CN103881128A - Preparation method of in-situ glucose detecting membrane based on fluorescence detection method - Google Patents
Preparation method of in-situ glucose detecting membrane based on fluorescence detection method Download PDFInfo
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- CN103881128A CN103881128A CN201410084483.8A CN201410084483A CN103881128A CN 103881128 A CN103881128 A CN 103881128A CN 201410084483 A CN201410084483 A CN 201410084483A CN 103881128 A CN103881128 A CN 103881128A
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- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
The invention relates to a preparation method of an in-situ glucose detecting membrane based on a fluorescence detection method. Specifically, the detecting membrane is applicable to an implantable in-situ (in vivo) glucose monitoring apparatus for novel minimally invasive, continuous and real-time glucose monitoring. The preparation method comprises the steps of 1) preparing glucose indicator molecules; 2) preparing a material carrier; 3) covalently connecting the glucose indicator molecules prepared in the step 1) to a carrier membrane prepared in the step 2). According to the preparation method of the in-situ glucose detecting membrane based on the fluorescence detection method disclosed by the invention, the glucose indicator molecules are covalently connected to the carrier membrane, so as to guarantee durability; moreover, specific sensitivity and fluorescence activity on glucose are maintained. The detection membrane carrier is free from toxicity or harm and is universal; the carrier can be safely implanted in bodies, so as to achieve a purpose of continuously monitoring glucose in real time.
Description
Technical field
The present invention relates to a kind of preparation method of the original position glucose detection film based on fluorescence detection method, particularly this kind detect film can be used for novel Wicresoft, continuously, implantable original position (in the body) blood sugar monitor of real time blood sugar monitoring.
Background technology
Diabetes are second killers in modern disease, and its harm to human body is only second to cancer, and as a kind of comparatively general endocrinopathy, it is increasing to people's physical and mental health harm, and have the trend of extension and rejuvenation.Result of study shows: the principal feature of diabetes is the blood sugar concentrations that slowly continue rising, in the time that blood sugar concentration rises to certain height, sugar can in urine, be detected higher than renal glucose threshold.Diabetic subject, because blood sugar concentration causes internal metabolism disorder extremely, easily causes the severe complications such as Diabetic ketosis, cardio cerebrovascular affection, ephrosis, illness in eye, acromelic gangrene and infection.In addition, the rising of blood sugar concentration can cause the complication of retina, kidney and neural system etc.Therefore, Main Means and the target for the treatment of diabetes are exactly the normal level that maintains blood sugar concentration.
Due to the complicacy of life science, also there is no thoroughly to effect a radical cure at present the medical means of diabetes.Treat now diabetes, mainly take to control blood sugar concentration with prevention or alleviate generation and the anti symptom treatment complication of complication, particularly by Monitoring Blood Glucose concentration continually and accurately, in time as the consumption according to adjusting orally-taken blood sugar reducing medicine and Regular Insulin.There are data to show to control blood sugar concentration can to reduce the complication of I type and type ii diabetes.
In addition, keep blood-sugar content to approach normal level is also an important problem in severe ward patient's nursing.Research shows: ICU patient's blood-sugar content is controlled at and approaches normal level, can significantly improve their clinical therapeutic efficacy.But the strict control of blood sugar is unapproachable and requires to measure quite frequently.Therefore the continuous monitoring of blood sugar will be also a large progress in diabetic subject and patient with severe symptoms's monitoring.
But, under existence conditions, in hospital, be to utilize biochemical reagents or enzyme and blood or blood plasma to have an effect to the detection method of blood sugar concentration, change come qualitative or represent quantitatively the height of blood sugar concentration by the consumption of reagent or with the shade after enzyme effect.The shortcoming of this method is constantly to consume biochemical reagents, and measuring speed is slower, reagent and consumables associated therewith are also dealt with improperly and may be polluted, can not obtain continuous blood glucose measurement result, sufferer by day, especially blood glucose fluctuation situation when evening just may be left in the basket, but only have lasting blood sugar monitoring just can guarantee best Regulation of blood glucose.
By U.S. FDA and Chinese CFDA authentication, be based on electrochemical principle for the sensor of continuous blood sugar monitoring in body now, use glucose oxidase to carry out molecular recognition.The unfavourable condition of these sensors comprises: 1. their electrochemistry selectivity is limited.Existing all equipment for lasting glucose detection is all to realize with the curent change causing that reacts of hydrogen peroxide by measuring glucose oxidase.The sensor of this class can be subject to the impact that in body, oxygen concn changes, and is also subject to urea, xitix (vitamins C, uric acid) simultaneously, and acetaminophen, waits the impact of electroactive species.Therefore, being used in severe case based on electrochemical glucose measurement is breakneck with it.2. in sensor, there is sizable drift current, reading was lost efficacy.3. due to the use of enzyme and protein, the life-span of their sensor is all extremely limited.
There are some researches show now that the glucose concn in subcutis liquid has positively related relation with the concentration of glucose in blood.By measuring the glucose concn in interstitial fluid, can indirectly measure the variation of glucose at blood middle concentration.Therefore, the developing direction of present most of sensors, is to be all positioned to continue to detect the glucose concn in interstitial fluid.In such cases, sensor is implanted in to subcutis, be most suitable.It can be realized by simple surgical operation.And sensor is impaired in the situation that, relatively easily replaces.
Based on the measuring method of fluorescent method, at present in the U.S. and Europe, be the main flow direction that carries out Wicresoft's blood sugar test research.At China, still do not have enterprise-like corporation to study successfully at present.Its concrete reason, is to lack successful sensor.Therefore,, under existence conditions, the sensor based on fluoroscopic examination that can be used in measuring blood concentration under research Wicresoft condition is very significant.It not only can replace biochemistry detection method traditionally, reaches the object of saving consumptive material and Quick Measurement, and is prerequisite and basis that blood sugar concentration detects in Wicresoft.The success of this technology, also will be widely applied in the fundamental research of diabetes.
Summary of the invention
The preparation method who the object of the invention is to provide for above-mentioned weak point a kind of original position glucose detection film based on fluorescence detection method, by detection film is implanted, realizes real-time continuous blood sugar monitoring.
The preparation method of the original position glucose detection film based on fluorescence detection method takes following technical scheme to realize:
The preparation method of the original position glucose detection film based on fluorescence detection method comprises the steps,
1) prepare to prepare material glucose indicator molecules
Described glucose indicator molecules can be specificly combined with glucose, and after being combined with glucose, the fluorescence intensity of this molecule reduces; When this molecular amounts one timing in system, by observing the total fluorescence intensity of this quasi-molecule, can learn the glucose concn of this molecule place environment; Described glucose indicator molecules is commercially available glucose indicator molecules;
2) prepare to prepare material support
Described carrier is glucose indicator molecules carrier, adopts polymer membrane;
Described carrier is by using the monomer mixture of macromolecular compound, in heating or the polymer carrier film that carries out polymerization UV-irradiation in the situation that and obtain copolymerization, for example, under atmospheric pressure at room, use 1.0M, 2.0M, the HEMA(hydroxyethyl methylacrylate of 2.5M or 3.0M) aqueous solution, wherein carrier monomer (HEMA) with have the corresponding regulation and control of ratio of monomer (AEMA) of active group for example, 1:3, 1:10, 1:20 and 1:33, for example, at pH6-9 (7.5, phosphate buffer solution) condition under, by above-mentioned HEMA aqueous solution spin coating monocrystalline silicon wafer crystal (wafer) surface, thickness is about 50-500 micron, 365 nm UV illumination polymerization in 6-36 hour, consequent hydrogel, water fully washs to remove unreacted monomer, this hydrogel is dried, obtains carrier film,
3) the glucose indicator molecules covalency of preparing in step 1) is linked to step 2) in prepare carrier film on;
Concrete covalency links step, and carrier film, under atmospheric pressure at room, is immersed and contains 0.1-1% formaldehyde, 0.005-0.10M hydrochloric acid, and in the solution of 10mM-100 mM glucose indicator molecules, stir 12-36 hour; Add PBS buffered soln and fully wash, obtain covalency and connect the polymeric membrane of glucose indicator molecules; The i.e. original position glucose detection film based on fluorescence detection method, this polymeric membrane, through cutting, can implant, and uses as the active membrane of glucose optical pickocff;
After glucose indicator molecules covalency is linked in carrier film, glucose indicator molecules still retains specificity and the fluorescent optics activity of cognitive glucose.
Glucose indicator molecules described in step 1) is the derivative based on boric acid, and this molecule can link with carrier polymeric membrane covalency by the Mannich reaction (Mannich reaction) of standard; Glucose indicator molecules is after connecting carrier, and its fluorescence activity is retained, and still can be used as the indicator of glucose.
The described derivative based on boric acid is 2,3'dihydroxyboron-4-hydroxy-azobenzene (2,3 '-dihydroxyl boron-4-hydroxyazobenzene).
Step 2) described polymer membrane is the polymeric membrane that can use in position.
Step 2) described in carrier, adopt Poly(Hydroxyethyl Methacrylate) hydroxyethylmethacrylate (HEMA) or other similar polymer carriers, comprise polyacrylic resin, for example, polymethylmethacrylate polymethylmethacrylate, polymethyl acrylic acid hydroxyl methyl esters polyhydroxypropylmethacrylate, and their multipolymer etc.
Step 2) described in active group in carrier film carry the monomer of active group from AEMA, other active class groups or other, described AEMA provides the amino of a suspension, thereby can covalency modify this polymer carrier film, the monomer that carries active group is aminopropylmethacrylate(APMA), except this type of uncle's Ammonia molecule, parahelium quasi-molecule also can be used; Other class active groups are sulfydryl (SH), carboxylic acid group (COOH), hydroxyl (OH), aldehyde radical (CHO) and cyano group (CN).
Step 2) in the carrier film that obtains be hydrophobic, in water, can expand, become soft; Due to this characteristic, it is one of first material, as the material of contact lens, and optics implant.This application demonstration, this type of material is safe as implant.
advantage of the present invention:the preparation method of the original position glucose detection film based on fluorescence detection method is glucose indicator molecules, being linked in carrier film of covalency, thereby acquisition persistence, and special susceptibility and fluorescence activity to glucose are kept, this detection membrane carrier toxicological harmless is always used, can be implanted safely, thereby for the object of real-time continuous blood sugar monitoring.
Accompanying drawing explanation
Below with reference to accompanying drawing, the invention will be further described:
Fig. 1 is wherein a kind of schematic arrangement of glucose indicator molecules that preparation method of the present invention uses.
Fig. 2 is wherein a kind of glucose indicator molecules of using of preparation method of the present invention fluorescence spectrum figure of (PH=7.5, standard phosphate buffer solution) under physiological condition.
Fig. 3 is wherein a kind of glucose indicator molecules (PH=7.5, standard phosphate buffer solution) under physiological condition that preparation method of the present invention uses, the fluorescence radiation total intensity figure in the glucose solution of different concns.
Fig. 4 is wherein a kind of molecular structure of the high-molecular copolymer as carrier that preparation method of the present invention uses.
Fig. 5 be preparation method of the present invention wherein a kind of glucose indicator molecules and a kind of high-molecular copolymer as carrier react, form covalency linked, diagram.
Fig. 6 be a kind of glucose indicator molecules of using of preparation method of the present invention be linked to by covalency a kind of as the high-molecular copolymer of carrier on after, (PH=7.5 under physiological condition, standard phosphate buffer solution), fluorescence radiation total intensity figure in the glucose solution of different concns, wherein carrier monomer (HEMA) regulates and controls at 1:3 with the ratio of the monomer (AEMA) that has active group.Carrier monomer polymerization concentration is controlled at 2.0M.When polymerization, pH is controlled at 7.5.
Fig. 7 be a kind of glucose indicator molecules of using of preparation method of the present invention be linked to by covalency a kind of as the high-molecular copolymer of carrier on after, (PH=7.5 under physiological condition, standard phosphate buffer solution), fluorescence radiation total intensity figure in the glucose solution of different concns, wherein carrier monomer (HEMA) regulates and controls at 1:10 with the ratio of the monomer (AEMA) that has active group.Carrier monomer polymerization concentration is controlled at 2.0M.When polymerization, pH is controlled at 7.5.
Fig. 8 be a kind of glucose indicator molecules of using of preparation method of the present invention be linked to by covalency a kind of as the high-molecular copolymer of carrier on after, (PH=7.5 under physiological condition, standard phosphate buffer solution), fluorescence radiation total intensity figure in the glucose solution of different concns, wherein carrier monomer (HEMA) regulates and controls at 1:20 with the ratio of the monomer (AEMA) that has active group.Carrier monomer polymerization concentration is controlled at 2.0M.When polymerization, pH is controlled at 7.5.
Fig. 9 be a kind of glucose indicator molecules of using of preparation method of the present invention be linked to by covalency a kind of as the high-molecular copolymer of carrier on after, (PH=7.5 under physiological condition, standard phosphate buffer solution), fluorescence radiation total intensity figure in the glucose solution of different concns, wherein carrier monomer (HEMA) regulates and controls at 1:33 with the ratio of the monomer (AEMA) that has active group.Carrier monomer polymerization concentration is controlled at 2.0M.When polymerization, pH is controlled at 7.5.
Figure 10 be a kind of glucose indicator molecules of using of preparation method of the present invention be linked to by covalency a kind of as the high-molecular copolymer of carrier on after, (PH=7.5 under physiological condition, standard phosphate buffer solution), fluorescence radiation total intensity figure in the glucose solution of different concns, wherein carrier monomer (HEMA) regulates and controls at 1:10 with the ratio of the monomer (AEMA) that has active group.Carrier monomer polymerization concentration is controlled at 1.0M.When polymerization, pH is controlled at 7.5.
Figure 11 be a kind of glucose indicator molecules of using of preparation method of the present invention be linked to by covalency a kind of as the high-molecular copolymer of carrier on after, (PH=7.5 under physiological condition, standard phosphate buffer solution), fluorescence radiation total intensity figure in the glucose solution of different concns, wherein carrier monomer (HEMA) regulates and controls at 1:10 with the ratio of the monomer (AEMA) that has active group.Carrier monomer polymerization concentration is controlled at 3.0M.When polymerization, pH is controlled at 7.5.
Figure 12 be a kind of glucose indicator molecules of using of preparation method of the present invention be linked to by covalency a kind of as the high-molecular copolymer of carrier on after, (PH=7.5 under physiological condition, standard phosphate buffer solution), fluorescence radiation total intensity figure in the glucose solution of different concns, wherein carrier monomer (HEMA) regulates and controls at 1:10 with the ratio of the monomer (AEMA) that has active group.Carrier monomer polymerization concentration is controlled at 2.0M.When polymerization, pH is controlled at 7.0.
Figure 13 be a kind of glucose indicator molecules of using of preparation method of the present invention be linked to by covalency a kind of as the high-molecular copolymer of carrier on after, (PH=7.5 under physiological condition, standard phosphate buffer solution), fluorescence radiation total intensity figure in the glucose solution of different concns, wherein carrier monomer (HEMA) regulates and controls at 1:10 with the ratio of the monomer (AEMA) that has active group.Carrier monomer polymerization concentration is controlled at 2.0M.When polymerization, pH is controlled at 8.0.
Embodiment
With reference to accompanying drawing 1 ~ 13, the preparation method of the original position glucose detection film based on fluorescence detection method comprises the steps,
1) prepare to prepare material glucose indicator molecules
Described glucose indicator molecules can be specificly combined with glucose, and after being combined with glucose, the fluorescence intensity of this molecule reduces; When this molecular amounts one timing in system, by observing the total fluorescence intensity of this quasi-molecule, can learn the glucose concn of this molecule place environment;
2) prepare to prepare material support
Described carrier is glucose indicator molecules carrier, adopts polymer membrane;
Described carrier is by using mix monomer, the in the situation that of heating or UV-irradiation, carry out polymerization and obtain carrier film, for example, under atmospheric pressure at room, use 1.0M, 2.0M, the HEMA(hydroxyethyl methylacrylate of 2.5M or 3.0M) aqueous solution, wherein carrier monomer (HEMA) with have the corresponding regulation and control of ratio of monomer (AEMA) of active group for example, 1:3, 1:10, 1:20 and 1:33, for example, at pH6-9 (7.5, phosphate buffer solution) condition under, by above-mentioned HEMA aqueous solution spin coating monocrystalline silicon wafer crystal (wafer) surface, thickness is about 50-500 micron, 365 nm UV illumination polymerization in 6-36 hour, consequent hydrogel, water fully washs to remove unreacted monomer, this hydrogel is dried, obtains carrier film,
3) the glucose indicator molecules covalency of preparing in step 1) is linked to step 2) in prepare carrier film on;
Concrete covalency links step, and carrier film, under atmospheric pressure at room, is immersed and contains 0.1-1% formaldehyde, 0.005-0.10M hydrochloric acid, and in the solution of 10mM-100 mM glucose indicator molecules, stir 12-36 hour; Add PBS buffered soln and fully wash, obtain covalency and connect the polymeric membrane of glucose indicator molecules; This polymeric membrane, through cutting, can implant, and uses as the active membrane of glucose optical pickocff;
After glucose indicator molecules covalency is linked in carrier film, glucose indicator molecules still retains specificity and the fluorescent optics activity of cognitive glucose.
Glucose indicator molecules described in step 1) is the derivative based on boric acid, and this molecule can link with carrier polymeric membrane covalency by the Mannich reaction of standard; Glucose indicator molecules is after connecting carrier, and its fluorescence activity is retained, and still can be used as the indicator of glucose.
The described derivative based on boric acid is 2,3'dihydroxyboron-4-hydroxy-azobenzene (2,3 '-dihydroxyl boron-4-hydroxyazobenzene).
Step 2) described polymer membrane is the polymeric membrane that can use in position.
Step 2) described in carrier, adopt Poly(Hydroxyethyl Methacrylate) hydroxyethylmethacrylate (HEMA) or other similar polymer carriers, comprise polyacrylic resin, for example, polymethylmethacrylate polymethylmethacrylate, polymethyl acrylic acid hydroxyl methyl esters polyhydroxypropylmethacrylate, and their multipolymer etc.
Glucose indicator molecules described in step 1) is the derivative based on boric acid, and this molecule can link with carrier polymeric membrane covalency in * position by the Mannich reaction of standard; Described derivative is 2,3'dihydroxyboron-4-hydroxy-azobenzene (2,3 '-dihydroxyl boron-4-hydroxyazobenzene).
Fig. 1 provides a kind of molecular structure (2,3'dihydroxyboron-4-hydroxy-azobenzene (2,3 '-dihydroxyl boron-4-hydroxyazobenzene)) of glucose indicator molecules.This molecule can be combined by the glucose molecule in environment, thereby causes its fluorescence intensity to reduce.The derivative of Fig. 1 molecule, for example, non-* position access function group on phenyl ring, for example, halogen, methyl, ester group, carboxylic acid, etc., should also there is this type of activity.Glucose indicator molecules is after connecting carrier, and its fluorescence activity is retained, and still can be used as the indication of glucose
Agent.
Fig. 2 has provided the fluorescence spectrum of (PH=7.5, standard phosphate buffer solution) under physiological condition of molecule shown in 100mM Fig. 1, and this fluorescence can be recorded by multiple photoelectric diode.
Fig. 3 has provided molecule shown in 100mM Fig. 1 (PH=7.5, standard phosphate buffer solution) under physiological condition, the fluorescence radiation total intensity in the glucose solution of different concns.This figure demonstration, by observing the total fluorescence intensity of this quasi-molecule, we can learn the glucose concn of this molecule place environment.
Step 2) described in active group in carrier film from AEMA, it provides the amino (see figure 4) of a suspension, modifies this polymer carrier film thereby we can covalency.Other carry the monomer of active group, for example, and aminopropylmethacrylate(APMA) etc., also can be used.Except this type of uncle's Ammonia molecule, parahelium quasi-molecule also can be used; Other carry active group is sulfydryl (SH), carboxylic acid group (COOH), and hydroxyl (OH), aldehyde radical (CHO), and cyano group (CN), also wait and can use.
The present invention adopts and in Poly(Hydroxyethyl Methacrylate) hydroxyethylmethacrylate (HEMA), adds a small amount of monomer aminoethylmethacrylate(AEMA that has active group) monomer, form multipolymer.In polymerization process, the object of introducing the monomer that has active group is in order to make glucose indicator molecules link with carrier polymeric membrane covalency.In polymerization process, AEMA provides the amino (Fig. 4) of a suspension, modifies this polymer carrier film thereby we can covalency.This mixed polymer film can be modified upper indicator molecules by the crosslinking reaction of standard, and covalency linked, diagram is shown in Fig. 5.
specific embodiment 1:
The preparation method of the original position glucose detection film based on fluorescence detection method comprises the steps,
1) prepare to prepare material glucose indicator molecules
Described glucose indicator molecules is 2,3'dihydroxyboron-4-hydroxy-azobenzene (2,3 '-dihydroxyl boron-4-hydroxyazobenzene);
Described glucose indicator molecules can be specificly combined with glucose, and after being combined with glucose, the fluorescence intensity of this molecule reduces; When this molecular amounts one timing in system, by observing the total fluorescence intensity of this quasi-molecule, can learn the glucose concn of this molecule place environment;
2) prepare to prepare material support
Described carrier is glucose indicator molecules carrier, adopts polymer membrane;
Described carrier is by using mix monomer, the in the situation that of heating or UV-irradiation, carry out polymerization and obtain carrier film, under atmospheric pressure at room, use the HEMA aqueous solution of 2.0M, carrier monomer (HEMA) regulates and controls at 1:3 with the ratio of the monomer (AEMA) that has active group, under the condition of pH 7.5 phosphate buffer solutions, by solution spin coating monocrystalline silicon wafer crystal (wafer) surface, thickness is 100 microns; 365 nm UV illumination polymerization in 12 hours, consequent hydrogel, water fully washs to remove unreacted monomer; This hydrogel is dried, obtains carrier film;
3) the glucose indicator molecules covalency of preparing in step 1) is linked to step 2) in prepare carrier film on;
Concrete covalency links step, and carrier film, under atmospheric pressure at room, is immersed and contains 0.1-1% formaldehyde, 0.005-0.10M hydrochloric acid, and in the solution of 10mM-100 mM glucose indicator molecules, stir 12-36 hour; Add PBS buffered soln and fully wash, obtain covalency and connect the polymeric membrane of glucose indicator molecules; This polymeric membrane, through cutting, can implant, and uses as the active membrane of glucose optical pickocff;
After glucose indicator molecules covalency is linked in carrier film, glucose indicator molecules still retains specificity and the fluorescent optics activity of cognitive glucose.
Fig. 6 represents to be linked to glucose indicator molecules in the carrier film fluorescence radiation total intensity in the glucose solution of different concns through step 3) covalency.
specific embodiment 2:
The preparation method of the original position glucose detection film based on fluorescence detection method comprises the steps,
1) prepare to prepare material glucose indicator molecules
Described glucose indicator molecules is 2,3'dihydroxyboron-4-hydroxy-azobenzene (2,3 '-dihydroxyl boron-4-hydroxyazobenzene);
Described glucose indicator molecules can be specificly combined with glucose, and after being combined with glucose, the fluorescence intensity of this molecule reduces; When this molecular amounts one timing in system, by observing the total fluorescence intensity of this quasi-molecule, can learn the glucose concn of this molecule place environment;
2) prepare to prepare material support
Described carrier is glucose indicator molecules carrier, adopts polymer membrane;
Described carrier is by using mix monomer, the in the situation that of heating or UV-irradiation, carry out polymerization and obtain carrier film, under atmospheric pressure at room, use the HEMA aqueous solution of 2.0M, carrier monomer (HEMA) regulates and controls at 1:10 with the ratio of the monomer (AEMA) that has active group, under the condition of pH 7.5 phosphate buffer solutions, by solution spin coating monocrystalline silicon wafer crystal (wafer) surface, thickness is 100 microns; 365 nm UV illumination polymerization in 12 hours, consequent hydrogel, water fully washs to remove unreacted monomer; This hydrogel is dried, obtains carrier film;
3) the glucose indicator molecules covalency of preparing in step 1) is linked to step 2) in prepare carrier film on;
Concrete covalency links step, and carrier film, under atmospheric pressure at room, is immersed and contains 0.1-1% formaldehyde, 0.005-0.10M hydrochloric acid, and in the solution of 10mM-100 mM glucose indicator molecules, stir 12-36 hour; Add PBS buffered soln and fully wash, obtain covalency and connect the polymeric membrane of glucose indicator molecules; This polymeric membrane, through cutting, can implant, and uses as the active membrane of glucose optical pickocff;
After glucose indicator molecules covalency is linked in carrier film, glucose indicator molecules still retains specificity and the fluorescent optics activity of cognitive glucose.
Fig. 7 represents to be linked to glucose indicator molecules in the carrier film fluorescence radiation total intensity in the glucose solution of different concns through step 3) covalency.
specific embodiment 3:
The preparation method of the original position glucose detection film based on fluorescence detection method comprises the steps,
1) prepare to prepare material glucose indicator molecules
Described glucose indicator molecules is 2,3'dihydroxyboron-4-hydroxy-azobenzene (2,3 '-dihydroxyl boron-4-hydroxyazobenzene);
Described glucose indicator molecules can be specificly combined with glucose, and after being combined with glucose, the fluorescence intensity of this molecule reduces; When this molecular amounts one timing in system, by observing the total fluorescence intensity of this quasi-molecule, can learn the glucose concn of this molecule place environment;
2) prepare to prepare material support
Described carrier is glucose indicator molecules carrier, adopts polymer membrane;
Described carrier is by using mix monomer, the in the situation that of heating or UV-irradiation, carry out polymerization and obtain carrier film, under atmospheric pressure at room, use the HEMA aqueous solution of 2.0M, carrier monomer (HEMA) regulates and controls at 1:20 with the ratio of the monomer (AEMA) that has active group, under the condition of pH 7.5 phosphate buffer solutions, by solution spin coating monocrystalline silicon wafer crystal (wafer) surface, thickness is 100 microns; 365 nm UV illumination polymerization in 12 hours, consequent hydrogel, water fully washs to remove unreacted monomer; This hydrogel is dried, obtains carrier film;
3) the glucose indicator molecules covalency of preparing in step 1) is linked to step 2) in prepare carrier film on;
Concrete covalency links step, and carrier film, under atmospheric pressure at room, is immersed and contains 0.1-1% formaldehyde, 0.005-0.10M hydrochloric acid, and in the solution of 10mM-100 mM glucose indicator molecules, stir 12-36 hour; Add PBS buffered soln and fully wash, obtain covalency and connect the polymeric membrane of glucose indicator molecules; This polymeric membrane, through cutting, can implant, and uses as the active membrane of glucose optical pickocff;
After glucose indicator molecules covalency is linked in carrier film, glucose indicator molecules still retains specificity and the fluorescent optics activity of cognitive glucose.
Fig. 8 represents to be linked to glucose indicator molecules in the carrier film fluorescence radiation total intensity in the glucose solution of different concns through step 3) covalency.
specific embodiment 4:
The preparation method of the original position glucose detection film based on fluorescence detection method comprises the steps,
1) prepare to prepare material glucose indicator molecules
Described glucose indicator molecules is 2,3'dihydroxyboron-4-hydroxy-azobenzene (2,3 '-dihydroxyl boron-4-hydroxyazobenzene);
Described glucose indicator molecules can be specificly combined with glucose, and after being combined with glucose, the fluorescence intensity of this molecule reduces; When this molecular amounts one timing in system, by observing the total fluorescence intensity of this quasi-molecule, can learn the glucose concn of this molecule place environment;
2) prepare to prepare material support
Described carrier is glucose indicator molecules carrier, adopts polymer membrane;
Described carrier is by using mix monomer, the in the situation that of heating or UV-irradiation, carry out polymerization and obtain carrier film, under atmospheric pressure at room, use the HEMA aqueous solution of 2.0M, carrier monomer (HEMA) regulates and controls at 1:33 with the ratio of the monomer (AEMA) that has active group, under the condition of pH 7.5 phosphate buffer solutions, by solution spin coating monocrystalline silicon wafer crystal (wafer) surface, thickness is 100 microns; 365 nm UV illumination polymerization in 12 hours, consequent hydrogel, water fully washs to remove unreacted monomer; This hydrogel is dried, obtains carrier film;
3) the glucose indicator molecules covalency of preparing in step 1) is linked to step 2) in prepare carrier film on;
Concrete covalency links step, and carrier film, under atmospheric pressure at room, is immersed and contains 0.1-1% formaldehyde, 0.005-0.10M hydrochloric acid, and in the solution of 10mM-100 mM glucose indicator molecules, stir 12-36 hour; Add PBS buffered soln and fully wash, obtain covalency and connect the polymeric membrane of glucose indicator molecules; This polymeric membrane, through cutting, can implant, and uses as the active membrane of glucose optical pickocff;
After glucose indicator molecules covalency is linked in carrier film, glucose indicator molecules still retains specificity and the fluorescent optics activity of cognitive glucose.
Fig. 9 represents to be linked to glucose indicator molecules in the carrier film fluorescence radiation total intensity in the glucose solution of different concns through step 3) covalency.
specific embodiment 5:
The preparation method of the original position glucose detection film based on fluorescence detection method comprises the steps,
1) prepare to prepare material glucose indicator molecules
Described glucose indicator molecules is 2,3'dihydroxyboron-4-hydroxy-azobenzene (2,3 '-dihydroxyl boron-4-hydroxyazobenzene);
Described glucose indicator molecules can be specificly combined with glucose, and after being combined with glucose, the fluorescence intensity of this molecule reduces; When this molecular amounts one timing in system, by observing the total fluorescence intensity of this quasi-molecule, can learn the glucose concn of this molecule place environment;
2) prepare to prepare material support
Described carrier is glucose indicator molecules carrier, adopts polymer membrane;
Described carrier is by using mix monomer, the in the situation that of heating or UV-irradiation, carry out polymerization and obtain carrier film, under atmospheric pressure at room, use the HEMA aqueous solution of 1.0M, carrier monomer (HEMA) regulates and controls at 1:10 with the ratio of the monomer (AEMA) that has active group, under the condition of pH 7.5 phosphate buffer solutions, by solution spin coating monocrystalline silicon wafer crystal (wafer) surface, thickness is 100 microns; 365 nm UV illumination polymerization in 12 hours, consequent hydrogel, water fully washs to remove unreacted monomer; This hydrogel is dried, obtains carrier film;
3) the glucose indicator molecules covalency of preparing in step 1) is linked to step 2) in prepare carrier film on;
Concrete covalency links step, and carrier film, under atmospheric pressure at room, is immersed and contains 0.1-1% formaldehyde, 0.005-0.10M hydrochloric acid, and in the solution of 10mM-100 mM glucose indicator molecules, stir 12-36 hour; Add PBS buffered soln and fully wash, obtain covalency and connect the polymeric membrane of glucose indicator molecules; This polymeric membrane, through cutting, can implant, and uses as the active membrane of glucose optical pickocff;
After glucose indicator molecules covalency is linked in carrier film, glucose indicator molecules still retains specificity and the fluorescent optics activity of cognitive glucose.
Figure 10 represents to be linked to glucose indicator molecules in the carrier film fluorescence radiation total intensity in the glucose solution of different concns through step 3) covalency.
specific embodiment 6:
The preparation method of the original position glucose detection film based on fluorescence detection method comprises the steps,
1) prepare to prepare material glucose indicator molecules
Described glucose indicator molecules is 2,3'dihydroxyboron-4-hydroxy-azobenzene (2,3 '-dihydroxyl boron-4-hydroxyazobenzene);
Described glucose indicator molecules can be specificly combined with glucose, and after being combined with glucose, the fluorescence intensity of this molecule reduces; When this molecular amounts one timing in system, by observing the total fluorescence intensity of this quasi-molecule, can learn the glucose concn of this molecule place environment;
2) prepare to prepare material support
Described carrier is glucose indicator molecules carrier, adopts polymer membrane;
Described carrier is by using mix monomer, the in the situation that of heating or UV-irradiation, carry out polymerization and obtain carrier film, under atmospheric pressure at room, use the HEMA aqueous solution of 3.0M, carrier monomer (HEMA) regulates and controls at 1:10 with the ratio of the monomer (AEMA) that has active group, under the condition of pH 7.5 phosphate buffer solutions, by solution spin coating monocrystalline silicon wafer crystal (wafer) surface, thickness is 100 microns; 365 nm UV illumination polymerization in 12 hours, consequent hydrogel, water fully washs to remove unreacted monomer; This hydrogel is dried, obtains carrier film;
3) the glucose indicator molecules covalency of preparing in step 1) is linked to step 2) in prepare carrier film on;
Concrete covalency links step, and carrier film, under atmospheric pressure at room, is immersed and contains 0.1-1% formaldehyde, 0.005-0.10M hydrochloric acid, and in the solution of 10mM-100 mM glucose indicator molecules, stir 12-36 hour; Add PBS buffered soln and fully wash, obtain covalency and connect the polymeric membrane of glucose indicator molecules; This polymeric membrane, through cutting, can implant, and uses as the active membrane of glucose optical pickocff;
After glucose indicator molecules covalency is linked in carrier film, glucose indicator molecules still retains specificity and the fluorescent optics activity of cognitive glucose.
Figure 11 represents to be linked to glucose indicator molecules in the carrier film fluorescence radiation total intensity in the glucose solution of different concns through step 3) covalency.
specific embodiment 7:
The preparation method of the original position glucose detection film based on fluorescence detection method comprises the steps,
1) prepare to prepare material glucose indicator molecules
Described glucose indicator molecules is 2,3'dihydroxyboron-4-hydroxy-azobenzene (2,3 '-dihydroxyl boron-4-hydroxyazobenzene);
Described glucose indicator molecules can be specificly combined with glucose, and after being combined with glucose, the fluorescence intensity of this molecule reduces; When this molecular amounts one timing in system, by observing the total fluorescence intensity of this quasi-molecule, can learn the glucose concn of this molecule place environment;
2) prepare to prepare material support
Described carrier is glucose indicator molecules carrier, adopts polymer membrane;
Described carrier is by using mix monomer, the in the situation that of heating or UV-irradiation, carry out polymerization and obtain carrier film, under atmospheric pressure at room, use the HEMA aqueous solution of 2.0M, carrier monomer (HEMA) regulates and controls at 1:10 with the ratio of the monomer (AEMA) that has active group, under the condition of pH 7.0 phosphate buffer solutions, by solution spin coating monocrystalline silicon wafer crystal (wafer) surface, thickness is 100 microns; 365 nm UV illumination polymerization in 12 hours, consequent hydrogel, water fully washs to remove unreacted monomer; This hydrogel is dried, obtains carrier film;
3) the glucose indicator molecules covalency of preparing in step 1) is linked to step 2) in prepare carrier film on;
Concrete covalency links step, and carrier film, under atmospheric pressure at room, is immersed and contains 0.1-1% formaldehyde, 0.005-0.10M hydrochloric acid, and in the solution of 10mM-100 mM glucose indicator molecules, stir 12-36 hour; Add PBS buffered soln and fully wash, obtain covalency and connect the polymeric membrane of glucose indicator molecules; This polymeric membrane, through cutting, can implant, and uses as the active membrane of glucose optical pickocff;
After glucose indicator molecules covalency is linked in carrier film, glucose indicator molecules still retains specificity and the fluorescent optics activity of cognitive glucose.
Figure 12 represents to be linked to glucose indicator molecules in the carrier film fluorescence radiation total intensity in the glucose solution of different concns through step 3) covalency.
specific embodiment 8:
The preparation method of the original position glucose detection film based on fluorescence detection method comprises the steps,
1) prepare to prepare material glucose indicator molecules
Described glucose indicator molecules is 2,3'dihydroxyboron-4-hydroxy-azobenzene (2,3 '-dihydroxyl boron-4-hydroxyazobenzene);
Described glucose indicator molecules can be specificly combined with glucose, and after being combined with glucose, the fluorescence intensity of this molecule reduces; When this molecular amounts one timing in system, by observing the total fluorescence intensity of this quasi-molecule, can learn the glucose concn of this molecule place environment;
2) prepare to prepare material support
Described carrier is glucose indicator molecules carrier, adopts polymer membrane;
Described carrier is by using mix monomer, the in the situation that of heating or UV-irradiation, carry out polymerization and obtain carrier film, under atmospheric pressure at room, use the HEMA aqueous solution of 2.0M, carrier monomer (HEMA) regulates and controls at 1:10 with the ratio of the monomer (AEMA) that has active group, under the condition of pH 8.0 phosphate buffer solutions, by solution spin coating monocrystalline silicon wafer crystal (wafer) surface, thickness is 100 microns; 365 nm UV illumination polymerization in 12 hours, consequent hydrogel, water fully washs to remove unreacted monomer; This hydrogel is dried, obtains carrier film;
3) the glucose indicator molecules covalency of preparing in step 1) is linked to step 2) in prepare carrier film on;
Concrete covalency links step, and carrier film, under atmospheric pressure at room, is immersed and contains 0.1-1% formaldehyde, 0.005-0.10M hydrochloric acid, and in the solution of 10mM-100 mM glucose indicator molecules, stir 12-36 hour; Add PBS buffered soln and fully wash, obtain covalency and connect the polymeric membrane of glucose indicator molecules; This polymeric membrane, through cutting, can implant, and uses as the active membrane of glucose optical pickocff;
After glucose indicator molecules covalency is linked in carrier film, glucose indicator molecules still retains specificity and the fluorescent optics activity of cognitive glucose.
Figure 13 represents to be linked to glucose indicator molecules in the carrier film fluorescence radiation total intensity in the glucose solution of different concns through step 3) covalency.
Claims (9)
1. a preparation method for the original position glucose detection film based on fluorescence detection method, is characterized in that, comprises the steps:
1) prepare to prepare material glucose indicator molecules
Described glucose indicator molecules can be specificly combined with glucose, and after being combined with glucose, the fluorescence intensity of this molecule reduces; When this molecular amounts one timing in system, by observing the total fluorescence intensity of this quasi-molecule, can learn the glucose concn of this molecule place environment;
2) prepare to prepare material support
Described carrier is glucose indicator molecules carrier, adopts polymer membrane;
Described carrier is by using the monomer mixture of macromolecular compound, carries out polymerization and obtain the polymer carrier film of copolymerization in the situation that of heating or UV-irradiation;
3) the glucose indicator molecules covalency of preparing in step 1) is linked to step 2) in prepare carrier film on;
Concrete covalency links step, and carrier film, under atmospheric pressure at room, is immersed and contains 0.1-1% formaldehyde, 0.005-0.10M hydrochloric acid, and in the solution of 10mM-100 mM glucose indicator molecules, stir 12-36 hour; Add PBS buffered soln and fully wash, obtain covalency and connect the polymeric membrane of glucose indicator molecules; The i.e. original position glucose detection film based on fluorescence detection method.
2. the preparation method of the original position glucose detection film based on fluorescence detection method according to claim 1, it is characterized in that: the original position glucose detection film based on fluorescence detection method making is through cutting, can implant, use as the active membrane of glucose optical pickocff.
3. the preparation method of the original position glucose detection film based on fluorescence detection method according to claim 1, it is characterized in that: after described glucose indicator molecules covalency is linked in carrier film, glucose indicator molecules still retains specificity and the fluorescent optics activity of cognitive glucose.
4. the preparation method of the original position glucose detection film based on fluorescence detection method according to claim 1, it is characterized in that: the glucose indicator molecules described in step 1) is the derivative based on boric acid, this molecule can link with carrier polymeric membrane covalency by the Mannich reaction of standard.
5. the preparation method of the original position glucose detection film based on fluorescence detection method according to claim 1, is characterized in that: the described derivative based on boric acid is 2,3 '-dihydroxyl boron-4-hydroxyazobenzene.
6. the preparation method of the original position glucose detection film based on fluorescence detection method according to claim 1, it is characterized in that: step 2) described in carrier, adopt Poly(Hydroxyethyl Methacrylate) hydroxyethylmethacrylate or other similar polymer carriers, comprise polyacrylic resin and multipolymer thereof.
7. the preparation method of the original position glucose detection film based on fluorescence detection method according to claim 1, it is characterized in that: step 2) in being prepared as of carrier film, described carrier is by using mix monomer, the in the situation that of heating or UV-irradiation, carry out polymerization and obtain carrier film, for example, under atmospheric pressure at room, use 1.0M, 2.0M, the HEMA aqueous solution of 2.5M or 3.0M, wherein carrier monomer HEMA with have the corresponding regulation and control of ratio of monomer A EMA of active group at 1:3, 1:10, 1:20 and 1:33, under the condition of pH6-9 phosphate buffer solution, by above-mentioned HEMA aqueous solution spin coating monocrystalline silicon wafer crystal surface, thickness is 50-500 micron, 365 nm UV illumination polymerization in 6-36 hour, consequent hydrogel, water fully washs to remove unreacted monomer, this hydrogel is dried, obtains carrier film.
8. the preparation method of the original position glucose detection film based on fluorescence detection method according to claim 7, it is characterized in that: the active group in described carrier film carries the monomer of active group from AEMA, other active class groups or other, described AEMA provides the amino of a suspension, thereby can covalency modify this polymer carrier film, the monomer that carries active group is that aminopropylmethacrylate is APMA, except this type of uncle's Ammonia molecule, parahelium quasi-molecule also can use; Other carry active group be sulfydryl-SH, carboxylic acid group-COOH, hydroxyl-OH, aldehyde radical-CHO and cyano group-CN.
9. the preparation method of the original position glucose detection film based on fluorescence detection method according to claim 7, is characterized in that: described carrier film is hydrophobic.
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| CN104597090A (en) * | 2014-12-10 | 2015-05-06 | 中国科学院烟台海岸带研究所 | Enzyme-free potentiometric glucose sensor and detection method thereof |
| CN105572090A (en) * | 2016-01-20 | 2016-05-11 | 大连理工大学 | Oxytetracycline fluorescence detection method based on graphene-based compound hydrogel |
| 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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| US6344360B1 (en) * | 1998-03-11 | 2002-02-05 | Sensors For Medicine And Science, Inc. | Detection of analytes by fluorescent lanthanide metal chelate complexes containing substituted ligands |
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| CN105572090A (en) * | 2016-01-20 | 2016-05-11 | 大连理工大学 | Oxytetracycline fluorescence detection method based on graphene-based compound hydrogel |
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