CN108801983A - A kind of preparation method of optical glucose sensitive membrane - Google Patents
A kind of preparation method of optical glucose sensitive membrane Download PDFInfo
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- CN108801983A CN108801983A CN201810785393.XA CN201810785393A CN108801983A CN 108801983 A CN108801983 A CN 108801983A CN 201810785393 A CN201810785393 A CN 201810785393A CN 108801983 A CN108801983 A CN 108801983A
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- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/55—Specular reflectivity
- G01N21/552—Attenuated total reflection
- G01N21/553—Attenuated total reflection and using surface plasmons
- G01N21/554—Attenuated total reflection and using surface plasmons detecting the surface plasmon resonance of nanostructured metals, e.g. localised surface plasmon resonance
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Abstract
The invention discloses a kind of preparation methods of optical glucose sensitive membrane.Glucose oxidase is fixed on using chemical crosslink technique on the silicon oxide mesoporous foam (SiMCF) and monox nanometer ball (SiNP) being mixed in a certain ratio, it is used in combination polyacrylamide (PAM), cellulose acetate (CA) and polyvinyl alcohol (PVA) with good biocompatibility to be used as embedded material, preparing refractive index type composite Nano glucose-sensitive film in golden film using spin-coating method or czochralski method constitutes glucose sensing probe.Glucose sensing probe is placed in glucose solution to be measured, glucose molecule in solution enters with dissolved oxygen in the glucose-sensitive film on sensing probe, and redox reaction occurs under GOD catalytic action causes sensitive membrane refractive index that dull change occurs with concentration of glucose.
Description
Technical field
The invention belongs to optical sensor technology fields, and in particular to a kind of preparation method of optical glucose sensitive membrane.
Background technology
For glucose as essential ingredient in vital movement, the metabolism that it can be directly in participant's body, is body
The required most of energy of interior each histocyte offer activity, so the blood glucose level stablized is to meet each organ and tissue in vivo
The premise of health operation.Fasting plasma glucose concentration is 80~120mg/dL to normal person in the morning.Blood sugar concentration it is excessively high or it is too low all
Health can be influenced, and is possible to cause a series of complication, so being detected to blood sugar concentration has great significance.
In existing glucose detection systems, common optical means has：Gas chromatography, colorimetric method, spectrophotometric
Method, Optical Rotation etc., however these detection methods can not achieve on-line real-time measuremen and remote detection.Therefore, research and development have
Miniaturization, the glucose sensor for the features such as on-line monitoring, remote monitor, high sensitivity, stability are good are imperative.It is based on
There is the glucose sensor of variations in refractive index highly sensitive, Miniaturized, anti-electromagnetic wave to interfere, can remote monitoring etc. it is excellent
Point, therefore there is wide development and application prospect in chemistry and bio-sensing field.
The core component of glucose sensor is glucose-sensitive film, in order to make glucose sensor have it is highly sensitive,
Wide detection range and fast-response time, it is desirable that glucose-sensitive film must have excellent absorption property and optical property, and shadow
The principal element for ringing glucose-sensitive film properties has the property of sensitive materials, process for fixation and basis material.
But the preparation of glucose-sensitive film faces many technical barriers.First, the mechanical property of sensitive membrane is poor, seriously
Affect the detection of concentration of glucose；Second, the sensitive membrane rough surface of preparation is in uneven thickness, to experimental result generate compared with
It is big to influence.
Invention content
Present invention aims at a kind of preparation method of optical glucose sensitive membrane is provided, the thickness peace of sensitive membrane is controlled
It is whole to improve the mechanical property of sensitive membrane while spend, there is detection range and the sensitivity of bigger to concentration of glucose.
In order to achieve the above objectives, as follows using technical solution：
A kind of preparation method of optical glucose sensitive membrane, includes the following steps：
1) by hydrochloric acid, 1,3,5- trimethylbenzenes, tetraethyl orthosilicate, polyethylene oxide-polypropylene oxide-polyethylene oxide three
Block copolymer and ammonium fluoride are mixed, and heat up aging, insulation reaction, and then high-temperature calcination obtains silica nanometer foam
(SiMCF)；
2) silica nanosphere (SiNP) is mixed with gained silica nanometer foam (SiMCF), is subsequently dispersed
Stirring at normal temperature in the methanol solution of 3-aminopropyltriethoxysilane (APTES) has been added dropwise and has obtained mixed raw material, washing centrifugation is simultaneously
PBS buffer solutions are added to vibrate to obtain the silica nanometer foam (SiMCF) of surface amination and silica nanosphere
(SiNP) suspending liquid A；
3) PBS buffer solutions are added in ethyldimethyl amine carbodiimide and N- hydroxysuccinimides to mix often
Temperature stirs to get solution B；Glucose oxidase (GOD) is added to stirring at normal temperature in the mixed solution of above-mentioned A and B to be mixed
Solution C；Obtain securing the SiMCF/SiNP (GOD@SiMCF/SiNP) of glucose oxidase (GOD) after centrifuge washing solution C
Mixed raw material is added PBS buffer solutions and obtains solution D, Cord blood；
4) will in acquired solution D be added initiator acrylamide and bisacrylamide stirring, then be added ammonium persulfate and
Tetramethylethylenediamine solution forms gel, gel is spin-coated on to the Au film surface of glass slide impregnated through PBS buffer solutions, room temperature is put
Cord blood after a period of time is set, optical glucose sensitive membrane is obtained.
By said program, silica nanometer foam aperture described in step 1 is 10~20nm, silica nanosphere
Grain size is 50~55nm.
By said program, hydrochloric acid, 1 in step 1,3,5- trimethylbenzenes, tetraethyl orthosilicate, ammonium fluoride and polyethylene oxide-
Polypropylene oxide-polyethylene oxide triblock copolymer mass ratio is：1:2.28:2.49:5.71:1.86；Heat up aging temperature
It is 12h for 40 DEG C of times；Insulation reaction temperature is 120 DEG C of times for for 24 hours；Calcination temperature is 500 DEG C, time 10h.
By said program, the mass ratio of silica nanosphere and silica nanometer foam is 1 in step 2:(1.2-
9)。
By said program, mixing time is centrifugal speed 1300r/min, centrifugation time 5min for 24 hours in step 2.
By said program, ethyldimethyl amine carbodiimide and N- hydroxysuccinimides in step 3 solution B
Concentration is respectively 0.1M, 0.01M；Centrifugal speed 1300r/min, centrifugation time 5min；Cord blood temperature is 4 DEG C, and the time is
12h。
By said program, Au film glass thickness is 0.2mm in step 4, and area is 18mm × 18mm；The thickness of Au films is
50nm, soaking time 6h.
By said program, gained optical glucose sensitive membrane pore diameter range is in 10.5~19.8nm.
The present invention is distributed in the silica nanometer foam (SiMCF) and dioxy of 10~20nm using hydro-thermal method synthetic aperture
SiClx nanosphere (SiNP) utilizes its preferable biocompatibility, huge mesoporous pore size and larger after its surface amination
Specific surface area chemically and physically adsorb preliminary fixing glucose oxidase molecule.To prevent glucose oxidase leakage from losing
Deactivation will wrap GOD in the present invention program using chemical crosslink technique with the PAM gel moulds compared with high-biocompatibility
SiMCF/SiNP nano-particles, glucose-sensitive film be made be spin-coated on the surfaces of Au film glass pieces and make sensing chip, be used for
The detection of concentration of glucose.Utilize silica nanometer foam (SiMCF) and the fixed GOD molecules of silica nanosphere (SiNP)
PAM gel moulds are prepared with chemical crosslink technique, greatly enhance the fixed amount of GOD molecules, it is more so as to be catalyzed
Enzymatic reaction occurs for glucose, expands the detection range of glucose sensor, and greatly improve its sensitivity.By prism-type SPR
Sensing technology is combined with immobilization GOD, is based on redox reaction, is caused the variation of glucose-sensitive film effective refractive index,
Concentration of glucose is detected by measuring SPR reflectance spectrums resonance angle.It is good etc. to realize the good, high sensitivity of specificity, stability
Target, and have many advantages, such as fast response time, it operates simple.
Beneficial effects of the present invention are：
While the present invention controls the thickness and flatness of sensitive membrane, the silica nanosphere of different ratio is utilized
(SiNP) and silica nanometer foam (SiMCF) is used as basis material, improves the mechanical property of sensitive membrane, adsorbs more Portugals
Grape oxidase molecules have concentration of glucose detection range and the sensitivity of bigger.
Immobilization GOD and SPR sensorgram technology are combined by the present invention for the first time, ensure that sensitive materials catalytic activity and stabilization
Property.
Glass prism SPR sensor of the present invention good, simple operation and other advantages with measurement accuracy and high sensitivity, stability, can
Real-time monitoring for concentration of glucose.
The spin-coating method that the present invention uses is simple and practicable, high activity, silica nanometer foam SiMCF and the dioxy of desmoenzyme
The excellent performance of SiClx SiNP nanospheres, the composite Nano sensitive membrane good biocompatibility of development, the fixation conducive to biological enzyme, system
For at low cost, simple and fast, stability is good.
Specific implementation mode
Following embodiment further illustrates technical scheme of the present invention, but not as limiting the scope of the invention.
Embodiment 1
A kind of refractive index type glucose-sensitive film based on glucose oxidase, preparation method includes the following steps：
1) preparation of SiMCF：Weigh the addition of 10g polyethylene oxide-polypropylene oxide-polyethylene oxide triblock copolymers
Into the HCl solution of the 2.0M of 150mL, mixing is heated to 40 DEG C, is then slowly added into 1,3,5- trimethylbenzenes stirring 2h, stirs
It is 1000rmp/min to mix speed.It is then slowly added to 30mM tetraethyl orthosilicates 5mL and forms gel；By the gel of generation 40
Aging 12h under DEG C water bath condition, is then added 30mg ammonium fluorides afterwards and water-bath is for 24 hours at 120 DEG C.At 60 DEG C after centrifuge washing
Lower drying for 24 hours, obtains white powder.White powder is calcined into 10h at 500 DEG C and obtains silica nanometer foam SiMCF.
2) preparation of GOD/SiMCF&SiNP：It is 1 in mass ratio：9 weigh monox nanometer ball SiNP and silica is received
Rice foam SiMCF is added in 8mL methanol solvates, and 3-aminopropyltriethoxysilane (APTES) solution of 80 μ L is added dropwise, stirs
It mixes for 24 hours, makes silica nanometer foam SiMCF and the abundant aminations of silica nanosphere SiNP；By amidized nanoparticle
Son is suspended in the PBS buffer solutions (0.1M, PH=8) of 1mL, and 12mg GOD are then added and obtain suspending liquid A；4mL is prepared to contain
The PBS of crosslinking agent ethyldimethyl amine carbodiimide EDC (0.1M) and N- hydroxysuccinimides NHS (0.01M) are slow
Rush solution B (0.1M, PH=5.0)；4mLB solution is taken to be added in solution A, and at room temperature with 200rmp/min weak vibrations
12h obtains mixed solution C；Solution C obtains the GOD/SiMCF& of immobilization with PBS buffer solutions (0.1M, PH=7.0) centrifuge washing
SiNP, the PBS buffer solutions that 3mL is added obtain mixed solution D.
3) preparation of GOD/SiMCF&SiNP@PAM sensitive membranes：Weigh 0.15g acrylamides and 0.06g bisacrylamides in
It is uniformly mixed in the beaker of 20mL, the solution D stirring at normal temperature 6h of 1mL is then added；Then 10 μ L 12wt% ammonium persulfates are added
Solution continues to stir；10 μ L tetramethylethylenediamines are eventually adding, initiation polymerisation in solution is made to form gel.
4) preparation of sensing chip：Au films are immersed in 6h in PBS solution before applying gel, so that its surface is had hydrophilic
Property.One layer of PAM gel mould for being coated with immobilised enzymes is then coated on its surface using spin-coating method, forms sensing chip.It will pass
Sense chip is placed in drying at 4 DEG C, prevents enzyme from losing activity.
5) sensing principle：Light source emergent light enters prism, and the GOD/ of censorchip surface is radiated at through circular aperture
In SiMCF&SiNP@PAM sensitive membranes, excitating surface plasma resonance, wherein grape glucose in solutions molecule to be measured
Enter in glucose-sensitive film with dissolved oxygen and redox reaction occur, sensitive membrane refractive index is made to change, incident light with it is quick
Feel film and interaction occurs after reflection, into optical detector and data processing unit, optical detector is used with data processing unit
In acquisition reflectance spectrum signal and calculate concentration of glucose in glucose solution to be measured.
The concentration of glucose that glucose-sensitive film prepared by the present embodiment can detect is in 0-200mg/mL, sensitivity
For 0.025degree/ (mg/mL).Silica nanometer foam after being mixed in a certain ratio and silica nanosphere, have
To the optimal adsorption potential points and absorption potential of GOD molecules.
Embodiment 2
A kind of refractive index type glucose-sensitive film based on glucose oxidase, preparation method includes the following steps：
1) preparation of SiMCF：Weigh the addition of 10g polyethylene oxide-polypropylene oxide-polyethylene oxide triblock copolymers
Into the HCl solution of the 2.0M of 150mL, mixing is heated to 40 DEG C, is then slowly added into 1,3,5- trimethylbenzenes stirring 2h, stirs
It is 1000rmp/min to mix speed.It is then slowly added to 30mM tetraethyl orthosilicates 5mL and forms gel；By the gel of generation 40
Aging 12h under DEG C water bath condition, is then added 30mg ammonium fluorides afterwards and water-bath is for 24 hours at 120 DEG C.At 60 DEG C after centrifuge washing
Lower drying for 24 hours, obtains white powder.White powder is calcined into 10h at 500 DEG C and obtains silica nanometer foam SiMCF.
2) preparation of GOD/SiMCF&SiNP：It is 2 in mass ratio：8 weigh monox nanometer ball SiNP and silica is received
Rice foam SiMCF is added in 8mL methanol solvates, and 3-aminopropyltriethoxysilane (APTES) solution of 80 μ L is added dropwise, stirs
It mixes for 24 hours, makes silica nanometer foam SiMCF and the abundant aminations of silica nanosphere SiNP；By amidized nanoparticle
Son is suspended in the PBS buffer solutions (0.1M, PH=8) of 1mL, and 12mg GOD are then added and obtain suspending liquid A；4mL is prepared to contain
The PBS of crosslinking agent ethyldimethyl amine carbodiimide EDC (0.1M) and N- hydroxysuccinimides NHS (0.01M) are slow
Rush solution B (0.1M, PH=5.0)；4mLB solution is taken to be added in solution A, and at room temperature with 200rmp/min weak vibrations
12h obtains mixed solution C；Solution C obtains the GOD/SiMCF& of immobilization with PBS buffer solutions (0.1M, PH=7.0) centrifuge washing
SiNP, the PBS buffer solutions that 3mL is added obtain mixed solution D.
3) preparation of GOD/SiMCF&SiNP@PAM sensitive membranes：Weigh 0.15g acrylamides and 0.06g bisacrylamides in
It is uniformly mixed in the beaker of 20mL, the solution D stirring at normal temperature 6h of 1mL is then added；Then 10 μ L 12wt% ammonium persulfates are added
Solution continues to stir；10 μ L tetramethylethylenediamines are eventually adding, initiation polymerisation in solution is made to form gel.
4) preparation of sensing chip：Au films are immersed in 6h in PBS solution before applying gel, so that its surface is had hydrophilic
Property.One layer of PAM gel mould for being coated with immobilised enzymes is then coated on its surface using spin-coating method, forms sensing chip.It will pass
Sense chip is placed in drying at 4 DEG C, prevents enzyme from losing activity.
The concentration of glucose that glucose-sensitive film prepared by the present embodiment can detect is in 0-160mg/mL, sensitivity
For 0.011degree/ (mg/mL).
Embodiment 3
1) preparation of SiMCF：Weigh the addition of 10g polyethylene oxide-polypropylene oxide-polyethylene oxide triblock copolymers
Into the HCl solution of the 2.0M of 150mL, mixing is heated to 40 DEG C, is then slowly added into 1,3,5- trimethylbenzenes stirring 2h, stirs
It is 1000rmp/min to mix speed.It is then slowly added to 30mM tetraethyl orthosilicates 5mL and forms gel；By the gel of generation 40
Aging 12h under DEG C water bath condition, is then added 30mg ammonium fluorides afterwards and water-bath is for 24 hours at 120 DEG C.At 60 DEG C after centrifuge washing
Lower drying for 24 hours, obtains white powder.White powder is calcined into 10h at 500 DEG C and obtains silica nanometer foam SiMCF.
2) preparation of GOD/SiMCF&SiNP：It is 3 in mass ratio：7 weigh monox nanometer ball SiNP and silica is received
Rice foam SiMCF is added in 8mL methanol solvates, and 3-aminopropyltriethoxysilane (APTES) solution of 80 μ L is added dropwise, stirs
It mixes for 24 hours, makes silica nanometer foam SiMCF and the abundant aminations of silica nanosphere SiNP；By amidized nanoparticle
Son is suspended in the PBS buffer solutions (0.1M, PH=8) of 1mL, and 12mg GOD are then added and obtain suspending liquid A；4mL is prepared to contain
The PBS of crosslinking agent ethyldimethyl amine carbodiimide EDC (0.1M) and N- hydroxysuccinimides NHS (0.01M) are slow
Rush solution B (0.1M, PH=5.0)；4mLB solution is taken to be added in solution A, and at room temperature with 200rmp/min weak vibrations
12h obtains mixed solution C；Solution C obtains the GOD/SiMCF& of immobilization with PBS buffer solutions (0.1M, PH=7.0) centrifuge washing
SiNP, the PBS buffer solutions that 3mL is added obtain mixed solution D.
3) preparation of GOD/SiMCF&SiNP@PAM sensitive membranes：Weigh 0.15g acrylamides and 0.06g bisacrylamides in
It is uniformly mixed in the beaker of 20mL, the solution D stirring at normal temperature 6h of 1mL is then added；Then 10 μ L 12wt% ammonium persulfates are added
Solution continues to stir；10 μ L tetramethylethylenediamines are eventually adding, initiation polymerisation in solution is made to form gel.
4) preparation of sensing chip：Au films are immersed in 6h in PBS solution before applying gel, so that its surface is had hydrophilic
Property.One layer of PAM gel mould for being coated with immobilised enzymes is then coated on its surface using spin-coating method, forms sensing chip.It will pass
Sense chip is placed in drying at 4 DEG C, prevents enzyme from losing activity.
The concentration of glucose that glucose-sensitive film prepared by the present embodiment can detect is in 0-80mg/mL, sensitivity
For 0.013degree/ (mg/mL).
Comparative example 1
A kind of refractive index type glucose-sensitive film based on glucose oxidase, preparation method and the substantially phase of embodiment 1
Together, the difference is that only weighing 10mg silica nanometer foams SiMCF in step 2).
The detectable glucose concentration range of glucose-sensitive film prepared by this comparative example is sensitive in 0-160mg/mL
Degree is 0.0135degree/ (mg/mL).
Comparative example 2
A kind of refractive index type glucose-sensitive film based on glucose oxidase, preparation method and the substantially phase of embodiment 1
Together, the difference is that only weighing 10mg silica nanometer nanospheres SiNP in step 2).
The detectable glucose concentration range of glucose-sensitive film prepared by this comparative example is sensitive in 0-80mg/mL
Degree is 0.019degree/ (mg/mL).
The foregoing is merely the preferred embodiment of the present invention, it is noted that comes for those of ordinary skill in the art
It says, without departing from the concept of the premise of the invention, makes several modifications and variations, these belong to the protection model of the present invention
It encloses.
Claims (8)
1. a kind of preparation method of optical glucose sensitive membrane, it is characterised in that include the following steps：
1) by hydrochloric acid, 1,3,5- trimethylbenzenes, tetraethyl orthosilicate, polyethylene oxide-polypropylene oxide-polyethylene oxide three block
Copolymer and ammonium fluoride are mixed, and heat up aging, insulation reaction, and then high-temperature calcination obtains silica nanometer foam；
2) it by silica nanosphere and gained silica nanometer foams mix, is subsequently dispersed and 3- aminopropyls three has been added dropwise
Stirring at normal temperature obtains mixed raw material in the methanol solution of Ethoxysilane, and washing, which centrifuges and PBS buffer solutions are added, to be vibrated to obtain table
The amidized silica nanometer foam in face and silica nanosphere suspending liquid A；
3) PBS buffer solution mixing room temperature is added in ethyldimethyl amine carbodiimide and N- hydroxysuccinimides to stir
It mixes to obtain solution B；Glucose oxidase is added to stirring at normal temperature in the mixed solution of above-mentioned A and B and obtains mixed solution C；From
The SiMCF/SiNP mixed raw materials for obtaining securing glucose oxidase after heart washing solution C, are added PBS buffer solutions and obtain solution
D, Cord blood；
4) initiator acrylamide will be added in acquired solution D and bisacrylamide stirs, ammonium persulfate and tetramethyl is then added
Base ethylenediamine solution forms gel, and gel is spin-coated on to the Au film surface of glass slide impregnated through PBS buffer solutions, is placed at room temperature for one
Cord blood after the section time, obtains optical glucose sensitive membrane.
2. the preparation method of optical glucose sensitive membrane as described in claim 1, it is characterised in that silica described in step 1
Nanometer foam aperture is 10~20nm, and the grain size of silica nanosphere is 50~55nm.
3. the preparation method of optical glucose sensitive membrane as described in claim 1, it is characterised in that hydrochloric acid, 1,3,5- in step 1
Trimethylbenzene, tetraethyl orthosilicate, ammonium fluoride and polyethylene oxide-polypropylene oxide-polyethylene oxide triblock copolymer amount of substance
Than for：1:2.28:2.49:5.71:1.86；It is 12h that the aging temperature that heats up, which is 40 DEG C of times,；When insulation reaction temperature is 120 DEG C
Between for for 24 hours；Calcination temperature is 500 DEG C, time 10h.
4. the preparation method of optical glucose sensitive membrane as described in claim 1, it is characterised in that silica nanometer in step 2
The mass ratio of ball and silica nanometer foam is 1:(1.2-9).
5. the preparation method of optical glucose sensitive membrane as described in claim 1, it is characterised in that mixing time is in step 2
For 24 hours, centrifugal speed 1300r/min, centrifugation time 5min.
6. the preparation method of optical glucose sensitive membrane as described in claim 1, it is characterised in that ethyl two in step 3 solution B
The concentration of methyl amine carbodiimide and N- hydroxysuccinimides is respectively 0.1M, 0.01M；Centrifugal speed 1300r/
Min, centrifugation time 5min；Cord blood temperature is 4 DEG C, time 12h.
7. the preparation method of optical glucose sensitive membrane as described in claim 1, it is characterised in that Au films glass thickness in step 4
For 0.2mm, area is 18mm × 18mm；The thickness of Au films is 50nm, soaking time 6h.
8. the preparation method of optical glucose sensitive membrane as described in claim 1, it is characterised in that gained optical glucose is sensitive
Membrane aperture range is in 10.5~19.8nm.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109596569A (en) * | 2018-12-20 | 2019-04-09 | 南开大学 | A kind of low concentration glucose detection chip and preparation method thereof |
| CN113249426A (en) * | 2021-05-11 | 2021-08-13 | 四川大学 | Preparation method of collagen-based glucose fluorescent sensitive membrane |
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| CN101140258A (en) * | 2007-10-18 | 2008-03-12 | 北京化工大学 | Glucose oxidase film with nitrate cellulose film as substrate and method of producing the same |
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| CN103399039A (en) * | 2013-05-31 | 2013-11-20 | 上海大学 | Preparation method of toluene sensor sensitive material diphenylsulfonyl meso-porous silica |
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| CN101140258A (en) * | 2007-10-18 | 2008-03-12 | 北京化工大学 | Glucose oxidase film with nitrate cellulose film as substrate and method of producing the same |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109596569A (en) * | 2018-12-20 | 2019-04-09 | 南开大学 | A kind of low concentration glucose detection chip and preparation method thereof |
| CN113249426A (en) * | 2021-05-11 | 2021-08-13 | 四川大学 | Preparation method of collagen-based glucose fluorescent sensitive membrane |
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