TW200407542A - Electro-chemical electrode sensing test sheet for screen printing and its manufacturing method - Google Patents

Abstract

The present invention discloses an electro-chemical electrode sensing test sheet for screen printing and its manufacturing method. In brief, the test sheet structure includes two or three electrodes on an insulating substrate; a response layer directly absorbed on the surface of electrode area; and a test-object diversion area above the electrode area, which is formed by assembling an intermediate slice with an U-shaped or T-shaped channel located above the electrode and an upper lid sheet thereon, respectively. The formed hollow trench provides capillary and siphon phenomenon capable of attracting an extremely small amount of test-object rapidly to the electrode reacting area. Then, the electrochemical method is utilized to detect the response generated by the reacting catalytic and electron transferring material in the test-object and reacting area, so as to analyze the subject matter composition in the predetermined amount of test-object. The upper lid sheet has an airtight protruded space facing the wire in the electrode area, which provides the function of controlling the volume of the test-object and filling and aligning the test-object effectively.

Description

200407542 V. Description of the invention (1) 1. [Technical field to which the invention belongs] The technical field to which the present invention belongs is the category of biosensors included under the heading "Biomedical Biomedical Materials and Originals": Structural design, preparation method and application of sensing test piece. The sensing test piece described in the present invention is based on the principle of electrochemical detection technology, and cooperates with the design of the test piece device structure, and performs corresponding chemical or biological chemistry by detecting the target and the reactants and catalysts coated on the electrode surface. The reaction results in a change in the amount of electron conduction to derive the concentration of the detection target. The structure of the test piece involves the design and manufacturing methods of liquid diversion pipes, and explains the control of the volume of the specimen and the possible pollution prevention methods. I. [Xianru Technology] In recent years, electrode-type test strips have been successfully commercialized in the production of various test products due to their popularity in easy-to-manufacture, low-cost, and portable, inexpensive detectors; for example, blood glucose, Various biochemical detectors such as uric acid and cholesterol. Take the largest and most widely used blood glucose meter in the market as an example. ’Technical leaders in the market include companies such as Roche, Abbott, Bayer, and Therasense, which produce blood glucose test strips electrochemically. The first generation of electrochemical blood glucose test strips have high blood volume requirements (more than 5-10 microliters) and long time (ranging from 30 to 60 seconds), although they have surpassed in terms of blood volume and test time. F-system methods such as optical colorimetric methods (c ο 1 〇rimetric), but are still not ideal in use. With the advancement of technology, the latest generation of Muyu's blood volume is only 0.3 microliters (Therasense Company

Freestyle product) or 1 microliter (Lifescan 〇neT〇uch Ultra product

5. Description of the invention on page 5 (2)), the time required has also been shortened to 5-15 seconds, so blood collection is small and fast, which has become a guideline for μ li > and detection electrode structure and technology development. , Tickets, and various years of US ntt indicators development. Designed by Diebold et al. In 1995 and unveiled at the two electric gates J, 屮 " No. 9-using a palm-type electrode port, i = = capillary guide area and a circular guide with an upward opening containing a single brush System, but no matter what kind of system, its composition is made of two-phase ancient Sh plate. It is not only complicated and costly, but also costs 1 breed of ΐ extreme dog 1 blood: US patent 57 79867 in 1988, It is also revealed that-, the middle Li, the invention uses two upper and lower corresponding electrodes F, which are the reaction area and a round hole as the sample drop inlet, and a blood cell separation film in the sandwich area to filter red blood cells. In order to control the blood volume and use the separation membrane to remove the barrier of the second blood cell filter membrane, the optimal blood volume and speed cannot be achieved. Asia. Division A (U.S. Patent 6, 129, 823) covers the surface of the working electrode with a single layer or multiple layers of net layers, and the top layer is covered with a cover. This cover is located directly above the working electrode or near the working electrode. Where the sample is injected " It is also possible to achieve the purpose of only 2 · 0-2 · 5 microliters of small volume samples. However, this invention also uses the mesh layer as a way to reduce blood volume and distribute samples. Ding 1 ^ "33 6 113 6 company in US patents 6,299,757 and 6,338,79〇 two patents also disclosed the use of the palm electrode design, through the fine structure between the two electrodes, put a piece with excellent water absorption The specimen is sucked between the two electrodes for reaction. The use of tight water-absorbing film volume control can reduce the minimum volume required for the specimen to 0.3 microliters, which is currently the world ’s blood 200407542. V. Description of the invention (3) The design with the least amount of demand, but the invention is in the packaging process The more complicated one has the disadvantage of extremely high cost. Domestic 'Mr. Shen Yanshi and others also used the method and application of the first patent of the Republic of China. First, the conductive film and the electrical insulation layer were completed by screen printing, and then the metal layer was completed by electroplating. A circular recessed portion is formed everywhere, and the so-called biological layer is adsorbed in the recessed portion, and the specimen is directly dropped here during detection, which requires about 10 microliters of the specimen. This method requires tedious steps such as plating and high blood volume requirements. In addition, Wuding Company disclosed in the Republic of China Patent No. 1 24 332 that a diversion region was formed above the electrode area, and a retinal membrane containing a surfactant was covered above it. The capillary and siphon principle were used to aspirate the sample onto the electrode to react. This method, like Abbott's method, generally uses the covered omentum as a diversion design. In addition to increasing costs, it is also limited by the minimum sample demand. Winarta et al., U.S. Patent 6,258,229, No. 1, 2001, disclose a type of detection device ' that claims to require less than i microliters of liquid specimen. Its tin / present 1 gold bar / polystyrene (g〇ld / PolyeSter) or oxygen 4 dagger, gold, / ethylene (tin 〇xide / g〇ld / poiyester) The surface layer is a vaporized carbon laser frni \ like three electrodes—two 2 er), the last piece is engraved to form a surface, the two non-conducting blocks; working on the working electrode; end A middle layer containing w openings is pasted on the middle layer of the upper clump gate of this shape of the ventilation hole. The paste-containing-cuboid entrance to the pass-through / top electrode forms a liquid channel between the liquids which is supported by the liquid. Liquid passages limited the amount of megu which could be accumulated to 1 microliter in 1992. This design, as disclosed by Nankai et al., Was earlier than that in Li 51 2 0 4 2 〇, but in the conductive layer of the two.

Page 7 200407542 When the specimen in the vent is left in the ventilation subtraction sheet, the opening control of the finger on the plate revealed by the finger and then attached to an electrode is controlled, such as an end sticker containing a five, invention description ( 4) The electrode manufacturing method is different. The printing method of Nankai and other editions is to attach two spacer layers on the insulation back. 1 This forms a horizontal use of work to enter the channel, which cannot cause pollution. Another "amplified design" is to form a liquid inlet to the structure on the working electrode and then paste a piece on it. When the vent is too small, there is still a moderate mouth, and the specimen will stop and shrink. Today, the outflow of the sample is caused by taking the air port by hand, and the electrode test strip is made by a conductive layer in the form of a mesh double electrode. The two front and rear covers without working holes at the working electrode, such as mouth channels. The specimen is tested in this way and will flow from the other end of the channel. As described above, Winarta et al. Have a U-shaped opening in the middle layer of the ventilation hole cover, and then the liquid channel between them. There is a risk that such a vent will flow out; when the edges of the vent, especially when trying to test, it is easy to accidentally touch the pollution. III. [Contents of the invention] From the above points k -c. V 4. n. Jin, σ understands that if you want to reduce the volume of the specimen, ^ k speed analysis must also avoid the possibility of contamination of the specimen, 1 army : The principle of fast thinning and siphon and the design of the electrode can be achieved :; of :; The invention of wool is based on the above-mentioned purpose and the design of the capillary and siphon principle is combined to obtain a fast-flowing electrode reaction zone. The zone sample (about 0.5-0.8 microliters) can make the electrode work normally, and the whole time is about 5 to 10 seconds. It is a fast and accurate electrochemical time-lapse film with a simple structure, easy production and complete It is not necessary to cover the omentum, and the test can be avoided. 200407542 V. Description of the invention (5) The pollution sensing test strip caused by the external leakage of the test strip The structure of the electrochemical tripolar screen printing biosensing test strip of the present invention is shown in FIG. The bottom plate 1, such as polyvinyl chloride (PVC), polyester (pe) / polycarbonate (pc), etc., is made of conductive material, such as silver, gold, and other gel-like materials, and is screen-printed to form wires 2 . Then, a conductive gel material such as carbon, gold, or platinum is printed on the wire 2 to form a long strip of the electrode. One end is the working electrode 3, the reference electrode 4, and the auxiliary electrode 5 (there is no reference electrode in the bipolar design), and the other end is 3, 4, and 5, and the knowledge of the opposite electrode is the same as the detector. Contact, and 6 is the automatic identification line of the test piece of the tester. A non-conductive intermediate layer or insulating glue layer 7 containing U-shaped holes is pasted or screen-printed on the substrate printed with electrodes, which is an insulating dielectric layer and has a spacer function. The groove 7a is a specimen guide groove, and the upper cover 8 forms a closed and raised space $ a at the bottom of the opposite guide groove, and its internal volume is about 2 microliters. The electrode reaction zone coating reagent formula includes reactants, reaction catalysts (such as enzymes), electron conducting substances (such as dimethyl ferrocene, tetrathiofulvalene, etc.), and wetting agents (cellulose ^ hydroxyethyl cellulose λpolyvinyl al_cohol, polyvinyl, pyrroiidone) , Gelatin, etc.) and surfactants (tween 20, triton X-100, surfynol, nie_ga8, etc.). After the spacer layer 7 and the upper cover layer 8 are bonded together, a capillary flow channel 7 a is formed around the electrode working area, allowing the specimen such as blood to contact the leading edge of 7 a and then quickly fill the electrode reaction area via capillary phenomenon. Samples and reactants

200407542

Through the action of the reaction catalyst and electron transfer material, the detector detects the current signal generated. Diversion ditch mentioned above ^ and

Hit "less than" withdraw "^) " 让 to make the electrode detect normally. The structure of another electrochemical tripolar electrode test strip according to the present invention is shown in Fig. 2 and 2. On the insulating base plate 1, a conductive material such as silver, vaporized silver-, or gold is used. Material = screen printing method, brush the conductive layer 2, and then cover the conductive layer with a conductive material such as Shishan Lei or platinum to form: J pole 4 and auxiliary electrode 5. The electrodes are opposite to each other-ends 3, 4, and 5, and the contact points to be tested; and 6 is the automatic identification of the test strip of the tester. The second line is = pasted on the base plate of the completed electrode layer or the last one. Space layer 7 of the font hole. The upper cover layer 8 includes the inter-partition 8a together with the plate-stamp I2. The internal volume of this protrusion is about 2 microliters, and 8a is just above the intersection of the 7th A ^ line. For the body diversion grooves 7b and 7C formed between the upper cover and the compartment layer, the exhaust ducts facing to both sides. Inspection, after contact by the leading edge of 7a, it quickly flows through the capillary

Sait ΞΓ, and the leading edge of the specimen is also shown in the figure-design. It does not exceed i. It can also be used to delete the exhaust channel on either side of 7b or 7c. The base plate plastic is composed of two kinds of insulating materials, such as polymers, and two kinds of application materials, such as polymer, and beams. The choice of materials must match the electrode material 4. If it is an intrusive use, it should be selected Soft material 4

200407542 V. Description of the invention (7) In order to reduce pain and avoid tissue damage, such applications can usually use insulating polymers such as polycarbonate, polyester (such as DuPont's My 1 ar series products), Polyethylene terephthalate (PET ), Polyvinyl chloride (pvc), polyether, polyamine, polyurethane, polyimide, and the like. On the other hand, rigid materials such as ceramic materials such as stone dioxide or alumina can be used. For in vitro detection purposes, the size and width of the bottom plate is generally between 3-15 mm, more accurately between 5-10 mm, and the thickness is about 50-800 microns or more accurately between 200-4. 〇 microns, the length of the bottom plate may be affected by various factors between 8 cm, more accurately 2-5 cm. The wire layer and the electrode are shown in Figure 1. Conductive materials such as silver, gold, and printed materials * e > 1 gold and other materials are used to form a wire area 2 on the base plate i by a brush method: Use high conductivity: Connect the resistance of each electrode and the measuring electrode, the material with ancient resistance characteristics can reduce Er to detect the current signal once. Conductivity; bh 1 α »brushed on the wire 2, 6 as the master, 9, and dwell * 14 materials, such as carbon glue, At will be used as the test piece of the detector; In addition to the electrode 4, the brushed wire 2 is used for identification purposes. In addition to using the electrochemical principle of the wire part, S 2 ^, 4, and silver can be exposed: the electrode, or brushed with silver / silver chloride ink. t Cover: The silver ginseng 2 is made of silver / gasified silver ink to brush the wire, or the wire is treated with silver. 'Wanwan is also made of a chlorinated insulating layer, and the intermediate edge layer 7' can be made of an insulating dielectric material 200407542 V. Description of the invention (8) Or it can be covered on the electrode surface by adhesive method, except that it is not necessary to shield The exposed carbon surface is outside and provides a fixed area reaction zone. Reaction reagent zone The reaction reagent is coated on the electrode zone and includes reaction catalysts, buffer solutions, fillers, electron transfer substances, and surfactants for the reaction. For example, the reaction catalyst used in glucose detection can be glucose oxidase or dehydrogenase. The filler components include polymers or wetting agents such as cellulose, polyethanol, gelatin, etc., and surfactant components such as Tween-80, Triton. X-100, Surfynol, or Mega8, etc., provide the function of dissolving and dispersing specimens and reagents, as well as the hydrophilicity and dispersing function of capillary diversion area. Therefore, the reaction reagent layer can provide both reaction and capillary functions, which not only allows the sample to fill the electrode area and allow the analytical reaction to proceed, but also provides the electrode reaction current as a function to quantify the composition of the sample. The more suitable electron-transporting substance should have an oxidation-reduction potential between -100 and +200 mV, and different electron-transporting substances should be applied according to different detection requirements. For example, dimethylferrocene and tetrathiafulvalene or derivatives or complexes of the above can be used in the detection of glucose. The lower potential can avoid the sample. Interfering substances in the high electron transfer efficiency can provide a stronger current signal. The buffer solution is used to maintain a certain range of pH value. Generally, the pH value is between 4-9, which is more suitable for pH value between 5-8. The components of the buffer that can be used include phosphate, salt, citrate, and the like, and the concentration range is from 10 to 10,000 millimoles & the optimum concentration is about 30 to 100 millimoles. eye

Page 12 200407542 V. Description of the invention (9) The capillary diversion layer and the diversion layer are formed by covering the grid sheet 7 and the upper cover 8 above the electrode; the capillary grooves of the specimen, such as those having the function of exhausting to both sides (The τ-shaped design L has 7b and 7c as exhaust ports, and 7b or 7c can exist separately. The size of the diversion area can be adjusted by using the thickness of the sheet 7 and the width of the groove 7a. Generally, the thickness is between 5 and 5. -200 micron, the optimal thickness is 10-50 microns, the length of the hollow area is between 2-8 mm, the width is between 0.5 and 5 mm, and the optimal width is between Bu Guang. The volume of the hollow area formed is between 0. ^ .6 microliters, actual inspection = the required amount is about 0.5-2 microliters during operation, and the time required for the sample to reach the edge of the diversion layer to be filled is less than 1 second. It can be round, square, or several other channels used as electrode guides, and it will stop after advancing in the full reaction zone. Interval: 7 points and Λ make; ί: Ξμχ materials and other materials, such as plastic or high For molecular materials such as PVC, the 8a of the area above the diversion area can be transparent. Use f to visually check whether the sample is normal. It is full and has protection. "The upper cover layer is the so-called upwardly raised space, which can be formed in two steps. The first step is to make the figure-the so-called upper cover is shown in Figure 5) '8,83 and 9 can be complete—take 9 (as shown in Figure 4, Figure 3, and Figure 6 and Figure 1 and a longitudinal section containing a thin sheet of 9 test strips. 200407542 V. Description of the invention (ίο) Fill sensor full sensor When the three-electrode assisted electrode and the working position or resistance value are monitored and the electrode is filled, the electrification is started at the same time. The distribution principle and the sample fluid are monitored by the same principle. The auxiliary electrode can also be used in the design of the device. If the working electrode can be monitored, the impedance between the auxiliary electrodes will be sharply divided. When the electric direction is consistent, the auxiliary electrode must be connected last to detect that the impedance of the electrode has not decreased in a filled body. 'That is to say, the test specimens are arranged in the three groups of conductive conductance areas to fill the three values, and this is the way to go' Infinite time The method must be used to determine the electrode design. The tester must be the most accurate point for the electrode. Fill the sample, use auxiliary current, electricity, and set the electricity. When the sample is filled, it can also be used to check whether the electrode is full. Electrochemical analysis 1 After the electrode assembly is cut by slitting or punching, an external palm-type electrochemical instrument can be used for &#, n ^ silk + 1 丨 g suitable specimen analysis, and the analysis can be performed in various ways. Carry out, for example, to determine the stability of the current, "fl Λ ,,. ^ ^ ^ '", The 疋 potential method (0 — 0 · 3 volts) to measure the steady-state current, the total power value in your moan room, the power value For the current detection Si, the current can be used as the proportional relationship between the concentration of the components in the sample. The detection electrode is equipped with the electrode M 徭 cold a >, min ~ go + electricity to the moon, the main full sensing function, when the test When the tester is filled with signals, the automatic age and personal expenses will be automatically activated. This will increase the accuracy of the test, which will increase the accuracy of the test, especially when the overall test time is less than 10 seconds. Errors often cause large variations.

200407542 V. Description of the invention (11) IV. [Implementation method] The following examples are based on blood glucose testing, the purpose of which is just to explain the situation in this area ^ ○ Example 1, the screen printing of glucose-sensitive test pieces made of polypropylene synthesis A conductive silver adhesive layer was printed on a paper base plate with a 300 mesh screen, and after drying at 50 ° C for 30 minutes, three sets of electrodes (working electrode, reference electrode and auxiliary electrode) were brushed with carbon glue. Bake again at 90 ° C for 15 minutes, and then use an insulating rubber brush to harden and dry under UV light to form an insulating layer, and expose the pilot flow reaction zones 7a, 7b, and 7c (design with vent holes). 1-2 microliters of reaction reagents, (containing 0.5-3 units of glucose oxidase, 1-1. 1% polyvinyl alcohol, ρΗ4.0-9.0-9, 10-iomM potassium phosphate buffer, 10-100 mM potassium chloride, difluorenyl ferrocene 0.005-0.5%, tween-20 0.001-0.2%, surfynol 0 (Π-0.2%), coated on the aforementioned The formed diversion area 7 a 'is dried at 4 5 C for 1 hour, and then taken out and attached to the upper cover sheet 8 containing the central hole. After pasting, a transparent upper cover sheet 9 is pressed and fixed, and then cut. The finished product. Example 2. Standard glucose solution and whole blood test configuration. Standard potassium phosphate buffer solution (pH 7.4) contains a glucose concentration of 040 mg / 100 ml. Extreme glucose test strip, under the condition of electrochemical analyzer (CHlnstrumerU cq.65 (U) at a test potential of 100 millivolts), the test time is 8 seconds, and the volume of the supplied sample is 3 microliters for each test Every time the test piece is inhaled

200407542 V. Description of the invention (12) Less than 3 microliters. The test results are shown in Table 1. Table 1. Standard glucose test results Glucose concentration (mg / dl) Electricity (microcoulomb) 0 0. 690 25 1.532 50 2. 952 100 5. 248 200 7. 400 400 9. 577

Whole blood specimens can also be measured by this test strip. For freshly collected venous whole blood, the test data obtained by adding standard glucose is shown in Table 2. The test potential is 100 millivolts and the test whole blood volume is 2 microliters. Table 2. Test results of whole blood with different glucose contents.

Example three: blood glucose measurement with different whole blood volume

Page 16 Demand concentration of a system. The method is ′ and when the supply is low, {the density of the method is all more, this body. So proof five. Description of the invention (13) The principle of this test is 30 Omg / d1 using the sample blood to test the principle of the inhalation of the test. Seven results show that when the glucose concentration is too low, the whole volume of the blood test will be more. Inhale more test junctions, the electrode test piece of the foot, the blood volume 'venous whole blood is different, such as dry cases-an improper sample volume is close to being inhaled; it means that the sample is inhaled. The detection of the volume of the invention is sufficient (the specimens provided by the specimens are designed to provide different volumes of whole blood prepared with standard glucose solutions, so that the test strips are tested under rainbow conditions. As shown in Figure 0 · 5 microliters), the detection volume reaches a concentration of more than 0.8 microliters, and the higher the volume of the sample provided, the remaining application area has reached saturation, and it cannot exceed 8b and the diversion area is made of specimens. Volume function

200407542 Brief description of the drawings Figure 1. A schematic view of the U-shaped hole structure of the screen printing electrode test piece of the present invention. 1. Insulating base plate; 2. Conductor layer; 3. Working electrode; 4. Participant thunder pole; 5. Auxiliary thunder pole; 3 ', 4' and 5 ', detector contact point; 6. Detector test Automatic identification line of the film; 7, middle layer; 7 a, detection diversion groove; 8, upper cover layer; 8 a closed upward space. Fig. 2. Schematic diagram of the structure of the T-shaped hole of the screen printing electrode test piece of the present invention. 1. Insulating base plate; 2. Wire layer; 3. Working electrode; 4. Reference electrode; 5. Auxiliary electrode; 3 ', 4', 5 ', contact point of the detector; 6. Automatic identification line of the test piece ; 7, middle layer; 7 a, detection diversion groove; 7b, 7c, exhaust channel; 8, upper cover layer; 8a closed upward space. FIG. 3 is a schematic longitudinal sectional view of a screen printing electrode test piece according to the present invention. 1. Insulating base plate; 2. Conductor layer; 3. Electrode layer; 7. Intermediate layer; 7a; Detection diversion groove; 8. Upper cover layer; 8a. Fig. 4. Schematic diagram of the structure of the upwardly-enclosed closed space cover of the present invention. 8, upper cover sheet; 8 a, holes; 9, thin sheet. The upper cover may be a one-piece structure. Fig. 5. Schematic cross-sectional view of the structure of the upwardly-enclosed closed space cover of the present invention. 8, upper cover sheet; 8a, holes; 9, thin sheet. This cover may be a one-piece structure.

200407542 Brief description of the drawing Figure 6. The schematic diagram of the longitudinal section of the test piece of the enclosed type upwardly-enclosed closed space screen printing electrode of the present invention. 1. Insulating base plate; 2. Conductor 1 layer; 3. Electrode layer; 7. Intermediate layer; 7a; Detection diversion groove; 8. Top cover layer; 8a; Hole; 9. Thin sheet. Figure 7. The effect of whole blood sample volume on the test.

Page 19

Claims (1)

200407542 It consists of the following components: 1 · An electrochemical biosensing test piece-an insulating substrate; or conductive; layer electrode ΠΓ brush method ... brushed on the substrate-an electrode layer, covered by screen printing A two-electrode type of a working electrode and a reference electrode or a three-electrode type of a working electrode, a reference electrode and an auxiliary electrode is formed on a wire layer other than the reference electrode area, and includes an electrode area and a place connected to the detector Both ends;-an electrically insulating intermediate layer, directly brushed with insulating glue or directly covering the conductive layer with an insulating material, so that the electrodes and contact portions are exposed, and this intermediate layer has A U-shaped or T-shaped opening; a reactive active layer containing a reactant, a catalytic catalyst, an electronic medium, a surfactant and the like coated on the starting point of the u-shaped or τ-shaped channel to the surface of the electrode; The upper part of the electrically insulating intermediate layer is raised upwardly. The closed space is covered with a cover. This upper cover is attached to the electrically insulating intermediate layer, so that the u-shaped or T-shaped area forms a capillary flow guide area, which is relatively upward. Confined space from the bottom of the diversion zone. 2 · As described in item 1 of the scope of the patent application, the electrically insulating base plate is polycarbonate, polyacetate, polyether, polyamide, polyurethane, polyimide, polyvinyl chloride (PVC), glass, fiberglass board, Ceramic and polyethylene (PET). 3 · As described in item 1 of the patent application scope, the conductive layer is silver, silver chloride or 200407542 VI. 4 in the patent application scope, such as any 5 in the application of the patent, etc., such as the application of a special substance such as enzyme 6, such as the application of the special 500 micron 7, the application of the length of the channel, and the application of the special projection. Secret 9 · If you apply for a special examination body to cover 1 0 · If you apply for a special test, it will automatically start 11. A kind of electrification is completed: Any kind of material. As described in item 1, the electrode layer is made of carbon, silver, gold, or any other material. According to item 1 of the scope of interest, the reaction catalyst is a biochemical catalyst or other abiotic catalyst. According to the first item of the profit range, the thickness of the electrically insulating intermediate layer is between 5 and 1 ', and the optimum thickness is 70 to 50 microns. According to the first item of the profit range, the u-shaped 戋 τ-type dielectric of the electrically insulating intermediate layer = 2-8 mm, and the width is between 0.5-5 mm. According to item 1 of the profit range, the volume of the closed space relative to the electrically insulating intermediate layer is between 0.5-10 microliters. As described in the first item of the scope of benefit, the working electrode and auxiliary electrode have a detection function. As described in the first item of the scope of interest, the electrode lead area may have a set of functions capable of detecting the detector. A 'The method of making bio-sensing test piece is as follows: a. Use screen printing technology to print a conductive layer on one side of a substrate, then bake it at 40 ° C to 120 t: B. Brush the electrode layer on the aforementioned conductive layer by screen printing technology, and then dry it at 40 t: to 120 ° C; c. Use the screen printing technology to print the insulating adhesive layer on the aforementioned An intermediate layer containing a U-shaped notch is brushed on the base plate of the electrode layer, so that the working electrode, auxiliary electrode and reference electrode are located in the U-shaped hole, and 200407542 VI. The end of the patent application is exposed at the contact with the instrument; d. A reaction layer is applied between the U-shaped holes; e. A space cover with a closed upward protrusion is pasted on the middle layer, and this cover The protrusion is located at the bottom end of the U-shape; the space upper cover with a closed upward protrusion can be formed by covering a surface layer on the upper cover containing holes, or an integrally formed structure. 1 2. As described in item 11 of the scope of patent application, the manufacturing method of the intermediate layer can directly paste the insulating intermediate layer to replace the screen printing method. 1 3. As described in item 11 of the scope of the patent application, the U-shaped notch in the middle layer may be replaced by a T-shaped notch, and the T-shaped horizontal notch is a discharge to both sides of the test piece. Page 22