temperature detection electrode block (3) is in a shape that a Pt wire roundly and repeatedly turns back, two ends of the temperature detection electrode block (3) are respectively connected with a 2-side outgoing line (4), and the temperature detection electrode block (3) is located at the top end of the 2-side outgoing line (4).
10. A method for manufacturing a cell multi-parameter detection micro-nano sensor comprises the steps of manufacturing a PDMS sheet (2) and a glass substrate (1) respectively in no sequence, and bonding the two, wherein the manufacturing of the PDMS sheet (2) firstly comprises the preparation of a silicon substrate (9) serving as a mould of the PDMS sheet and the manufacturing of the PDMS sheet (2), and is characterized in that the manufacturing of the PDMS sheet (2) specifically comprises the following steps: step 1, pretreatment of a silicon substrate (9): from 98% H2SO4、30%H2O2Mixing the components in a ratio of 7:3 to prepare piranha solution, continuously soaking the silicon substrate (9) in the piranha solution at 120 ℃ for 30min, then taking out the silicon substrate (9), repeatedly washing the silicon substrate with deionized water, putting the silicon substrate (9) in a spin dryer, and spin-drying the surface water for later use; step 2, gluing: uniformly coating the LC100A photoresist (10) on the surface of a silicon substrate (9) by using a gumming machine; step 3, photoetching: developing after exposure for 15s to obtain a required pattern; step 4, etching: etching the silicon substrate (9) downwards to a depth of 5um by using a deep reactive ion etching method; step 5, removing the photoresist: removing the LC100A photoresist (10) by using piranha solution; step 6, gluing: throwing SU8-3050 photoresist (11) on a cleaned silicon substrate (9) to form a layer with the thickness of 50um, and horizontally standing for 2 hours; and 7, photoetching: after exposure for 10s, developing and hard baking to obtain a required silicon substrate (9); and 8: pouring vacuumized PDMS on a silicon substrate (9), standing for half an hour, and then drying in a 90 ℃ oven for 1 h; step 9, demolding: cutting the PDMS into single PDMS pieces (2) for later use by a cutter;
The manufacturing method of the glass substrate (1) specifically comprises the following steps: step 10, sputtering: sputtering a Ti/Pt layer (17) with the thickness of 500/1500a on the whole glass substrate; step 11, gluing: uniformly coating LC100A photoresist (10) on the metal surface by using a gumming machine; step 12, photoetching: developing after exposure for 15s to obtain a required pattern; step 13, etching: etching 2000a downwards on the glass substrate by using a deep reactive ion etching method, removing the redundant Ti/Pt layer (17), and only keeping the temperature detection electrode block (3), the conductivity detection electrode block (13) and the Ti/Pt layer (17) of the electrode lead; step 14, removing the photoresist: removing the photoresist (10) of the LC100A on the surface of each electrode by using acetone;step 15, sputtering: sputtering a Ti layer (18) with a thickness of 2500a on the basis of the above pattern; step 16, gluing: uniformly coating LC100A photoresist (10) on the metal surface by using a gumming machine; step 17, photoetching: developing after exposure for 15s to obtain a required pattern; step 18, etching: etching 2500a downwards on the glass substrate by using a deep reactive ion etching method, only keeping the Ti layer (18) on the pH value detection electrode block (14), and removing the Ti layer (18) at other positions; step 19, removing the photoresist: removing the photoresist (10) of the LC100A on the surface of the electrode by using acetone; step 20, gluing: uniformly coating LC100A photoresist (10) on the above pattern by using a gumming machine; step 21, photoetching: developing after exposure for 15s to obtain a required pattern; step 22, vacuum plating: plating an Ag layer (19) with the thickness of 2500a on the reference electrode block (15) by using a vacuum plating method, and then performing electrolytic chlorination by using an electrochemical workstation to form an Ag/AgCl layer (20); step 23, removing the photoresist: removing the photoresist by using dry etching, and removing the LC100A photoresist (10); step 24, PDCVD: depositing a layer of Si with the thickness of 5000a by a vapor deposition method 3N4A layer (12) as an insulating layer; step 25, gluing: the LC100A photoresist (10) is uniformly coated on Si by using a coater3N4A surface of the layer (12); step 26, photoetching: developing after exposure for 15s to obtain a required pattern; step 27, etching: etching down to 5000a by deep reactive ion etching to remove excessive Si3N4Layer (12), i.e. Si on top of the pH value detection electrode block (14) and the reference electrode block (15) with the lead wires removed3N4A layer (12), the remainder remaining; step 28, removing the photoresist: dry stripping is used to remove the excess LC100A photoresist (10); step 29, preparing a layer of Ag/AgCl film on the Ag layer (19) of the reference electrode block (15) by using an electrochemical constant potential deposition method to form an Ag/AgCl reference electrode; obtaining a processed glass substrate (1) for later use;
step 30, bonding: and cleaning the processed PDMS sheet and the glass substrate together, taking out, quickly aligning and bonding under a microscope in a structure-to-structure manner, and heating on a hot plate at 105 ℃ for 2h to ensure that the PDMS sheet and the glass substrate are firmly bonded.
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