CN1297992A - Composite enzymatic sensitive film containing nanometer silver sol and its preparation and use - Google Patents
Composite enzymatic sensitive film containing nanometer silver sol and its preparation and use Download PDFInfo
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Abstract
Nanometer silver sol solution of 1x10 to the minus sixth -10 to the minus first mol/Kg concentration in 0.1-10 ml is mixed with aqueous oxidase solution of 50-1000 activity unit/ml concentration in 0.05-2 ml; the mixture is first mixed with polymer gel solution of 0.5-5 % concentration in 10-200 ml through stirring and then cross-linked with glutaraldehyde solution of 1-10 % concentration and in 0.1-5 ml; the mixture solution is painted onto solid carrier to form one layer of composite sensitive oxidase film containing nanometer silver sol after volatilization. The oxidase film of the present invention results in less oxidase consumption in biological product, and is used in biological sensor and biological enzymatic engineering widely.
Description
The invention belongs to the enzyme technical field of bioengineering, particularly relate to the compound high sensitivity sensitive membrane of the enzyme function that contains nano silver colloidal sol and method for making and purposes.
The product that utilizes the enzyme biotechnology to produce is widely used in human body and the industry, for example: utilize the glucose biological sensor of glucose oxidase assembling, can be used for measuring the sugar in people's blood sugar, trade effluent, fermentation or the beverage.Biosensor has very significantly advantage, as: high specificity, in short-term low cost consumption, amalyzing substances is not had special requirement, operational safety, is convenient in situ measurement or the like, in the existing a lot of achievements of many countries.Transmitter the research worker all understand, and the development enzyme biologic sensor at first is the biosensor unit of development with the reaction of the molecular recognition of efficiency operation, to the target of this pursuit is: sensitive, inexpensive, microminiaturized.But the biosensor development so far, can enter the few in number of actual use.With the glucose biological sensor is example, and the electric current of the hydrogen peroxide that glucose biological sensor when work normally produces based on to the glucose oxidase catalytic oxidation of glucose time detects.This method has following limitation: when the concentration of dissolved oxygen is not enough big, be difficult to the high-content substrate is measured; And enzyme dosage when too much the response current transfer efficiency low.In recent years, metal, physical property that the metal-oxide semiconductor (MOS) nano particle is peculiar attract much attention.Nano particle when the certain size, has the new property and the new effect that or not with its respective masses material when little, as: surface effects, volume effect, tunnel effect, quantum effect etc.Because its surface effects, surface of metal particles has very high negative charge, is easy to cause great interest of people and concern by finishing regulation and control optics, electrology characteristic etc.Wherein metallic particles electroconductibility is preferable, can transmit electronics between enzyme and electrode.Improve the catalytic activity of immobilized enzyme.People such as U.S. A.L.Crumbliss. once introduced hydrophilic gold grain in biosensor, proved the biocompatibility of gold grain, but their usefulness is the hydrophilic gold grain of 50 nanometers, current-responsive susceptibility to enzyme electrodes does not have help, this is at " biotechnology and biotechnology " magazine, 1992,40 volumes, the article of 483-490 page or leaf " is applicable to a kind of physiologically acceptable mounting medium-Radioactive colloidal gold as electric precipitation method assembling enzyme electrode " and has report (A.L.Crumbliss.S.C.Perine, J.Stonehuerner, K.R.Tubergen, Junguo Zhao, andR.W.Henkens, Biotechnology and Bioengineering, Vol.40 Pp.483-490 (1992)).
Reported the oxidase function compound sensitive film that contains hydrophobic nanometre prill in the Chinese patent application 97116989.6, the enzyme membrane electrode current response sensitivity that contains glucose oxidase has usually been improved at least 10 times.At present Study on Biosensor just towards high-performance, microminiaturization, enter into family's direction and develop, be a kind of hi-tech that is generally believed most possible formation industry.
The objective of the invention is to nano silver colloidal sol particularly the hydrophobic nanometre silver sol introduce in the composite enzymatic sensitive film, provide a kind of and can save enzyme dosage, improve the composite enzymatic sensitive film that contains nano silver colloidal sol and the method for making and the purposes of the sensitivity of enzyme biologic sensor current-responsive.This composite enzymatic sensitive film that contains nano silver colloidal sol, enzyme dosage only are the parts of common consumption, and improve tens times of the current-responsive susceptibilitys of enzyme biologic sensor.
The objective of the invention is to be achieved through the following technical solutions:
The composite enzymatic sensitive film that contains nano silver colloidal sol of the present invention is characterized in that obtaining by following method: with weight-molality is 1 * 10
-6～10
-1Nano silver colloidal sol solution 0.1～10ml of mol/kg is the oxydase aqueous solution 0.05～2ml mixing of 50～1000 unit of activity/milliliters with concentration; Then add weight percent concentration and be high molecular gelating soln 10～200ml of 0.5～5%, stir, add weight percent concentration again and be glutaraldehyde 0.1～5ml of 1～10% and carry out crosslinked; Above-mentioned mixing solutions is coated on the solid carrier, vapors away solvent under the room temperature, form the oxidase function compound sensitive film that one deck contains nano silver colloidal sol at solid carrier surface.
Wherein nano silver colloidal sol solution is hydrophilic nano silver colloidal sol solution or hydrophobic nanometre silver sol solution, and the particle diameter of silver sol is 1～25 nanometer, is preferably 1～12 nanometer; Oxydase is glucose oxidase, Lactate Oxidase, alcohol oxidase or XOD etc.; Polymer is cellulose acetate, polyvinyl butyral acetal, urethane, polyoxyethylene glycol, polysulfoxides, polyureas or ethylene-vinyl alcohol copolymer etc.; Solid carrier is a platinum filament.
The method for making that contains the composite enzymatic sensitive film of nano silver colloidal sol of the present invention is characterized in that carrying out as follows:
The first step: adopt sol-gel method to prepare hydrophilic nano silver colloidal sol solution, concrete preparation method is as follows:
1.. prepare the aqueous solution of Silver Nitrate and reductive agent respectively, its weight-molality is respectively 1 * 10
-6-1 * 10
-1Mol/Kg and 1 * 10
-6-1mol/Kg; Wherein reductive agent is sodium borohydride, citric acid, Trisodium Citrate, ethanol or hydrazine etc.;
2.. prepare hydrophilic nano silver colloidal sol solution: room temperature or under 25-90 ℃, add the aqueous solution of silver nitrate aqueous solution and reductive agent in the reaction vessel respectively, wherein the mol ratio of the aqueous solution of silver nitrate aqueous solution and reductive agent is 1: 1～1: 30, mixing solutions under agitation reacted 0.5～10 hour, promptly obtain particle diameter in 1～25 nanometer, be preferably the hydrophilic nano silver colloidal sol solution of 1～12 nanometer, its weight-molality is 5 * 10
-6-1 * 10
-1Mol/kg.
Or adopt reverse micelle method to prepare hydrophobic nanometre silver sol solution:
Reverse micelle is meant surfactant dissolves in non-polar organic solvent, when its concentration surpasses micelle-forming concentration (CMC), forms the hydrophilic polar group head inwardly, the liquid particle structure that hydrophobic chain is outside.Endorse the ionogen of a large amount of water molecules of solubilising and water soluble and different compounds in the reverse micelle, the water nuclear of formation is one good " microreactor ".
Concrete preparation method is as follows:
1. prepare the non-polar organic solvent solution of tensio-active agent, its weight-molality is 50-300 mmole/kilogram solvent; Wherein tensio-active agent is that (molecular formula is for nonionogenic tenside such as alkylphenol polyoxyethylene
Alkyl R is C
7～C
9Alkyl) series, Span or sorbitan fatty acid ester series; Anion surfactant such as dioctyl succinate sodium sulfonate (being commonly called as AOT); Natural surface active agent such as Yelkin TTS etc.; Non-polar organic solvent is normal hexane, hexanaphthene, octane-iso or chloroform etc.
2. prepare the silver nitrate aqueous solution and the reductive agent aqueous solution respectively, its weight-molality is 1 * 10
-6～1 * 10
-1Mole/kilogram, and all according to the big or small desired water of hydrophobic nanometre silver sol and the mol ratio (R of tensio-active agent
wValue) and the mol ratio (R of ionogen Silver Nitrate or reductive agent and tensio-active agent
EValue) determines R
EValue comprises R
AgValue and R
AlsoValue.Work as R
wWhen value is 1-15, R
EValue is 0.0001-0.015; Or work as R
wWhen value is 22-50, R
EValue is 0.01-0.6; Wherein reductive agent is citric acid, Trisodium Citrate, sodium borohydride, ethanol or hydrazine etc.;
Water nuclear size in the reverse micelle is by the amount decision of solubilising water, that is: R
wThe size of value has been represented the diameter of the water nuclear that is surrounded by tensio-active agent, with R
wThe increase of value, water nuclear diameter increase, and therefore carry out chemical reaction when preparing ultra-fine grain in water nuclear, and the size of water nuclear directly limits the size of particle size, is the main factor of control granular size.R
EThe size of value has been represented the electrolytical amount of solubilising in the water nuclear that is surrounded by tensio-active agent, with R
EThe increase of value, the Silver Nitrate of institute's solubilising or the amount of reductive agent increase.Its consumption decides with different solvents with used tensio-active agent.Among the present invention, grain diameter is with R
AgValue increases and increases.But the investigator who does this respect work should know that this variation is compared with the water droplet volume that causes colloidal solid to increase, and this variation is gentle.
3. the inverse micellar solution for preparing Silver Nitrate and reductive agent: the step that the Silver Nitrate for preparing in getting 2. and the aqueous solution of reductive agent join 0.01～1 liter of equal volume respectively 1. in the non-polar organic solvent solution of tensio-active agent of preparation, the consumption of the aqueous solution of Silver Nitrate and reductive agent is according to the big or small desired R of hydrophobic nanometre silver sol
wValue and R
EValue is determined, wherein works as R
wWhen value is 1-15, R
EValue is 0.0001-0.015; Or work as R
wWhen value is 22-50, R
EValue is 0.01-0.6;
4.. preparation hydrophobic nanometre silver sol solution: under room temperature or 25-35 ℃, by equal volume with step in 3. Silver Nitrate and the inverse micellar solution of reductive agent under agitation mixed 1～8 hour, promptly obtain particle diameter in 1～25 nanometer, be preferably the hydrophobic nanometre silver sol solution of 1～12 nanometer, its weight-molality is 5 * 10
-6～1 * 10
-1Mole/kilogram.
Second step: prepare high molecular gelating soln:
Polymer substance is dissolved in corresponding organic solvent or the water, makes that the weight percent concentration of solution is 0.5～5%; Wherein polymer substance is cellulose acetate, polyvinyl butyral acetal, urethane, polyoxyethylene glycol, polysulfoxides, polyureas, hydroxypropylcellulose or ethylene-vinyl alcohol copolymer etc.; Organic solvent is acetone, dehydrated alcohol, epoxy six alkane or Virahol etc.;
The 3rd step: the oxidase function compound sensitive film that preparation contains nano silver colloidal sol carries out as follows:
With weight-molality is 5 * 10
-6～1 * 10
-1Nano silver colloidal sol solution 0.1～10ml of mol/kg is the oxydase aqueous solution 0.05～2ml mixing of 50～1000 unit of activity/milliliters with concentration; Then add weight percent concentration and be high molecular gelating soln 10～200ml of 0.5～5%, stir, add weight percent concentration again and be glutaraldehyde 0.1～5ml of 1～10% and carry out crosslinking Treatment; Above-mentioned mixing solutions is coated on the solid carrier, under the room temperature, vapors away solvent, form the oxidase function compound sensitive film that one deck contains nano silver colloidal sol at solid carrier surface.
Wherein silver sol solution is hydrophilic nano silver colloidal sol solution or hydrophobic nanometre silver sol solution, and the particle diameter of silver sol is 1～25 nanometer, is preferably 1～12 nanometer; Oxydase is glucose oxidase, Lactate Oxidase, alcohol oxidase, XOD etc.; Polymer is cellulose acetate, polyvinyl butyral acetal, urethane, polyoxyethylene glycol, polysulfoxides, polyureas, hydroxypropylcellulose or ethylene-vinyl alcohol copolymer etc.; Solid carrier is a platinum filament.
Purposes of the present invention:
The composite enzymatic sensitive film that the present invention is prepared can be used for enzyme biologic sensor, bioseparation film and biocatalysis engineering.Especially glucose biological sensor is specially adapted to measurement of sugar in blood sugar, trade effluent, nutrient solution, fermentation and the beverage in the human body etc.
Advantage of the present invention and effect:
The prepared nano silver colloidal sol microbial film that goes out of the present invention has the incomparable superiority of conventional organic-biological film, just can find out from the biological sensor electrode of making of film of the present invention.
The glucose oxidase microbial film of the nano silver colloidal sol that will make with aforesaid method is coated in the electrode made on the platinum filament as working electrode, and Ag/AgCl electrode or mercurous chloride electrode are formed the glucose oxidase enzyme biologic sensor as reference electrode.It is in the phosphate buffered solution battery of 6-7 that the two electrodes of glucose oxidase enzyme biologic sensor is placed pH value, survey its matrix electric current, the D/W that adds different concns then successively, survey its response current, make the electrode current response curve corresponding to corresponding glucose concn (every liter of 2-35mmol glucose).
Because the response that glucose is had feature of this kind biological sensor electrode enzyme membrane, so it is specially adapted to the blood sugar in the human body, trade effluent, fermentation, the measurement of sugar etc. in the beverage.
Because the used enzyme membrane of this kind biological sensor electrode contains particle diameter 1-25 nanometer, particularly 1-12 nano silver colloidal sol (again particularly hydrophobic silver sol), so that its electrode current response ratio is not introduced the electrode current of silver sol is high tens times.And enzyme dosage is 1/5th～50 of a common consumption.As shown in Figure 1: curve A is to contain the electrode current response curve that the glucose oxidase electrode of hydrophobic nanometre silver sol is done glucose concn, and curve B is not contain the electrode current response curve that the glucose oxidase electrode of hydrophobic nanometre silver sol is done glucose concn.The current-responsive value of curve A has improved 50 times than the current-responsive value of curve B.
Because the introducing of nano level hydrophobic silver sol, the hydrophobic chain effect of hydrophobic silver sol surface hydrophobicity chain and glucose oxidase makes the glucose oxidase conformation become more stable, and the result of this hydrophobic interaction has also increased the sensitivity of glucose oxidase.Simultaneously, at the high polar organic solvent miscible with water, in ethanol, methyl alcohol, acetone, it on the surface of electrode, still keeps very high enzymic activity with the high-molecular gel Covalent Immobilization.And usually, hydrophilic oxydase very fast inactivation of meeting in these high polar organic solvents, and lose catalytic activity.The glucose sensor that contains particle diameter 1-10nm hydrophobic nanometre silver sol has reduced the consumption of glucose oxidase simultaneously more, because the electron transport katalysis of hydrophobic silver sol, only use several-1/tens of common enzyme dosage in every 1ml high-molecular gel, the i.e. glucose oxidase of tens even several units, just can obtain several thousand even a few Wan Naan/centimetre
2(nA/cm
2) response current.In addition, the stability that contains the glucose sensor of hydrophobic silver sol also improves greatly, experiment METHOD FOR CONTINUOUS DETERMINATION 50 times, and the electrode current response results repeats very much.And the transmitter of not introducing the nano hydrophobic silver sol only can replication about 7,8 times.
Just can find out that from The above results microbial film of the present invention is specially adapted to make the used product of biotechnology, its effect has outstanding qualitative change.
In a word, the present invention has following advantage:
1. prepare simplely, do not need chemical after-treatment reason, be easy to apply.
2. because nano level silver sol, the particularly introducing of hydrophobic silver sol, the product that can make product stability and life-span not introduce the nano hydrophobic silver sol improves greatly, used enzyme amount also significantly reduces than common consumption, and this just greatly reduces the cost of the composite enzymatic sensitive film that contains nano silver colloidal sol.
3. at organic solvent, though have with the mixable organic solvent of water in, still can guarantee the immobilization of zymoprotein not damaged.
Description of drawings:
Fig. 1-electrode response electric current is corresponding to the glucose concn relation curve
X-coordinate---glucose concn (unit: mmol/L)
Ordinate zou---response current (unit: nA/cm
2)
Curve A---contain that the glucose sensor of hydrophobic nanometre silver sol does glucose concn
The stream response curve
Curve B---electric current that the glucose sensor of hydrophobic silver sol is done glucose concn do not contained
Response curve
Fig. 2--contain the system of hydrophilic nano silver colloidal sol and glucose oxidase composite enzymatic sensitive film bioelectrode
The electrode response electric current is corresponding to the glucose concn relation curve among the method embodiment 1
X-coordinate---glucose concn (unit: mmol/L)
Ordinate zou---response current (unit: nA/cm
2)
Curve C---contain that the glucose sensor of hydrophilic nano silver colloidal sol does glucose concn
The stream response curve
Fig. 3--contain the system of hydrophobic nanometre silver sol and glucose oxidase composite enzymatic sensitive film bioelectrode
The electrode response electric current is corresponding to the glucose concn relation curve among the method embodiment 2
X-coordinate---glucose concn (unit: mmol/L)
Ordinate zou---response current (unit: nA/cm
2)
Curve D---contain that the glucose sensor of hydrophobic nanometre silver sol does glucose concn
The stream response curve
Fig. 4--contain among the method for making embodiment 3 of hydrophobic nanometre silver sol and glucose oxidase composite enzymatic sensitive film bioelectrode the electrode response electric current corresponding to glucose concn relation curve X-coordinate--glucose concn (unit: mmol/L) ordinate zou---response current (unit: nA/cm
2) curve E---contains that the glucose sensor of hydrophobic nanometre silver sol does glucose concn
The stream response curve
The invention will be further described below in conjunction with drawings and Examples: embodiment 1
The first step: adopt sol-gel method to prepare hydrophilic nano silver colloidal sol solution:
Prepare the aqueous solution of Silver Nitrate and borane reducing agent sodium hydride respectively, its weight-molality is respectively 3.9 * 10
-4Mol/Kg and 3.9 * 10
-3Mol/Kg;
Prepare hydrophilic nano silver colloidal sol solution: 80 ℃ of aqueous solution with silver nitrate aqueous solution and borane reducing agent sodium hydride add in the reaction vessel respectively, wherein the mol ratio of the aqueous solution of silver nitrate aqueous solution and borane reducing agent sodium hydride is 1: 10, mixing solutions under agitation reacted 0.5 hour, promptly obtain the hydrophilic nano silver colloidal sol solution of particle diameter in 6 nanometers, its weight-molality is 3.9 * 10
-4Mol/Kg.
Second step: prepare high molecular gelating soln:
The polymer substance polyvinyl butyral acetal is dissolved in the dehydrated alcohol, makes that the weight percent concentration of solution is 2%;
The 3rd step: the oxidase function compound sensitive film that preparation contains nano silver colloidal sol carries out as follows:
With weight-molality is 3.9 * 10
-4The nano silver colloidal sol solution 2ml of mol/Kg is the glucose oxidase enzyme aqueous solution 0.3ml mixing of 500 unit of activity/milliliters with concentration; Then add weight percent concentration and be the gelating soln 10ml of 2% high-molecular polyivnyl alcohol butyral, stir, add concentration expressed in percentage by weight again and be 1% glutaraldehyde 1ml and carry out crosslinked; Above-mentioned mixing solutions is coated on the solid carrier platinum filament, under the room temperature, vapors away solvent, form the oxidase function compound sensitive film that one deck contains nano silver colloidal sol on solid carrier platinum filament surface.
With the Ag/AgCl electrode as reference electrode, it is in 6.86 the phosphate buffered solution battery that two electrodes is placed the 5ml pH value, survey its matrix electric current, add molarity then successively and be 2.8,5.6,11.1,16.7,22.2,27.8, β-D D/W of 33.3mmol/L, survey its response current (Y-axis), make response curve C corresponding to corresponding glucose concn (X-axis), as shown in Figure 2, glucose concn is 33mmol/L, and the electrode response electric current can reach 7875nA/cm
2And do not contain the nano-hydrophilic silver sol, but the electrode current that contains the glucose oxidase electrode of same enzyme consumption responds only 500nA/cm
2Embodiment 2
The first step: adopt reverse micelle method to prepare hydrophobic nanometre silver sol solution:
1). the cyclohexane solution of preparation anion surfactant dioctyl succinate sodium sulfonate (AOT), its weight-molality is 200 mmoles/kilogram solvent;
2). prepare silver nitrate aqueous solution and borane reducing agent sodium hydride aqueous solution respectively, its weight-molality is respectively 3.9 * 10
-4Mole/kilogram, 7.8 * 10
-4Mole/kilogram, desired R
wValue is 8, R
AgValue is 0.005 and R
AlsoIt is 0.01 value.
3). the inverse micellar solution of preparation Silver Nitrate and borane reducing agent sodium hydride: the aqueous solution of getting Silver Nitrate and borane reducing agent sodium hydride joins respectively in the bad hexane solution of non-polar organic solvent of the tensio-active agent of preparing in the step 1) of 0.1 liter of equal volume, and the consumption of the aqueous solution of Silver Nitrate and borane reducing agent sodium hydride is according to the big or small desired R of hydrophobic nanometre silver sol
wValue 8, R
AgValue is 0.005 and R
AlsoBe 0.01.
4). preparation hydrophobic nanometre silver sol solution: under 25 ℃, under agitation mixed 8 hours by the inverse micellar solution of equal volume Silver Nitrate in the step 3) and borane reducing agent sodium hydride, promptly obtain the hydrophobic nanometre silver sol solution of particle diameter in 8 nanometers, its weight-molality is 3.9 * 10
-4Mole/kilogram.
Second step: prepare high molecular gelating soln:
The polymer substance polyvinyl butyral acetal is dissolved in the dehydrated alcohol, makes that the weight percent concentration of solution is 2%;
The 3rd step: the oxidase function compound sensitive film that preparation contains nano silver colloidal sol carries out as follows:
With weight-molality is 3.9 * 10
-4The nano silver colloidal sol solution 5ml of mole/kilogram is the glucose oxidase enzyme aqueous solution 0.3ml mixing of 500 unit of activity/milliliters with concentration; Then add weight percent concentration and be the gelating soln 10ml of 2% high-molecular polyivnyl alcohol butyral, stir, add weight percent concentration again and be 1% glutaraldehyde 1.2ml and carry out crosslinked; Above-mentioned mixing solutions is coated on the solid carrier platinum filament, vapors away solvent under the room temperature, form the oxidase function compound sensitive film that one deck contains nano silver colloidal sol on solid carrier platinum filament surface.
Survey its response current by the mode of embodiment 1, make response curve D corresponding to corresponding glucose concn.As shown in Figure 3, glucose concn is 33mmol/l, and the electrode response electric current can reach 32365nA/cm
2Embodiment 3
The first step: adopt reverse micelle method to prepare hydrophobic nanometre silver sol solution:
1). the cyclohexane solution of preparation nonionogenic tenside polyoxyethylene octylphenol ether (TritonX-45), its weight-molality is 300 mmoles/kilogram solvent;
2). prepare silver nitrate aqueous solution and reductive agent sodium citrate aqueous solution respectively, its weight-molality is respectively 2.3 * 10
-5Mole/kilogram, 6.9 * 10
-4Mole/kilogram, desired R
wValue is 6, R
AgValue is 0.0003 and R
AlsoBe 0.009.
3). the inverse micellar solution of preparation Silver Nitrate and reductive agent Trisodium Citrate: the aqueous solution of getting Silver Nitrate and reductive agent Trisodium Citrate joins respectively in the cyclohexane solution of the tensio-active agent TritonX-45 for preparing in the step 1) of 0.1 liter of equal volume, and the consumption of the aqueous solution of Silver Nitrate and reductive agent Trisodium Citrate is according to the big or small desired R of hydrophobic nanometre silver sol
wValue 6, R
AgValue is 0.0003 and R
AlsoBe 0.009.
4). preparation hydrophobic nanometre silver sol solution: under 30 ℃, under agitation mixed 8 hours by the inverse micellar solution of equal volume Silver Nitrate in the step 3) and reductive agent Trisodium Citrate, promptly obtain the hydrophobic nanometre silver sol solution of particle diameter in 8 nanometers, its weight-molality is 2.3 * 10
-5Mole/kilogram.
Second step: prepare high molecular gelating soln:
The polymer substance cellulose acetate is dissolved in the acetone, makes that the weight percent concentration of solution is 1%;
The 3rd step: the oxidase function compound sensitive film that preparation contains nano silver colloidal sol carries out as follows:
With weight-molality is 2.3 * 10
-5The nano silver colloidal sol solution 30ml of mole/kilogram is the glucose oxidase enzyme aqueous solution 5ml mixing of 500 unit of activity/milliliters with concentration; Then add weight percent concentration and be the gelating soln 100ml of 1% polymer cellulose acetate, stir, add weight percent concentration again and be 10% glutaraldehyde 2ml and carry out crosslinking Treatment; Above-mentioned mixing solutions is coated on the solid carrier platinum filament, under the room temperature, vapors away solvent, form the oxidase function compound sensitive film that one deck contains nano silver colloidal sol on solid carrier platinum filament surface.
Survey its response current by the mode of embodiment 1, make response curve E corresponding to corresponding glucose concn.As shown in Figure 4, glucose concn is 33mmol/l, and the electrode response electric current can reach 14526nA/cm
2Embodiment 4
The first step: adopt reverse micelle method to prepare hydrophobic nanometre silver sol solution:
1). the non-polar organic solvent chloroformic solution of preparing natural tensio-active agent Yelkin TTS, its weight-molality are 100 mmoles/kilogram solvent;
2). prepare silver nitrate aqueous solution and reductive agent sodium citrate aqueous solution respectively, its weight-molality is respectively 1.6 * 10
-3Mole/kilogram, 1.6 * 10
-2Mole/kilogram, desired R
wValue is 10, R
AgValue is 0.002 and R
AlsoIt is 0.015 value.
3). the inverse micellar solution of preparation Silver Nitrate and reductive agent Trisodium Citrate: the aqueous solution of getting Silver Nitrate and reductive agent Trisodium Citrate joins respectively in the non-polar organic solvent chloroformic solution of the tensio-active agent of preparing in the step 1) of 0.1 liter of equal volume, and the consumption of the aqueous solution of Silver Nitrate and reductive agent Trisodium Citrate is according to the big or small desired R of hydrophobic nanometre silver sol
wValue 10, R
AgValue is 0.002 and R
AlsoBe 0.015.
4). preparation hydrophobic nanometre silver sol solution: under 25 ℃, under agitation mixed 8 hours by the inverse micellar solution of equal volume Silver Nitrate in the step 3) and reductive agent Trisodium Citrate, promptly obtain the hydrophobic nanometre silver sol solution of particle diameter in 9 nanometers, its weight-molality is 1.6 * 10
-3Mole/kilogram.
Second step: prepare high molecular gelating soln:
Polymer substance urethane is dissolved in epoxy six alkane, makes that the weight percent concentration of solution is 1%;
The 3rd step: the oxidase function compound sensitive film that preparation contains nano silver colloidal sol carries out as follows:
With weight-molality is 1.6 * 10
-3The nano silver colloidal sol solution 1ml of mole/kilogram is the Lactate Oxidase aqueous solution 0.1ml mixing of 50 unit of activity/milliliters with concentration; Then add weight percent concentration and be the gelating soln 10ml of 1% macromolecule polyurethane, stir, add concentration expressed in percentage by weight again and be 1% glutaraldehyde 0.2ml and carry out crosslinked; Above-mentioned mixing solutions is coated on the solid carrier platinum filament, vapors away solvent under the room temperature, form the oxidase function compound sensitive film that one deck contains nano silver colloidal sol on solid carrier platinum filament surface.
Survey its response current by the mode of embodiment 1, the electrode response electric current can reach 2436nA/cm when glucose concn was 10mmol/L
2Embodiment 5
The first step: adopt reverse micelle method to prepare hydrophobic nanometre silver sol solution:
1). the hexane solution of preparation polyoxyethylene nonylphenol ether (6), its weight-molality is 200 mmoles/kilogram solvent;
2). prepare the aqueous solution of silver nitrate aqueous solution and hydrazine reducing agent respectively, its weight-molality is respectively 4.7 * 10
-2Mole/kilogram, 7.1 * 10
-1Mole/kilogram, desired R
wValue is 45, R
AgValue is 0.3 and R
AlsoIt is 0.6 value.
3). the inverse micellar solution of preparation Silver Nitrate and reductive agent: the aqueous solution of getting Silver Nitrate and hydrazine reducing agent joins respectively in the hexane solution of the polyoxyethylene nonylphenol ether of preparing in the step 1) of 0.1 liter of equal volume (6), and Silver Nitrate reaches the big or small desired R of the consumption of the aqueous solution of going back agent according to the hydrophobic nanometre silver sol
wValue is 45, R
AgValue is 0.3 and R
AlsoBe 0.6.
4). preparation hydrophobic nanometre silver sol solution: under 25 ℃, under agitation mixed 8 hours by the inverse micellar solution of equal volume Silver Nitrate in the step 3) and hydrazine reducing agent, promptly obtain the hydrophobic nanometre silver sol solution of particle diameter in 23 nanometers, its weight-molality is 4.7 * 10
-2Mole/kilogram.
Second step: prepare high molecular gelating soln:
The polymer substance polyvinyl butyral acetal is dissolved in the Virahol, makes that the weight percent concentration of solution is 5%;
The 3rd step: the oxidase function compound sensitive film that preparation contains nano silver colloidal sol carries out as follows:
With weight-molality is 4.7 * 10
-2The nano silver colloidal sol solution 5ml of mole/kilogram is the water-soluble 0.6ml mixing of glucose oxidase of 100 unit of activity/milliliters with concentration; Then add weight percent concentration and be the gelating soln 10ml of 2% high-molecular polyivnyl alcohol butyral, stir, add weight percent concentration again and be 1% glutaraldehyde 1.2ml and carry out crosslinked; Above-mentioned mixing solutions is coated on the solid carrier platinum filament, vapors away solvent under the room temperature, form the oxidase function compound sensitive film that one deck contains nano silver colloidal sol on solid carrier platinum filament surface.
Survey its response current by the mode of embodiment 1, the electrode response electric current can reach 11565nA/cm when glucose concn was 33mmol/L
2Embodiment 6
The first step: adopt reverse micelle method to prepare hydrophobic nanometre silver sol solution:
1). the isooctane solution of preparation anion surfactant dioctyl succinate sodium sulfonate (AOT), its weight-molality is 50 mmoles/kilogram solvent;
2). prepare silver nitrate aqueous solution and reductive agent aqueous citric acid solution respectively, its weight-molality is respectively 7.8 * 10
-6Mole/kilogram, 7.8 * 10
-6Mole/kilogram, desired R
wValue is 15, R
AgValue is 0.0001 and R
AlsoIt is 0.0001 value.
3). the inverse micellar solution of preparation Silver Nitrate and reductive agent citric acid: get in the isooctane solution that Silver Nitrate and reductive agent lemon aqueous acid join the tensio-active agent dioctyl succinate sodium sulfonate of preparing in the step 1) of 0.02 liter of equal volume (AOT) respectively, the consumption of Silver Nitrate and reductive agent lemon aqueous acid is according to the big or small desired R of hydrophobic nanometre silver sol
wValue 15, R
AgValue is 0.0001 and R
AlsoBe 0.0001.
4). preparation hydrophobic nanometre silver sol solution: under the room temperature, under agitation mixed 6 hours by the inverse micellar solution of equal volume Silver Nitrate in the step 3) and reductive agent citric acid, promptly obtain the hydrophobic nanometre silver sol solution of particle diameter in 3 nanometers, its weight-molality is 7.8 * 10
-6Mole/kilogram.
Second step: prepare high molecular gelating soln:
The polymer substance polyvinyl butyral acetal is dissolved in the dehydrated alcohol, makes that the weight percent concentration of solution is 2%;
The 3rd step: the oxidase function compound sensitive film that preparation contains nano silver colloidal sol carries out as follows:
With weight-molality is 7.8 * 10
-6The molten 5ml of the nano silver colloidal sol of mole/kilogram is the water-soluble 0.3ml mixing of alcohol oxidase of 100 unit of activity/milliliters with concentration; Then add weight percent concentration and be the gelating soln 10ml of 2% high-molecular polyivnyl alcohol butyral, stir, add weight percent concentration again and be 1% glutaraldehyde 1.2ml and carry out crosslinking Treatment; Above-mentioned mixing solutions is coated on the solid carrier platinum filament, under the room temperature, vapors away solvent, form the oxidase function compound sensitive film that one deck contains nano silver colloidal sol on solid carrier platinum filament surface.
Survey its response current by the mode of embodiment 1, glucose concn is 10mmol/l, and the electrode response electric current can reach 3054nA/cm
2Embodiment 7
The first step: adopt sol-gel method to prepare hydrophilic nano silver colloidal sol solution:
Prepare Silver Nitrate and reductive agent lemon aqueous acid respectively, its weight-molality is respectively 5.6 * 10
-4, mol/Kg and 2.8 * 10
-3Mol/Kg;
Prepare hydrophilic nano silver colloidal sol solution: 60 ℃ add silver nitrate aqueous solution and reductive agent lemon aqueous acid in the reaction vessel respectively, wherein the mol ratio of silver nitrate aqueous solution and reductive agent lemon aqueous acid is that 1: 5 mixing solutions under agitation reacted 0.5 hour, promptly obtain the hydrophilic nano silver colloidal sol solution of particle diameter in 12 nanometers, its weight-molality is 5.6 * 10
-4Mol/Kg.
Second step: prepare high molecular gelating soln:
The polymer substance polyvinyl butyral acetal is dissolved in the dehydrated alcohol, makes that the weight percent concentration of solution is 2%;
The 3rd step: the oxidase function compound sensitive film that preparation contains nano silver colloidal sol carries out as follows:
With weight-molality is 5.6 * 10
-4The molten 2ml of the nano silver colloidal sol of mol/Kg is the glucose oxidase enzyme aqueous solution 0.3ml mixing of 1000 unit of activity/milliliters with concentration; Then add weight percent concentration and be 2% high molecular gelating soln 20ml, stir, add concentration expressed in percentage by weight again and be 1% glutaraldehyde 3ml and carry out crosslinked; Above-mentioned mixing solutions is coated on the solid carrier platinum filament, under the room temperature, vapors away solvent, form the oxidase function compound sensitive film that one deck contains nano silver colloidal sol on solid carrier platinum filament surface.
Survey its response current by the mode of embodiment 1, glucose concn is 10mmol/l, and the electrode response electric current can reach 8112nA/cm
2Embodiment 8
The first step: adopt reverse micelle method to prepare hydrophobic nanometre silver sol solution:
1). the cyclohexane solution of preparation nonionogenic tenside TritonX-100, its weight-molality is 200 mmoles/kilogram solvent;
2). prepare silver nitrate aqueous solution and reductive agent sodium citrate aqueous solution respectively, its weight-molality is respectively 5.4 * 10
-5Mole/kilogram, 2.7 * 10
-4Mole/do restraining desired R
wValue is 2, R
AgValue is 0.0007 and R
AlsoIt is 0.0035 value.
3). the inverse micellar solution of preparation Silver Nitrate and reductive agent Trisodium Citrate: the aqueous solution of getting Silver Nitrate and reductive agent Trisodium Citrate joins respectively in the cyclohexane solution of the nonionogenic tenside TritonX-100 for preparing in the step 1) of 0.1 liter of equal volume, and the consumption of the aqueous solution of Silver Nitrate and reductive agent Trisodium Citrate is according to the big or small desired R of hydrophobic nanometre silver sol
wValue 2, R
AgValue is 0.0007 and R
AlsoBe 0.0035.
4). preparation hydrophobic nanometre silver sol solution: under 25 ℃, under agitation mixed 5 hours by the inverse micellar solution of equal volume Silver Nitrate in the step 3) and reductive agent Trisodium Citrate, promptly obtain the hydrophobic nanometre silver sol solution of particle diameter in 2 nanometers, its weight-molality is 5.4 * 10
-4Mole/kilogram.
Second step: prepare high molecular gelating soln:
The polymer substance polyvinyl butyral acetal is dissolved in the dehydrated alcohol, makes that the weight percent concentration of solution is 2%;
The 3rd step: the oxidase function compound sensitive film that preparation contains nano silver colloidal sol carries out as follows:
With weight-molality is 5.4 * 10
-4The molten 5ml of the nano silver colloidal sol of mole/kilogram is the water-soluble 0.4ml mixing of glucose oxidase of 500 unit of activity/milliliters with concentration; Then add weight percent concentration and be 2% high molecular gelating soln 10ml, stir, add concentration expressed in percentage by weight again and be 1% glutaraldehyde 1.1ml and carry out crosslinked; Above-mentioned mixing solutions is coated on the solid carrier platinum filament, vapors away solvent under the room temperature, form the oxidase function compound sensitive film that one deck contains nano silver colloidal sol on solid carrier platinum filament surface.
Survey its response current by the mode of embodiment 1, making the response curve glucose concn corresponding to corresponding glucose concn is 20mmol/l, and the electrode response electric current can reach 8353nA/cm
2
Claims (12)
1. a composite enzymatic sensitive film that contains nano silver colloidal sol is characterized in that getting by following method
Arrive:
With weight-molality is 1 * 10
-6～10
-1Nano silver colloidal sol solution 0.1～10ml of mol/kg is the oxydase aqueous solution 0.05～2ml mixing of 50～1000 unit of activity/milliliters with concentration; Then add weight percent concentration and be high molecular gelating soln 10～200ml of 0.5～5%, stir, add weight percent concentration again and be glutaraldehyde 0.1～5ml of 1～10% and carry out crosslinked; Above-mentioned mixing solutions is coated on the solid carrier, vapors away solvent under the room temperature, form the oxidase function compound sensitive film that one deck contains nano silver colloidal sol at solid carrier surface.
2. a kind of composite enzymatic sensitive film that contains nano silver colloidal sol as claimed in claim 1 is characterized in that described nano silver colloidal sol solution is hydrophobic nanometre silver sol solution.
3. a kind of composite enzymatic sensitive film that contains nano silver colloidal sol as claimed in claim 1 is characterized in that described nano silver colloidal sol solution is hydrophilic nano silver colloidal sol solution.
4. as claim 1 or 2 or 3 described a kind of composite enzymatic sensitive films that contain nano silver colloidal sol, the particle diameter that it is characterized in that described silver sol is 1～25 nanometer.
5. a kind of composite enzymatic sensitive film that contains nano silver colloidal sol as claimed in claim 4, the particle diameter that it is characterized in that described silver sol is 1～12 nanometer.
6. a kind of composite enzymatic sensitive film that contains nano silver colloidal sol as claimed in claim 1 is characterized in that described oxydase is glucose oxidase, Lactate Oxidase, alcohol oxidase or XOD; Polymer is cellulose acetate, polyvinyl butyral acetal, urethane, polyoxyethylene glycol, polysulfoxides, polyureas or ethylene-vinyl alcohol copolymer; Solid carrier is a platinum filament.
7. method for making that contains the composite enzymatic sensitive film of nano silver colloidal sol is characterized in that carrying out as follows:
The first step: adopt sol-gel method to prepare hydrophilic nano silver colloidal sol solution, concrete preparation method is as follows:
1.. prepare the aqueous solution of Silver Nitrate and reductive agent respectively, its weight-molality is respectively 1 * 10
-6-1 * 10
-1Mol/Kg and 1 * 10
-6-1mol/Kg;
2.. prepare hydrophilic nano silver colloidal sol solution: room temperature or under 25-90 ℃, add the aqueous solution of silver nitrate aqueous solution and reductive agent in the reaction vessel respectively, wherein the mol ratio of the aqueous solution of silver nitrate aqueous solution and reductive agent is 1: 1～1: 30, mixing solutions under agitation reacted 0.5～10 hour, promptly obtain the hydrophilic nano silver colloidal sol solution of particle diameter in 1～25 nanometer, its weight-molality is 5 * 10
-6-1 * 10
-1Mol/kg;
Or adopt reverse micelle method to prepare hydrophobic nanometre silver sol solution:
1) the non-polar organic solvent solution of preparation tensio-active agent, its weight-molality is 50-300 mmole/kilogram solvent,
2) prepare the silver nitrate aqueous solution and the reductive agent aqueous solution respectively, its weight-molality is 1 * 10
-6～1 * 10
-1Mole/kilogram, and be R according to the big or small desired water of hydrophobic nanometre silver sol and the mol ratio of tensio-active agent all
wThe mol ratio of value and ionogen Silver Nitrate or reductive agent and tensio-active agent is R
EValue is determined R
EValue comprises R
AgValue and R
AlsoValue; Work as R
wWhen value is 1-15, R
EValue is 0.0001-0.015; Or work as R
wWhen value is 22-50, R
EValue is 0.01-0.6;
3) Silver Nitrate for preparing the inverse micellar solution of preparation Silver Nitrate and reductive agent: get 2) and the aqueous solution of reductive agent join respectively in the non-polar organic solvent solution of the tensio-active agent of preparing in the step 1) of 0.01～1 liter of equal volume, and the consumption of the aqueous solution of Silver Nitrate and reductive agent is according to the big or small desired R of hydrophobic nanometre silver sol
wValue and R
EValue is determined, wherein works as R
wWhen value is 1-15, R
EValue is 0.0001-0.015; Or work as R
wWhen value is 22-50, R
EValue is 0.01-0.6;
4) preparation hydrophobic nanometre silver sol solution: under room temperature or 25-35 ℃, under agitation mixed 1～8 hour by the inverse micellar solution of equal volume Silver Nitrate in the step 3) and reductive agent, promptly obtain the hydrophobic nanometre silver sol solution of particle diameter in 1～25 nanometer, its weight-molality is 5 * 10
-6～1 * 10
-1Mole/kilogram;
Second step: prepare high molecular gelating soln:
Polymer substance is dissolved in corresponding organic solvent or the water, makes that the weight percent concentration of solution is 0.5～5%;
The 3rd step: the oxidase function compound sensitive film that preparation contains nano silver colloidal sol carries out as follows: with weight-molality is 5 * 10
-6～1 * 10
-1Nano silver colloidal sol solution 0.1～10ml of mol/kg is the oxydase aqueous solution 0.05～2ml mixing of 50～1000 unit of activity/milliliters with concentration; Then add weight percent concentration and be high molecular gelating soln 10～200ml of 0.5～5%, stir, add weight percent concentration again and be glutaraldehyde 0.1～5ml of 1～10% and carry out crosslinking Treatment; Above-mentioned mixing solutions is coated on the solid carrier, under the room temperature, vapors away solvent, form the oxidase function compound sensitive film that one deck contains nano silver colloidal sol at solid carrier surface.
8. a kind of method for making that contains the composite enzymatic sensitive film of nano silver colloidal sol as claimed in claim 7, the particle diameter that it is characterized in that described nano silver colloidal sol is 1～12 nanometer.
9. a kind of method for making that contains the composite enzymatic sensitive film of nano silver colloidal sol as claimed in claim 7 is characterized in that described reductive agent is sodium borohydride, citric acid, Trisodium Citrate, ethanol or hydrazine; Described tensio-active agent is nonionogenic tenside, anion surfactant or natural surface active agent; Described non-polar organic solvent is normal hexane, hexanaphthene, octane-iso or chlorine; Described polymer substance is cellulose acetate, polyvinyl butyral acetal, urethane, polyoxyethylene glycol, polysulfoxides, polyureas, hydroxypropylcellulose or ethylene-vinyl alcohol copolymer; Described organic solvent is acetone, dehydrated alcohol, epoxy six alkane or Virahol.
10. as claim 7 or 9 described a kind of method for makings that contain the composite enzymatic sensitive film of nano silver colloidal sol, it is characterized in that described nonionogenic tenside is alkylphenol polyoxyethylene series or Span or sorbitan fatty acid ester series; Anion surfactant is a dioctyl succinate sodium sulfonate; Natural surface active agent is a Yelkin TTS.
11. the described purposes that contains the composite enzymatic sensitive film of nano silver colloidal sol of claim 1 is characterized in that being used for enzyme biologic sensor, bioseparation film and biocatalysis engineering.
12. the purposes that contains the composite enzymatic sensitive film of nano silver colloidal sol as claimed in claim 11, it is characterized in that described enzyme biologic sensor is a glucose biological sensor, be used for the measurement of sugar in blood sugar, trade effluent, nutrient solution, fermentation and the beverage of human body.
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|---|---|---|---|
| CN99125072A CN1131301C (en) | 1999-11-25 | 1999-11-25 | Composite enzymatic sensitive film containing nanometer silver sol and its preparation and use |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN99125072A CN1131301C (en) | 1999-11-25 | 1999-11-25 | Composite enzymatic sensitive film containing nanometer silver sol and its preparation and use |
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| Publication Number | Publication Date |
|---|---|
| CN1297992A true CN1297992A (en) | 2001-06-06 |
| CN1131301C CN1131301C (en) | 2003-12-17 |
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|---|---|---|---|
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102374982A (en) * | 2011-09-16 | 2012-03-14 | 厦门大学 | Method for enhancing glucose sensing film fluorescent signal by chemically depositing micro-nano silver |
| CN102642362A (en) * | 2012-04-23 | 2012-08-22 | 东北林业大学 | Method for preparing transparent Raman effect film |
| CN105588867A (en) * | 2015-12-24 | 2016-05-18 | 天津市职业大学 | Preparation method of gelatin glucose sensor coated with AgNPS (Ag nanoparticles) and GOx (glucose oxidase) |
| CN113275586A (en) * | 2021-05-31 | 2021-08-20 | 广东省江门市质量计量监督检测所 | Method for preparing nano silver with different particle sizes by using surfactant |
| WO2021207994A1 (en) * | 2020-04-16 | 2021-10-21 | 三诺生物传感股份有限公司 | Biocompatible membrane, preparation method therefor, and implantable biosensor |
-
1999
- 1999-11-25 CN CN99125072A patent/CN1131301C/en not_active Expired - Fee Related
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102374982A (en) * | 2011-09-16 | 2012-03-14 | 厦门大学 | Method for enhancing glucose sensing film fluorescent signal by chemically depositing micro-nano silver |
| CN102642362A (en) * | 2012-04-23 | 2012-08-22 | 东北林业大学 | Method for preparing transparent Raman effect film |
| CN102642362B (en) * | 2012-04-23 | 2014-07-02 | 东北林业大学 | Method for preparing transparent Raman effect film |
| CN105588867A (en) * | 2015-12-24 | 2016-05-18 | 天津市职业大学 | Preparation method of gelatin glucose sensor coated with AgNPS (Ag nanoparticles) and GOx (glucose oxidase) |
| WO2021207994A1 (en) * | 2020-04-16 | 2021-10-21 | 三诺生物传感股份有限公司 | Biocompatible membrane, preparation method therefor, and implantable biosensor |
| CN113275586A (en) * | 2021-05-31 | 2021-08-20 | 广东省江门市质量计量监督检测所 | Method for preparing nano silver with different particle sizes by using surfactant |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1131301C (en) | 2003-12-17 |
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