CN1461810A - Biological testing strip - Google Patents
Biological testing strip Download PDFInfo
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- CN1461810A CN1461810A CN 02110617 CN02110617A CN1461810A CN 1461810 A CN1461810 A CN 1461810A CN 02110617 CN02110617 CN 02110617 CN 02110617 A CN02110617 A CN 02110617A CN 1461810 A CN1461810 A CN 1461810A
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Abstract
A biologic testing strip is composed of a basic strip, a working electrode and a counter electrode. On said working electrode there is the envelope substance prepared from cyclodextrin, bioactive enzyme, and electron transferring medium chosen from quinone, methyl blue, methyl violet, and ferrocene or its derivatives. Its advantages are low cost, and high exchangeability and anti-interference power.
Description
Invention field
The present invention relates to electrochemical analysis and clinical chemistry field.More particularly, the present invention relates to a kind of new biological testing strip and preparation method thereof.
Background technology
Present existing commercially available biological testing strip, the test strip for blood-sugar of test blood glucose level for example, normally bioactive enzyme (as Hexose phosphate dehydrogenase or glucose oxidase) is mixed with carbon paste, adopt screen printing technique to produce test strip then based on carbon paste electrode (being commonly referred to thick membrane electrode).
Yet there are following shortcomings in this carbon paste electrode.At first, therefore higher with the enzyme amount in the electrode make the cost of biological testing strip raise.The second, in the electrode that makes with method for printing screen, the electron transport mesosome runs off easily, thereby makes sensitivity, linear response and the freedom from jamming variation of biological testing strip.The 3rd, the storage time of this biological testing strip is shorter, needs stored refrigerated usually, has brought many difficulties and inconvenience so just for the transportation and the storage of product.In addition, its size of electrode of producing with screen printing technique is difficult to keep stable, and therefore, the qualification rate of product is lower.
In order to overcome above-mentioned shortcoming, need still in this area that a kind of new cost is low, interchangeability good, good linearity, anti-interference, can the biological testing strip of preserving steady in a long-term.
Summary of the invention
The purpose of this invention is to provide that a kind of new cost is low, interchangeability good, good linearity, anti-interference and/or can the biological testing strip of preserving steady in a long-term.
Another object of the present invention provides the preparation method of above-mentioned biological testing strip.
First aspect present invention provides a kind of biological testing strip, it comprises substrate, working electrode and counter electrode (double as reference electrode) are arranged on this substrate, the inclusion complex that has cyclodextrin, electron transport mesosome and bioactive enzyme to form on the described working electrode, wherein said electron transport mesosome is selected from quinone, Methylene blue, methyl violet, ferrocene and ferrocene deriv.
The substrate of described biological testing strip can adopt any conventional material well known by persons skilled in the art.These materials are nonconducting, stable materials mechanically.These materials include, but are not limited to stupalith, polyester material etc.This strip can be made various suitable shapes, preferably can be made into strip or sheet.
In the present invention, on-chip working electrode can be made with identical gold or platinum material with counter electrode.Unlike the prior art be in the present invention, to be to adopt microelectronics processes that working electrode and counter electrode are applied on the substrate.Be applicable to that microelectronics processes of the present invention for example comprises methods such as vapour deposition (for example vacuum-evaporation, sputter coating and chemical vapour deposition), ion sputtering deposition, cathodic arc deposition, plating, electroless plating, vacuum plating and photoetching.These methods are the methods that are applied to metal on the base material in the microelectronic to be used always.In order to obtain the settled layer/film of certain pattern, also can adopt methods known in the art such as mask, photoetching, directed ion beam and deposition source bundle.
Described bioactive enzyme is the enzyme that can react with chemical substance to be measured.Therefore, the type of enzyme depends on the type of chemical substance to be measured.In the present invention, preferable bioactive enzyme is selected from glucose oxidase, rCO and galactose oxidase.These enzymes all can commercially availablely be buied.Yet those skilled in the art also can be improved content disclosed herein, so that the present invention can adopt the enzyme of other type or test other chemical substance.All these changes include within the scope of the invention.
The present invention has used the supramolecular chemistry principle, form inclusion complex (inclusive) by cyclodextrin and electron transport mesosome, and then mix with the biological activity enzyme solution, make that institute's fixed enzyme can be than the long-term down high biological activity that keeps of mal-condition (for example high temperature).In addition, this method can also make transfer transport (migration) mesosome of inclusion in the enzyme layer not run off medium-term and long-term the stablizing of the aqueous solution, thereby the externally-applied potential when testing is reduced by a relatively large margin, reduces the interference of other material in the blood sample when measuring.
Cyclodextrin is a class ring-type oligosaccharide molecular, and it comprises the Glucopyranose subunit, has ring-shaped cylinder shape space structure.Molecule Semi-polarity (wetting ability) hydroxyl is outside structure, and nonpolar (lipophilicity) main chain carbon and ether oxygen lining are in the inner chamber of ring.This cavity can hold (acceptance) active ingredient in conjunction with forming inclusion complex by non-covalent mode.
In the present invention, adoptable cyclodextrin for example is the cyclodextrin that contains 6,7 and 8 Glucopyranose molecules, promptly is respectively alpha-cylodextrin, beta-cyclodextrin and γ-Huan Hujing.In addition, can be improved the outside hydroxyl substituent of cyclodextrin molecular, to be formed with the derivative of other required reinforced effects.The example of these cyclodextrin derivative is: alkyl derivative, and as 2, the 6-DM-; The hydroxy alkylated derivative is as hydroxypropyl-beta-cyclodextrin; Side chain derivative such as didextrose group-beta-cyclodextrin; The sulfoalkyl derivative is as sulfo group butyl ether-beta-cyclodextrin; And carboxyl methylation derivant, as carboxymethyl-beta-cyclodextrin.The use of these derivatives also within the scope of the present invention.
The electron transport mesosome that adopts among the present invention is selected from quinone, Methylene blue, methyl violet, ferrocene and ferrocene deriv (as methyl ferrocene, ferrocenecarboxylic acid, ferrocene ethanol etc.).Cyclodextrin that the present invention is used and electron transport mesosome all can obtain from commercial channels.
Those skilled in the art need not the consumption that too much experiment can be determined cyclodextrin, electron transport mesosome and bioactive enzyme.Usually, the concentration of cyclodextrin is preferably the 0.05-0.1 grams per milliliter, and preferable is the 0.06-0.09 grams per milliliter, and best is 0.08 grams per milliliter.The consumption of electron transport mesosome need not strict control, can be excessive, and its concentration usually can be between the 0.5-2 mg/ml.
In a preferable embodiment of the present invention, also can randomly cover the last layer interference rejection membrane on the described test strip.This interference rejection membrane is the permanent stability of being maintained fixed enzyme layer further, and interference free performance is provided.Described interference rejection membrane should have following feature: 1) have controllable aperture; 2) has autohension; 3) has biocompatibility.The material for preparing described interference rejection membrane includes, but are not limited to, and gelatin, chitosan, cellulose acetate membrane etc. wherein are best with the chitosan.
The present invention provides a kind of the present invention of preparation the method for above-mentioned biological testing strip on the other hand, and this method comprises the following steps:
A) on substrate, form working electrode and counter electrode;
B) hybrid ring dextrin, electron transport mesosome and bioactive enzyme form inclusion complex, and wherein said electron transport mesosome is selected from quinone, Methylene blue, methyl violet, ferrocene and ferrocene deriv;
C) inclusion complex that step b) is obtained is applied on the working electrode surface;
D) make the inclusion complex drying that applies.
Cyclodextrin and electron transport mesosome and bioactive enzyme usually can be under heating or the conditions that does not heat, and reaction forms inclusion complex in appropriate device.The inclusion complex that makes can be applied on the working electrode with any usual manner or device, wherein preferably adopts microsyringe to carry out application of sample.
This strip can be in (normally under the room temperature) drying under not active condition of destructive enzyme and the temperature.
Compared with prior art, the present invention has following several advantage: at first, the formation of inclusion complex makes that institute's fixed enzyme can be than the down long-term high biological activity that keeps of mal-condition (for example high temperature), products obtained therefrom can be under room temperature even comparatively high temps prolonged preservation and do not lose enzymic activity.Secondly, compare with method for printing screen, the used enzyme amount of the inventive method is less, thereby has reduced the production cost of biological testing strip.The 3rd, because the present invention adopts inclusion complex to fix the transfer transport mesosome, therefore prevented the loss of transfer transport mesosome, thereby the externally-applied potential when testing is reduced by a relatively large margin, reduce the interference of other material in the blood sample when measuring.
The present invention adopts platinum to make counter electrode double as reference electrode, and its surface need not chlorination.The used microelectronics plane surface processing method of the present invention has been simplified complete processing, makes product size keep stable, and product qualified rate is higher than 95%, and this microelectronics processes also is suitable for inclusion complex enzyme fixing means of the present invention.In addition, on biological testing strip, be covered with one deck biological interference rejection membrane, especially chitosan film, thereby can further improve selectivity, freedom from jamming and the biocompatibility of mensuration.Biological testing strip of the present invention can be used for every field such as clinical detection, food test.
Description of drawings
Fig. 1 is the synoptic diagram of plane table roller electrode, number in the figure (1) expression reference electrode (double as counter electrode), (2) expression working electrode.
Embodiment
Further specifically describe the present invention below in conjunction with embodiment.Yet, should be appreciated that the present invention is not limited to these specific embodiments.
The preparation of embodiment 1 glucose test strip
1) at first, with 4 * 7cm
2Ceramic plate is a matrix, vacuum sputtering titanium (50nm) and platinum (200nm).Again with peeling off etching, make diameter and be 2 millimeters platinum dish working electrode, and around the platinum circular arc reference electrode (see figure 1) of working electrode.
2) take by weighing 3.5 gram cyclodextrin (Shanghai biochemical reagents company), add 50 ml distilled waters, in 60 ℃ of water-baths, dissolve.0 milliliter of ferrocene ethanol of Dropwise 5 (Tokyo changes into) solution (/ 100 milliliters of 1.2 grams) stirred 4 hours under 60 ℃ of water-baths while stirring.Precipitation is taken out in cooling back centrifugation, with four cyanogen furans washing three times, uses three final vacuum dryings of distilled water wash again.
3) medical cotton stick dips in and gets analytical pure acetone and clean electrode surface gently for several times, cleans electrode surface with the analytical pure dehydrated alcohol again.Then, it is standby to place culture dish to dry at this gained electrode.
4) electrode is accurately alignd with the silk screen figure, stamp the extremely viscous platen glue of water-based, then, electrode and silk screen are peeled off.
5) take by weighing 21 milligrams of glucose oxidases (U.S. SIGMA company, 10000 units/0.42 gram), with dissolved in distilled water and be diluted to 0.5 milliliter, making enzyme content is 1000U/ml.
6) take by weighing 20 milligrams and 100 milligrams chemical pure gelatin of the above-mentioned ferrocene cyclodextrin inclusion complex that makes, add about 5 milliliters distilled water (be about cumulative volume 30%), make it dissolving with low baking temperature heated and stirred in water-bath.
7) get the above-mentioned enzyme solution for preparing 10 microlitres and ferrocene solution 100 microlitres, fully stir, drip these mixing solutions 1.5 microlitres in working electrode surface.Make solution cover the whole working electrode surface.Make test strip in culture dish under the room temperature after dry 2～3 days.
8) the micropore cellulose acetate filter membrane with the 0.22U aperture covers on the electrode surface, and compresses with clean slide glass flicking.Cut filter membrane with cutter along the ceramic plate line of cut.
As on the electrode of the above-mentioned immobilized enzyme that makes, being covered with chitosan when making interference rejection membrane, can save operation steps 4).
9) enzyme electrodes of making is placed the phosphoric acid buffer of pH=7 soaked dried overnight 2 hours.
The preparation of embodiment 2 cholesterol test bars
Repeat each step of embodiment 1, just in step 5), replace glucose oxidase with rCO (available from U.S. SIGMA company).The result makes the test strip that is used to test cholesterol.
Embodiment 3 strip performance tests
A) strip interchangeability
50 strips that make with embodiment 1 are a collection of, and therefrom randomly drawing 10 is one group, 6.0 mmoles/liter glucose solution in measure response current.Draw after learn handling by statistics, four batches the relative deviation that records of totally 17 groups of strips less than 10%.Simultaneously, the relative standard deviation of strip in serum is 7%.These results show that protein does not have a significant impact strip interchangeability, and the strip that the present invention makes is suitable for blood sample analysis.
B) linear response of strip
Get the strip of embodiment 1 and test in glucose solution and standard serum sample, the result shows, it has good linear response at 0-16.0 mmole/rise in glucose concn scope.
C) longevity test
Choose some strip samples from embodiment 1 different criticizing, strip and potentiostat are linked, measuring current potential is 0.65V vs Pt.Earlier strip is inserted in the phosphate buffered saline buffer, when electric current was reduced to 0.25 μ A, taking-up was wiped away dried, again strip is inserted in 6.0 mmoles/rise in the glucose solution, writes down 1.5 minutes response current, and write down room temperature, after taking out the flushing of strip water, wipe away dried, room temperature preservation.Spend some days and carry out the test second time, the data when used data all are corrected to 20 ℃.Detected current value is compared with last detected current value for the first time, calculates the sign of the active percentage ratio of residual enzyme as the strip life-span.Longevity test the results are shown in Table 1a and table 1b, data are unit with μ A in the table, the numerical value in the bracket is the current value when being corrected to 20 ℃.
The test in work-ing life of table 1a strip
| Group number | Shelf time (my god)/survey periodic temperature (℃) | Remaining activity % | |||||
| 69 days/26 ℃ | 80 days/21 ℃ | 99 days/23 ℃ | 130 days 18 ℃ | 168 days 21 ℃ | 196 days 17 ℃ | ||
| ????1 | ??2.23(1.74) | ?1.92(1.84) | ??1.89(1.63) | ??1.43(1.57) | ??1.74(1.67) | ??1.32(152) | ????87.4 |
| ????2 | ??2.28(1.78) | ?1.93(1.85) | ??1.98(1.7) | ??1.52(1.67) | ??1.91(1.84) | ??1.42(1.63) | ????91.6 |
| ????3 | ??2.25(1.76) | ?1.97(1.89) | ??2.02(1.73) | ??1.51(1.66) | ??1.92(1.84) | ??1.37(1.58) | ????89.8 |
The test in work-ing life of table 1b strip
| Group number | Shelf time (my god)/survey periodic temperature (℃) | Remaining activity % | |||||
| 30 days/26 ℃ | 41 days/21 ℃ | 60 days/23 ℃ | 91 days 18 ℃ | 130 days/21 ℃ | 158 days 17 ℃ | ||
| ????4 | ??2.83(2.21) | ??2.49(2.82) | ??2.45(2.11) | ??1.94(2.13) | ??2.52(2.42) | ??1.9(2.19) | ????99.1 |
| ????5 | ??2.74(2.14) | ??2.28(2.29) | ??2.57(2.21) | ??1.93(2.12) | ??2.42(2.32) | ??1.84(2.12) | ????99.1 |
| ????6 | ??2.93(2.29) | ??2.54(2.44) | ??2.58(2.22) | ??1.99(2.19) | ??2.61(2.51) | ??1.81(2.08) | ????90.8 |
The result shows, after strip was at room temperature preserved half a year (once go through 38 ℃ high temperature a couple of days), the strip enzymic activity was 85～99% of an original enzyme activity, and this shows that strip can be at room temperature or even day with high temperature (36 ℃-40 ℃) prolonged preservation and do not lose enzyme and live down.
D) anti-interference test
A. glucose solution test
Get vitamins C (Vc) and uric acid as representational interfering substance in the blood, Vc and uric acid be added in 6 mmoles/the rise concentration in the glucose near the highest normal value in the blood (the Vc=0.08 mmole/liter, uric acid=0.3 mmole/liter).Sample drawn from the strip that embodiment 1 makes at random, sample strip and potentiostat are linked, getting 0.65VvsPt is test potential, drip phosphate buffered saline buffer on strip, when electric current is reduced to 0.25 μ A, disconnect potentiostat, wipe away dried, drip 6.00 mmoles/rise glucose solution again, measure 1.5 minutes response current.Other gets strip and replaces 6.00 mmoles/rise glucose solution with containing 6.00 mmoles of vitamins C (or uric acid)/rise glucose solution respectively, the response current when measuring 1.5 minutes respectively, and both differences are the response current of strip to vitamins C (or uric acid).Many groups are measured, the average percentage that the calculated response electric current increases.
B. serum test
The serum test method basically as mentioned above, but with standard serum and the standard serum that contains vitamins C (or uric acid) replace 6.0 mmoles/liter glucose solution and contain 6.0 mmoles of vitamins C (or uric acid)/rise glucose solution.
Test result shows that the adding of vitamins C or uric acid is measured can for hardly actual blood sample and be brought result error.
Although the invention describes concrete example, having a bit is significantly to those skilled in the art, promptly can do various variations and change to the present invention under the premise without departing from the spirit and scope of the present invention.Therefore, claims have covered all these changes within the scope of the present invention.
Claims (10)
1. biological testing strip, it comprises substrate, working electrode and counter electrode are arranged on this substrate, it is characterized in that, the inclusion complex that has cyclodextrin, electron transport mesosome and bioactive enzyme to form on the described working electrode, wherein said electron transport mesosome is selected from quinone, Methylene blue, methyl violet, ferrocene and ferrocene deriv.
2. biological testing strip according to claim 1 is characterized in that described bioactive enzyme is selected from glucose oxidase, rCO and galactose oxidase.
3. biological testing strip according to claim 1 is characterized in that, described on-chip working electrode and counter electrode are made with the method that is selected from chemical vapour deposition, ion sputtering deposition, cathodic arc deposition, plating, electroless plating, vacuum plating and photoetching.
4. biological testing strip according to claim 1 is characterized in that, has also covered one deck interference rejection membrane on the described test strip.
5. biological testing strip according to claim 4 is characterized in that described interference rejection membrane is selected from gelatin, chitosan, cellulose acetate membrane.
6. biological testing strip according to claim 5 is characterized in that described interference rejection membrane is a chitosan film.
7. a method for preparing the described biological testing strip of claim 1 is characterized in that, this method comprises the following steps:
A) on substrate, make working electrode and counter electrode;
B) hybrid ring dextrin, electron transport mesosome and bioactive enzyme form inclusion complex, and wherein said electron transport mesosome is selected from quinone, Methylene blue, methyl violet, ferrocene and ferrocene deriv;
C) inclusion complex that step b) is obtained is applied on the working electrode surface;
D) make the inclusion complex drying that applies.
8. method according to claim 7 is characterized in that, after the step d) interference rejection membrane is being covered on the gained test strip.
9. method according to claim 8 is characterized in that described interference rejection membrane is selected from gelatin, chitosan, cellulose acetate membrane.
10. method according to claim 7 is characterized in that, adopts the method that is selected from chemical vapour deposition, ion sputtering deposition, cathodic arc deposition, plating, electroless plating, vacuum plating and photoetching to form working electrode and counter electrode in step a).
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 02110617 CN1218046C (en) | 2002-01-23 | 2002-01-23 | Biological testing strip |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 02110617 CN1218046C (en) | 2002-01-23 | 2002-01-23 | Biological testing strip |
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| Publication Number | Publication Date |
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| CN1461810A true CN1461810A (en) | 2003-12-17 |
| CN1218046C CN1218046C (en) | 2005-09-07 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 02110617 Expired - Fee Related CN1218046C (en) | 2002-01-23 | 2002-01-23 | Biological testing strip |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103630593A (en) * | 2012-08-21 | 2014-03-12 | 苏州宇钿医疗器械有限公司 | Two-electrode glucolase electrode sensor |
| CN112067827A (en) * | 2020-11-16 | 2020-12-11 | 天津德祥生物技术有限公司 | Antibody diluent and blood type test card comprising same |
-
2002
- 2002-01-23 CN CN 02110617 patent/CN1218046C/en not_active Expired - Fee Related
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103630593A (en) * | 2012-08-21 | 2014-03-12 | 苏州宇钿医疗器械有限公司 | Two-electrode glucolase electrode sensor |
| CN112067827A (en) * | 2020-11-16 | 2020-12-11 | 天津德祥生物技术有限公司 | Antibody diluent and blood type test card comprising same |
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| Publication number | Publication date |
|---|---|
| CN1218046C (en) | 2005-09-07 |
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