Summary of the invention
The objective of the invention is to overcome deficiency of the prior art; supporting test paper of a kind of blood glucose monitoring system and preparation method thereof is provided; after making this test paper in a single day contact human body fluid; can on corresponding supporting testing tool, conveniently read test result intuitively; its making method is simple; make things convenient for post-production and encapsulation, help large-scale production.
The present invention is achieved by the following technical solutions, and test paper of the present invention comprises: a substrate, as the PVC substrate; Be printed on a working electrode on the substrate; Be printed on a reference electrode on the substrate; Be printed on electric loop on the substrate, so that with the continuous blood sugar test of finishing of testing tool; End at substrate is printed on the lead that connects with the readout meter epiphase.Working electrode and reference electrode are respectively to be imprinted on the carbon coating that forms on the substrate conductive silver layer, be coated with the biological enzyme of catalysis blood-glucose reaction on the working electrode, as glucose oxidase, and a kind of medium and tamanori that can between enzymic catalytic reaction center and electrode, carry out electron transport, so constitute a circuit loop, according to the height of glucose concn in the blood, produce the current signal of corresponding size during reaction, and the detection of tested instrument is read.This test paper has omitted sealing coat and insulation layer, and is simple in structure, and tested material can be lactic acid, cholesterol or glucose, and the enzyme of use can be Lactate Oxidase, rCO and glucose oxidase.
Working electrode is the double-deck conductive film of a kind of silk screen printing on upholder, comprises the enzyme of catalysis biochemical reaction and the medium that can carry out electron transport between enzymic catalytic reaction is with electrode in spraying printing on the conductive film.This working electrode can transmit the electric current that produces behind the enzymic catalytic reaction.Working electrode and reference electrode are close, and two electrodes constitute an effecting reaction zone, after connecting with tester, form effective body fluid components test macro.
The present invention adopts bio-sensing and electrochemical principle, make above-mentioned test paper by the method that silk screen and spraying printing combine, silk screen printing is confined to print the conducting wire, only play the conducting effect, relatively more responsive biological elements such as enzyme, conducting medium and tamanori etc. are separated from wire printing material, adopt non-contacting spraying method to be printed on the electrode support substrate, the quantity for spray and the spray area of spray material are accurately controlled by computer fully.
Below the inventive method is further described, concrete steps are as follows:
(1) Natvosol of adding 1.2-2.5% in the silk-screen conductive carbon ink, the electronic conduction solid support material of adding 10-20%, as the benzoquinones or the Tripotassium iron hexacyanide, mixing is standby;
(2) metal of 200-400 order number or nylon polyester material silk screen are fixed on square light-weight metal frame and add, in the darkroom, coat Kodak's photoresists, drying for standby;
(3) working electrode and the reference electrode negative film with computer drawing places on the photoresists, and under 20-40 watt of ultraviolet lamp exposure 15-30 minute, remove the unhardened photoresists that covered by the electrode negative film with the water blast gun flushing, dry back forms the silk-screen template;
(4) the silk-screen template that will have working electrode and a reference electrode figure is installed on the automatic screen printer standby;
(5) be the Lactate Oxidase of 10-30 international unit with activity or active be the glucose oxidase of 200-500 international unit for the rCO of 12-25 international unit or activity, allocate in the phosphoric acid buffer of every liter of 50-120 mmole, add every liter of 200-800 milligram of the benzoquinones or the Tripotassium iron hexacyanide simultaneously, it is standby fully to shelve 4 ℃ of refrigerators after the dissolving.
Working electrode adopts screen printing technique that conductive silver material and conductive carbon material are imprinted on the substrate earlier; Enzyme and electron transit mediator such as the benzoquinones or the Tripotassium iron hexacyanide are printed in spraying then, and reference electrode is also made with identical method.
The printing ink that is used for spraying printing comprises: liquid adhesive, conducting objects solution and enzyme.The material of substrate can be a kind of good toughness, the polymeric material that insulativity is high, and such as polyvinyl chloride, polyester or polycarbonate.
The present invention has substantive distinguishing features and marked improvement, and the inventive method is confined to print the conducting wire with silk screen printing, only plays the conducting effect, makes requirement thereby reduced technology; Wire printing material and screen printing device needn't specially be processed, and can obtain by commercially available channel, and cost is very low; Relatively more responsive biological elements such as enzyme, conducting medium and tamanori etc. are separated from wire printing material, be printed on the electrode support substrate with non-contacting spraying method, eliminated the disadvantageous effect that composite filament India side formula is brought biological sensitive materials because of contact printing repeatedly; Because the spraying printing technology is reached perfection, the material that spray can accurately be controlled its quantity for spray and area by computer fully, and the precision of the finished product and repeatability are fully ensured.After in a single day test paper of the present invention contacts human body fluid, can conveniently on corresponding supporting testing tool read test result intuitively, making method is simple, the precision height, and cost is low, makes things convenient for post-production and encapsulation, helps large-scale production.
Embodiment
As shown in Figure 1, test paper of the present invention comprises: a substrate 1, be printed on a working electrode 2 on the substrate 1, be printed on a reference electrode 3 on the substrate 1, be printed on electric loop on the substrate 1, end at substrate 1 is printed on the lead 4 that connects with the readout meter epiphase, working electrode 2 and reference electrode 3 are respectively to be imprinted on the carbon coating that forms on substrate 1 conductive silver layer, be coated with the biological enzyme of catalysis blood-glucose reaction on the working electrode 2, as glucose oxidase, and a kind of medium and tamanori that can between enzymic catalytic reaction center and electrode, carry out electron transport, so constitute a circuit loop, according to the height of glucose concn in the blood, produce the current signal of corresponding size during reaction, and the detection of tested instrument is read.Tested material can be lactic acid, cholesterol or glucose, and the enzyme of use can be Lactate Oxidase, rCO and glucose oxidase.
Working electrode 2 is a kind of silk screen printing double-deck conductive films 5 on upholder, and the spraying printing comprises the enzyme of catalysis biochemical reaction and the medium that can carry out electron transport between enzymic catalytic reaction is with electrode on conductive film 5.Working electrode 2 is close with reference electrode 3, and two electrodes constitute an effecting reaction zone, after connecting with tester, forms effective body fluid components test macro.
Content in conjunction with the inventive method provides following examples, and the present invention is further illustrated.
Embodiment 1: polyethylene (PVC) sheet material, it is standby to be cut into 150X150 centimetre substrate in advance.
(1) Natvosol of adding 1.2% in 1 gram silk-screen conductive carbon ink (ACHESON Acheson423 SS), the electronic conduction solid support material benzoquinones of adding 10%, mixing is standby.
(2) metal of 200 order numbers or nylon polyester material silk screen are fixed on square light-weight metal frame and add, in the darkroom, coat Kodak's photoresists, drying for standby.
(3) working electrode and the reference electrode negative film with computer drawing places on the photoresists, and exposure is 15 minutes under 20 watts of ultraviolet lamps.Remove the unhardened photoresists that covered by the electrode negative film with water blast gun flushing.Dry back forms the silk-screen template.
(4) the silk-screen template that will have working electrode and a reference electrode figure is installed on (moral gram DEK-245) type automatic screen printer standby.
(5) be that the Lactate Oxidase of 10 international unit is allocated in the phosphoric acid buffer of every liter of 50 mmole with activity, add 200 milligrams every liter of benzoquinones simultaneously.It is standby fully to shelve 4 ℃ of refrigerators after the dissolving.
On above-mentioned automatic screen printing machine, add conductive silver ink (ACHESON Acheson 427 SS) and carry out print conductive circuits; Dry back adds above-mentioned hybrid conductive silk-screen carbon ink on automatic screen printing machine, the silver-colored line of ink marker road of before having printed is covered, and makes working electrode and reference electrode respectively.The electrode of having printed need be held in 60 ℃ of baking ovens and solidify 30 minutes, forms 50 pairs of work and reference electrode on each sheet PVC substrate, takes out standby then.Making can be tested lactic acid, during the enzyme test peper of cholesterol and glucose, take out Manufactured element task electrode and reference electrode, mixed solution in above-mentioned (5) is added in the holding bottle of the automatic accurate spraying instrument in hundred roads (BIODOT-3000), by computer installation spacing and drop size, evenly be sprayed on the working electrode made and the setting position of reference electrode then.This position is positioned at the interval of the about 5X5mm of working reaction end of working electrode and reference electrode.
After above-mentioned electrode was made drying, the polyester nethike embrane that an about 10mm is wide covered on the reaction zone, was printed on the non-reactive site of work and reference electrode then with the empire silk printing ink, exposed reaction interval.The electrode slice that prints of spraying drying at room temperature 3 hours under drying conditions.It is long with shears the exsiccant testing plate to be cut into 35mm, the test strip finished product that 0.8mm is wide.
With Manufactured test strip, place tester and apply 220 mV operating voltage for two sections at working electrode and reference electrode, measure the lactic acid of different concns, the result shows that reaction took place after 10 seconds, shows to make good test repeatability and the dependency of electrode.Through relatively with Biochemical Analyzer, get 50 parts of lactic acid control liquids and 50 parts of patient's blood sample lactic acid contrast tests respectively, the relative standard deviation that its dependency γ reaches 0.9567 test strip is CV≤6.5%.
Embodiment 2: polyester (PET) sheet material, it is standby to be cut into 150X150 centimetre substrate in advance.
(1) Natvosol of adding 1.8% in silk-screen conductive carbon ink (ACHESON Acheson423 SS), the electronic conduction solid support material benzoquinones of adding 15%, mixing is standby.
(2) metal of 300 order numbers or nylon polyester material silk screen are fixed on square light-weight metal frame and add, in the darkroom, coat Kodak's photoresists, drying for standby.
(3) working electrode and the reference electrode negative film with computer drawing places on the photoresists, and exposure is 25 minutes under 20 watts of ultraviolet lamps.Remove the unhardened photoresists that covered by the electrode negative film with water blast gun flushing.Dry back forms the silk-screen template.
(4) the silk-screen template that will have working electrode and a reference electrode figure is installed on (moral gram DEK-245) type automatic screen printer standby.
(5) be that the rCO of 25 international unit is allocated in the phosphoric acid buffer of every liter of 50 mmole with activity, add 800 milligrams every liter of benzoquinones simultaneously.It is standby fully to shelve 4 ℃ of refrigerators after the dissolving.
Making methods such as spraying are with example 1, with the test strip of making, place tester and apply 300 mV operating voltage for two sections at working electrode and reference electrode, measure the cholesterol of different concns respectively, the result shows that reaction took place after 20 seconds, shows to make good test repeatability and the dependency of electrode.Through relatively with Biochemical Analyzer, get 30 parts of cholesterol control liquids and 20 parts of patient's blood sample cholesterol contrast tests, its dependency γ reaches 0.967, and the relative standard deviation of test strip is CV≤5.7%.
Embodiment 3: polyphenol ester (PP) sheet material, it is standby to be cut into 150X150 centimetre substrate in advance.
(1) Natvosol of adding 2.5% in the silk-screen conductive carbon ink, the electronic conduction solid support material Tripotassium iron hexacyanide of adding 20%, mixing is standby;
(2) metal of 400 order numbers or nylon polyester material silk screen are fixed on square light-weight metal frame and add, in the darkroom, coat Kodak's photoresists, drying for standby;
(3) working electrode and the reference electrode negative film with computer drawing places on the photoresists, and exposure is 30 minutes under 40 watts of ultraviolet lamps, removes the unhardened photoresists that covered by the electrode negative film with water blast gun flushing, and dry back forms
The silk-screen template;
(4) the silk-screen template that will have working electrode and a reference electrode figure is installed on the automatic screen printer standby;
(5) be that the glucose oxidase of 30 international unit is allocated in the phosphoric acid buffer of every liter of 120 mmole with activity, add 600 milligrams every liter of the Tripotassium iron hexacyanides simultaneously.It is standby fully to shelve 4 ℃ of refrigerators after the dissolving.
Making methods such as spraying are with embodiment 1, with the test strip of making, place tester and apply 400 mV operating voltage for two sections at working electrode and reference electrode, measure the glucose of different concns respectively, the result shows that reaction took place after 30 seconds, shows to make good test repeatability and the dependency of electrode.Through relatively with hospital's Biochemical Analyzer, get 50 parts of glucose control liquids and 50 parts of diabetics's blood sample glucose contrast tests respectively, its dependency γ reaches 0.97, and the relative standard deviation of test strip is CV≤8.1%.