CN112378972B

CN112378972B - Non-mark biosensor and manufacturing method thereof

Info

Publication number: CN112378972B
Application number: CN202011062164.9A
Authority: CN
Inventors: 冀健龙; 田海平; 王靖宵; 张强; 李强; 桑胜波
Original assignee: Taiyuan University of Technology
Current assignee: Taiyuan University of Technology
Priority date: 2020-09-30
Filing date: 2020-09-30
Publication date: 2023-01-10
Anticipated expiration: 2040-09-30
Also published as: CN112378972A

Abstract

The invention provides an unidentified biosensor and a manufacturing method thereof, belonging to the technical field of biosensors; the technical problem to be solved is as follows: an improvement of a non-marking biosensor structure and a manufacturing method thereof are provided; the technical scheme for solving the technical problem is as follows: the sensor chip assembly comprises an amplifying chip assembly and a sensing chip assembly, wherein at least one amplifying chip is arranged in the amplifying chip assembly, at least one sensing chip is arranged in the sensing chip assembly, and the amplifying chip is connected with the sensing chip through a bridging structure; the amplifying chip comprises an organic semiconductor film, a bridging medium and a first micro-electrode group, wherein the first micro-electrode group comprises a substrate, a source electrode and a drain electrode, a current carrier running channel between the source electrode and the drain electrode is arranged in parallel to the plane of the substrate, and the first micro-electrode group is horizontally connected with the organic semiconductor film through an electrode; the invention is applied to biosensors.

Description

Non-mark biosensor and manufacturing method thereof

Technical Field

The invention provides an unmarked biosensor and a manufacturing method thereof, belonging to the technical field of biosensors.

Background

Organic electrochemical transistors are widely used for label-free biosensing, such as detection of dopamine, epinephrine, ascorbic acid, adenosine triphosphate, cell activity and the like. Organic electrochemical transistors are generally prepared by a solvation method, i.e., an organic semiconductor polymer is dissolved in a solvent, and then spin-coated and dried. And finally, patterning the polymer film through a photoetching process to realize the preparation of the channel layer. In the manufacturing process, water is often used as a solvent in order to reduce environmental pollution and toxicity to the operator. Therefore, hydrophilization of polymers is often beneficial to improve the efficiency and accuracy of the manufacturing process.

Biological samples to be tested, such as DNA, protein, cells, etc., at different levels generally need to be stored in an aqueous environment. Therefore, the sensing process also needs to be performed in a solution environment rich in water molecules. At this time, the hydrophilic characteristic for improving the processability of the polymer becomes a negative factor affecting the stability of the device. Specifically, the interaction between the hydrophilic polymer and water molecules can cause the organic semiconductor layer to have structural damage phenomena such as chapping, desorption and the like.

Furthermore, on the micrometer scale, liquids exhibit laminar flow, while biological fluids, such as blood, generally have a greater viscosity. Therefore, the biosensor based on the planar microelectrode is often lack of an effective convective mass transfer means, so that the biochemical reaction speed is low and the detection time is long. In addition, for a cell sample to be tested, it is often necessary to move the cells to a specific position and perform adherent culture and observation. The process depends on efficient manipulation and accurate positioning of cells, especially at the level of single cells.

In order to solve the above problems, the present invention provides a biosensor. The sensor separates the polymer hydrophilic phase from the aqueous solution, thereby improving the stability of the device. A plurality of groups of microelectrode pairs are introduced around the measuring electrodes of the sensing chip and used for inducing and generating electrokinetic convection (such as alternating current electroosmosis and alternating current electrothermal) and dielectrophoresis force. On the one hand, the detection efficiency of the molecular biosensor can be improved. On the other hand, the positioning accuracy of the cell biosensor can be improved. In addition, the chip utilizes the potential change generated by biochemical reaction to carry out sensing detection, thereby being a label-free biosensor with aqueous solution stability.

Disclosure of Invention

In order to overcome the defects in the prior art, the invention aims to solve the technical problems that: improvements in a non-identifying biosensor structure and method of making the same are provided.

In order to solve the technical problems, the invention adopts the technical scheme that: a non-mark biosensor comprises an amplification chip assembly and a sensing chip assembly, wherein at least one amplification chip is arranged in the amplification chip assembly, at least one sensing chip is arranged in the sensing chip assembly, and the amplification chip is connected with the sensing chip through a bridging structure;

the amplifying chip comprises an organic semiconductor film, a bridging medium and a first micro-electrode group, wherein the first micro-electrode group comprises a substrate, a source electrode and a drain electrode, a current carrier running channel between the source electrode and the drain electrode is arranged in parallel to the plane of the substrate, and the first micro-electrode group is horizontally connected with the organic semiconductor film through an electrode;

the sensing chip comprises a second micro-electrode group for improving the control efficiency and precision of the biological sample to be detected.

The first micro-electrode group comprises: the amplification chip comprises an amplification chip substrate, a first lead layer, a first insulating layer, a first through hole and a first microelectrode layer, wherein the first lead layer is arranged on the upper side of the amplification chip substrate, the first insulating layer covers the first lead layer and the upper side of the amplification chip substrate, and the first microelectrode layer covers the upper side of the first insulating layer;

the first lead layer comprises electrode leads which are not connected with each other, and the first lead layer comprises a voltage-controlled electrode lead, a source electrode lead and a drain electrode lead;

the first micro-electrode layer includes: the voltage-controlled electrode, the source electrode, the drain electrode, the voltage-controlled electrode PAD electrode, the source PAD electrode and the drain PAD electrode;

the voltage-controlled electrode is connected with a voltage-controlled electrode PAD electrode through a voltage-controlled electrode lead which passes through the first through hole;

the source electrode is connected with the source PAD electrode through a source electrode lead penetrating through the first through hole;

the drain electrode is connected with a drain electrode PAD electrode through a drain electrode lead penetrating through the first through hole;

an organic semiconductor film is arranged on the upper side of the first micro electrode layer, and the organic semiconductor film is only connected with a source electrode and a drain electrode;

the bridging medium covers the voltage-controlled electrode and the upper side of the organic semiconductor film, the voltage-controlled electrode is connected with the organic semiconductor film through the bridging medium, the bridging medium is in contact with the upper surface of the organic semiconductor film, the bridging medium is in contact with the upper surface and/or the side surface of the voltage-controlled electrode, and the bridging medium is not in contact with the source electrode and the drain electrode.

The sensing chip comprises: the sensing chip comprises a sensing chip substrate, a second lead layer, a second insulating layer, a second through hole and a second microelectrode layer, wherein the second lead layer is arranged on the upper side of the sensing chip substrate, the second insulating layer covers the second lead layer and the upper side of the sensing chip substrate, the second microelectrode layer covers the upper side of the second insulating layer, and the second through hole is formed in the second insulating layer;

the second microelectrode layer comprises a measuring electrode, a measuring PAD electrode, a pressure supply electrode and a pressure supply PAD electrode;

the second wire layer includes electrode wires that are not connected to each other, and the second wire layer includes: a measuring electrode lead and a voltage supply electrode lead;

the measuring electrode is connected with the measuring PAD electrode through a measuring electrode lead which passes through the second through hole;

and the pressure supply electrode is connected with the pressure supply PAD electrode through a pressure supply electrode lead which passes through the second through hole.

The second micro electrode layer further comprises at least one pair of convection electrodes and a pair of convection PAD electrodes, and the second lead layer further comprises a convection electrode lead;

the convection electrode is connected with the convection PAD electrode through a convection electrode lead which passes through the second through hole;

the convection electrodes are uniformly arranged around the measuring electrode.

A voltage-controlled electrode PAD electrode arranged in the amplification chip is connected with a measurement PAD electrode arranged in the sensing chip through a bridging structure;

the bridging medium is a liquid, solid, or gel having ionic conductivity.

A method for manufacturing a non-identification biosensor is characterized in that: the method comprises the following steps:

the method comprises the following steps: preparing an amplifying chip by using an MEMS (micro-electromechanical systems) process;

step two: preparing an organic semiconductor film between a source electrode and a drain electrode of the first micro-electrode group, and covering the organic semiconductor film and the voltage-controlled electrode with a bridging medium to form an amplification chip;

step three: preparing a sensing chip by using an MEMS (micro-electromechanical systems) process;

step four: and the voltage-controlled electrode PAD electrode of the amplification chip is connected with the measurement PAD electrode of the sensing chip through the bridging structure.

The specific process for preparing the amplification chip by using the MEMS process in the first step comprises the following steps:

selecting an amplification chip substrate made of an insulating material, and processing a first electrode layer on the surface of the amplification chip substrate by utilizing photoetching and lift-off or photoetching and corrosion processes to realize patterning of a voltage-controlled electrode, a source electrode, a voltage-controlled electrode lead and a source electrode lead on the first electrode layer;

depositing a first insulating layer and patterning, specifically growing a silicon dioxide insulating layer with the thickness of 100-500nm on a substrate by adopting a vapor deposition method, photoetching and developing, and corroding the first insulating layer by using a dry etching method or a wet etching method so as to leak out an electrode window;

depositing and patterning the first microelectrode layer to form a voltage-controlled electrode, a source electrode, a drain electrode, a voltage-controlled electrode PAD electrode, a source PAD electrode and a drain PAD electrode, and ensuring that no physical contact exists between the source electrode and the drain electrode.

The specific process for preparing the sensing chip in the third step is as follows:

selecting a sensing chip substrate made of insulating materials, and processing a second lead layer on the surface of the sensing chip substrate by utilizing photoetching and lift-off or photoetching and corrosion processes to realize patterning of a measuring electrode lead, a voltage supply electrode lead and a convection electrode lead;

depositing a second insulating layer and patterning, specifically growing a silicon dioxide insulating layer with the thickness of 100-500nm on the substrate by adopting a vapor deposition method, exposing by using a photoetching plate, and leaking out of the electrode window by dry etching or wet etching;

depositing a second microelectrode layer and patterning to form a measuring electrode, a pressure supply electrode, a convection electrode and a measuring PAD electrode.

Compared with the prior art, the invention has the following beneficial effects:

1. according to the invention, the amplification chip is separated from the sensing chip, so that the hydrophilic polymer of the amplification chip is not in direct contact with the aqueous solution of the biological sample to be detected, and the stability of the biosensor is improved;

2. the biochemical reaction signal in the sensing chip is transmitted to the amplifying chip of the sensor in the form of potential change. The optical component is not involved, and the pointer molecule (such as a fluorescent group) label is not needed, so the method is a low-cost and label-free sensing method;

3. the sensing part of the biosensing chip is provided with the positioning electrode, so that a biological sample (nucleotide sequence, protein, cell and the like) can be controlled to be positioned on the measuring electrode more quickly and accurately, and the testing efficiency and precision are improved;

4. the amplification chip and the sensing chip are both in an array structure, and can be used for testing the flux of cell biosensing.

Drawings

The invention is described in further detail below with reference to the accompanying drawings:

FIG. 1 is a general structural diagram of an unidentified biochip with high stability according to the first, second and fourth embodiments of the present invention;

FIG. 2 is a schematic diagram of an overall structure of a non-labeled biochip with high stability according to a third embodiment of the present invention;

FIG. 3 is a structural diagram of an amplification chip 4 in an unidentified biochip with high stability according to an embodiment of the invention;

FIG. 4 is a structural diagram of a sensor chip 5 in a non-labeled biochip with high stability according to an embodiment of the present invention;

FIG. 5 is a structural diagram of a sensor chip 5 in a non-labeled biochip with high stability according to the second and fourth embodiments of the present invention;

FIG. 6 is a structural diagram of a sensor chip 5 in a non-labeled biochip with high stability according to a third embodiment of the present invention;

fig. 7 to 10 are flow charts of the manufacturing process for the amplification chip 4 according to the present invention;

fig. 7 is a process diagram of a first wiring layer 42 of an amplifying chip 4 prepared by a MEMS process according to an embodiment of the present invention;

fig. 8 is a process diagram of the first insulating layer 43 of the enlarged chip 4 having the first via hole 44 prepared in the example of the present invention;

FIG. 9 is a process diagram of the first microelectrode layer 45 in the amplifying chip 4 prepared by the MEMS process according to the embodiment of the present invention;

FIG. 10 is a process diagram of an organic semiconductor film 6 and a bridging medium 7 employed in an embodiment of the present invention;

fig. 11 to 13 are flow charts of a manufacturing process for a sensor chip according to an embodiment of the present invention;

fig. 11 is a process diagram of a second conductive line layer 52 of the sensor chip 5 manufactured by the MEMS process according to the embodiment of the present invention;

FIG. 12 is a process diagram of a second insulating layer 53 of a sensor chip 5 having a second via 54, prepared in accordance with an embodiment of the present invention;

FIG. 13 is a process diagram of a second microelectrode layer 55 in a sensor chip 5 fabricated by MEMS process according to an embodiment of the present invention;

FIG. 14 is a schematic structural diagram of a molecular recognition biosensor system according to the second, third and fourth embodiments of the present invention;

in the figure: 1 is an amplifying chip component, 2 is a sensing chip component, 3 is a bridging structure, 4 is an amplifying chip, 5 is a sensing chip, 6 is an organic semiconductor film, 7 is a bridging medium, and 8 is a flexible substrate material;

41 is an amplifying chip substrate, 42 is a first lead layer, 43 is a first insulating layer, 44 is a first through hole, and 45 is a first microelectrode layer;

421 is a voltage-controlled electrode wire, 422 is a source wire, 423 is a drain wire;

451 is a voltage-controlled electrode, 452 is a source, 453 is a drain, 454 is a voltage-controlled electrode PAD electrode, 455 is a source PAD electrode, 456 is a drain PAD electrode;

51 is a sensing chip substrate, 52 is a second lead layer, 53 is a second insulating layer, 54 is a second through hole, and 55 is a second microelectrode layer;

551 is a measuring electrode, 552 is a voltage supply electrode, 553 is a convection electrode, 554 is a measuring PAD electrode, 555 is a voltage supply PAD electrode, 556 is a convection PAD electrode;

521 is a measuring electrode lead and 522 is a voltage supply electrode lead; 523 is a counter electrode lead.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described below in detail and completely with reference to the drawings in the embodiments of the present invention, and it is obvious that the embodiments are some embodiments of the present invention, but not all embodiments; all other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.

Example one

Structural description:

fig. 1 is a schematic structural diagram of a biosensor according to a first embodiment of the present invention. The biosensor comprises an amplification chip component 1 and a sensing chip component 2, wherein the amplification chip component 1 is connected with the sensing chip component 2 through five bridging structures 3, five amplification chips 4 are arranged in the amplification chip component 1, each amplification chip 4 comprises a micro-electrode group, an organic semiconductor film 6 and a bridging medium 7, five sensing chips 5 are arranged in the sensing chip component 2, and each sensing chip 5 consists of a micro-electrode.

Specifically, the bridge structure 3 is a lead made of gold wire, the organic semiconductor film 6 is specifically a conductive polymer (PEDOT: PSS) formed of poly (3,4-ethylenedioxythiophene) and styrene sulfonate, and the bridging medium 7 is specifically an ionic liquid formulated of 1-ethyl-3-methylimidazolium trifluoroacetate (EMIMT/TfA).

FIG. 3 is a schematic structural diagram of an amplification chip 4 in the biosensor of the present invention. The amplification chip 4 includes: the structure comprises an amplification chip substrate 41, a first lead layer 42, a first insulating layer 43, a first through hole 44, a first micro electrode layer 45, an organic semiconductor film 6 and a bridging medium 7, wherein the first lead layer 42 is positioned on the amplification chip substrate 41, the first insulating layer 43 covers the first lead layer 42 and the upper surface of the amplification chip substrate 41 at the same time, and the first micro electrode layer 45 is positioned on the first insulating layer 43.

The source 452, the drain 453, the voltage-controlled electrode PAD electrode 454, the source PAD electrode 455, and the drain PAD electrode 456 included in the first micro electrode layer 45 are all disposed on the upper surface of the first insulating layer 43, and the voltage-controlled electrode wire 421, the source wire 422, and the drain wire 423 included in the first wire layer 42 are disposed on the upper surface of the amplification chip substrate 41;

specifically, the amplification chip substrate 41 uses quartz glass as a base material, the first insulating layer 43 is silicon dioxide, and the first wire layer 42 and the first microelectrode layer 45 are gold.

The first conductive line layer 42 is composed of a voltage-controlled electrode line 421, a source line 422, and a drain line 423, which are not connected to each other.

The first micro electrode layer 45 is composed of a voltage-controlled electrode 451 for voltage input, a pair of source 452 and drain 453 for current output, a voltage-controlled electrode PAD electrode 454, a source PAD electrode 455, and a drain PAD electrode 456.

Specifically, the voltage-controlled electrode 451, the voltage-controlled electrode PAD electrode 454 are connected to the voltage-controlled electrode wire 421 through the first via hole 44, and the source electrode 452, the source electrode PAD electrode 455 are connected to the source wire 422 through the first via hole 44; the drain 453 and the drain PAD electrode 456 are connected to the drain wire 423 through the first via hole 44.

Specifically, the voltage-controlled electrode 451, the source 452 and the drain 453 of the present embodiment have an area of 400 μm ² The source electrode 452 and the drain electrode 453 are tip electrode pairs, the distance between the electrode pairs is set to 10 μm, and the voltage-controlled electrode PAD electrode 454, the source PAD electrode 455 and the drain PAD electrode 456 are all 4mm ² The rectangular electrode of (1).

An organic semiconductor film 6 is provided on the upper side of the first micro electrode layer 45, the organic semiconductor film 6 is to be connected only to the source electrode 452 and the drain electrode 453, the bridging medium 7 covers the upper surfaces of the organic semiconductor film 6 and the voltage control electrode 451, and the voltage control electrode 451 is connected to the organic semiconductor film 6 through the bridging medium 7.

Fig. 4 is a schematic structural diagram of the sensor chip 5 of the present invention, where the sensor chip 5 includes: the sensor chip comprises a sensing chip substrate 51, a second lead layer 52, a second insulating layer 53, a second through hole 54 and a second microelectrode layer 55, wherein the second lead layer 52 is located on the sensing chip substrate 51, the second insulating layer 53 covers the second lead layer 52 and the upper surface of the sensing chip substrate 51, and the second microelectrode layer 55 is located on the second insulating layer 53.

Specifically, the substrate 51 of the sensor chip uses quartz glass as a base material, the second insulating layer 53 uses silicon dioxide as a base material, and the second wire layer 52 and the second microelectrode layer 55 are gold.

The second conductive line layer 52 is composed of a measuring electrode wire 521 and a voltage supply electrode wire 522 which are not connected with each other, and the second electrode layer 55 is composed of a voltage supply electrode 552 for regulating and controlling an input voltage signal and a corresponding voltage supply PAD electrode 555 thereof, and a measuring electrode 551 for biosensing and a corresponding measuring PAD electrode 554 thereof.

Specifically, the measurement electrode 551 and the measurement PAD electrode 554 are connected to the measurement electrode lead 521 through the second through hole 54, and the pressure supply electrode 552 and the pressure supply PAD electrode 555 are connected to the pressure supply electrode lead 522 through the second through hole 54.

More specifically, the measuring electrode 551, the voltage supply electrode 552, the measuring PAD electrode 554 and the voltage supply PAD electrode 555 are all rectangular electrodes, and the area of the measuring electrode 551 is 2mm ² The area of the voltage supply electrode 552 is 4mm ² The areas of the PAD electrode 554 and the PAD electrode 555 are 4mm ² 。

Description of the preparation method:

accordingly, a method for manufacturing a label-free biosensor with high stability comprises the following steps:

s10, preparing a first micro-electrode group by using an MEMS (micro-electromechanical system) process;

s20, preparing an organic semiconductor film 6 between a source electrode 452 and a drain electrode 453 of the first micro-electrode group by utilizing a piezoelectric ink-jet printing process, and manually placing to enable a bridging medium 7 to cover the organic semiconductor film 6 and the voltage-controlled electrode 451 to form a complete amplification chip 4;

s30, preparing a sensing chip 5 by using an MEMS (micro-electromechanical systems) process;

s40, connecting the voltage-controlled electrode PAD electrode 454 of the amplification chip 4 with the measurement PAD electrode 554 of the sensing chip 5 through the bridging structure 3, so as to communicate the amplification chip component 1 and the sensing chip component 2 to form a complete biosensor.

Specifically, in step S10, the amplification chip 4 is prepared by using the MEMS process, which specifically includes:

s101, depositing a first conductive line layer 42 on the amplification chip substrate 41, and patterning the first conductive line layer to form a voltage-controlled electrode conductive line 421, a source conductive line 422, and a drain conductive line 423, as shown in fig. 7;

specifically, before step S101, quartz glass is selected as a substrate, the substrate is soaked in chromic acid solution 24h, washed with deionized water, dried for later use, and then the first conductive line layer 42 is processed by photolithography and lift-off process;

more specifically, the photolithography and lift-off process is: spin coating and drying on a quartz glass sheet, carrying out photoetching development by using a mask, sputtering titanium (Ti) with the thickness of 30nm as an adhesion layer of the glass sheet and metal, sputtering gold (Au) with the thickness of 200 nm, and finally putting the wafer into an ultrasonic groove filled with acetone for 30 min to finish lift-off and realize the patterning of the first lead layer 42;

s102, depositing and etching a first insulating layer 43 to expose the electrode window to form a first through hole 44, as shown in FIG. 8;

specifically, a silicon dioxide insulating layer with a thickness of 300nm is grown on the substrate 41 and the first wiring layer 42 by PECVD, exposure is performed using a photolithography mask, and the insulating layer is etched with a mixed solution of hydrofluoric acid and ammonium fluoride to form a first via hole 44 to leak out of the electrode window.

S103, depositing a first micro electrode layer 45, and forming a voltage-controlled electrode 451, a source 452, a drain 453, a voltage-controlled electrode PAD electrode 454, a source PAD electrode 455, and a drain PAD electrode 456, as shown in FIG. 9;

specifically, the first microelectrode layer 45 is processed and manufactured by adopting photoetching and lift-off processes, and specifically, titanium with the thickness of 30nm and gold with the thickness of 400nm are adopted.

In step S20, the organic semiconductor film 6 is prepared between the source 452 and the drain 453 of the amplifier chip 4 by using a piezoelectric inkjet printing process, and the specific steps are as follows:

s201, placing an amplifying chip only comprising a microelectrode structure in a piezoelectric ink-jet printing device, adjusting the position of the amplifying chip, and ensuring that a printing nozzle is positioned above a position between a source 452 and a drain 453 and is vertical to the upper surface of the amplifying chip;

s202, inputting a periodic voltage pulse signal with pulse voltage of 25V and rise time of 1 mu S according to the required droplet size, expanding and contracting the printing nozzle to different degrees to form a droplet with a certain ejection speed, depositing the droplet between the source electrode 452 and the drain electrode 453, and enabling the source electrode 452 and the drain electrode 453 to be communicated through a small droplet;

specifically, the voltage pulse signal is divided into four stages in one cycle: adding a low voltage signal to fill the nozzle with the aqueous solution in the first stage, removing the voltage signal in the second stage, then refluxing the aqueous solution, adding a high voltage to extrude and spray the aqueous solution to form liquid drops in the third stage, and reducing the voltage signal in the fourth stage to prevent the excessive aqueous solution from being sprayed out;

more specifically, the components of the liquid drops and the aqueous solution are organic semiconductor aqueous solution formed by poly (3,4-ethylenedioxythiophene) and styrene sulfonate;

s203, the printed chip is heated in a vacuum oven at 120 ℃ for 10 minutes, and after being cooled naturally, the chip is taken out from the vacuum oven, and at this time, the organic semiconductor film 6 having stable physicochemical properties is formed between the source electrode 452 and the drain electrode 453.

In step S20, the step of forming the whole amplification chip 4 by manually placing the bridging medium 7 to cover the organic semiconductor film 6 and the voltage-controlled electrode 451 specifically includes:

s204, sucking a certain amount of solution by using a micro-injector, and then moving a needle of the micro-injector to be right above an amplification chip comprising an organic semiconductor film and a microelectrode structure and be positioned between the organic semiconductor film 6 and a voltage-controlled electrode 451;

specifically, in this embodiment, the droplet component is an ionic liquid prepared from 1-ethyl-3-methylimidazole trifluoroacetate (EMIMT/TfA);

s205, the solution is dropped between the organic semiconductor film 6 and the voltage-controlled electrode 451 at a constant speed, and the solution is ensured to cover the organic semiconductor film 6 and the voltage-controlled electrode 451, but not to contact other microelectrodes of the amplification chip.

In step S30, the preparing of the sensing chip 5 by using the MEMS process may specifically include:

s301, depositing a second lead layer 52 on the sensor chip substrate 51 to form a measuring electrode lead 521 and a voltage supply electrode lead 522, as shown in FIG. 11;

specifically, before step S301, quartz glass is selected as a substrate, the substrate is soaked in chromic acid solution for 24 hours, washed with deionized water, dried for standby, and then the second wire layer 52 is processed by photolithography and lift-off process;

more specifically, the photoetching and lift-off process comprises the following steps: spin coating and drying on a quartz glass sheet, carrying out photoetching development by using a mask, then sputtering titanium (Ti) with the thickness of 30nm as an adhesion layer of the glass sheet and metal, then sputtering gold (Au) with the thickness of 200 nm, finally putting the wafer into an ultrasonic groove filled with acetone for 30 min, finishing lift-off, and realizing the patterning of the second lead layer 52;

s302, depositing and etching the second insulating layer 53 to form the second through hole 54 and expose the electrode window, as shown in FIG. 12.

Specifically, a silicon dioxide insulating layer with the thickness of 300nm grows on the substrate by adopting PECVD (plasma enhanced chemical vapor deposition), a photoetching plate is used for exposure, and a mixed solution of hydrofluoric acid and ammonium fluoride is used for corroding the insulating layer so as to enable the insulating layer to leak out of an electrode window to form a second through hole 54;

s303, depositing a second microelectrode layer 55 to form a measuring electrode 551, a voltage supply electrode 552, a measuring PAD electrode 554 and a voltage supply PAD electrode 555, as shown in FIG. 13;

specifically, the second microelectrode layer 55 is processed and manufactured by adopting a photoetching and lift-off process, specifically, titanium with the thickness of 30nm and gold with the thickness of 400nm are adopted.

Example two

Structural description:

FIG. 1 is a schematic diagram of an overall structure of a label-free biochip with high stability according to the second embodiment of the present invention. The biological sensor chip comprises an amplifying chip component 1 and a sensing chip component 2, wherein the amplifying chip component 1 is connected with the sensing chip component 2 through five bridging structures 3, five amplifying chips 4 are arranged in the amplifying chip component 1, each amplifying chip 4 comprises a micro-electrode group, an organic semiconductor film 6 and a bridging medium 7, five sensing chips 5 are arranged in the sensing chip component 2, and each sensing chip 5 is composed of a micro-electrode.

Specifically, the bridging structure 3 is a wire made of gold wire, the organic semiconductor film 6 is specifically a conductive polymer (PEDOT: PSS) formed by poly (3,4-ethylenedioxythiophene) and styrene sulfonate, and the bridging medium 7 is specifically an ionic gel formed by polyvinylidene fluoride-hexafluoropropylene poly (vinylidene fluoride-co-hexafluoropropylene), ((P (VDF-HFP))) and 1-butyl-3-methylimidazolium bis (trifluoromethylsulfonyl) imide (1-butyl-3-methylimidazolium bis (trifluoromethyl sulfonyl)) and [ EMI ] [ TFSA ].

FIG. 3 is a schematic structural diagram of an amplification chip 4 in a label-free biochip having high stability according to a second embodiment of the present invention. The amplification chip 4 includes: the organic light-emitting diode comprises a substrate 41, a first lead layer 42, a first insulating layer 43, a first through hole 44, a first microelectrode layer 45, an organic semiconductor film 6 and a bridging medium 7, wherein the first lead layer 42 is positioned on the substrate 41, the first insulating layer 43 covers the first lead layer 42 and the substrate 41 at the same time, and the first microelectrode layer 45 is positioned on the first insulating layer 43.

Specifically, the amplification chip substrate 41 uses quartz glass as a base material, the first insulating layer 43 is silicon dioxide, the first insulating layer 43 uses quartz glass and silicon dioxide as base materials, and the first lead layer 42 and the first microelectrode layer 45 are gold.

Specifically, the voltage-controlled electrode 451, the source 452 and the drain 453 of the present embodiment have an area of 400 μm ² The source electrode 452 and the drain electrode 453 form a tip electrode pair, the distance between the electrode pairs is set to 10 μm, and the voltage-controlled electrode PAD electrode 454, the source PAD electrode 455 and the drain PAD electrode 456 are all 4mm in area ² The rectangular electrode of (1).

The organic semiconductor film 6 is located on the first microelectrode layer 45, connects and covers the source electrode 452 and the drain electrode 453, and does not contact the remaining electrodes in the first microelectrode layer 45.

The bridging medium 7 connects and covers the organic semiconductor film 6 and the voltage-controlled electrode 451 and does not contact the remaining electrodes in the first microelectrode layer 45.

Fig. 5 is a schematic structural diagram of a sensor chip 5 in a label-free biochip with high stability according to an embodiment of the present invention, where the sensor chip 5 includes: the substrate 51, the second wire layer 52, the second insulating layer 53, the second via 54, and the second microelectrode layer 55, wherein the second wire layer 52 is located on the substrate 51, the second insulating layer 53 covers the second wire layer 52 and the upper surface of the substrate 51, and the second microelectrode layer 55 is located on the second insulating layer 53.

The second conductive line layer 52 is composed of a measurement electrode line 521, a voltage supply electrode line 522 and a convection electrode line 523, which are not connected to each other, the second microelectrode layer 55 is composed of a voltage supply electrode 552 for regulating and controlling an input voltage signal and a corresponding voltage supply PAD electrode 555, a measurement electrode 551 for biosensing and a corresponding measurement PAD electrode 554, and at least one pair of a convection electrode 553 for forming an electrokinetic current and a corresponding convection PAD electrode 556.

In particular, two pairs of convection electrodes 553 are used in this example and are evenly distributed around the measurement electrode 551;

specifically, the measurement electrode 551 and the measurement PAD electrode 554 are connected to the measurement electrode wire 521 through the second through hole 54, the pressure supply electrode 552 and the pressure supply PAD electrode 555 are connected to the pressure supply electrode wire 522 through the second through hole 54, and the convection electrode 553 and the convection PAD electrode 556 are connected to the convection electrode wire 523 through the second through hole 54.

More specifically, the measuring electrode 551, the voltage supply electrode 552, the convection electrode 553, the measuring PAD electrode 554, the voltage supply PAD electrode 555, and the convection PAD electrode 556 are all rectangular electrodes, and the area of the measuring electrode 551 is 2mm ² The area of the voltage supply electrode 552 is 4mm ² The area of the counter electrode 553 is 1 mm ² The areas of the measurement PAD electrode 554, the pressure supply PAD electrode 555 and the convection PAD electrode 556 are all 4mm ² 。

Description of the preparation:

accordingly, a method for manufacturing a label-free biochip with high stability comprises the following steps:

s20, preparing an organic semiconductor film 6 between a source electrode 452 and a drain electrode 453 of the first micro-electrode set by utilizing a piezoelectric ink-jet printing process, and manually placing to enable a bridging medium 7 to cover the organic semiconductor film 6 and the voltage-controlled electrode 451 to form a complete amplification chip 4;

s101, a first conductive line layer 42 is deposited on the substrate 41 and patterned to form a voltage-controlled electrode line 421, a source line 422 and a drain line 423, as shown in fig. 7.

Specifically, before step S101, quartz glass is selected as a substrate, the substrate is soaked in chromic acid solution 24h, washed with deionized water, dried for standby, and then the first conductive line layer 42 is processed by photolithography and lift-off process.

More specifically, the photolithography and lift-off process is: the method comprises the steps of coating glue on a quartz glass sheet, drying, carrying out photoetching development by using a mask, sputtering titanium (Ti) with the thickness of 30nm as an adhesion layer of the glass sheet and metal, sputtering gold (Au) with the thickness of 200 nm, and finally putting a wafer into an ultrasonic groove filled with acetone for 30 min to finish lift-off, thereby realizing the patterning of the first lead layer 42.

S102, depositing the first insulating layer 43 and etching to expose the electrode window, as shown in fig. 8.

Specifically, a silicon dioxide insulating layer with a thickness of 300nm is grown on the substrate 41 and the first wiring layer 42 by PECVD, exposure is performed using a photolithography mask, and the insulating layer is etched by a mixed solution of hydrofluoric acid and ammonium fluoride to form the first via hole 44 to leak out of the electrode window.

S103, depositing and patterning the first micro electrode layer 45 to form a voltage-controlled electrode 451, a source 452, a drain 453, a voltage-controlled electrode PAD electrode 454, a source PAD electrode 455, and a drain PAD electrode 456, as shown in fig. 9.

Specifically, the first microelectrode layer 45 is processed and manufactured by adopting a photoetching and lift-off process, the metal of the first microelectrode layer is a Ti/Au material, and the thickness of the first microelectrode layer is (30 nm/400 nm).

In step S20, the preparing the organic semiconductor film 6 between the source electrode 452 and the drain electrode 453 of the amplifier chip by using the piezoelectric inkjet printing process may specifically include:

s201, placing the amplification chip containing only the micro-electrode structure in the piezoelectric inkjet printing apparatus, and adjusting the position of the amplification chip to ensure that the printing nozzle is above the space between the source 452 and the drain 453 and perpendicular to the upper surface of the amplification chip.

S202, inputting a periodic pulse signal with a pulse voltage of 25V and a rise time of 1 mu S according to the required droplet size, enabling the printing nozzle to expand and contract in different degrees to form a droplet with a certain ejection speed, depositing the droplet between the source electrode 452 and the drain electrode 453, and enabling the source electrode 452 and the drain electrode 453 to be communicated through a small droplet.

Specifically, the voltage pulse signal is divided into four stages in one cycle: adding a low voltage signal in the stage 1 to fill the nozzle with the aqueous solution, removing the voltage signal in the stage 2 and then refluxing the aqueous solution, adding a high voltage in the stage 3 to extrude and spray to form liquid drops, and reducing the voltage signal in the stage 4 to prevent the excessive aqueous solution from being sprayed.

More specifically, the components of the liquid drops and the aqueous solution are organic semiconductor aqueous solution formed by poly (3,4-ethylenedioxythiophene) and styrene sulfonate.

S203, the printed chip is placed in a vacuum oven and heated at 120 ℃ for 10 minutes, and after being cooled naturally, the chip is taken out from the vacuum oven, and at this time, an organic semiconductor film 6 having stable physicochemical properties is formed between the source electrode 452 and the drain electrode 453.

In step S20, the manual placement of the bridging medium 7 to cover the organic semiconductor film 6 and the voltage-controlled electrode 451 forms the complete amplification chip 4, which specifically includes:

s204, mixing P (VDF-HFP), [ EMI ] [ TFSA ] and acetone according to the mass ratio of 1.

S205, the mixed solution obtained in the step S204 is coated on a glass slide in a spinning mode, and is dried in a vacuum drying oven at 70 ℃ for 24 hours, residual solvent is removed, and ionic gel is formed.

S206, placing the bridging medium 7 between the organic semiconductor film 6 and the voltage-controlled electrode 451 by using tweezers, and ensuring that the bridging medium 7 is not in contact with other microelectrodes of the amplification chip 4.

s301, depositing and patterning a second lead layer 52 on the substrate 51 to form a measuring electrode lead 521, a voltage supply electrode lead 522 and a convection electrode lead 523, as shown in FIG. 11.

Specifically, before step S301, quartz glass is selected as a substrate, the substrate is soaked in chromic acid solution for 24 hours, washed with deionized water, dried for standby, and then the second conductive line layer 52 is processed by photolithography and lift-off process.

More specifically, the photolithography and lift-off process is: and (3) glue is uniformly distributed on a quartz glass sheet and dried, photoetching and developing are carried out by using a mask, then titanium (Ti) with the thickness of 30nm is sputtered to be used as an adhesion layer of the glass sheet and metal, gold (Au) with the thickness of 200 nm is sputtered, finally the wafer is placed into an ultrasonic groove filled with acetone for 30 min, lift-off is completed, and the patterning of the second lead layer 52 is realized.

Specifically, a silicon dioxide insulating layer with a thickness of 300nm is grown on the substrate by PECVD, exposure is performed using a photolithography mask, and the insulating layer is etched by a mixed solution of hydrofluoric acid and ammonium fluoride to leak out of the electrode window, thereby forming the second via 54.

S303, depositing and patterning the second microelectrode layer 55 to form a measuring electrode 551, a voltage supplying electrode 552, two pairs of convection electrodes 553, a measuring PAD electrode 554, a voltage supplying PAD electrode 555, and a convection PAD electrode 556, as shown in fig. 13.

Specifically, the second microelectrode layer 55 is processed and manufactured by photolithography and lift-off process, and the metal of the layer is Ti/Au material with a thickness of (30 nm/400 nm).

The practical application is as follows:

when detecting a biological sample in a solution, the sensing chip can be placed in the micro flow channel, and the sample to be detected is conveyed to the surface of the sensing chip through the liquid feeding system. After the convection electrode is electrified, an electric current is formed on the sensing chip, so that the object to be measured is positioned on the surface of the measuring electrode 551 more quickly and accurately. When the sample to be measured reacts on the sensor chip 5, the potentials of the measurement electrode 551 and the measurement PAD electrode 554 connected thereto change. The potential change signal is transmitted to the voltage-controlled electrode 451 of the amplification chip 4 through the bridging structure 3, and the voltage-controlled electrode 451 controls the electrochemical doping degree of the organic semiconductor film 6 through the bridging medium 7, so that the output current signal between the source 452 and the drain 453 is changed, and the state of the sample to be detected is determined according to the change of the output current.

Specifically, taking DNA detection as an example, the measurement system is shown in fig. 14. Before detection, probe DNA molecules are modified on the surface of the measuring electrode 551 of the sensor chip 5. During detection, liquid to be detected is conveyed to the surface of the sensing chip 5, the liquid is ensured to cover the measuring electrode 551 and the pressure supply electrode 552, 2V direct-current voltage signals are input to the pressure supply electrode 552, and 2V alternating-current voltages are input to the four convection electrodes 553. When the complementary DNA in the solution hybridizes to the probe DNA, the potential of the measurement electrode 551 and the measurement PAD electrode 554 connected thereto changes.

The measurement PAD electrode 554 and the voltage-controlled electrode PAD electrode 454 are connected by the bridge structure 3, so that the potential variation signal is transmitted to the amplification chip 4. And applying a constant voltage of-0.6V to the two ends of the source 452 and the drain 453 of the amplification chip 4, and detecting the current change between the source PAD electrode 455 and the drain PAD electrode 456 to realize the detection of the concentration of the complementary DNA sequence in the liquid to be detected.

EXAMPLE III

Structural description:

FIG. 2 is a schematic diagram of the overall structure of a label-free biochip with high stability according to the third embodiment of the present invention. The biological sensor chip is including enlargiing chip subassembly 1 and sensing chip subassembly 2, enlarge chip subassembly 1 with sensing chip subassembly 2 links to each other perpendicularly through flexible substrate material 8, be equipped with five and enlarge chip 4 in the enlarged chip subassembly 1, it includes microelectrode group, organic semiconductor film 6 and bridging medium 7 to enlarge chip 4, be equipped with five sensing chip 5 in the sensing chip subassembly 2, sensing chip 5 comprises the microelectrode, enlarge chip subassembly 1 the microelectrode with sensing chip subassembly 2's microelectrode is connected through five bridging structure 3.

The flexible substrate 8 may be Polydimethylsiloxane (PDMS), parylene (PE), polyimide (PI), polyetherimide (PEI), polyvinyl alcohol (PVA), polyethylene naphthalate (PEN), and various fluoropolymers and copolymers, in this example, PDMS is used as the flexible substrate 8, the bridging structure 3 is specifically a dupont line, the organic semiconductor film 6 is specifically a conductive polymer (PPY: PSS) formed by polypyrrole and styrene sulfonate, the bridging medium 7 is specifically a polystyrene triblock copolymer, poly (styrene-block-methyl methacrylate-styrene) (PS-PMMA-PS), and 1-ethyl-3-methylimidazoline bis (trifluoromethylsulfonyl) imide, 1-ethyl-3-methyl-lithium bis (trifluoromethylsulfonyl) tfis ([ EMIM ] [ si ]).

FIG. 3 is a schematic structural diagram of an amplification chip 4 in a non-labeled biochip with high stability according to an embodiment of the present invention. The amplification chip 4 includes: the organic light-emitting diode comprises a substrate 41, a first lead layer 42, a first insulating layer 43, a first through hole 44, a first microelectrode layer 45, an organic semiconductor film 6 and a bridging medium 7, wherein the first lead layer 42 is positioned on the substrate 41, the first insulating layer 43 covers the first lead layer 42 and the substrate 41 at the same time, and the first microelectrode layer 45 is positioned on the first insulating layer 43.

Specifically, the amplification chip substrate 41 uses quartz glass as a base material, the first insulating layer 43 is silicon dioxide, the first insulating layer 43 uses quartz glass and silicon dioxide as base materials, and the first wire layer 42 and the first microelectrode layer 45 are gold.

Specifically, the voltage-controlled electrode 451 and the voltage-controlled electrode PAD electrode 454 are connected to the voltage-controlled electrode wire 421 through the first via 44, and the source electrode 452 and the source electrode PAD electrode 455 are connected to the source wire 422 through the first via 44; the drain 453 and the drain PAD electrode 456 are connected to the drain wire 423 through the first via hole 44.

Specifically, the voltage-controlled electrode 451, the source 452 and the drain 453 of the present embodiment have an area of 400 μm ² The rectangular electrode of (1), the source electrode452 and the drain 453 are tip electrode pairs with a distance of 10 μm, and the voltage-controlled PAD electrode 454, the source PAD electrode 455 and the drain PAD electrode 456 are all 4mm in area ² The rectangular electrode of (1).

Fig. 6 is a schematic structural diagram of a sensor chip 5 in a label-free biochip with high stability according to an embodiment of the present invention, where the sensor chip 5 includes: the substrate 51, the second wire layer 52, the second insulating layer 53, the second via 54, and the second microelectrode layer 55, wherein the second wire layer 52 is located on the substrate 51, the second insulating layer 53 covers the second wire layer 52 and the substrate 51, and the second microelectrode layer 55 is located on the second insulating layer 53.

Specifically, three pairs of counter electrodes 553 are employed in this example and are evenly distributed around the measuring electrode 551;

Description of the preparation:

s40, connecting the prepared amplification chip assembly 1 and the prepared sensing chip assembly 2 through the flexible substrate material 8, and then connecting the voltage-controlled electrode PAD electrode 454 of the amplification chip 4 with the measurement PAD electrode 554 of the sensing chip 5 by adopting the bridging structure 3.

Specifically, before step S101, quartz glass is selected as a substrate, the substrate is soaked in chromic acid solution for 24 hours, washed with deionized water, dried for standby, and then the first wire layer 42 is processed by photolithography and lift-off process.

More specifically, the photolithography and lift-off process is: the method comprises the steps of coating glue on a quartz glass sheet, drying, carrying out photoetching development by using a mask, sputtering titanium (Ti) with the thickness of 30nm as an adhesion layer of the glass sheet and metal, sputtering gold (Au) with the thickness of 200 nm, finally putting a wafer into an ultrasonic groove filled with acetone for 30 min, finishing lift-off, and realizing the patterning of a first lead layer 42.

In step S20, the preparing the organic semiconductor film 6 between the source 452 and the drain 453 of the amplifier chip by using the electrohydrodynamic inkjet printing process may specifically include:

s201, placing an amplifying chip only containing a microelectrode structure in the ink-jet printing device, and adjusting the position of the amplifying chip to ensure that a printing nozzle is positioned above the position between the source electrode 452 and the drain electrode 453 and is vertical to the upper surface of the amplifying chip.

S202, adjusting the jetting height according to the required droplet size, setting the pressure of an injection pump, inputting a periodic pulse signal with the pulse voltage of 30V and the time of 2 mu S, expanding and contracting the printing nozzle to form a droplet with a certain jetting speed, depositing the droplet between the source 452 and the drain 453, and enabling the source 452 and the drain 453 to be communicated through a small droplet.

Specifically, the voltage pulse signal is divided into 2 stages in one cycle: the low voltage signal is added in stage 1 to make the water solution fill the nozzle, and the high voltage is added in stage 2 to extrude and spray to form liquid drops.

More specifically, the components of the liquid drops and the aqueous solution are organic semiconductor aqueous solution formed by polypyrrole and styrene sulfonate.

S203, the printed chip is placed in a vacuum drying oven and heated at 120 ℃ for 10 minutes, and after being cooled naturally, the chip is taken out from the vacuum drying oven, and at this time, an organic semiconductor film 6 having stable physicochemical properties is formed between the source electrode 452 and the drain electrode 453.

s204, mixing PS-PMMA-PS, [ EMI ] [ TFSA ] and ethyl acetate according to a mass ratio of 0.1.

S205, the mixed solution obtained in the step S204 is coated on a glass slide in a spinning mode, and is dried in a vacuum drying box for 24 hours at room temperature, residual solvent is removed, and ionic gel is formed.

s301, depositing and patterning a second conductive line layer 52 on the substrate 51 to form a measuring electrode conductive line 521, a voltage supply electrode conductive line 522, and a convection electrode conductive line 523, as shown in fig. 11.

Specifically, before step S301, quartz glass is selected as a substrate, the substrate is soaked in chromic acid solution for 24 hours, washed with deionized water, dried for standby, and then the second wire layer 52 is processed by photolithography and lift-off process.

More specifically, the photoetching and lift-off process comprises the following steps: and (3) coating and drying quartz glass sheets, carrying out photoetching development by using a mask, sputtering titanium (Ti) with the thickness of 30nm as an adhesion layer of the glass sheets and metal, sputtering gold (Au) with the thickness of 200 nm, and finally putting the wafers into an ultrasonic groove filled with acetone for 30 min to finish lift-off and realize the patterning of the second lead layer 52.

Specifically, a silicon dioxide insulating layer with a thickness of 300nm is grown on the substrate by PECVD, exposure is performed by using a photolithography mask, and the insulating layer is etched by a mixed solution of hydrofluoric acid and ammonium fluoride so as to leak out of the electrode window to form the second via hole 54.

S303, depositing and patterning the second microelectrode layer 55 to form a measuring electrode 551, a voltage supply electrode 552, three pairs of convection electrodes 553, a measuring PAD electrode 554, a voltage supply PAD electrode 555 and a convection PAD electrode 556, as shown in FIG. 13.

In step S40, the connecting of the prepared amplification chip 4 and the sensor chip 5 through the flexible substrate material 8 may specifically include:

s401, steaming the prepared glass positive film for 3min by using a trimethyl chloride silanization reagent (TMCS), and placing the silanized glass positive film in a required container.

S402, pouring the prepared PDMS on an anode film glass sheet, wherein the thickness of the PDMS layer is about 2mm, and then placing the glass sheet in a vacuum drying oven to be dried for 2h at the temperature of 80 ℃ to cure the PDMS.

And S403, taking the cured PDMS cover plate off the male mold, cutting the PDMS cover plate into required sizes by using a scalpel, and ensuring that the sizes of the PDMS cover plate are the same as those of the chip to finish the preparation of the flexible substrate material 8.

S404, placing the amplification chip, the sensing chip and the flexible substrate material in a plasma cleaning machine, cleaning for 60S by oxygen plasma, and then respectively connecting the bottom surfaces of the amplification chip and the sensing chip with the upper surface and the lower surface of the flexible substrate material 8 to enable the flexible substrate material to be respectively bonded with the amplification chip and the sensing chip to complete connection.

The practical application is as follows:

when detecting sweat components, the chip is attached to the surface of the skin. After sampling, the convection electrode is electrified to form an electrokinetic flow in the micro-pool, so that uric acid, glutamic acid, lactic acid and the like in sweat can be rapidly transported to the surface of the measuring electrode 551.

When the analyte reacts with a probe molecule (e.g., lactate oxidase) on the surface of the measuring electrode 551, the potential of the measuring electrode 551 and the measuring PAD electrode 554 connected thereto changes. The potential change signal is transmitted to the voltage-controlled electrode 451 of the amplification chip 4 through the bridging structure 3, and the voltage-controlled electrode 451 controls the electrochemical doping degree of the organic semiconductor film 6 through the bridging medium 7, so that the output current signal between the source 452 and the drain 453 is changed, and the concentration of the substance to be detected is determined according to the change of the output current.

Specifically, taking the detection of lactic acid as an example, lactate oxidase is modified on the surface of the measurement electrode 551 of the sensor chip 5 before the detection. In the detection, sweat is transported to the surface of the sensor chip 5, and is secured to cover the measurement electrode 551 and the voltage supply electrode 552, and 2V dc voltage signals are input to the voltage supply electrode 552 and 2V ac signals are input to the six counter electrodes 553. When lactate oxidase oxidizes lactate to produce pyruvic acid, the potentials of the measurement electrode 551 and the measurement PAD electrode 554 connected thereto change. The measurement PAD electrode 554 and the voltage-controlled electrode PAD electrode 454 are connected by the bridge structure 3, so that the potential variation signal is transmitted to the amplification chip. And applying a constant voltage of-0.6V to the two ends of the source 452 and the drain 453 of the amplification chip 4, and detecting the current change between the source PAD electrode 455 and the drain PAD electrode 456 to realize the detection of the concentration of the lactic acid to be detected.

Example four

Structural description:

FIG. 1 is a schematic diagram of the overall structure of a label-free biochip with high stability according to the second embodiment of the present invention. The biological sensor chip comprises an amplifying chip component 1 and a sensing chip component 2, wherein the amplifying chip component 1 is connected with the sensing chip component 2 through five bridging structures 3, five amplifying chips 4 are arranged in the amplifying chip component 1, each amplifying chip 4 comprises a micro-electrode group, an organic semiconductor film 6 and a bridging medium 7, five sensing chips 5 are arranged in the sensing chip component 2, and each sensing chip 5 is composed of a micro-electrode.

Specifically, the bridging structure 3 is a wire made of gold wire, the organic semiconductor film 6 is a conductive polymer (PEDOT: PSS) formed by poly (3,4-ethylenedioxythiophene) and styrene sulfonate, and the bridging medium 7 is an ionic gel (EMI/TFSI- [ SEAS ]) synthesized by polymer poly (styrene-b-ethyl acrylate-b-styrene) (SEAS) and ionic liquid 1-ethyl-3-methylimidazolium bis (trifluoromethylsulfonyl) imide, 1-ethyl-3-methylimidazolium bis- (trifluoromethylsulfonyl) imide ([ EMI ] [ SI ]).

Specifically, the voltage-controlled electrode 451, the source 452 and the drain 453 of the present embodiment have an area of 400 μm ² The source 452 and the drain 453The distance between the tip electrode pair is set to 10 μm, and the voltage-controlled PAD electrode 454, the source PAD electrode 455 and the drain PAD electrode 456 are all 4mm in area ² The rectangular electrode of (1).

Fig. 5 is a schematic structural diagram of a sensor chip 5 in an unidentified biochip with high stability according to an embodiment of the present invention, where the sensor chip 5 includes: the substrate 51, the second wire layer 52, the second insulating layer 53, the second via 54, and the second microelectrode layer 55, wherein the second wire layer 52 is located on the substrate 51, the second insulating layer 53 covers the second wire layer 52 and the upper surface of the substrate 51, and the second microelectrode layer 55 is located on the second insulating layer 53.

Specifically, two pairs of counter electrodes 553 are employed in this example and are evenly distributed around the measuring electrode 551;

Description of the preparation:

s20, preparing an organic semiconductor film 6 between the source electrode 452 and the drain electrode 453 of the first micro-electrode set, and covering the organic semiconductor film 6 and the voltage-controlled electrode 451 with a bridging medium 7 to form an amplification chip 4;

More specifically, the photolithography and lift-off process is: the method comprises the steps of coating glue on a quartz glass sheet, drying, carrying out photoetching development by using a mask, sputtering titanium (Ti) with the thickness of 3-0nm as an adhesion layer of the glass sheet and metal, sputtering gold (Au) with the thickness of 200 nm, and finally putting a wafer into an ultrasonic groove filled with acetone for 30 min to finish lift-off, thereby realizing the patterning of the first lead layer 42.

In step S20, the preparing the organic semiconductor film 6 between the source electrode 452 and the drain electrode 453 of the amplifier chip by using the ac electrodeposition method may specifically include:

s201, preparing electrolyte according to requirements. Adding sodium polystyrene sulfonate (PSS), 3,4 ethylene dioxythiophene monomer (EDOT) and a doping agent into a solvent, and fully stirring and uniformly mixing to obtain an electrolyte;

specifically, firstly, adding a proper amount of solvent into a volumetric flask, and then adding a certain amount of sodium polystyrene sulfonate (NaPSS) and a doping agent into the volumetric flask; then, dropwise adding a quantitative EDOT monomer into the solution, and continuously stirring until the EDOT monomer is completely dissolved to obtain the required electrolyte;

the solvent can be an aqueous solution, ethanol, acetonitrile or propylene carbonate and other organic solutions, and the aqueous solution is selected as the solvent in the embodiment; the concentration range of the NaPSS is 10 mmol to 0.5 mol/L; the concentration range of the EDOT monomer is 10 mmol to 0.5 mol/L; in this example, the concentrations of the aqueous solutions of EDOT and NaPSS were 10 mmol/L and 0.1 mol/L, respectively; the dopant can be graphene quantum dots, ethylene glycol, dimethyl sulfoxide and the like;

s202, an electrolyte is dropped on the surface of the amplification chip 4 manufactured in step S10, and an organic semiconductor film is deposited electrochemically so that the source electrode 452 and the drain electrode 453 which are prepared are in contact with each other.

Specifically, a micro syringe is used to drop an electrolyte between the source 452 and the drain 453 of the amplification chip 4; applying an electric signal to the source and drain electrodes using an electrochemical workstation to cause polymerization of the electrolyte to form an organic semiconductor film 6 connecting the source electrode 452 and the drain electrode 453; the remaining electrolyte was then rinsed clean with deionized water and dried in a dry box.

The electric signal types can specifically adopt square waves, triangular waves and sine waves: when a square wave alternating current signal is adopted, the voltage range is 1.6V to 6V, and the frequency is 50 Hz to 2 MHz; when a triangular wave alternating current signal is adopted, the voltage range is 1.6V to 10V, and the frequency is 200 Hz to 5 MHz; when a sine wave alternating current signal is adopted, the voltage range is 1.6V to 8V, and the frequency is 50 Hz to 3 MHz. In this embodiment, sinusoidal signals are used, and the amplitude and frequency are 6V and 100 Hz, respectively.

s203, mixing the polymer SEAS and the ionic liquid [ EMI ] [ TFSI ] according to the weight ratio of 1: the mass ratio of 9 was dissolved in dichloromethane.

And S204, spin-coating the mixed solution obtained in the step S204 on a glass slide, standing for 24 hours in a nitrogen flow, then carrying out vacuum drying for 48 hours at 80 ℃, and removing residual organic solvent to form ionic gel.

S205, placing the bridging medium 7 between the organic semiconductor film 6 and the voltage-controlled electrode 451 by using tweezers, and ensuring that the bridging medium 7 is not in contact with other microelectrodes of the amplification chip 4.

More specifically, the photolithography and lift-off process is: and (3) coating and drying quartz glass sheets, carrying out photoetching development by using a mask, sputtering titanium (Ti) with the thickness of 30nm as an adhesion layer of the glass sheets and metal, sputtering gold (Au) with the thickness of 200 nm, and finally putting the wafers into an ultrasonic groove filled with acetone for 30 min to finish lift-off and realize the patterning of the second lead layer 52.

S303, depositing and patterning the second microelectrode layer 55 to form a measuring electrode 551, a voltage supply electrode 552, two pairs of counter electrodes 553, a measuring PAD electrode 554, a voltage supply PAD electrode 555 and a counter PAD electrode 556, as shown in FIG. 13.

The practical application is as follows:

when detecting cell samples, the sensing chip can be placed in the micro-channel, and a sample to be detected is conveyed to the surface of the sensing chip through the liquid feeding system. When the convection electrode is energized, an electromotive current is formed on the sensor chip, so that the cell is more accurately positioned on the surface of the measurement electrode 551. When the physical and chemical properties of the cells change on the sensor chip 5, the potential of the measuring electrode 551 and the measuring PAD electrode 5534 connected thereto changes. The potential change signal is transmitted to the voltage-controlled electrode 451 of the amplification chip 4 through the bridging structure 3, and the voltage-controlled electrode 451 controls the electrochemical doping degree of the organic semiconductor film 6 through the bridging medium 7, so that the output current signal between the source 452 and the drain 453 is changed, and the physiological characteristics of the cell are determined according to the change of the output current.

Specifically, taking Hela cells as an example, the measurement system is shown in fig. 14. The cell suspension is delivered to the surface of the sensor chip 5, ensuring that the liquid covers the measuring electrode 551, the pressure supply electrode 552, and the counter electrode 553. A 2V dc voltage signal is input to the voltage supply electrode 552, and a 20V 10MHz sinusoidal ac signal is input to the four counter current electrodes 553. And a phase difference between the adjacent convection electrodes 553 is maintained to be 180 degrees. Monitoring of the response of the cells to an external stimulus can be accomplished by a change in the potential of measurement electrode 551 and measurement PAD electrode 554 connected thereto. The measurement PAD electrode 554 and the voltage-controlled electrode PAD electrode 454 are connected by the bridging structure 3, so that the potential variation signal is transmitted to the amplification chip. A constant voltage of-0.6V is applied to both ends of the source 452 and the drain 453 of the amplifier chip 4, and the change in current between the source PAD electrode 455 and the drain PAD electrode 456 is detected, whereby the physiological state of the cell can be obtained. Typically, an AC signal with a frequency of (0.1 Hz-1 MHz) is also input to the voltage supply electrode 552, so that an amplified electrochemical impedance signal is obtained from the source-drain current.

Claims

1. An identification-free biosensor comprising an amplification chip assembly (1) and a sensing chip assembly (2), characterized in that: at least one amplification chip (4) is arranged in the amplification chip component (1), at least one sensing chip (5) is arranged in the sensing chip component (2), and the amplification chip (4) is connected with the sensing chip (5) through a bridging structure (3);

the amplification chip (4) comprises an organic semiconductor film (6), a bridging medium (7) and a first micro-electrode group, wherein the first micro-electrode group comprises a substrate, a source electrode and a drain electrode, and a carrier running channel between the source electrode and the drain electrode is arranged in parallel to the plane of the substrate;

the sensing chip (5) comprises a second micro-electrode group for improving the control efficiency and precision of the biological sample to be detected;

the first micro-electrode group comprises: the chip amplification structure comprises an amplification chip substrate (41), a first lead layer (42), a first insulating layer (43), a first through hole (44) and a first micro electrode layer (45), wherein the first lead layer (42) is arranged on the upper side of the amplification chip substrate (41), the first insulating layer (43) covers the first lead layer (42) and the upper side of the amplification chip substrate (41), and the first micro electrode layer (45) covers the upper side of the first insulating layer (43);

the first lead layer (42) comprises electrode leads which are not connected with each other, and the first lead layer (42) comprises a voltage-controlled electrode lead (421), a source lead (422) and a drain lead (423);

the first micro-electrode layer (45) comprises: a voltage-controlled electrode (451), a source electrode (452), a drain electrode (453), a voltage-controlled electrode PAD electrode (454), a source PAD electrode (455), and a drain PAD electrode (456);

the voltage-controlled electrode (451) is connected with a voltage-controlled electrode PAD electrode (454) through a voltage-controlled electrode lead (421) of the first through hole (44);

the source (452) is connected to a source PAD electrode (455) by a source wire (422) passing through a first via (44);

the drain (453) is connected to a drain PAD electrode (456) by a drain wire (423) passing through the first via (44);

an organic semiconductor film (6) is arranged on the upper side of the first micro electrode layer (45), and the organic semiconductor film (6) is only connected with a source electrode (452) and a drain electrode (453);

the bridging medium (7) covers the voltage-controlled electrode (451) and the upper side of the organic semiconductor film (6), the voltage-controlled electrode (451) is connected with the organic semiconductor film (6) through the bridging medium (7), the bridging medium (7) is in contact with the upper surface and/or the side surface of the organic semiconductor film (6), the bridging medium (7) is in contact with the upper surface and/or the side surface of the voltage-controlled electrode (451), and the bridging medium (7) is not in contact with the source electrode (452) and the drain electrode (453);

the sensing chip (5) comprises: the sensor comprises a sensing chip substrate (51), a second lead layer (52), a second insulating layer (53), a second through hole (54) and a second micro electrode layer (55), wherein the second lead layer (52) is arranged on the upper side of the sensing chip substrate (51), the second insulating layer (53) covers the second lead layer (52) and the upper side of the sensing chip substrate (51), the second micro electrode layer (55) covers the upper side of the second insulating layer (53), and the second through hole (54) is formed in the second insulating layer (53);

the second microelectrode layer (55) comprises a measuring electrode (551), a measuring PAD electrode (554), a pressure supply electrode (552) and a pressure supply PAD electrode (555);

the second lead layer (52) includes electrode leads that are not connected to each other, and the second lead layer (52) includes: a measuring electrode lead (521) and a pressure supply electrode lead (522);

the measuring electrode (551) is connected to a measuring PAD electrode (554) by a measuring electrode lead (521) passing through the second through hole (54);

the pressure supply electrode (552) is connected to the pressure supply PAD electrode (555) by a pressure supply electrode lead (522) passing through the second through hole (54).

2. An unidentified biosensor as claimed in claim 1, wherein: the second micro electrode layer (55) further comprises at least one pair of convection electrodes (553) and a pair of convection PAD electrodes (556), and the second lead layer (52) further comprises a convection electrode lead (523);

the convection electrode (553) is connected to the convection PAD electrode (556) by a convection electrode wire (523) passing through the second via (54);

the counter electrode (553) is arranged evenly around the measuring electrode (551).

3. An unidentified biosensor as claimed in claim 2, wherein: a voltage-controlled electrode PAD electrode (454) arranged in the amplification chip (4) is connected with a measurement PAD electrode (554) arranged in the sensing chip (5) through a bridging structure (3);

the bridging medium (7) is a liquid, solid, or gel having ionic conductivity.

4. A method for manufacturing a non-identification biosensor is characterized in that: the method comprises the following steps:

the method comprises the following steps: the method is characterized in that an amplifying chip (4) is prepared by using an MEMS (micro-electromechanical systems) process, and the specific process comprises the following steps:

an amplification chip substrate (41) made of an insulating material is selected, a first electrode layer (42) is processed on the surface of the amplification chip substrate (41) by utilizing photoetching and lift-off or photoetching and corrosion processes, and patterning of a voltage-controlled electrode (451), a source electrode (452), a voltage-controlled electrode lead (423) and a source electrode lead (424) on the first electrode layer (42) is realized;

depositing a first insulating layer (43) and patterning, specifically growing a silicon dioxide insulating layer with the thickness of 100-500nm on the substrate by adopting a vapor deposition method, photoetching and developing, and corroding the first insulating layer (43) by using a dry etching method or a wet etching method so as to leak out the electrode window;

depositing and patterning a first micro electrode layer (45) to form a voltage-controlled electrode (451), a source electrode (452), a drain electrode (453), a voltage-controlled electrode PAD electrode (454), a source PAD electrode (455) and a drain PAD electrode (456), and ensuring that no physical contact exists between the source electrode (452) and the drain electrode (453);

step two: preparing an organic semiconductor film (6) between a source electrode (452) and a drain electrode (453) of the first microelectrode group, and covering the organic semiconductor film (6) and the voltage-controlled electrode (451) with a bridging medium (7) to form an amplification chip (4);

step three: the MEMS technology is utilized to prepare the sensing chip (5), and the specific process is as follows:

selecting a sensing chip substrate (51) made of an insulating material, and processing a second lead layer (52) on the surface of the sensing chip substrate (51) by utilizing photoetching and lift-off or photoetching and corrosion processes to realize the patterning of a measuring electrode lead (521), a pressure supply electrode lead (522) and a convection electrode lead (523);

depositing a second insulating layer (53) and patterning, specifically growing a silicon dioxide insulating layer with the thickness of 100-500nm on the substrate by adopting a vapor deposition method, exposing by using a photoetching plate, and enabling the silicon dioxide insulating layer to leak out of the electrode window by dry etching or wet etching;

depositing and patterning a second microelectrode layer (55) to form a measuring electrode (551), a pressure supply electrode (552), a convection electrode (553) and a measuring PAD electrode (554);

step four: a voltage-controlled electrode PAD electrode (454) of the amplifying chip (4) is connected with a measurement PAD electrode (554) of the sensing chip (5) through the bridging structure (3).