Biological detection device, biological chip and preparation method thereof

Abstract

The present disclosure provides a biological detection device, a biochip and a method for preparing the biochip. The biochip may include a substrate, an electrode layer, an insulating layer and a microfluidic layer. The electrode layer is arranged on one side of the substrate and includes a plurality of electrode regions arranged at intervals. The insulating layer covers the electrode layer and is provided with a first opening exposing the plurality of electrode regions. The microfluidic layer covers the insulating layer, and the microfluidic layer includes a first liquid inlet, a microfluidic channel and a second opening which are connected in sequence, and the second opening exposes at least a portion of the first opening. The present disclosure has the advantage of being easy to carry.

Description

Technical Field

[0001] The present disclosure relates to the field of sensor technology, and in particular to a biological detection device, a biological chip and a preparation method thereof. Background Art

[0002] Electrochemical biological detection devices refer to detection devices that use electrodes as signal conversion elements and potential or current as detection signals. Due to the advantages of high sensitivity, simple operation, and low cost, biological detection devices have been widely used in clinical testing, environmental analysis, food safety, drug analysis and other fields in recent years. However, existing biological detection devices are large in size and not easy to carry. Summary of the invention

[0003] The purpose of the present disclosure is to provide a biological detection device, a biochip and a method for preparing the biochip, which have the advantage of being easy to carry.

[0004] According to one aspect of the present disclosure, there is provided a biochip, comprising:

[0005] substrate;

[0006] An electrode layer, disposed on one side of the substrate and comprising a plurality of electrode regions disposed at intervals;

[0007] an insulating layer, covering the electrode layer and having a first opening exposing a plurality of the electrode regions;

[0008] A microfluidic channel layer covers the insulating layer, and the microfluidic channel layer includes a first liquid inlet, a microfluidic channel, and a second opening that are connected in sequence, and the second opening exposes at least a portion of the first opening.

[0009] Furthermore, there are a plurality of the first openings, and the plurality of the first openings expose a plurality of the electrode regions in a one-to-one correspondence, and at least a portion of an area of ​​each of the first openings is exposed through the second opening.

[0010] Furthermore, the plurality of electrode regions include a reference electrode region and one or more working electrode regions.

[0011] Furthermore, a sensitive functional layer is provided on the surface of the working electrode region facing away from the substrate.

[0012] Furthermore, the plurality of electrode areas include a plurality of working electrode areas, and two of the plurality of working electrode areas have different sensitive functional layers disposed on their surfaces.

[0013] Furthermore, the sensitive functional layer is a sodium ion sensitive functional layer, a potassium ion sensitive functional layer, a calcium ion sensitive functional layer, a hydrogen ion sensitive functional layer or a chloride ion sensitive functional layer.

[0014] Furthermore, the plurality of electrode regions include a plurality of working electrode regions, and the plurality of working electrode regions are arranged around the reference electrode region.

[0015] Furthermore, the distance between the working electrode area and the reference electrode area is 0.8 mm-1.2 mm.

[0016] Furthermore, the working electrode area and / or the reference electrode area is rectangular in shape, with a long side dimension of 5.5 mm-6.5 mm and a short side dimension of 2.5 mm-3.5 mm.

[0017] Furthermore, the area of ​​the second opening is larger than the area of ​​the first liquid inlet.

[0018] Furthermore, the first liquid inlet is circular and has a diameter of 1 mm-2 mm.

[0019] Furthermore, the second opening is rectangular, with a long side dimension of 14 mm-20 mm and a short side dimension of 8 mm-9 mm.

[0020] Furthermore, the number of the microchannels and the number of the first liquid inlets are both two, and the two first liquid inlets are connected to the second openings through the two microchannels in a one-to-one correspondence.

[0021] Furthermore, the two microfluidic channels are distributed in an axisymmetric manner, the two first liquid inlets are distributed in an axisymmetric manner, and the symmetry axes of the two microfluidic channels coincide with the symmetry axes of the two first liquid inlets.

[0022] Furthermore, the second opening is rectangular, and the symmetry axes of the two microchannels are parallel to the short side or the long side of the second opening.

[0023] Furthermore, the second opening is rectangular, and the two microchannels are connected to two adjacent corners of the second opening in a one-to-one correspondence.

[0024] Furthermore, the width of the microchannel gradually decreases from the second opening to the first liquid inlet.

[0025] Furthermore, the microchannel layer also includes a liquid outlet communicated with the second opening.

[0026] Furthermore, the liquid outlet and the first liquid inlet are located on two opposite sides of the second opening in a one-to-one correspondence.

[0027] Furthermore, the second opening is rectangular, and the liquid outlet and the first liquid inlet are located on two sides of the second opening in a short side direction.

[0028] Furthermore, the liquid outlet is a liquid outlet groove, and the width of the liquid outlet groove is 1.5mm-2.5mm.

[0029] Furthermore, the substrate is a flexible substrate.

[0030] Furthermore, the biochip also includes:

[0031] The attachment layer is arranged on a side of the microchannel layer away from the substrate, and the attachment layer is provided with a second liquid inlet communicated with the first liquid inlet.

[0032] According to one aspect of the present disclosure, there is provided a method for preparing a biochip, comprising:

[0033] providing a substrate;

[0034] forming an electrode layer on one side of the substrate, wherein the electrode layer includes a plurality of electrode regions arranged at intervals;

[0035] forming an insulating layer covering the electrode layer, wherein the insulating layer is provided with a first opening exposing a plurality of the electrode regions;

[0036] A microchannel layer covering the insulating layer is formed, wherein the microchannel layer comprises a first liquid inlet, a microchannel and a second opening which are connected in sequence, and the second opening exposes at least a part of the first opening.

[0037] According to one aspect of the present disclosure, there is provided a biological detection device, comprising the above-mentioned biochip.

[0038] The biological detection device, biochip and method for preparing the biochip disclosed in the present invention, during use, the first liquid inlet can receive the sample liquid, the microfluidic channel constitutes a capillary, and under the action of the capillary, the sample liquid in the first liquid inlet enters the second opening through the microfluidic channel. Since the electrode area is exposed through the first opening, and the second opening exposes at least a part of the first opening, the sample liquid entering the second opening can contact the electrode area, and then the detection is carried out. In addition, the biochip is small in size and has the advantage of being easy to carry. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 Schematic cross-sectional view of a biochip according to an embodiment of the present disclosure.

[0040] Figure 2 Schematic diagram of a plan view of an electrode layer in a biochip according to an embodiment of the present disclosure.

[0041] Figure 3 Schematic diagram of a plan view of an insulating layer in a biochip according to an embodiment of the present disclosure.

[0042] Figure 4 It is a schematic plan view of a microfluidic layer in a biochip according to an embodiment of the present disclosure.

[0043] Figure 5 It is a plan view schematically showing an attachment layer in a biochip according to an embodiment of the present disclosure.

[0044] Figure 6 It is a schematic diagram of the coordination between the microfluidic layer and the attachment layer in the biochip according to the embodiment of the present disclosure.

[0045] Figure 7 4 is a flow chart of a method for preparing a biochip according to an embodiment of the present disclosure.

[0046] Figure 8 Schematic diagram of the voltage signal of the biochip according to the embodiment of the present disclosure.

[0047] Fig. 9 Schematic diagram of sodium ion detection results according to an embodiment of the present disclosure.

[0048] Fig.10 Schematic diagram of potassium ion detection results according to an embodiment of the present disclosure.

[0049] Fig.11 Schematic diagram of calcium ion detection results according to an embodiment of the present disclosure.

[0050] Fig.12 Schematic diagram of chloride ion detection results according to an embodiment of the present disclosure.

[0051] Fig.13 is a schematic diagram of hydrogen ion detection results according to an embodiment of the present disclosure.

[0052] Explanation of the reference numerals: 1. substrate; 2. electrode layer; 201. working electrode area; 202. reference electrode area; 203. signal line; 3. insulating layer; 301. first opening; 4. microfluidic layer; 401. second opening; 402. microfluidic channel; 403. first liquid inlet; 404. liquid outlet; 5. attachment layer; 501. second liquid inlet. DETAILED DESCRIPTION

[0053] Exemplary embodiments will be described in detail herein, examples of which are shown in the accompanying drawings. When the following description refers to the drawings, the same numbers in different drawings represent the same or similar elements unless otherwise indicated. The embodiments described in the following exemplary embodiments do not represent all embodiments consistent with the present disclosure. Instead, they are merely examples of devices consistent with some aspects of the present disclosure as detailed in the appended claims.

[0054] The terms used in the present disclosure are only for the purpose of describing specific embodiments and are not intended to limit the present disclosure. Unless otherwise defined, the technical terms or scientific terms used in the present disclosure should be understood by people with ordinary skills in the field to which the present disclosure belongs. The words "first", "second" and similar words used in the present disclosure specification and claims do not indicate any order, quantity or importance, but are only used to distinguish different components. Similarly, words such as "one" or "one" do not indicate a quantity limit, but indicate that there is at least one. "Multiple" or "several" means two and more than two. Unless otherwise specified, words such as "front", "rear", "lower" and / or "upper" are only for the convenience of explanation and are not limited to one position or one spatial orientation. Words such as "include" or "comprise" mean that the elements or objects appearing in front of "include" or "comprise" include the elements or objects listed after "include" or "comprise" and their equivalents, and do not exclude other elements or objects. Words such as "connect" or "connected" are not limited to physical or mechanical connections, and can include electrical connections, whether direct or indirect. As used in this disclosure and the appended claims, the singular forms "a", "an", "said", and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It should also be understood that the term "and / or" as used herein refers to and encompasses any or all possible combinations of one or more of the associated listed items.

[0055] The present disclosure provides a biochip. Figure 1 and Figure 4 As shown, the biochip may include a substrate 1, an electrode layer 2, an insulating layer 3 and a microfluidic layer 4, wherein:

[0056] The electrode layer 2 is disposed on one side of the substrate 1 and includes a plurality of electrode regions disposed at intervals. The insulating layer 3 covers the electrode layer 2 and is provided with a first opening 301 exposing the plurality of electrode regions. The microfluidic layer 4 covers the insulating layer 3 and includes a first liquid inlet 403, a microfluidic channel 402 and a second opening 401 which are connected in sequence, and the second opening 401 exposes at least a portion of the first opening 301.

[0057] In the biochip of the disclosed embodiment, during use, the first liquid inlet 403 can receive the sample liquid, and the microfluidic channel 402 constitutes a capillary. Under the action of the capillary, the sample liquid in the first liquid inlet 403 enters the second opening 401 through the microfluidic channel 402. Since the electrode area is exposed through the first opening 301, and the second opening 401 exposes at least a part of the first opening 301, the sample liquid entering the second opening 401 can contact the electrode area, and then be detected. In addition, the biochip is small in size and easy to carry.

[0058] The following is a detailed description of each part of the biochip according to the embodiment of the present disclosure:

[0059] The substrate 1 may be a rigid substrate. The rigid substrate may be a glass substrate or a PMMA (Polymethylmethacrylate) substrate, etc. Of course, the substrate 1 may also be a flexible substrate, so that the biochip disclosed in the present invention has the advantages of being thin, soft and comfortable to wear. The flexible substrate may be a PET (Polyethyleneterephthalate) substrate, a PEN (Polyethylene naphthalate twoformic acid glycol ester) substrate or a PI (Polyimide) substrate.

[0060] The material of the electrode layer 2 may be a metal material, such as Au, Ag, Pd, Pt, etc. The electrode layer 2 may include a plurality of electrode regions arranged at intervals. Figure 2 As shown, the plurality of electrode regions may include a reference electrode region 202 and a working electrode region 201. The number of the reference electrode region 202 may be one. The number of the working electrode regions 201 may be one, two, three or more. The shapes of any two working electrode regions 201 in the plurality of working electrode regions 201 may be the same. The sizes of any two working electrode regions 201 in the plurality of working electrode regions 201 may be the same. The shape and size of the reference electrode region 202 may be the same as the shape and size of the working electrode region 201. For example, the working electrode area 201 is in the shape of a rectangle, the long side dimension b of the working electrode area 201 can be 5.5mm-6.5mm, for example 6mm, and the short side dimension a of the working electrode area 201 can be 2.5mm-3.5mm, for example 3mm; the reference electrode area 202 is in the shape of a rectangle, the long side dimension of the reference electrode area 202 can be 5.5mm-6.5mm, for example 6mm, and the short side dimension of the reference electrode area 202 can be 2.5mm-3.5mm, for example 3mm.

[0061] like Figure 2As shown, the multiple working electrode areas 201 can be arranged around the reference electrode area 202 to reduce the influence of the reference electrode area 202 on the detection result. Taking the shape of the working electrode area 201 and the reference electrode area 202 as a rectangle and the number of the working electrode areas 201 as an example, the three working electrode areas 201 are arranged around the reference electrode area 202, the long side of one working electrode area 201 among the three working electrode areas 201 is parallel to the long side of the reference electrode area 202, the long side of another working electrode area 201 is parallel to the short side of the reference electrode area 202, and the long side of the remaining working electrode area 201 is parallel to the short side of the reference electrode area 202. In addition, the distance c between the working electrode area 201 and the reference electrode area 202 can be 0.8mm-1.2mm, for example 0.8mm, 0.9mm, 1mm, 1.1mm, 1.2mm, etc., and since the long side dimension b of the working electrode area 201 and the long side dimension of the reference electrode area 202 are both 5.5mm-6.5mm, the short side dimension a of the working electrode area 201 and the short side dimension of the reference electrode area 202 are both 2.5mm-3.5mm, the voltage signal stability of the biochip disclosed in the present invention is optimal, and the voltage signal response error is within 10%. Specifically, taking the distance c between the working electrode area 201 and the reference electrode area 202 as 1 mm, the long side dimension b of the working electrode area 201 and the long side dimension of the reference electrode area 202 as 6 mm, and the short side dimension a of the working electrode area 201 and the short side dimension of the reference electrode area 202 as 3 mm as an example, the biochip of the present disclosure is used to measure potassium ions, and the schematic diagram of the voltage signal is as follows: Figure 8 As shown in the figure, S1, S2, and S3 correspond to the three measurement results of potassium ions, and their slopes are basically the same, indicating that the stability of the voltage signal is excellent. Figure 2 As shown, the electrode layer 2 may further include a plurality of signal lines 203. The plurality of signal lines 203 are electrically connected to the plurality of electrode regions in a one-to-one correspondence. In addition, the thickness of the electrode region is relatively small, and there is no stimulation to the skin during operation, so that the skin is not damaged.

[0062] The biochip may also include a sensitive functional layer. The sensitive functional layer may be disposed on the surface of the working electrode area 201 facing away from the substrate 1. The sensitive functional layer may include ion carriers so that the working electrode area 201 forms an ion selective electrode, thereby enabling the biochip to be used to measure ions. For example, the sensitive functional layer may be a sodium ion sensitive functional layer, a potassium ion sensitive functional layer, a calcium ion sensitive functional layer, a hydrogen ion sensitive functional layer or a chloride ion sensitive functional layer, so that the biochip may be used to measure sodium ions, potassium ions, calcium ions, hydrogen ions, chloride ions, and the like. In the working electrode area 201, there may be two working electrode areas 201 with different sensitive functional layers disposed on their surfaces, so that the biochip of the present disclosure can simultaneously detect multiple ions in the sample solution. Through the sensitive functional layer, high sensitivity and high accuracy detection can be achieved with only a very small amount of sample solution.

[0063] like Figure 1 and Figure 3 As shown, the insulating layer 3 can cover the electrode layer 2 and the substrate 1. The insulating layer 3 is provided with a first opening 301 exposing multiple electrode regions. In one embodiment, the number of the first opening 301 is one, and the multiple electrode regions are exposed through the first opening 301. In another embodiment, the number of the first opening 301 is multiple, and the multiple first openings 301 expose the multiple electrode regions one by one.

[0064] like Figure 1 As shown, the material of the microchannel layer 4 can be an insulating material. The microchannel layer 4 covers the insulating layer 3. Figure 4 As shown, the microfluidic layer 4 may include a first liquid inlet 403, a microfluidic channel 402, a second opening 401, and a liquid outlet 404. The first liquid inlet 403 is used to receive the sample liquid. The first liquid inlet 403 and the second opening 401 are spaced apart, and the microfluidic channel 402 connects the first liquid inlet 403 and the second opening 401, so that the sample liquid received by the first liquid inlet 403 enters the second opening 401 through the microfluidic channel 402. Figure 1 As shown, at least part of the areas of the plurality of first openings 301 are exposed through the second openings 401, and since the electrode area is exposed through the first openings 301, the sample liquid entering the second openings 401 can contact the electrode area. The liquid outlet 404 is connected to the second opening 401, so that the sample liquid after detection flows out from the liquid outlet 404.

[0065] The shape of the first liquid inlet 403 can be circular, rectangular, etc. The shape of the second opening 401 can be circular, rectangular, etc. The liquid outlet 404 can be a liquid outlet trough, etc. For example, Figure 4As shown, the first liquid inlet 403 is circular in shape, and the diameter n of the first liquid inlet 403 can be 1mm-2mm, for example, 1.5mm; the second opening 401 is rectangular in shape, and the long side dimension d of the second opening 401 can be 14mm-20mm, for example, 14.4mm, and the short side dimension e of the second opening 401 can be 8mm-9mm, for example, 8.5mm; the liquid outlet 404 is a liquid outlet groove, and the width f of the liquid outlet groove can be 1.5mm-2.5mm, for example, 2.0mm; under this preset value, after simulation, the efficiency of sample liquid injection, flow and outflow is optimal. The time required from sample liquid injection to detection completion is less than 10min. Among them, the area of ​​the second opening 401 can be larger than the area of ​​the first liquid inlet 403. The width of the microchannel 402 gradually decreases from the second opening 401 to the first liquid inlet 403 . This arrangement can ensure the injection efficiency of the sample liquid, so that the sample liquid can quickly enter the second opening 401 from the first liquid inlet 403 through the microchannel 402 .

[0066] like Figure 4 As shown, the number of the first liquid inlet 403 and the microchannel 402 can be multiple, the number of the second opening 401 can be one, and the multiple first liquid inlets 403 are connected to the second opening 401 through the multiple microchannels 402 in a one-to-one correspondence. For example, the number of the first liquid inlet 403 and the microchannel 402 is two. Taking the shape of the second opening 401 as a rectangle as an example, the two microchannels 402 are connected to two adjacent corners of the second opening 401 in a one-to-one correspondence. In addition, the two microchannels 402 are axially symmetrically distributed, and the two first liquid inlets 403 are also axially symmetrically distributed, and the symmetry axes of the two microchannels 402 can coincide with the symmetry axes of the two first liquid inlets 403. Among them, the symmetry axes of the two microchannels 402 can be parallel to the short sides of the rectangular second opening 401, and of course, can also be parallel to the long sides. The above-mentioned liquid outlet 404 can be located on the opposite sides of the second opening 401 in a one-to-one correspondence with the first liquid inlet 403. Taking the second opening 401 as a rectangular shape as an example, the liquid outlet 404 and the first liquid inlet 403 are located on both sides of the short side direction of the second opening 401. According to the pressure drop formula of the microchannel 402: And the capillary pressure formula It can be seen that reducing the cross section of the microchannel 402 increases the pressure drop; increasing the cross section of the microchannel 402 increases the volume and prolongs the filling time.

[0067] like Figure 5 and Figure 6As shown, the biochip of the present disclosure may further include an attachment layer 5. Through the attachment layer 5, the biochip can be attached to human skin or other animal skin. The attachment layer 5 can be arranged on the side of the microfluidic layer 4 away from the substrate 1, and the attachment layer 5 is provided with a second liquid inlet 501 connected to the first liquid inlet 403. When the attachment layer 5 is in contact with human skin or the skin of other animals, the sweat produced by the human body or the animal can enter the second liquid inlet 501. Since the second liquid inlet 501 is connected to the first liquid inlet 403, the sweat enters the first liquid inlet 403, and finally enters the second opening 401 of the microfluidic layer 4 through the microfluidic channel 402 to contact the electrode area.

[0068] The present disclosure also provides a method for preparing a biochip, which is used to prepare the above-mentioned biochip. Figure 7 As shown, the method for preparing the biochip may include steps S100 to S130, wherein:

[0069] Step S100: providing a substrate.

[0070] Step S110: forming an electrode layer on one side of the substrate, wherein the electrode layer includes a plurality of electrode regions arranged at intervals.

[0071] Step S120: forming an insulating layer covering the electrode layer, wherein the insulating layer is provided with a first opening exposing a plurality of electrode regions.

[0072] Step S130, forming a microchannel layer covering the insulating layer, wherein the microchannel layer includes a first liquid inlet, a microchannel, and a second opening that are sequentially connected, and the second opening exposes at least a portion of the first opening.

[0073] The biochip prepared by the method for preparing the biochip according to the embodiment of the present disclosure is the same as the biochip in the above-mentioned embodiment of the biochip, and therefore, it has the same beneficial effects, which will not be described in detail in the present disclosure.

[0074] The following is a detailed description of each step of the method for preparing a biochip according to an embodiment of the present disclosure:

[0075] In step S100 , a substrate is provided.

[0076] Taking the substrate as a flexible substrate as an example, the flexible substrate is supported on a carrier. The process of supporting the flexible substrate on the carrier includes: spin coating polydimethylsiloxane (PDMS) on the surface of the carrier; cleaning the flexible substrate with acetone, ethanol, ultrapure water and oxygen plasma in sequence, and bonding the cleaned flexible substrate to the carrier by spin coating the dimethylsiloxane on the surface of the carrier, thereby supporting the flexible substrate on the carrier.

[0077] In step S110 , an electrode layer is formed on one side of a substrate, wherein the electrode layer includes a plurality of electrode regions that are spaced apart from each other.

[0078] The present disclosure can form the electrode layer by a magnetron sputtering process. Specifically, step S110 may include: forming a first photoresist layer on one side of the substrate; patterning the first photoresist layer to form a plurality of through holes exposing the substrate on the first photoresist layer; using the patterned first photoresist layer as a mask, magnetron sputtering the substrate to form an electrode material layer, which fills the plurality of through holes and covers the surface of the first photoresist layer facing away from the substrate; immersing the electrode material layer in a stripping solution for 30 minutes, stripping the portion of the electrode material layer outside the through holes to obtain the electrode layer, wherein the portion of the electrode material layer located in each through hole constitutes the electrode region of the electrode layer. The photoresist used for the first photoresist layer may be AZ1500 photoresist, but the present disclosure is not limited thereto. The thickness of the first photoresist layer may be 2μm-3μm, for example, 2μm, 2.2μm, 2.5μm, 3μm, etc.

[0079] In step S120 , an insulating layer covering the electrode layer is formed, and the insulating layer is provided with a first opening exposing a plurality of electrode regions.

[0080] For example, step 120 may include: forming a second photoresist layer covering the electrode layer and the first photoresist layer; patterning the second photoresist layer to form a plurality of first openings exposing a plurality of electrode regions on the second photoresist layer, wherein the first photoresist layer and the patterned second photoresist layer constitute the insulating layer of the present disclosure. The photoresist used for the second photoresist layer may be AZ1500 photoresist, but the present disclosure is not limited thereto. The thickness of the second photoresist layer may be 2 μm-3 μm, for example, 2 μm, 2.2 μm, 2.5 μm, 3 μm, etc.

[0081] In step S130, a microchannel layer covering the insulating layer is formed, the microchannel layer comprising a first liquid inlet, a microchannel and a second opening which are connected in sequence, and the second opening exposes at least a portion of the first opening.

[0082] For example, step S130 may include: forming a third photoresist layer covering the insulating layer, patterning the third photoresist layer to form a first liquid inlet, a microchannel, and a second opening that are sequentially connected, wherein the patterned third photoresist layer constitutes the microchannel layer of the present disclosure. The third photoresist layer may be SU8 photoresist, but the present disclosure is not limited thereto.

[0083] The preparation method of the biochip disclosed in the present invention may also include: forming a sensitive functional layer on the surface of the working electrode area facing away from the substrate. Taking the sensitive functional layer as a sodium ion sensitive functional layer as an example, the preparation process of the sodium ion sensitive functional layer is: preparing a membrane mixture containing sodium ion carrier X (1% w / w), Na-TFPB (0.5% w / w), PVC (33% w / w) and DOS (65.5% w / w), and dissolving 100 mg of the membrane mixture in 660 μL of THF to form an ion selection solution; drop-casting 10 μL of the ion selection solution onto the surface of the working electrode area facing away from the substrate to form a sensitive functional layer. Wherein, the sodium ion carrier X is 4-tert-butyl cup [4]-arene-tetraethyl tetraacetate.

[0084] The principle of sodium ion detection includes: a sodium ion selective carrier X is provided on the working electrode area, and the carrier generates a response potential by identifying the sodium ions in the substance to be tested. In the reaction, the response potential is proportional to the logarithm of the sodium ion concentration.

[0085] Taking the potassium ion sensitive functional layer as an example, the preparation process of the potassium ion sensitive functional layer is: prepare a membrane mixture containing valinomycin (2% w / w), sodium tetraphenylborate Na-TFPB (0.5% w / w), PVC (33% w / w) and DOS (64.5% w / w), and dissolve 100 mg of the membrane mixture in 350 μL of cyclohexanone to form an ion selective solution; drop-cast 10 μL of the ion selective solution onto the surface of the working electrode area facing away from the substrate to form a sensitive functional layer.

[0086] The principle of potassium ion detection includes: a potassium ion selective carrier valinomycin is fixed on the working electrode area, and the carrier generates a response potential by identifying potassium ions in the substance to be tested. The response potential in the reaction is proportional to the logarithm of the sodium ion concentration.

[0087] Taking the chloride ion sensitive functional layer as an example, the preparation process of the chloride ion sensitive functional layer is: coating Ag / AgCl ink on the surface of the working electrode area facing away from the substrate to form the sensitive functional layer.

[0088] The principle of chloride ion detection includes: Ag / AgCl ink is fixed on the working electrode area. When the electrode is immersed in a solution containing chloride ions, a corresponding membrane potential will be generated, and the response potential is proportional to the logarithm of the chloride ion concentration.

[0089] Taking the sensitive functional layer as a calcium ion sensitive functional layer as an example, the preparation process of the calcium ion sensitive functional layer is: prepare a membrane mixture containing ETH129 (1% w / w), Na-TFPB (0.5% w / w), PVC (33% w / w) and DOS (65.5% w / w), and dissolve 100 mg of the membrane mixture in 660 μL of THF to form an ion selective solution; drop-cast 10 μL of the ion selective solution onto the surface of the working electrode area facing away from the substrate to form a sensitive functional layer.

[0090] The principle of calcium ion detection includes: a calcium ion selective carrier ETH129 is provided on the working electrode area, and the carrier generates a response potential by identifying the calcium ions in the substance to be tested. The response potential in the reaction is proportional to the logarithm of the sodium ion concentration.

[0091] Taking the sensitive functional layer as a hydrogen ion sensitive functional layer as an example, the preparation process of the hydrogen ion sensitive functional layer is: distilling aniline at a vapor temperature of 100°C and a pressure of 13 mmHg; polymerizing polyaniline (PANI) in a 0.1M aniline and 0.1M HCl solution; first modifying the surface of the working electrode area by depositing Au at 0V for 30s, and then using cyclic voltammetry to perform 25 cycles of PANI deposition from -0.2V to 1V at 200mV / s to form a sensitive functional layer. In addition, after forming the hydrogen ion sensitive functional layer, the present disclosure can drop-cast 20μL of Nafion solution onto the hydrogen ion sensitive functional layer to form a gel layer.

[0092] The principle of hydrogen ion (pH value) detection includes: the working electrode area is modified with polyaniline (PANI). When the electrode is immersed in a solution, the PANI contacts the hydrogen ions in the solution and generates a corresponding membrane potential. The response potential is proportional to the pH value. At the same time, the Nafion solution is drop-cast on the sensitive functional layer, and the gel layer formed has selective permeability, which improves the utilization rate of the electrode, and at the same time plays a role in protecting the electrode and increasing the reliability of the electrode.

[0093] In addition, before forming any of the above-mentioned sensitive functional layers, the present invention can perform pre-treatment on the working electrode, specifically including: depositing a solution containing 0.01M 3,4-ethylenedioxythiophene (EDOT) and 0.1M sodium polystyrene sulfonate (NaPSS) onto the surface of the working electrode area facing away from the substrate through constant current electrochemical polymerization of an external Ag / AgCl electrode to reduce the influence of interfering ions.

[0094] The surface of the reference electrode facing away from the substrate can also be covered with a functional layer. The preparation process of the functional layer is as follows: 79.1 mg PVB and 50 mg NaCl are dissolved in 1 mL Methanol to prepare a mixture, 2 mg F127 and 0.2 mg Multiwall carbon nanotubes are added to the membrane mixture solution, and 10 μL of ion selective solution is drop-casted onto the surface of the reference electrode area facing away from the substrate to form a functional layer. By forming a functional layer on the surface of the reference electrode area, the signal drift of the biochip can be reduced and the accuracy of detection can be improved.

[0095] like Figures 9 to 13 It can be seen that through the above-mentioned sensitive functional layer, the concentration range of sodium ion detection is 10-160mM, and the sensitivity is 101mV / decade; the concentration range of potassium ion detection is 1-20mM, and the sensitivity is 81mV / decade; the concentration range of calcium ion detection is 0.1-20mM, and the sensitivity is 30mV / decade; the concentration range of chloride ion detection is 10-120mM, and the sensitivity is 30mV / decade; the range of pH value detection is 3-8mM, and the sensitivity is 50mV / decade; at the same time, in the repeatability test, the error range of the sensor response value is within 10%. The gel layer formed by the Nafion solution effectively avoids the reduction of detection sensitivity caused by the dissolution and consumption of the sensitive functional layer under long-term detection.

[0096] The present disclosure also provides a biological detection device. The biological detection device may include the biochip described in any of the above embodiments. Since the biochip included in the biological detection device is the same as the biochip in the above embodiment of the biochip, it has the same beneficial effects, and the present disclosure will not repeat them here.

[0097] The above is only a preferred embodiment of the present disclosure and does not limit the present disclosure in any form. Although the present disclosure has been disclosed as a preferred embodiment as above, it is not used to limit the present disclosure. Any technician familiar with this profession can make some changes or modify the technical contents disclosed above into equivalent embodiments without departing from the scope of the technical solution of the present disclosure. However, any simple modification, equivalent change and modification made to the above embodiments based on the technical essence of the present disclosure without departing from the content of the technical solution of the present disclosure are still within the scope of the technical solution of the present disclosure.

Claims

1. A biochip, characterized in that: include: substrate; An electrode layer, disposed on one side of the substrate and comprising a plurality of electrode regions disposed at intervals; The plurality of electrode regions include a reference electrode region and a plurality of working electrode regions; the plurality of working electrode regions are arranged around the reference electrode region; the distance between the working electrode region and the reference electrode region is 0.8 mm-1.2 mm; the shape of the working electrode region and / or the reference electrode region is rectangular, and the long side dimension is 5.5 mm-6.5 mm, and the short side dimension is 2.5 mm-3.5 mm; an insulating layer, covering the electrode layer and having a first opening exposing a plurality of the electrode regions; A microfluidic channel layer covers the insulating layer, and the microfluidic channel layer includes a first liquid inlet, a microfluidic channel, and a second opening that are connected in sequence, and the second opening exposes at least a portion of the first opening.

2. The biochip according to claim 1, characterized in that: There are multiple first openings, and the multiple first openings expose multiple electrode regions one by one, and at least a portion of each first opening is exposed through the second opening.

3. The biochip according to claim 1, characterized in that: A sensitive functional layer is disposed on the surface of the working electrode region facing away from the substrate.

4. The biochip according to claim 3, characterized in that: The plurality of electrode areas include a plurality of working electrode areas, and the sensitive functional layers arranged on the surfaces of two of the plurality of working electrode areas are different.

5. The biochip according to claim 3 or 4, characterized in that: The sensitive functional layer is a sodium ion sensitive functional layer, a potassium ion sensitive functional layer, a calcium ion sensitive functional layer, a hydrogen ion sensitive functional layer or a chloride ion sensitive functional layer.

6. The biochip according to claim 1, characterized in that: The area of ​​the second opening is larger than the area of ​​the first liquid inlet.

7. The biochip according to claim 6, characterized in that: The first liquid inlet is circular and has a diameter of 1 mm to 2 mm.

8. The biochip according to claim 6, characterized in that: The second opening is rectangular, with a long side dimension of 14 mm-20 mm and a short side dimension of 8 mm-9 mm.

9. The biochip according to claim 1, characterized in that: The number of the microchannels and the number of the first liquid inlets are both two, and the two first liquid inlets are connected to the second openings through the two microchannels in a one-to-one correspondence.

10. The biochip according to claim 9, characterized in that: The two microfluidic channels are distributed in an axisymmetric manner, and the two first liquid inlets are distributed in an axisymmetric manner, and the symmetry axes of the two microfluidic channels coincide with the symmetry axes of the two first liquid inlets.

11. The biochip according to claim 10, characterized in that: The second opening is rectangular, and the symmetry axes of the two microchannels are parallel to the short side or the long side of the second opening.

12. The biochip according to claim 9, characterized in that: The second opening is rectangular, and the two microchannels are connected to two adjacent corners of the second opening in a one-to-one correspondence.

13. The biochip according to claim 1, characterized in that: The width of the microchannel gradually decreases from the second opening to the first liquid inlet.

14. The biochip according to claim 1, characterized in that: The microchannel layer further includes a liquid outlet communicated with the second opening.

15. The biochip according to claim 14, characterized in that: The liquid outlets and the first liquid inlets are located on two opposite sides of the second opening in a one-to-one correspondence.

16. The biochip according to claim 15, characterized in that: The second opening is rectangular, and the liquid outlet and the first liquid inlet are located on two sides of the second opening in a short side direction.

17. The biochip according to claim 14, characterized in that: The liquid outlet is a liquid outlet groove, and the width of the liquid outlet groove is 1.5mm-2.5mm.

18. The biochip according to claim 1, characterized in that: The substrate is a flexible substrate.

19. The biochip according to claim 1, characterized in that: The biochip further comprises: The attachment layer is arranged on a side of the microchannel layer away from the substrate, and the attachment layer is provided with a second liquid inlet communicated with the first liquid inlet.

20. A method for preparing a biochip, characterized in that: include: providing a substrate; forming an electrode layer on one side of the substrate, wherein the electrode layer includes a plurality of electrode regions arranged at intervals; An insulating layer covering the electrode layer is formed, wherein the insulating layer is provided with a first opening exposing a plurality of the electrode regions; the plurality of the electrode regions include a reference electrode region and a plurality of working electrode regions; the plurality of working electrode regions are arranged around the reference electrode region; the distance between the working electrode region and the reference electrode region is 0.8 mm-1.2 mm; the shape of the working electrode region and / or the reference electrode region is rectangular, and the long side dimension is 5.5 mm-6.5 mm, and the short side dimension is 2.5 mm-3.5 mm; A microchannel layer covering the insulating layer is formed, wherein the microchannel layer comprises a first liquid inlet, a microchannel and a second opening which are connected in sequence, and the second opening exposes at least a part of the first opening.

21. A biological detection device, characterized in that: A biochip comprising any one of claims 1-19.

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