CN101144782A - Quick test strip for detecting hydrogen peroxide in food - Google Patents
Quick test strip for detecting hydrogen peroxide in food Download PDFInfo
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- CN101144782A CN101144782A CNA2007100471546A CN200710047154A CN101144782A CN 101144782 A CN101144782 A CN 101144782A CN A2007100471546 A CNA2007100471546 A CN A2007100471546A CN 200710047154 A CN200710047154 A CN 200710047154A CN 101144782 A CN101144782 A CN 101144782A
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Abstract
The invention relates to a quick test strip used for detecting the hydrogen peroxide in food, and belongs to the biological detection technical field. The invention comprises an insulation waterproofing substrate, a reaction substrate substance membrane piece, a filtering layer, an isolation piece and a cover piece, wherein, the insulation waterproofing substrate adopts a foundation base, and round hole is arranged on the insulation waterproofing substrate, and the reaction area of the test strip is assembled on above the round hole according to the order of the reaction substrate substance membrane piece and the filtering layer and by covered in sequence, the isolation piece is covered on the two ends of the reaction area, and the cover piece is arranged above the reaction substrate substance membrane piece, the filtering layer and the isolation piece, a round hole is arranged on the cover piece, and the round hole is arrayed to the round hole on the insulation waterproofing substrate. Detecting the hydrogen peroxide by the test strip, the pre-treatment of the sample is simple, and the detection time is short, being generally less than 90 seconds; the detection limit reaches 0.5 ppm, the reproducibility is good, the effective preservation time of the test strip is one year, and the quick in-place detection of the hydrogen peroxide in the food can be realized.
Description
Technical field
The present invention relates to a kind of test-strips of technical field of biological, specifically is hydrogen peroxide quick test strip in a kind of detection food.
Background technology
At present, for the look of killing the microorganism in the food and increasing food is pleased sense, food production enterprise excess or illegally add the phenomenon ubiquity of oxygenant or bleaching agent.Domestic food quality inspection report shows all have the hydrogen peroxide (hydrogen peroxide) of excess residual in part beans fabricated product, Flour product, fish ball, boiled salted duck, the Boiled chicken with salt etc.Also detected the hydrogen peroxide up to 1.5% in the shark's fin of the Hong Kong Special Administrative Region, " Chinese food health legislation " regulation must not have residual peroxide in food and the health products.That over hydrogenation hydrogen has is carcinogenic, it is old and feeble to quicken or bring out toxic action such as angiocardiopathy.The national standard method of hydrogen peroxide is chemical titration (GB1616-88 permanganimetric method or an iodimetric titration) in current detection food and the health products, the sample pretreatment process complexity of this method, detection time is long, titration end-point is difficult to be grasped, and often cause testing result higher because of the existence that reducing substances (as vitamin C etc.) is arranged in the food, and detectability is generally about 100ppm, does not reach many trace or trace requirement.Also have research to adopt the HPLC method to detect hydrogen peroxide in the food, the method need adopt large-scale precision instrument, and the pre-treatment process complexity of sample, and detection time is long.The utilization electrochemical method detects hydrogen peroxide, has very high sensitivity, but because the modification of enzyme electrode and testing conditions limit, can cause the poor stability of electrode, is difficult for preserving.China does not also set up corresponding hydrogen peroxide fast detecting national standard at present.
Find by prior art documents, Chinese patent application (patent) number is 03132054.6, name is called " a kind of method for preparing the TiO 2 gel film electrochemica biological sensor ", the gordian technique of this patent is to adopt TiO 2 gel film that electrode is modified, and adopts electrochemical method to detect hydrogen peroxide then.The process that this method prepares modified electrode is quite complicated, and form the thickness of TiO 2 gel film in the process of modifying and the enzyme amount of absorption can't effectively be controlled, cause the modified electrode poor stability prepared, and gel mould is unfavorable for preserving the biologically active of enzyme, cause the life-span of modified electrode to be no more than 14 days, thereby can not promote the field quick detection of hydrogen peroxide on a large scale.
Summary of the invention
The objective of the invention is at the deficiencies in the prior art, hydrogen peroxide quick test strip in a kind of detection food is provided, it is enhanced productivity, reduce production costs, and shorten sample preparation and detection time greatly, realize the field quick detection of sample, detect hydrogen peroxide with this strip, sample pre-treatments is simple, and detection time is short, generally is lower than 90 seconds; Detectability reaches 0.5ppm, good reproducibility, and the effective storage life of strip is 1 year, can realize the field quick detection of hydrogen peroxide in the food.
The present invention is achieved by the following technical solutions, test-strips of the present invention, it is a kind of photoelectric type biology sensor, comprise the insulative water-proof substrate, the reaction substrate diaphragm, filtering layer, spacer and cover plate, the insulative water-proof substrate is substrate, on the insulative water-proof substrate, be provided with a circular hole, above circular hole according to the reaction substrate diaphragm, the order of filtering layer covers the conversion zone that is assembled into test-strips successively, spacer on the conversion zone two ends cover, cover plate is arranged on the reaction substrate diaphragm, on filtering layer and the spacer, cover plate is provided with a circular hole, and this circular hole aligns with circular hole on the insulative water-proof substrate.
Described reaction substrate diaphragm, filtering layer width are 5mm, and length is identical with the insulative water-proof substrate width, and cover plate is consistent with the insulative water-proof sizes of substrate.
Described reaction substrate diaphragm is made up of reaction substrate rete and transparent substrate film slide glass, the sticking on the transparent substrate film slide glass of reaction substrate even film layer.
Described reaction substrate rete, thickness is 200～240nm, and peroxidase is arranged on it.
Described insulative water-proof substrate and cover plate, its thickness is 0.3～0.5mm, be polyester sheet, POLYCARBONATE SHEET, polystyrene sheet, polyimide piece, polyvinyl chloride (PVC) sheets, polythene strip or tygon terephthalate sheet, preferably polyethylene terephthalate sheet, this polymkeric substance are sheet material or coiled material.
The circular hole of opening on described insulative water-proof substrate and the cover plate is to make in the mode of punching press, and Circularhole diameter is 0.3～0.4mm, and hole wall is smooth, no burr, and its size should be consistent with the checkout equipment interface.
Described substrate film slide glass is transparent polyvinyl chloride sheet, polyester sheet, POLYCARBONATE SHEET, polystyrene sheet, polyimide piece, polythene strip or tygon terephthalate sheet, polyvinyl chloride (PVC) sheets preferably, and optimum thickness is 80～200nm.
Described spacer, its double-sided belt sticker sheet, thickness homogeneous, its thickness is 0.6mm, be polyester sheet, POLYCARBONATE SHEET, polystyrene sheet, polyimide piece, polyvinyl chloride (PVC) sheets, polythene strip or tygon terephthalate sheet, preferred polyvinyl chloride (PVC) sheets, this polymkeric substance is sheet material or coiled material.
Described filtering layer by do not absorb water, glass fiber sheets a little less than the absorption affinity forms thickness 0.5mm.
Compare with the existing technology that detects hydrogen peroxide, the invention has the advantages that, utilize the selection catalysis characteristics of biology enzyme under suitable reagent environment, to keep good biologically active, and carry out machinery and film and produce the thickness homogeneous, the fireballing substrate diaphragm of detection reaction, the production of whole strip is further simplified, enhance productivity, reduce production costs, and shorten the sample preparation time greatly, on average each sample detection time is 45 seconds, sensing range is 0.5mg/L-20mg/L, repeat samples detects relative standard deviation (RSD) 8.2%, and testing result rate of accuracy reached to 98% can reach the requirement of the field quick detection of sample.
Description of drawings
Fig. 1 detects principle schematic for the use of fast detecting hydrogen peroxide strip of the present invention
Fig. 2 is the STRUCTURE DECOMPOSITION synoptic diagram of fast detecting hydrogen peroxide strip of the present invention
Fig. 3 is the horizontal section synoptic diagram of fast detecting hydrogen peroxide strip of the present invention
Embodiment
Below in conjunction with accompanying drawing embodiments of the invention are elaborated: present embodiment is being to implement under the prerequisite with the technical solution of the present invention, provided detailed embodiment and concrete operating process, but protection scope of the present invention is not limited to following embodiment.
Fig. 1 detects principle schematic for the use of fast detecting hydrogen peroxide strip of the present invention, and as can be seen from Figure 1 change in optical signal derives from the reaction substrate diaphragm change in color of strip.Comprise insulative water-proof substrate 1, reaction substrate diaphragm 2, filtering layer 3, spacer 4 and the cover plate 5 of fast detecting hydrogen peroxide strip as can be seen from Figure 2 of the present invention are formed.The punching of mechanical stamping punch method is adopted in insulative water-proof substrate 1 and cover plate 5 superimposed backs, aperture 0.3～0.4mm, hole wall is apart from substrate edge 2mm, the requirement hole wall is smooth, no burr, the adsorption effect of hole wall in the time of reducing application of sample like this, sample is evenly distributed at well, punch position will match with the checkout equipment interface, and circular hole is a well on the cover plate 5, and circular hole is an instrument connection on the substrate 1.Insulative water-proof substrate 1 adopts the polymkeric substance of choosing any one kind of them in polyester, polycarbonate, polystyrene, polyimide, Polyvinylchloride, tygon, the tygon terephthalate composition to make the preferably polyethylene terephthalate.
The reaction substrate rete comprises developer, developer protective agent, water soluble polymer, peroxidase and enzyme live keeping agent, adopts the coating film forming method to make.Described developer is a kind of in aniline, hydrochloric acid o-tolidine, the tetramethyl hydrochloric acid o-tolidine etc., wherein, and hydrochloric acid o-tolidine preferably.During making, take by weighing hydrochloric acid o-tolidine 0.285g, add absolute ethyl alcohol 2mL, and then adding dioxane 2mL, the citrate buffer solution constant volume with pH5.0 is 100mL at last, adds water soluble chitosan 4.5g then, skimmed milk power 1g, sucrose 5g is dissolved in the above-mentioned solution, and 4 ℃ leave standstill 12h, forms gel.Add lemon yellow 50g then, 1mg/mL horseradish peroxidase 1mL stirs, and vacuumizes 30min then in vacuum dryer, putting it into 4 ℃ again leaves standstill 12h. and carries out coating film forming at last on substrate film carrying tablet, after the vacuum freeze drying, carry out filming vacuum freeze drying the second time.
Hydrogen peroxide can develop the color with a lot of substrate reactions under the catalytic action of horseradish peroxidase, according to substrate self stability and color stability and the comprehensive reagent environment that forms of reagent, suggestion is adopted in aniline, hydrochloric acid o-tolidine, the tetramethyl hydrochloric acid o-tolidine any one.In order to improve the contrast of change color, in the reaction substrate layer, add lemon yellow background color as the substrate film, hydrogen peroxide and developer reaction show blue, and blue and yellow background color is overlapped into the green with more obvious discrimination, helps improving the precision of sensor.The hydrophilic high mol material can make sample liquids enter enzyme reaction layer apace to react, and can stop lyophobic dust to enter enzyme reaction layer, reduces and disturbs, and can also allow the substrate film firmly be adsorbed on the substrate film carrying tablet.Described water soluble polymer is carboxymethyl cellulose, hydroxy ethyl cellulose, hydroxy propyl cellulose, methylcellulose and ethyl cellulose, aminomethylethyl cellulose, gelatin, agarose, a kind of in the water soluble chitosan, wherein, be preferably water soluble chitosan.In order effectively to prolong the life-span of strip, in substrate, add suitable developer protective agent and enzymatic protective reagent.Because strip is to adopt photoelectric sensor to detect, the thickness of reaction substrate diaphragm 2 and homogeneity determine its light transmission and reflection function, adopt mechanical rubbing method, and the film thickness homogeneous of making can drop to the difference of the disposable test strips produced minimum like this.
The reaction substrate rete is to take by weighing hydrochloric acid o-tolidine 0.285g, add absolute ethyl alcohol 2mL, and then adding dioxane 2mL, citrate buffer solution constant volume with pH5.0 is 100mL at last, add water soluble chitosan 4.5g then, skimmed milk power 1g, sucrose 5g are dissolved in the above-mentioned solution, 4 ℃ leave standstill 12h, form gel.Add lemon yellow 50g then, 1mg/mL horseradish peroxidase 1mL stirs, and vacuumizes 30min then in vacuum dryer, putting it into 4 ℃ again leaves standstill 12h. and carries out coating film forming at last on substrate film carrying tablet, after the vacuum freeze drying, carry out filming vacuum freeze drying the second time.Machinery is cut into the small pieces more bigger than substrate openings then, covers on the aperture of substrate 1.
The polymkeric substance of choosing any one kind of them during spacer 4 is made up of polyester, polycarbonate, polystyrene, polyimide, Polyvinylchloride, tygon, tygon terephthalate is made, preferred Polyvinylchloride.
When strip inserts the checkout equipment port, in the well of cover plate, add the sample solution that 20 microlitres contain hydrogen peroxide, solution sees through filtering layer, with reagent generation chromogenic reaction in the reaction substrate rete, the colour developing depth is relevant with the concentration of hydrogen peroxide, change color is an electric signal by conversion of photoelectric sensor, and electric signal shows the concentration of hydrogen peroxide through microprocessor processes by display.
Introduce the use of hydrogen peroxide test-strips below in conjunction with practical operation.
Sample preparation: (1) fluid sample: accurately pipette sample 0.9mL with the 1mL pipettor, add hydrogen peroxide agent (), stir, left standstill 5 minutes, centrifugal then, get supernatant, add hydrogen peroxide agent (two), the back that stirs is as solution to be measured.
(2) solid sample: take by weighing sample 1.00g with electronic balance, add distilled water 2mL then and stirred 1 minute, add hydrogen peroxide agent () then, stir, left standstill 5 minutes, centrifugal then, get supernatant, add hydrogen peroxide agent (two), the back that stirs is as solution to be measured.
Superoxol with 30% adopts the permanganimetric method titration, is diluted to following concentration: 0.0mg/L, 0.5mg/L, 1mg/L, 2mg/L, 3mg/L, 4mg/L, 6mg/L, 8mg/L, 12mg/L, 16mg/L, 20mg/L then respectively as titer.
Employing contains developer 0.01mol/L, and horseradish peroxidase 0.01mg/L prescription is produced test-strips, tests.The hydrogen peroxide titer is dropped in the well of the hydrogen peroxide test-strips made from said method, measure the reflectivity of strip after 30 seconds, 45 seconds, 60 seconds, 75 seconds, 90 seconds, 105 seconds, 120 seconds, 135 seconds, 150 seconds (behind the application of sample before strip reflective light intensity/application of sample strip reflective light intensity), each concentration of hydrogen peroxide replicate determination four times, the reflectivity that concentration of hydrogen peroxide and sensor are surveyed become good proportional relationship 75 seconds the time behind application of sample.
Test-strips is made up of insulative water-proof substrate 1, reaction substrate diaphragm 2, filtering layer 3, spacer 4 and cover plate 5, insulative water-proof substrate 1 is substrate, on insulative water-proof substrate 1, be provided with a circular hole, order according to reaction substrate diaphragm 2, filtering layer 3 above circular hole covers the conversion zone that is assembled into test-strips successively, spacer 4 on the conversion zone two ends cover, cover plate 5 is arranged on reaction substrate diaphragm 2, filtering layer 3 and the spacer 4, cover plate 5 is provided with a circular hole, and this circular hole aligns with circular hole on the insulative water-proof substrate 1.
The insulative water-proof substrate 1 of test-strips, filtering layer 3, spacer 4 and cover plate 5 material therefors are with embodiment 1, and concrete manufacturing process is as follows:
1. insulative water-proof substrate 1 and cover plate 5 punchings.Drilling method, aperture and requirement are with embodiment 1.
2. the making of reaction substrate rete.Agent prescription adopts developer 0.143g, horseradish peroxidase 0.01mg/L, and other reagent dosages and method for making are with embodiment 1.Reaction substrate rete machinery with preparation is cut into the small pieces more bigger than substrate circular hole then, covers on the aperture of insulative water-proof substrate 1.
3. glass fiber sheets is cut into the identical small pieces of size and reaction substrate diaphragm 2, superimposed being covered on the reaction substrate diaphragm 2 forms filtering layer 3.
4. paste spacer 4, with reaction substrate diaphragm 2 and filtering layer 3 card jails.
5. cover cover plate 5, guarantee that the cover plate circular hole should overlap fully with the substrate circular hole.
After making strip as stated above, the configuration of sample preparation and hydrogen peroxide standard solution is tested then with embodiment 1.Hydrogen peroxide titer 20 microlitres are dropped in the well of the hydrogen peroxide test-strips made from said method, measure the reflectivity of strip after 30 seconds, 45 seconds, 60 seconds, 75 seconds, 90 seconds, 105 seconds, 120 seconds (behind the application of sample before strip reflective light intensity/application of sample strip reflective light intensity), each concentration of hydrogen peroxide replicate determination four times, the reflectivity that concentration of hydrogen peroxide and sensor are surveyed become good proportional relationship 45 seconds the time behind application of sample.
The insulative water-proof substrate of test-strips, filtering layer, spacer and cover plate material therefor are with embodiment 1, and concrete manufacturing process is as follows:
1. insulative water-proof substrate and cover plate punching.Drilling method, aperture and requirement are with embodiment 1.
2. the making of reaction substrate rete 2.Agent prescription adopts developer 0.285g, horseradish peroxidase 0.001mg/L, and other reagent dosages and method for making are with embodiment 1.Reaction substrate rete machinery with preparation is cut into the small pieces more bigger than substrate circular hole then, covers on the aperture of insulative water-proof substrate 1.
3. glass fiber sheets is cut into the identical small pieces of size and reaction substrate diaphragm 2, superimposed being covered on the reaction substrate diaphragm 2 forms filtering layer 3.
4. paste spacer 4, with reaction substrate diaphragm 2 and filtering layer 3 card jails.
5. cover cover plate 5, guarantee that cover plate 5 circular holes should overlap fully with insulative water-proof substrate 1 circular hole.
After making strip as stated above, the configuration of sample preparation and hydrogen peroxide standard solution is tested then with embodiment 1.Hydrogen peroxide titer 20 microlitres are dropped in the well of the hydrogen peroxide test-strips made from said method, measure the reflectivity of strip after 30 seconds, 45 seconds, 60 seconds, 75 seconds, 90 seconds, 105 seconds, 120 seconds (behind the application of sample before strip reflective light intensity/application of sample strip reflective light intensity), each concentration of hydrogen peroxide replicate determination four times, the reflectivity that concentration of hydrogen peroxide and sensor are surveyed become good proportional relationship 150 seconds the time behind application of sample.
According to the linear and detection time of concentration of hydrogen peroxide in colour developing degree and the sample to be checked, cost consideration, employing contains developer 0.005mol/L, it is optimal case that horseradish peroxidase 0.01mg/L prescription is produced test-strips, test-strips is inserted micro photo electric formula sensing instrument, add sample then, the direct just concentration of hydrogen peroxide in the sample as can be known of reading after 45 seconds, this shows the content that adopts fast detecting hydrogen peroxide strip of the present invention to detect hydrogen peroxide in the food, sample-pretreating method is simple, easy to operate, be 45 seconds detection time, sensing range is 0.5mg/L-20mg/L, repeat samples detects relative standard deviation (RSD) 8.2%, and testing result rate of accuracy reached to 98% can realize the field quick detection of hydrogen peroxide.
Claims (10)
1. one kind is detected hydrogen peroxide quick test strip in the food, comprise insulative water-proof substrate, reaction substrate diaphragm, filtering layer, spacer and cover plate, it is characterized in that, the insulative water-proof substrate is substrate, on the insulative water-proof substrate, be provided with a circular hole, order according to reaction substrate diaphragm, filtering layer above circular hole covers the conversion zone that is assembled into test-strips successively, spacer on the conversion zone two ends cover, cover plate is arranged on reaction substrate diaphragm, filtering layer and the spacer, cover plate is provided with a circular hole, and this circular hole aligns with circular hole on the insulative water-proof substrate.
2. hydrogen peroxide quick test strip in the detection food according to claim 1 is characterized in that, described reaction substrate diaphragm and filtering layer, and width is 5mm, and length is all identical with the insulative water-proof substrate width, and cover plate is identical with the insulative water-proof sizes of substrate.
3. hydrogen peroxide quick test strip in the detection food according to claim 1 is characterized in that, described reaction substrate diaphragm is made up of reaction substrate rete and transparent substrate film slide glass, the sticking on the transparent substrate film slide glass of reaction substrate even film layer.
4. hydrogen peroxide quick test strip in the detection food according to claim 3 is characterized in that, described reaction substrate rete, and thickness is 200～240nm, and peroxidase is arranged on it.
5. hydrogen peroxide quick test strip in the detection food according to claim 3, it is characterized in that, described substrate film slide glass is transparent polyvinyl chloride sheet, polyester sheet, POLYCARBONATE SHEET, polystyrene sheet, polyimide piece, polythene strip or tygon terephthalate sheet.
6. according to hydrogen peroxide quick test strip in claim 3 or the 5 described detection food, it is characterized in that described substrate film slide glass is a polyvinyl chloride (PVC) sheets, thickness is 80～200nm.
7. hydrogen peroxide quick test strip in the detection food according to claim 1, it is characterized in that, described insulative water-proof substrate and cover plate, its thickness is 0.3～0.5mm, is polyester sheet, POLYCARBONATE SHEET, polystyrene sheet, polyimide piece, polyvinyl chloride (PVC) sheets, polythene strip or tygon terephthalate sheet.
8. according to hydrogen peroxide quick test strip in claim 1 or the 7 described detection food, it is characterized in that, described insulative water-proof substrate and cover plate, its Circularhole diameter is 0.3～0.4mm, hole wall is smooth.
9. hydrogen peroxide quick test strip in the detection food according to claim 1, it is characterized in that, described spacer, its double-sided belt sticker sheet, thickness is 0.6mm, is polyester sheet, POLYCARBONATE SHEET, polystyrene sheet, polyimide piece, polyvinyl chloride (PVC) sheets, polythene strip or tygon terephthalate sheet.
10. hydrogen peroxide quick test strip in the detection food according to claim 1 is characterized in that described filtering layer is made up of glass fiber sheets, and thickness is 0.5mm.
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| CNB2007100471546A CN100510717C (en) | 2007-10-18 | 2007-10-18 | Quick test strip for detecting hydrogen peroxide in food |
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| CNB2007100471546A CN100510717C (en) | 2007-10-18 | 2007-10-18 | Quick test strip for detecting hydrogen peroxide in food |
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