TWI239397B - 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

1239397 _ ^ " —----- — ^ ---- 5. Description of the invention (1) 1. [Technical field to which the invention belongs]

The technical field to which the present invention pertains is the scope of the biosensors under the heading "Biomedical Materials and Originals of Biotechnology": structure design, preparation methods, and applications of sensory test pieces. 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. Second, [prior art]

In recent years, electrode test strips have been successfully commercialized for the production of various test products due to their popularity of easy-to-manufacture, low-cost, and portable, inexpensive detectors; Device. Taking the largest and most widely used blood glucose meter in the market as an example, technology leaders on the market, including Roche, Abbott, Bayer, and Therasense, all produce blood glucose test strips electrochemically. The first generation of electrochemical blood glucose test strips had high blood volume requirements (more than 5 to 10 microliters) and long time (ranging from 30 to 60 seconds), although it has surpassed optics in terms of blood volume and detection time. Traditional methods such as color rendering (c ο 1 〇rimetric), but still not ideal in use. With the advancement of technology, the current latest generation of products requires only 0.3 microliters (Therasense Freestyle product) or 1 microliter (Lifesc an OneTouch Ultra product)

Page 6 1239397 V. Description of the invention (2)

), The time required has also been shortened to 5 to 15 seconds, so less blood collection and fast detection have become indicators of the development of such products and technologies, and the research and development of various electrode structures are also moving towards this indicator. D i ebo 1 d et al., US Patent No. 5,4 3,79,99, 195, discloses a design using a pair of palm electrodes and a capillary guide area between the two electrodes and an upward opening With a circular air port, the specimen can enter the reaction zone precisely and in small amounts. The invention can adopt photomask and screen printing processes, but no matter which method is used, the composition is made of two pieces of substrates containing single-strand electrodes, which are not only complicated and costly. Shieh's U.S. Patent No. 5 7 798 6 7 in 1988 also disclosed an electrode type blood glucose test strip design. The invention uses two upper and lower corresponding electrodes, the middle sandwich is the reaction area and a circular hole is left as At the entrance of the specimen, a blood cell separation membrane is included in the dissection area to filter red blood cells. This method uses the membranes sandwiched by the upper and lower test strips to control the blood volume and hopes to separate the membranes to remove the interference of blood cells. Achieve the best state in volume and speed. Abbott Corporation (U.S. Patent No. 6, 1 2 9, 8 2 3) 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, which is located directly above the working electrode or adjacent to the working electrode. A hole, where the specimen is injected. This design also achieves the purpose of only 2.0-2.5 microliters of small volume specimens. However, this invention also uses the mesh layer as a method to reduce blood volume and distribute samples. Ding 1161 ^ 361136 in U.S. Patent Nos. 6,299,757 and 6,338,790 discloses that the same palm electrode design is used to pass a fine structure between the two electrodes and put a sheet with excellent water absorption , Aspirate the specimen between the two electrodes for reaction. Utilizing tight water-absorptive film volume control, the minimum volume required for the specimen can be reduced to 0.3 microliters, which is currently the world's blood 1239397 V. Description of the invention (3)

The design with the least amount of demand, but this invention has the disadvantage of extremely high cost when the packaging process is more complicated. In China, Mr. Shen Yanshi and others also disclosed the method and application of screen printing electrodes in the Republic of China Patent No. 032398, which emphasized that the conductive film and electrical insulation layer were completed by screen printing, and then the metal layer was completed by electroplating, and A circular recess is formed at the working and reference electrodes with insulating glue. The so-called biological layer is absorbed in this recess, and the sample is directly dropped here during testing, which requires about 0 microliters of sample. 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 24332 that a diversion area was formed above the electrode area, and an omentum containing a surface active agent was covered on the top. The capillary and siphon principle were used to aspirate the specimen onto the electrode to respond to the problem. As with Abbott's method, the design of the covering omentum as a diversion method is generally limited by the minimum sample requirement in addition to increasing costs.

Winarta et al., U.S. Patent No. 62,582,291, 2001 discloses a disposable detection device ' that claims less than 1 microliter of liquid sample. Its preparation method is on the surface layer of gold / polystyrene (tin / ox / g〇u / polyester)

At one end, a carbon dioxide laser (C02 User) was used to etch the strip to form a three-electrode three-phase non-conducting block; on top of the working electrode, a paste containing U was pasted along the front end of the test strip. In the middle layer of the opening, the working electrode is located in the U-shaped groove, and an upper cover containing a square ventilation hole is pasted on the middle layer. Therefore, a liquid channel is formed at the upper end of the working electrode from the liquid inlet to the middle of the vent hole. This liquid channel limits the volume of liquid that can be withheld to less than 1 microliter. This design is as disclosed by Nankai et al. In 1992 U.S. Patent 5,120,420, but in the conductive layer of the two.

Page 8 1239397 V. Description of the invention (4) Different electrode printing methods The printing method is on an insulating back and its liquid channel group is pasted with two pieces of space

Nankai et al. Formed a layer on top of this way, which formed a lateral passage of working volume that could not be used to form another composition. The working electrode's access channel caused pollution. The design is to paste a structure on it. When the air vent is moderate, the current air port of the specimen piece is reduced, which causes the specimen to be printed on the plate, and the electrode is attached to the work. The control method is shown as making a two-piece opening and if the upper part is pasted with a tablet containing a liquid inlet in the middle to too small, it still happens to stop the sky and take outflow by hand, and the vent sample is caused by leaving the test piece. The ventilation port of the middle hand means that the method of making the dirty electrode test strip is a conductive layer in the form of a mesh electrode. As the upper cover without holes at the front and back ends of the electrode, such as a channel. In this way, the specimen will flow from the other end of the channel. According to Winarta et al., There is a U-shaped opening in the middle layer of the air hole cover, so the liquid channel is. This kind of mouth may flow out; when the edge of ventilation, especially when trying, it is easy to accidentally touch the infection.

Page 9 l239397 Checking the contaminated clam test strips caused by the leakage of Zhao ~ The structure of the invented electrochemical tripolar screen-printed biosensing test strip, such as, poly stone 7, on the insulating base plate 1, such as polyvinyl chloride (p I * Acid_ (pc), etc., rv ^ x polyester (pe (P c), etc., with a conductive material, such as silver, gold and other colloidal barges — 'a ... section, soil Materials, such as silver and silver-shaped holding section 2, are printed on the wire 2 by screen printing. Then conductive conductive adhesive such as carbon, gold or platinum is printed on the wire 2 to form electricity, two hammers 4 One end is the working electrode 3, the reference electrode 4, and the auxiliary electrode 5 phases, there is no reference electrode when designing); the other end is 3, 4, and 5, and at the end of the test strip self-distortion is the detection The connection point of the instrument, and 6 is the long strip of the detector ... ~, HIJ U way gallery 丨 L identification line. Paste on the substrate printed with electrodes or use screen printing as insulation. Bayi contains U-shaped holes. Conductive intermediate layer or insulating adhesive layer 7, 8 of which has a separation function in the phase material and electrical layer. The groove 7a is a sample guide groove, which is formed at the bottom of the upper cover, the sub & flow groove. A closed and raised space 8 a, the internal volume of the two is about 2 microliters. The electrode reaction zone is coated with a reagent formulation, including reactants, reaction catalysts (such as enzymes), and electron-conducting substances (such as dimethyl ferrocene, tetrathi). fulvalene, etc.), wetting agents (cellulose 'hydroxyethyl cellulose' polyvinyl alcohol Λ polyvinyl-pyrroli done-gelatin, etc.), and surfactants (tween 20, triton X-100, surfynol me ga8, etc.). The spacer layer 7 and the cap layer 8 Capillary diversion channels 7 a are formed around the electrode working area after lamination, allowing specimens such as blood to contact the leading edge of 7 a and then quickly fill the electrode reaction area via capillary phenomenon. At this time, the specimen and reactants

Page 10 1239397 V. Description of the invention (6) Signals generated by the reaction of catalysts and electron transfer substances through electrodes and detectors. The above-mentioned diversion groove can allow the electrode to perform the normal detection operation under the condition of very fast (less than 1 second) and requiring little blood (less than 1 microliter). The structure of another electrochemical diode electrode strip according to the present invention is shown in Figure 2. Briefly, the conductive substrate 2 is coated with a conductive material such as silver, silver chloride, gold and the like by screen printing on the insulating substrate 1, and the conductive material such as carbon, gold or platinum is brushed to cover the conductive layer. A working electrode 3, a reference electrode 4 and an auxiliary electrode 5 are formed on the layer. The opposite ends 3 ', 4', and 5 'of each electrode are the contact points connected to the detector; and 6 is the test strip automatic identification line of the detector. The next step is to paste a spacer layer 7 of a T-shaped hole on the base plate of the completed electrode layer or by screen printing with insulating glue. The upper cover layer 8 includes a closed and upwardly raised space 8a. The internal volume of the protrusion is about 2 microliters. 8a is located directly above the intersection of the T-shaped vertical and horizontal lines. The groove 7a formed between the upper cover and the compartment layer is a sample guide groove, and 7b and 7c are exhaust channels facing to both sides. The specimen, such as blood, is filled with the leading edge of the electrode quickly after flowing through the leading edge of 7 a to the electrode reaction area. The leading edge of the specimen is also shown in Figure ^ 1 and does not exceed the leading edge of 8 a. Moreover, this design can delete the exhaust channel on either side of 7b or 7c, and can also obtain the same function. Base plate substrate The base plate 1 can be composed of various insulating materials, such as polymers, plastics, and ceramic materials. The type of material selected must match the electrode material and application requirements. If it is an intrusive application, a soft material should be used.

Page 1239397 '--_____ V. Description of the invention (7) To reduce pain and avoid damage to tissues, such applications can usually use insulating plutonium molecules such as polycarbonate, polyester (such as DuPont's My 1 ar series) Products) 'polyethylene terephthalate (PET), polyvinyl chloride (PVC), polyether, polyamide, polyurethane, polyimide, etc. On the other hand, rigid materials can be used such as ceramic materials such as stone dioxide or monolithic gas. 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 micron, the length of the bottom plate is affected by various factors can be between 1-g cm, more accurately between 2-5 cm.

Lead layer and electrode

As shown in the figure, 'electrical materials such as silver, gold, platinum and other materials are screen-printed to form a lead region 2 on the base plate 1 for the purpose of connecting various electrodes to the test device, using high conductivity and low resistance characteristics. The material can reduce the impedance of the electrode and increase the detection current signal. Conductive materials such as carbon glue are brushed on the wire 2 '6 for automatic identification of the test piece of the detector. In addition to the reference electrode 4, the brushed wire 2 is completely covered, and the exposed silver wire is used in part 4: Chemically, the surface of silver can be treated as the reference electrode of silver chloride, or the silver / gasified silver ink is brushed on the silver wire or the wire is directly brushed with silver / gasified silver ink, so there is no need to Also treated with gasification silver. Insulating layer The insulating intermediate layer 7 can be made of an insulating dielectric material.

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1239397 V. Description of the invention (8) Or cover the surface of the electrode in an adhesive manner, in addition to shielding the carbon surface which does not have to be exposed 'and provide a reaction area with a fixed area. … Reaction reagent area

The reaction reagent is coated on the electrode area, and includes a reaction catalyst, a buffer solution, a filler, an electron transporting substance, and a surfactant 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 the sample and reagent, as well as the hydrophilicity and dispersing function of the 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 used 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 buffer components that can be used include phosphate, acetate, citrate, etc. The concentration range is from 10 to 100 millimoles, and the optimum concentration is about 30 to 100 millimoles.

Page 13 1239397 V. Explanation of the invention (9) The capillary diversion layer The diversion layer is covered by the grid sheet 7 and the upper cover 8 above the electrode. 7a is the capillary groove of the specimen. / ，)) 7b and 7C are exhaust ports, and the two or more can exist separately. Using the sigh: the thickness and the width of the 7a groove can adjust the volume of the diversion region. The thickness of the glycoside is between 5-200 microns, the optimal thickness is 10-50 microns, and the hollow degree is between 2- 8 square meters, with a width between 0.5 and 5 units, and a length of 2 mm, forming a hollow area? The appropriate width is between 0.5 to 2 buckets when the body is in operation; the touch is slightly raised, and the time required for the actual inspection to be full is less than 1 second. -The closed protrusion 8a of the contact layer 8 on the edge of the diversion layer may be a β-shaped industry, a wood figure, an eighth nr, a 0-shaped, a square or other geometric shape, and the appropriate size is between 0.5 and 5%. In the ancient method, the gap is set up after you make the electrode diversion channel at t. This gap is located at the position ^, and the gap is to stop the advance of the blood sample after it fills the reaction area. The 7 spacers and the 8 diaphragms can be composed of transparent or opaque insulating materials, such as plastic, plastic, or rubber molecular materials such as PVC or My 1 ar. The 8a of the upper region of the upper region can be transparent, in addition to the user can visually check whether the sample is filled successfully, and the test result is β-, Ma Man, and has the function of protecting the test piece. The upper layer is the so-called upward convex space, V,, and two, which can be formed in one piece or formed in two steps. The first step is to open a hole on the upper end of the upper cap layer as shown in Figure _ 所 士 田 # Μ , That is, 8 a, and then overlay a 6 sheet, a 70 sheet (as shown in Figures 4 and 5), 8, 8a and 9 are OK! ^ Η & The thin and integrally formed sheet. Figures 3 and 6 show the longitudinal section of Figure 1 and the test sheet containing sheet 9. 1239397 V. Description of the invention αο) Filling the sensor In the polar design, the monitoring position of the auxiliary electrode and the working electrode or the resistance value is monitored by the same principle as when the impedance is started when the electrode is filled. The auxiliary electrode must be the same to distribute and flow the sample. The auxiliary design can also be used to detect that if the working electrode is fully filled, when the detection electrodes are sharply divided, the electrical direction will be consistent and the impedance of the auxiliary electrode body will not be reduced. The test specimens were filled in the top three groups The value should be passed. When the filling is full, the electrode can be connected to the electrode area first. The three functions are not available. When the two inductors work correctly, the contact is already above the outside. The electrode time limit is large. It is full of specimens. It uses auxiliary current and electricity to set the electricity. When the specimens are filled, it can also be used to check whether the electrodes are first contacted and filled. Electrochemical analysis is cut by slitting or punching when the electrode assembly is completed. After that, the sample analysis can be performed by using an external palm-type electrochemical instrument. The analysis method can be performed in various ways, such as the potentiostat method (0-0.3 volts) to measure the steady-state current, or the potentiostat to measure a certain time. The total power value, the power value value, the integral of current and time, and the steady-state current can be used as the proportional measurement relationship of the component concentration in the sample. The detector is equipped with the filling function of the electrode pad. When the detector reads When the signal is filled, the electrochemical analysis parameters are automatically started, which increases the accuracy of the detection, especially when the overall detection time is less than 10 seconds, a small time error often results. To great variation.

Page 15 1239397 V. Description of the invention (11) IV. [Implementation method] The following examples are based on blood glucose testing, the purpose of which is only to illustrate the feasibility of the invention and not to limit the scope of the invention. For example one, let's make an electrode clock at 50, and then expose it. Reaction 0.1-1%, 1 0-1 0.01-0 Dividing of recessed guide holes, ie, screen printing of glucose sensing test sheet. Production of acrylic synthetic paper. After 30 minutes of dryness, brush three sets of electrodes (work, reference and auxiliary electrodes) with carbon gel. Once again at 90, it is baked for 15 minutes, then it is made of insulating glue, and then hardened and dried under ultraviolet light to form an insulating layer. Examination and guide flow reaction zones 7a, 7b, and 7c (design with vents). '(Contains 0.5 to 3 units of glucose oxidizing scale, polyvinyl alcohol, ρΗ4.0--9-9, 10-10 mM potassium phosphate buffer solution OOmM potassium chloride, dimethylferrocene. · 〇5 10 · 5%, tween_2〇 · 2%, surfynol 0 · 01—〇 · 2%), coated on the flow area for 7 years, dried at 4 5 C for 1 hour, and then removed and then attached with a lid The sheet 8 is a finished product after being pasted and fixed by a transparent upper cover sheet 9. Example two, standard glucose solution and whole blood test

A standard potassium phosphate buffer solution (pH 7 4) containing grape bran 0-400 mg / 100 ml was prepared. The screen printing ^ polar glucose test strip prepared in Example 1 was used in an electrochemical analyzer (CHInstrument 65. A) At a test potential of 100 millivolts, the test time is 8 seconds. At each test, the volume of the supplied sample is 3 microliters, and the test piece is inhaled every 1239397. V. Description of the invention (12). The test results are shown in the table below. 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. Glucose concentration (mg / dl) Electricity (microcoulomb) 80 1.556 105 2. 636 1 30 3. 440 180 5. 946 280 9. 707 380 11.733 480 12.464 580 13.945 Different examples Blood glucose test

Page 17 1239397 V. Description of the invention (13) The electrode test strips made in Example 1 are used to supply different volumes of whole blood to test the required blood volume of the present invention. The venous whole blood is formulated with a standard glucose solution to 300 mg / dl. Its concentration. The method of this test is to provide different volumes of whole blood, so that the test piece is inhaled by the principle of siphon, and the test conditions are tested as in Example 2. The results shown in Figure 7 show that when the amount of samples provided is insufficient (such as 0.5 microliters), the detected glucose concentration is low; but when the volume of the provided samples exceeds 0.8 microliters, the glucose detection concentration is It is close to the prepared concentration, and the whole blood sample provided cannot be fully inhaled; the higher the volume of the sample provided, the more the remaining volume, which means that the diversion reaction area of the sample has reached saturation, No more samples can be inhaled. The front edge of the inhaled specimen does not exceed 8 b. It is sufficient to prove that the design of the present invention has the function of restricting the volume of the specimen. 1239397 Schematic illustration of the screen printing electrode test piece of the present invention Schematic diagram with u-shaped holes. 1. Insulating base plate; 2. Conductor layer; 3. Working electrode; 4. Reference electrode; 5. Auxiliary electrode; 3 ', 4', 5 ', contact point of the detector; 6. Automatic identification line of test piece ; 7, the middle layer; 7 a, the detection diversion groove; 8, the upper cover layer; 8 a closed upward space. Figure 2. Schematic diagram of the T-hole structure of the screen printing electrode test piece of the present invention. 1. Insulating base plate; 2. Conductor layer; 3. Working electrode; 4. Reference electrode; 5. Auxiliary electrode; 3 ', 4', 5 ', contact point of the detector; 6. Automatic identification line of test piece ; 7, middle layer; 7 a, detection diversion groove; 7b, 7c, exhaust channel; 8, upper cover layer; 8a closed upward space.

Page 19 1239397 Brief description of the drawings 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 layer; 3. Electrode layer; 7. Intermediate layer; 7 a. Detection diversion groove; 8. Overlay layer; 8 a. Hole; 9. Thin sheet. Figure 7. The effect of whole blood sample volume on the test.

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Claims (1)

1 · An electrochemical biosensing test strip, ^-an insulating substrate; is composed of the following components:-a wire layer, screen-printed on the knife double or three electrode type wire; x; on the insulating substrate Brush-made-an electrode layer on the wire layer outside of screen printing to form _ electrode type or a working electrode, three electrode type, containing an electrode area, an electrically insulating intermediate layer, and covering the conductive layer with an insulating adhesive This intermediate layer is opposite to the openings printed with electrical or T-types; the method covers the working electrode in the reference electrode area and a reference electrode, a double reference electrode and an auxiliary electrode, and the two ends connected to the detector; Brush directly or use insulating material to make the electrode and contact part exposed. The insulating base of the electrode part has — U A reaction active layer, which contains reactants, reaction catalysts, electron mediators and surfactants and is coated on the U-shaped or T-shaped channels from the starting point to the electrode surface; An upper cover with an upwardly-enclosed closed space opposite to the electrically insulating intermediate layer, and the upper cover is adhered to the electrically insulating intermediate layer, so that the U-shaped or T-shaped area forms a capillary flow-conducting area, and the relatively upwardly convex closed area The space is between 0 · 5 ~ 4 microliters and is located at the bottom of the diversion zone. 2 · The electrochemical biosensing test sheet as described in item 1 of the scope of patent application, the electrically insulating bottom plate is polycarbonate, polyester, polyether, polyamide, polyvinyl chloride, polyimide, polyvinyl chloride (PVC), One of glass, fiberglass board, ceramic and polyethylene (PET). 3 · The electrochemical biosensing test strip as described in the first patent application, conductive Page 21 1239397 Work 8.: Application: The electrochemical biological electrode and auxiliary electrode of item No. of the patent scope have the function of detecting and covering the specimen. Electrochemical type biosensor test strip 1 surrounding the electrode 1. =: i! A set of functions that can automatically start the detector. The method for producing the electrochemical test strips is based on the following steps: a. Using screen printing technology on a substrate— The conductive layer is then dried under the core of ". ⑶: brush out-layer b. Use screen printing technology on the aforementioned conductive brush at 40. (: to 120. (: dried under; electrode layer, Re-C · Use screen printing technology to brush the insulating rubber layer on the first plate-containing u-shaped notches in Erda "^ auxiliary electrode and reference electrode position hole ^ as the electrode, the hole, and the base plate another 1239397 VI. The end of the patent application exposes 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 1 · If the method for manufacturing an electrochemical biosensing test piece under the scope of application for patent No. 10, the manufacturing method of the intermediate layer can directly paste the insulating intermediate layer to replace the screen printing method. 1 2 · For the manufacture of electrochemical biosensor test strips in the scope of patent application No. 10, the U-shaped notch in the middle layer can be replaced by a T-shaped notch, and the horizontal notch of the T-type is a discharge to both sides of the test piece. Page 23