CN108375616A - A kind of liquid crystal biosensor of detection of alkaline phosphatase and its preparation method and application - Google Patents
A kind of liquid crystal biosensor of detection of alkaline phosphatase and its preparation method and application Download PDFInfo
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- CN108375616A CN108375616A CN201810107545.0A CN201810107545A CN108375616A CN 108375616 A CN108375616 A CN 108375616A CN 201810107545 A CN201810107545 A CN 201810107545A CN 108375616 A CN108375616 A CN 108375616A
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- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
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- G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
Abstract
The present invention relates to liquid crystal biosensors of a kind of detection of alkaline phosphatase and its preparation method and application, belong to technical field of analysis and detection.The liquid crystal biosensor includes the upper slide that liquid crystal sensitive membrane, the 5CB liquid crystal molecules being added dropwise in the liquid crystal sensitive membrane and DMOAP are modified, the liquid crystal sensitive membrane is to first pass through silylation modification, recycle glutaraldehyde that decorated by nano-gold is prepared to surface of glass slide, the 5 CB liquid crystal molecules of silver nano-grain pair for depositing to liquid crystal sensitivity film surface generate upset to realize the detection to alkaline phosphatase.Enzymatic metallization reaction, nanogold induction deposition of silver are applied to liquid crystal biosensor by the present invention, with easy to operate, high specificity, high sensitivity and the advantages that do not need complex instrument, result visualization can be applied to the highly sensitive Visual retrieval of blood serum sample alkaline phosphatase.
Description
Technical field
The present invention relates to a kind of liquid crystal biosensors, and in particular to a kind of liquid crystal biology for detection of alkaline phosphatase
Sensor further relates to preparation method and application, belongs to technical field of analysis and detection.
Background technology
Liquid crystal（Liquid Crystal, abbreviation LC）It is the abbreviation of liquid crystal, is one between liquid and solid
Kind particular matter form.Since liquid crystal molecule has the special natures such as birefringence, ordered orientation and photoelectric activity, thus it
Not only it is widely used in electronics, photoelectric display field, the signal conversion element being also used as in biological and chemical field.
Researchers develop many liquid crystal biosensors using liquid crystal molecule as signal conversion element, and will
It is applied to many kinds of substance such as：The detection of protein, antibody, heavy metal ion, nucleic acid, pesticide.Its basic principle is to utilize to wait for
It surveys object to be reacted in the specific recognition of sensitive film surface to upset the ordered arrangement of liquid crystal molecule, and then realizes the qualitative of object
And semi-quantitative analysis.Liquid crystal biosensor can be built using common slide, be not necessarily to complex instrument equipment and special light
Source only provides for biochemical reaction information by the variation of liquid crystal pond color, in clinical medicine, food inspection and biological section
Field has broad application prospects.But since the size of many biomolecule is little, utilize biologic specificity recognition reaction
（Such as antigen-antibody reaction）The upset effect of the liquid crystal molecule caused by sensitive film surface is captured than relatively limited, to shadow
The detection sensitivity of method is rung.Therefore, how to improve the detection sensitivity of liquid crystal biosensor becomes the key of problem.
Invention content
To solve the above-mentioned problems, the object of the present invention is to provide a kind of highly sensitive, Visual retrieval alkaline phosphatase
Liquid crystal biosensor and its preparation method and application.
To achieve the above object, the technical solution that the present invention specifically uses is as follows：
A kind of liquid crystal biosensor of detection of alkaline phosphatase, including liquid crystal sensitive membrane, dropwise addition are in the liquid crystal sensitive membrane
5CB liquid crystal molecules and DMOAP modifications upper slide, the liquid crystal sensitive membrane is to first pass through silylation modification, recycles penta 2
Decorated by nano-gold is prepared aldehyde to surface of glass slide, using depositing to the silver nano-grain of liquid crystal sensitivity film surface to 5-CB liquid
Brilliant molecule, which generates, to be upset to realize the detection to alkaline phosphatase.
The preparation method of liquid crystal biosensor of the present invention, includes the following steps：
Step（1）, prepare glutathione be ligand nanogold；
Step（2）, slide cleaning；
Step（3）, DMOAP modification upper slide and amido modified lower slide preparation；
Step（4）, liquid crystal sensitive membrane preparation
By step（3）Lower slide immerse in the glutaraldehyde water solution of a concentration of 0.5-1%, react at room temperature 1-2h, it is clear with ultra-pure water
Wash clean, N2Drying, obtains the slide substrate of aldehyde radical function；
Then 200 μ L steps are added dropwise in the slide substrate surface of the aldehyde radical function（1）Nanogold, room temperature reaction 1 h or so,
Unreacted nanogold, N are washed away with ultra-pure water2Drying, obtains liquid crystal sensitive membrane；
Step（5）, liquid crystal biosensor preparation
The alkaline phosphatase of various concentration to be detected is added in detection liquid, it is quick to be added drop-wise to above-mentioned liquid crystal after mixing rapidly
Feel film surface, be protected from light, ultrapure water, N are used after enzymatic metallization reaction2Drying, it is spare；
1 ~ 2 μ L 5CB liquid crystal molecules are added dropwise in liquid crystal sensitive membrane after above-mentioned enzymatic metallization reaction, cover step
（3）DMOAP modifications upper slide to get to the liquid crystal biosensor for detection of alkaline phosphatase.
Further, step（1）In, a concentration of the 1.41 × 10 of nanogold-8 mol/L。
Further, step（2）In, slide is immersed in Piranha solution, be placed in 80 DEG C of water-bath stand 1 ~
2 h.It is rinsed after reaction with a large amount of ultra-pure water and absolute ethyl alcohol, then uses N2Drying is positioned in exsiccator and does for 110 DEG C
Dry 2 ~ 4h is positioned over drying in exsiccator, obtains clean slide.
Further, step（3）In, the preparation method of the upper slide of DMOAP modifications is：
Clean slide is immersed in the DMOAP aqueous solutions containing 0.5-1%, reaction 20-30min is stood under room temperature, with nothing
Water-ethanol cleans unreacted DMOAP, then clean, the N with a large amount of ultrapure water2Drying is positioned over drying in baking oven, obtains
The upper slide substrate of DMOAP modifications；
The preparation method of amido modified lower slide is：
Clean slide is immersed in the Acetic acid-sodium acetate buffer solution containing 1%DMOAP and 4-6%APS, 80 DEG C in water-bath
1.5 h of isothermal reaction cleans unreacted DMOAP and APS with ethyl alcohol, then is cleaned up with a large amount of ultra-pure waters, N2Drying is placed
It dries in an oven, makes the upper chain alkyl of surface of glass slide modification and amido functional group.
Further, step（5）In, detection liquid is to contain 2-8mmol/L ascorbic acid 2- phosphates and 2-8mmol/L nitre
Diethanol amine-nitric acid buffer solution that 0.1-0.2mol/L, pH of sour silver are 9.5-10；1-2 μ are added dropwise in liquid crystal sensitivity film surface
L5CB liquid crystal molecules.
Further, step（1）It is specific as follows to prepare the nanogold that glutathione is ligand：
0.10mL glutathione (GSH, 0.24 mol/L) is added into the gold chloride of 1mL 1%, at room temperature stirring 2 ~ 5 minutes, so
1 mol/L NaOH to the pH of solution are slowly added dropwise in backward solution and occur largely light yellow sink in 2.5 ~ 3.0, solution
It forms sediment.Then it utilizes centrifuge to be centrifuged 1 minute using 6000 ~ 8000 revs/min of rotating speed, discards supernatant liquid and obtain Au (I)-GSH
Precipitation.Then precipitation 5mmol/L sodium hydroxides are dissolved, 11 mL ultra-pure waters is added, then adjust solution ph to 7.0
Left and right.It is slowly added dropwise into reaction solution into 0.1 mg/mL sodium borohydrides, 100 μ L, until solution becomes brown color, is stirred in magnetic force
The rotating speed using 600 revs/min on device is mixed, reacts 4 h at room temperature, uses molecular cut off to be purified for the super filter tube of 30kD, most
Obtained solution is dispersed in 4 mL ultra-pure waters afterwards, 4 DEG C save backup.
The application of the liquid crystal biosensor of above-mentioned detection of alkaline phosphatase of the present invention, includes the following steps：
Step（1）, the alkaline phosphatase of various concentration is added in detection liquid, is added drop-wise to claim after mixing rapidly
The liquid crystal sensitivity film surface of the decorated by nano-gold of one of 1-6, is protected from light；The detection liquid is to contain ascorbic acid 2-
The diethanol amine of phosphate and silver nitrate-nitric acid buffer solution；
Step（2）, by step（1）Liquid crystal sensitive membrane ultrapure water, N2Then 1-2 μ L5CB liquid crystal point is added dropwise in drying
Son covers the upper slide of DMOAP modifications, obtains liquid crystal biosensor；
Step（3）, then above-mentioned liquid crystal biosensor is placed on 25 DEG C ~ 27 DEG C of heated at constant temperature platform, it is aobvious using polarisation
Micro mirror is observed under cross-polarized light pattern, is taken pictures, and obtains the optic response image of various concentration alkaline phosphatase, utilizes this
The variation of the quantity and color of white hot spot carries out quantitative and semi-quantitative detection to alkaline phosphatase on optical imagery.
The present invention utilizes target alkaline phosphatase in the liquid crystal sensitivity film surface enzymatic deposition of silver occurred of decorated by nano-gold
Reaction, using deposit to the silver nano-grain of liquid crystal sensitivity film surface the upset of 5-CB liquid crystal molecules is acted on and the optics that generates
Signal realizes the highly sensitive detection to alkaline phosphatase.
The presence of alkaline phosphatase can be catalyzed its substrate ascorbic acid 2- phosphate and phosphate radical is gone to generate ascorbic acid, and
Silver ion reduction in reaction solution can be rapidly silver-colored simple substance and deposit to decorated by nano-gold by the ascorbic acid of strong reducing property
Liquid crystal sensitivity film surface upsets 5-CB liquid crystal molecules and generates birefringence signal in the ordered arrangement of the sensitivity film surface, then
The interference of different level light further occurs and obtains the petrographic microscope image of different colours, eventually by the optics detected
The quantity and color of white hot spot realizes the detection to target alkalinity alkaline phosphatase on image.
Compared with prior art, the present invention has the beneficial effect that：
（1）, establish a kind of liquid crystal bio-sensor signal amplification method to metallize based on enzymatic, utilize decorated by nano-gold
It is sensitive that slide promotees metallization reaction as liquid crystal sensitivity membrane bound enzyme, nanogold induces deposition of silver reaction that can greatly improve detection
Degree, only by liquid crystal optics image number of spots and color can realize the detection to alkaline phosphatase, do not need
Complex instrument, testing result visualization, solve the problems, such as that liquid crystal biosensor detection sensitivity is not high.
（2）, provide a kind of preparation method of the liquid crystal sensitive membrane of decorated by nano-gold, the nanogold with the sweet peptide of Guang be with
Body has colloidal stability very good, and surface has abundant amino and carboxyl, the features such as can directly be modified.We
It further utilizes glutaraldehyde by the surface of glass slide of its covalent coupling to amino functional, is prepared for the uniformly modified liquid crystal of nanogold
Sensitive membrane, the liquid crystal biosensor which is suitable for metallizing based on enzymatic.
（3）, the present invention for other liquid crystal biosensors, realize for the first time multi-color LCD sensing, detected
The optical imagery arrived is bright-colored and abundant, and color change is apparent, is very suitable for Visual retrieval.
（4）, the present invention it is very wide to the detection range of alkaline phosphatase, it can be achieved that 5 × 10-9The U/mL of U/mL ~ 0.5 8
The Visual retrieval of order of magnitude alkaline phosphatase.
（5）, this method detection limit down to 5 × 10-9U/mL, the visible detection method phase with other alkaline phosphatases
Than detection sensitivity improves 5 ~ 7 orders of magnitude.
（6）, the present invention it is easy to operate, do not need complex instrument, result visualization, and high sensitivity, specificity is good, can
To realize the highly sensitive Visual retrieval of human serum alkaline phosphatase, there is extraordinary application prospect in clinical detection.
Description of the drawings
Fig. 1 is the principle schematic of the liquid crystal biosensor detection of alkaline phosphatase to be metallized based on enzymatic；Wherein：
（A）The preparation process of the liquid crystal sensitive membrane of decorated by nano-gold；（B）The testing principle of alkaline phosphatase；
Fig. 2 is optical imagery of the different solutions in the liquid crystal sensitivity film surface of decorated by nano-gold；
Wherein：（A）1×10-6 U/mL alkaline phosphatase+5mM silver nitrates；（B）+ 5 mM nitric acid of 5mM ascorbic acid 2- phosphates
Silver；（C）1×10-6 U/mL alkaline phosphatase+5mM ascorbic acid 2- phosphates；（D）10 μM of sodium vanadate+1 × 10-6 U/mL alkalinity
Phosphatase+5mM ascorbic acid 2- phosphate+5mM silver nitrates；（E）1×10-6 U/mL alkaline phosphatase+5mM ascorbic acid 2- phosphorus
Acid esters+5mM silver nitrates；
Fig. 3 is the atomic force microscope characterization result of different surface of glass slide；
Wherein：（A）Clean slide；（B）The slide of silylating reagent DMOAP and APS modification；（C）The slide of decorated by nano-gold；
（D）The later slide of enzymatic metallization reaction；
Fig. 4 is the scanning electron microscope characterization result of different surface of glass slide；
Wherein：（A）Clean slide；（B）The slide of decorated by nano-gold；（C）The later slide of enzymatic metallization reaction；
Fig. 5 is the optimization that liquid crystal sensitive membrane modifies condition；
Wherein：（A）The ratio of APS and DMOAP：(a)1:1；(b)5:1；(c)10:1；(d)20:1；（B）Glutaraldehyde dosage it is excellent
Change (a) 1%；(b) 5%；(c) 10%；(d) 20%；（C）The optimization of nanometer gold concentration： (a) 1.41×10-8mol/L；(b)
2.82×10-8mol/L；(c) 4.24×10-8 mol/L；(d) 6.84×10-8 mol/L。
Fig. 6 is the optimization of testing conditions；
Wherein：（A）Detect the concentration of substrate AA-2P in liquid：(a) 1 mmol/L; (b) 5 mmol/L；(c) 10 mmol/L；
(d)20mmol/L；（B）Detect the concentration of silver nitrate in liquid：(a) 1 mmol/L；(b) 5mmol/L；(c)10mmol/L；(d)
20mmol/L；（C）Detect the influence of liquid pH：(a) 8.0；(b) 9.5；(c) 9.8；(d) 11.0；（D）The shadow in reaction time
It rings：(a)30 min; (b) 1 h; (c) 1.5 h ; (d) 2 h；
Fig. 7 is the petrographic microscope that various concentration alkaline phosphatase is detected using the liquid crystal biosensor to metallize based on enzymatic
Optical imagery；
Wherein,（A）Control；(B) 5×10-9 U/mL；(C) 5×10-8U/mL；(D) 5×10-7 U/mL；(E) 5×10-6
U/mL；(F) 5×10-5 U/mL；(G) 5×10-4U/mL；(H) 5×10-3U/mL；(I) 5×10-2U/mL；(J) 5×
10-1U/mL；Figure A-D white bright spots gradually increase, and figure E whole visual fields brighten and start to occur a small amount of orange interference colours, and figure F is
Orange, figure G is bluish violet, and figure H is red（The a small amount of blue of doping）, figure I is blue（Doping is a small amount of red）, figure J is green；
Fig. 8 is to be detected obtained optical imagery using the liquid crystal sensitive membrane of unmodified nanogold；
Wherein：（A）Control；(B) 5×10-9 U/mL；(C) 5×10-8U/mL；(D) 5×10-7 U/mL；(E) 5×10-6
U/mL；(F) 5×10-5 U/mL；(G) 5×10-4U/mL；(H) 5×10-3U/mL；(I) 5×10-2U/mL；(J) 5×
10-1U/mL.Figure A-H white bright spots gradually increase, and figure I and J white bright spots are gradually linked to be piece.
Fig. 9 is the specific detection image of method；
Wherein：（A）50 μ g/mL horseradish peroxidases；（B）1.5 U/mL pepsins；（C）1.5 U/mL carbonic anhydrases；
（D）50 μ g/mL glutathione；（E）1.5 U/mL trypsase；（F）1×10-6U/mL alkaline phosphatases.
Figure 10 is the testing result of blood serum sample alkaline phosphatase；
Wherein：（A）50000 times of dilution,（B）Dilution 10000 times and（C）The blood serum sample of 1000 times of dilution；It dilutes（D）
50000 times and（E）1 × 10 is added in 10000 times of blood serum sample-6The alkaline phosphatase of U/mL；（F）The blood of 1000 times of dilution
Final proof product utilize 120 DEG C of high-temperature process 30 minutes；Wherein, figure C is yellow, and figure E is a small amount of red, blue of yellow doping.
Specific implementation mode
With reference to embodiment, the present invention is described in further detail.
The following example is merely to illustrate the present invention, and should not be taken as limiting the scope of the invention.It is not specified in embodiment
Particular technique or condition person carry out according to technology or condition described in document in the art or according to product description.
Production firm person is not specified in material therefor or equipment, and being can be by buying the conventional products obtained.
1, the detection of alkaline phosphatase
Embodiment 1
The liquid crystal biosensor of the detection of alkaline phosphatase of the present embodiment, including liquid crystal sensitive membrane, be added dropwise it is quick in the liquid crystal
Feel the upper slide of the 5CB liquid crystal molecules and DMOAP modifications on film, the liquid crystal sensitive membrane is to first pass through silylation modification, then profit
Decorated by nano-gold is prepared to surface of glass slide with glutaraldehyde, utilizes the silver nano-grain pair for depositing to liquid crystal sensitivity film surface
5-CB liquid crystal molecules, which generate, to be upset to realize the detection to alkaline phosphatase.
As shown in Figure 1, Fig. 1 is to detect alkaline phosphatase using the liquid crystal biosensor amplified based on enzymatic metalized signal
The principle schematic of enzyme.
Figure 1A is the preparation process of liquid crystal sensitive membrane：First in clean surface of glass slide self assembly silylating reagent APS and
DMOAP（Two kinds of silylating reagents have the function of different：N containing long alkyl chain, N- dimethyl-N-octadecyl base -3- ammonia third
Base trimethyoxysilane（DMOAP）Nematic 5CB liquid crystal molecules can be effectively induced to be arranged vertically；3- aminopropyl-triethoxies
Silane（APS）Contain-NH2Functional group can be used for the covalent coupling of nanogold）, then will be with glutathione using glutaraldehyde
For ligand nanogold covalent coupling to surface of glass slide, obtain the liquid crystal sensitive membrane of decorated by nano-gold.
Figure 1B is to utilize the sensitive membrane combination liquid crystal bio-sensing detection of alkaline phosphatase（ALP）Principle：Alkaline phosphatase
The presence of enzyme can be catalyzed its substrate ascorbic acid 2- phosphate（AA-2P）The product ascorbic acid of hydrolysis, generation has strong reduction
Property, deposition of silver reaction can be induced to be attached to nanogold by the silver ion reduction detected in liquid at silver-colored simple substance and by nanogold and repaiied
The sensitive film surface of decorations generates birefringence signal to upset the ordered arrangement of 5CB liquid crystal molecules, can using petrographic microscope
To realize the highly sensitive Visual retrieval of alkaline phosphatase.
Specifically, the preparation method of the liquid crystal biosensor of the present embodiment, includes the following steps：
Step（1）, prepare glutathione be ligand nanogold
0.10mL glutathione (GSH, 0.24 mol/L) is added into the gold chloride of 1mL 1%, at room temperature stirring 2 minutes, then
There is a large amount of light-yellow precipitate in 2.5 ~ 3.0, solution in the pH that 1 mol/L NaOH are slowly added dropwise to solution into solution.
Then it utilizes centrifuge to be centrifuged 1 minute using 8000 revs/min of rotating speed, discards supernatant liquid and obtain Au (I)-GSH precipitations.So
Precipitation 5mmol/L sodium hydroxides are dissolved afterwards, 11 mL ultra-pure waters are added, then adjust solution ph to 7.0 or so.To
It is slowly added dropwise in reaction solution into 0.1 mg/mL sodium borohydrides, 100 μ L, until solution becomes brown color, on magnetic stirring apparatus
Using 600 revs/min of rotating speed, 4 h are reacted at room temperature, molecular cut off is used to be purified for the super filter tube of 30kD, are finally incited somebody to action
To solution be dispersed in 4 mL ultra-pure waters, 4 DEG C save backup.
Step（2）, slide cleaning
The coverslip that size is the mm of 18 mm × 18 is immersed in Piranha solution, Piranha solution is dense H2SO4And H2O2
It is 7 by volume:3 mixing, are placed in 80 DEG C of water-bath and stand 1.5 h.After reaction with a large amount of ultra-pure water and anhydrous
Ethyl alcohol rinses, and then uses N2Drying, is positioned over 110 DEG C of dry 3h in exsiccator, and it is spare to obtain clean slide.Piranha is molten
There is liquid strong oxidizing property, operation to need great care.
Step（3）, DMOAP modification upper slide and amido modified lower slide preparation
Carry out the modification of silanised glass slides：
The modification of upper slide：Clean slide is immersed in the DMOAP aqueous solutions containing 0.5%, stands reaction 20 under room temperature
Min, with the unreacted DMOAP of washes of absolute alcohol, then clean, the N with a large amount of ultrapure water2Drying, is positioned in baking oven,
110 DEG C of dry 1h obtain the upper slide substrate of DMOAP modifications, spare.
The modification of lower slide：Clean slide is immersed into the Acetic acid-sodium acetate buffer solution containing 1% DMOAP and % APS
（10mM, pH5.0）In, 80 DEG C of isothermal reaction 1.5h in water-bath clean unreacted DMOAP and APS with ethyl alcohol, then with greatly
Amount ultra-pure water cleans up, N2Drying is placed in 110 DEG C of baking ovens dry 1h, makes the upper amido functional group of surface of glass slide modification,
It is spare.
Step（4）, liquid crystal sensitive membrane preparation
The lower slide of above-mentioned amino functional is immersed in 1% glutaraldehyde water solution, react at room temperature 1h, cleaned with ultra-pure water dry
Only, N2Drying, obtains the slide substrate of aldehyde radical function；Then 1.41 × 10 are added dropwise in surface of glass slide-8 mol/L GSH-AuNPs
200 μ L react at room temperature 1 h, unreacted nanogold, N are washed away with ultra-pure water2Drying, obtains the slide substrate of decorated by nano-gold
It is spare as liquid crystal sensitive membrane.
Step（5）, liquid crystal biosensor preparation
The ALP of various concentration is added in detection liquid, detection liquid be containing 5mmol/L ascorbic acid 2- phosphates AA-2P and
The diethanol amine of the 0.1mol/L of 5mmol/L silver nitrates, pH 9.8-nitric acid buffer solution, is added drop-wise to rapidly after mixing
Liquid crystal sensitivity film surface is stated, 37 DEG C are protected from light 1.5 h, and ultrapure water, N are used after enzymatic metallization reaction2Drying,
It is spare.
2 μ L 5CB liquid crystal molecules are added dropwise in liquid crystal sensitive membrane after above-mentioned enzymatic metallization reaction, cover step
（3）DMOAP modifications upper slide to get to the liquid crystal biosensor for detection of alkaline phosphatase.
The method that the present embodiment uses above-mentioned liquid crystal biosensor detection of alkaline phosphatase, includes the following steps：
Step（1）, the alkaline phosphatase of various concentration is added in detection liquid, is added drop-wise to above-mentioned receive after mixing rapidly
The liquid crystal sensitivity film surface of rice gold modification, is protected from light；The detection liquid is to contain ascorbic acid 2- phosphates and nitre
Diethanol amine-nitric acid buffer solution of sour silver；
Step（2）, by step（1）Liquid crystal sensitive membrane ultrapure water, N2Drying, is then added dropwise 2 μ L5CB liquid crystal molecules,
The upper slide for covering DMOAP modifications, obtains liquid crystal biosensor；
Step（3）, then above-mentioned liquid crystal biosensor is placed on 25 DEG C ~ 27 DEG C of heated at constant temperature platform, it is aobvious using polarisation
Micro mirror is observed under cross-polarized light pattern, is taken pictures, and obtains the optic response image of various concentration alkaline phosphatase, utilizes this
The variation of the quantity and color of white hot spot carries out quantitative and semi-quantitative detection to alkaline phosphatase on optical imagery.
Embodiment 2
The preparation method of the liquid crystal biosensor of the present embodiment, includes the following steps：
Step（1）, prepare glutathione be ligand nanogold
0.10mL glutathione (GSH, 0.24 mol/L) is added into the gold chloride of 1mL 1%, at room temperature stirring 5 minutes, then
There is a large amount of light-yellow precipitate in 2.5 ~ 3.0, solution in the pH that 1 mol/L NaOH are slowly added dropwise to solution into solution.
Then it utilizes centrifuge to be centrifuged 1 minute using 6000 revs/min of rotating speed, discards supernatant liquid and obtain Au (I)-GSH precipitations.Then
Precipitation 5mmol/L sodium hydroxides are dissolved, 11 mL ultra-pure waters are added, then adjust solution ph to 7.0 or so.To anti-
It answers and is slowly added dropwise in liquid into 0.1 mg/mL sodium borohydrides, 100 μ L, until solution becomes brown color, adopted on magnetic stirring apparatus
With 600 revs/min of rotating speed, 4 h are reacted at room temperature, are used molecular cut off to be purified for the super filter tube of 30kD, will finally be obtained
Solution be dispersed in 4 mL ultra-pure waters, 4 DEG C save backup.
Step（2）, slide cleaning
The coverslip that size is the mm of 18 mm × 18 is immersed in Piranha solution, Piranha solution is dense H2SO4And H2O2
It is 7 by volume:3 mixing are placed in 1 ~ 2 h of standing in 80 DEG C of water-bath.After reaction with a large amount of ultra-pure water and anhydrous
Ethyl alcohol rinses, and then uses N2Drying, is positioned over 110 DEG C of dry 2h in exsiccator, and it is spare to obtain clean slide.Piranha is molten
There is liquid strong oxidizing property, operation to need great care.
Step（3）, DMOAP modification upper slide and amido modified lower slide preparation
Carry out the modification of silanised glass slides：
The modification of upper slide：Clean slide is immersed in the DMOAP aqueous solutions containing 1%, stands reaction 30 under room temperature
Min, with the unreacted DMOAP of washes of absolute alcohol, then clean, the N with a large amount of ultrapure water2Drying, is positioned in baking oven,
110 DEG C of 2 h of drying obtain the upper slide substrate of DMOAP modifications, spare.
The modification of lower slide：Clean slide is immersed into the Acetic acid-sodium acetate buffer solution containing 1% DMOAP and 4% APS
（10mM, pH5.0）In, 80 DEG C of isothermal reaction 1.5h in water-bath clean unreacted DMOAP and APS with ethyl alcohol, then with greatly
Amount ultra-pure water cleans up, N2Drying is placed in 110 DEG C of baking ovens dry 2 h, makes the upper amido functional group of surface of glass slide modification,
It is spare.
Step（4）, liquid crystal sensitive membrane preparation
The lower slide of above-mentioned amino functional is immersed in 0.5% glutaraldehyde water solution, reacts at room temperature 2 h, it is clear with ultra-pure water
Wash clean, N2Drying, obtains the slide substrate of aldehyde radical function；Then 1.41 × 10 are added dropwise in surface of glass slide-8 mol/L GSH-
200 μ L of AuNPs react at room temperature 1 h, unreacted nanogold, N are washed away with ultra-pure water2Drying, obtains the glass of decorated by nano-gold
Piece substrate is spare as liquid crystal sensitive membrane.
Step（5）, liquid crystal biosensor preparation
The ALP of various concentration is added in detection liquid, detection liquid be containing 2mmol/L ascorbic acid 2- phosphates AA-2P and
The diethanol amine of the 0.2mol/L of 2mmol/L silver nitrates, pH 9.5-nitric acid buffer solution, is added drop-wise to rapidly after mixing
Liquid crystal sensitivity film surface is stated, 37 DEG C are protected from light 0.5h, and ultrapure water, N are used after enzymatic metallization reaction2Drying, it is standby
With.
1 μ L 5CB liquid crystal molecules are added dropwise in liquid crystal sensitive membrane after above-mentioned enzymatic metallization reaction, cover step
（3）DMOAP modifications upper slide to get to the liquid crystal biosensor for detection of alkaline phosphatase.
The method that the present embodiment uses above-mentioned liquid crystal biosensor detection of alkaline phosphatase, includes the following steps：
Step（1）, the alkaline phosphatase of various concentration is added in detection liquid, is added drop-wise to above-mentioned receive after mixing rapidly
The liquid crystal sensitivity film surface of rice gold modification, is protected from light；The detection liquid is to contain ascorbic acid 2- phosphates and nitre
Diethanol amine-nitric acid buffer solution of sour silver；
Step（2）, by step（1）Liquid crystal sensitive membrane ultrapure water, N2Drying, is then added dropwise 1 μ L5CB liquid crystal molecules,
The upper slide for covering DMOAP modifications, obtains liquid crystal biosensor；
Step（3）, then above-mentioned liquid crystal biosensor is placed on 25 DEG C ~ 27 DEG C of heated at constant temperature platform, it is aobvious using polarisation
Micro mirror is observed under cross-polarized light pattern, is taken pictures, and obtains the optic response image of various concentration alkaline phosphatase, utilizes this
The variation of the quantity and color of white hot spot carries out quantitative and semi-quantitative detection to alkaline phosphatase on optical imagery.
Remaining is same as Example 1.
Embodiment 3
The preparation method of the liquid crystal biosensor of the present embodiment, includes the following steps：
Step（1）, prepare glutathione be ligand nanogold
0.10mL glutathione (GSH, 0.24 mol/L) is added into the gold chloride of 1mL 1%, at room temperature stirring 3 minutes, then
There is a large amount of light-yellow precipitate in 2.5 ~ 3.0, solution in the pH that 1 mol/L NaOH are slowly added dropwise to solution into solution.
Then it utilizes centrifuge to be centrifuged 1 minute using 7500 revs/min of rotating speed, discards supernatant liquid and obtain Au (I)-GSH precipitations.Then
Precipitation 5mmol/L sodium hydroxides are dissolved, 11 mL ultra-pure waters are added, then adjust solution ph to 7.0 or so.To anti-
It answers and is slowly added dropwise in liquid into 0.1 mg/mL sodium borohydrides, 100 μ L, until solution becomes brown color, adopted on magnetic stirring apparatus
With 600 revs/min of rotating speed, 4 h are reacted at room temperature, are used molecular cut off to be purified for the super filter tube of 30kD, will finally be obtained
Solution be dispersed in 4 mL ultra-pure waters, 4 DEG C save backup.
Step（2）, slide cleaning
The coverslip that size is the mm of 18 mm × 18 is immersed in Piranha solution, Piranha solution is dense H2SO4And H2O2
It is 7 by volume:3 mixing, are placed in 80 DEG C of water-bath and stand 1.5h.After reaction with a large amount of ultra-pure water and anhydrous second
Alcohol rinses, and then uses N2Drying, is positioned over 110 DEG C of dry 4h in exsiccator, and it is spare to obtain clean slide.Piranha solution
With strong oxidizing property, operation needs great care.
Step（3）, DMOAP modification upper slide and amido modified lower slide preparation
Carry out the modification of silanised glass slides：
The modification of upper slide：Clean slide is immersed in the DMOAP aqueous solutions containing 0.75%, stands reaction under room temperature
25min, with the unreacted DMOAP of washes of absolute alcohol, then clean, the N with a large amount of ultrapure water2Drying, is positioned over baking oven
In, 110 DEG C of dry 1h obtain the upper slide substrate of DMOAP modifications, spare.
The modification of lower slide：Clean slide is immersed into the Acetic acid-sodium acetate buffer solution containing 1% DMOAP and 6% APS
（10mM, pH5.0）In, 80 DEG C of isothermal reaction 1.5h in water-bath clean unreacted DMOAP and APS with ethyl alcohol, then with greatly
Amount ultra-pure water cleans up, N2Drying is placed in 110 DEG C of baking ovens dry 2 h, makes the upper amido functional group of surface of glass slide modification,
It is spare.
Step（4）, liquid crystal sensitive membrane preparation
The lower slide of above-mentioned amino functional is immersed in 0.5% glutaraldehyde water solution, reacts at room temperature 1.5h, it is clear with ultra-pure water
Wash clean, N2Drying, obtains the slide substrate of aldehyde radical function；Then 1.41 × 10 are added dropwise in surface of glass slide-8 mol/LGSH-
200 μ L of AuNPs react at room temperature 1 h, unreacted nanogold, N are washed away with ultra-pure water2Drying, obtains the glass of decorated by nano-gold
Piece substrate is spare as liquid crystal sensitive membrane.
Step（5）, liquid crystal biosensor preparation
The ALP of various concentration is added in detection liquid, detection liquid be containing 8mmol/L ascorbic acid 2- phosphates AA-2P and
The diethanol amine of the 0.2mol/L of 8mmol/L silver nitrates, pH10.0-nitric acid buffer solution, are added drop-wise to rapidly after mixing
Liquid crystal sensitivity film surface is stated, 37 DEG C are protected from light 1h, and ultrapure water, N are used after enzymatic metallization reaction2Drying, it is standby
With.
1.5 μ L 5CB liquid crystal molecules are added dropwise in liquid crystal sensitive membrane after above-mentioned enzymatic metallization reaction, cover step
Suddenly（3）DMOAP modifications upper slide to get to the liquid crystal biosensor for detection of alkaline phosphatase.
The method that the present embodiment uses above-mentioned liquid crystal biosensor detection of alkaline phosphatase, includes the following steps：
Step（1）, the alkaline phosphatase of various concentration is added in detection liquid, is added drop-wise to above-mentioned receive after mixing rapidly
The liquid crystal sensitivity film surface of rice gold modification, is protected from light；The detection liquid is to contain ascorbic acid 2- phosphates and nitre
Diethanol amine-nitric acid buffer solution of sour silver；
Step（2）, by step（1）Liquid crystal sensitive membrane ultrapure water, N2Then 1.5 μ L5CB liquid crystal point are added dropwise in drying
Son covers the upper slide of DMOAP modifications, obtains liquid crystal biosensor；
Step（3）, then above-mentioned liquid crystal biosensor is placed on 25 DEG C ~ 27 DEG C of heated at constant temperature platform, it is aobvious using polarisation
Micro mirror is observed under cross-polarized light pattern, is taken pictures, and obtains the optic response image of various concentration alkaline phosphatase, utilizes this
The variation of the quantity and color of white hot spot carries out quantitative and semi-quantitative detection to alkaline phosphatase on optical imagery.
Remaining is same as Example 1.
2, feasibility analysis
In order to verify the feasibility that the Liquid Crystal Sensor detects alkaline phosphatase, different detection drops are added to nanometer by us
It is protected from light in golden slide substrate and is incubated 1.5 h, rinsed well using ultra-pure water, 2 μ L 5CB liquid crystal molecules are added dropwise and are fabricated to liquid
Brilliant pond, using its optical signalling of polarized light microscope observing.When detect lack in liquid alkaline phosphatase, ascorbic acid phosphoric acid esters,
When silver nitrate any type substance, such as (A) 1 × 10-6 U/mL alkaline phosphatase+5mM silver nitrates, (B) 5mM ascorbic acid phosphoric acid
Ester+5mM silver nitrates, (C) 1 × 10-6 U/mL alkaline phosphatase+5mM ascorbic acid phosphoric acid esters, polarised light cannot by analyzer,
Optical imagery is the background image of grey black, as shown in Fig. 2 A-C, only contains alkaline phosphatase simultaneously, resists when detect in liquid
It just will produce stronger optical signalling when bad hematic acid phosphate, silver nitrate, optical imagery is the texture image of green, such as Fig. 2 E
It is shown.In addition final concentration of 10 μM of sodium vanadate is also further added to above-mentioned containing 1 × 10 by we-6 U/mL alkaline phosphatases
It is incubated in enzyme detection liquid, testing result is as shown in Figure 2 D, since the presence of sodium vanadate can inhibit the work of alkaline phosphatase
Property, we equally can only obtain the background image of grey black.It is possible thereby to prove that the optical signalling detected by us is derived from liquid
The catalytic action of brilliant sensitive membrane surface alkalinty phosphatase, the signal can be used for the detection of alkaline phosphatase.
3, the characterization of substrate of glass
In order to further prove that enzymatic gold has occurred in the liquid crystal biosensor surface to metallize based on enzymatic that we are established
Categoryization is reacted, we characterize the surface topography of slide substrate using atomic force microscope, scanning electron microscope.By scheming
The surface of glass slide that 3A can be seen that cleaning is more smooth, surface Root Mean Square roughness（RMS）For 0.29 nm.Using silanization
After reagent modification, the roughness of substrate surface increases, and as shown in Figure 3B, RMS value increases to 0.32 nm.It is repaiied in substrate surface
After adoring nanogold, the roughness of surface of glass slide significantly increases, and as shown in Figure 3 C, RMS value becomes 2.14 nm, it was demonstrated that nanogold quilt
Success modifies and has arrived surface of glass slide.After the surface of glass slide of decorated by nano-gold carries out enzymatic metallization reaction, metal nano
Grain is obviously grown up, and as shown in Figure 3D, the roughness on surface further increases, and RMS value becomes 3.73 nm.Proof passes through enzymatic
Metallization reaction can be such that the surface topography of the liquid crystal sensitive membrane of decorated by nano-gold substantially change.
Fig. 4 is the characterization result of scanning electron microscope.Its surface smoother of the slide of silylating reagent modification is flat,
As shown in Figure 4 A, by Fig. 4 B it is obvious that gold nano grain is evenly distributed in entire surface of glass slide, it was demonstrated that gold nano
Particle arrives basement membrane surface by successfully modification.With the generation of enzymatic metallization reaction, nanogold can induce silver nanoparticle
Grain is deposited on surface of glass slide and forms larger Nano silver grain, can see by Fig. 4 C, the ruler of the nano particle of surface of glass slide
It is very little obviously to become larger.It can prove that we are successfully prepared by the characterization result of atomic force microscope and scanning electron microscope to receive
The liquid crystal sensitive membrane of rice gold modification can make the table of sensitive membrane by enzymatic metallization reaction and nanogold induction deposition of silver reaction
Significant change occurs for face pattern.
4, the optimization of liquid crystal sensitive membrane modification condition
Oriented of the liquid crystal molecule in liquid crystal pond is mainly influenced by liquid crystal sensitivity membrane superficial tissue, therefore, design and system
The liquid crystal sensitive membrane of standby inducible liquid crystal molecule vertical arrangement is the key that structure liquid crystal biosensor.We use containing length
The silylating reagent DMOAP of alkyl chain and contain-NH2The silylating reagent APS of functional group carries out Hybrid assembling to slide substrate,
Wherein the DMOAP silylating reagents containing chain alkyl chain can induce liquid crystal molecule to be arranged vertically, and contain-NH2Work(
The silylating reagent APS that can be rolled into a ball can be used for the fixation of biomolecule or nano material, be used for liquid crystal biosensor.
We optimize the ratio of sensitive membrane surface A PS and DMOAP first, as shown in Figure 5A, with APS/DMOAP ratios
Increase, background signal gradually increases, and the white bright spot in liquid crystal optics image gradually increases.Such as Fig. 5 A a and Fig. 5 A b institutes
Show, the ratio of APS/DMOAP is 1:1 and 5:When 1, optical imagery is the background of grey black, when ratio reaches 10：When 1, liquid crystal
There is a small amount of white speck in optical imagery, as shown in Fig. 5 A c.The amount that surface of glass slide fixes APS is very little, can influence nanogold
Fixed amount, therefore it is 5 that we, which select the ratio of APS/DMOAP,:1.
The dosage of glutaraldehyde can also affect to background signal, and therefore, we carry out the dosage of glutaraldehyde
Optimization, as shown in Figure 5 B, when glutaraldehyde concentration is 1%, background signal is uniform grey black, with glutaraldehyde concentration
Increase, background signal gradually increases, therefore we select 1% glutaraldehyde to carry out covalent coupling to nanogold.
The fixed nanogold of liquid crystal sensitivity film surface helps to induce silver nano-grain deposition, improves the sensitivity of detection.
It is covalently fixed to sensitive film surface by we using GSH-AuNPs using glutaraldehyde, and the nanogold synthesized by us is averaged
Grain size is about 6.9 ± 1.4 nm, and colloidal stability is very good（Even if will not all be rolled into a ball in 1 mol/L NaCl solutions
It is poly-）, surface ligand is glutathione can directly carry out covalent coupling containing abundant amino and carboxyl.We are to fixation
Concentration to sensitive film surface GSH-AuNPs is optimized, as a result as shown in Figure 5 C, when nanogold a concentration of 1.41 ×
10-8When mol/L (Fig. 5 C a), background signal is uniform grey black, and the concentration of nanogold reaches 2.82 × 10-8 mol/L
After (Fig. 5 C b), occur a small amount of white bright spot on background optical image, and background signal with the increase of nanometer gold concentration and
Increase.It proves when the nanogold of modification to surface of glass slide is less, it is smaller to the upset effect of the ordered arrangement of liquid crystal molecule, when
When nanometer gold concentration reaches a certain level, the ordered arrangement of liquid crystal molecule can be upset, to generate background signal.Therefore, we
Selection 1.41 × 10-8The GSH-AuNPs of mol/L carries out covalent modification.
5, the optimization of testing conditions
In order to improve detection sensitivity, we are the concentration to substrate A A-2P, Ag+Concentration, buffer solution pH, reaction time etc.
The factor for influencing enzymatic metallization reaction optimizes.
The AA-2P of various concentration is added to containing 1 × 10 by we-6 U/mL ALP and 5 mM Ag+Detection liquid in, incubate
Liquid crystal pond is assembled after educating 1.5 h, with determination of polarized light microscopy optical signalling.Its testing result is as shown in Figure 6A, as AA-2P is dense
The increase optical signalling first increases and then decreases of degree, when AA-2P concentration reaches 5 mM, detection letter is maximum, as shown in Fig. 6 A b.
A concentration of 5 mmol/L of AA-2P, pH of buffer 9.8, enzymatic metallization reaction time are in fixed test liquid
A concentration of the 1 × 10 of 1.5 h, ALP-6 U/mL changes the concentration of silver nitrate in detection liquid.Testing result is as shown in Figure 6B, with
Ag+The increase of concentration, optical signalling increase, and work as Ag+Concentration when being more than 5 mmol/L, the optical signalling detected gradual drop again
It is low.This is because Ag+When concentration is higher, the activity of alkaline phosphatase can on the one hand reduced, on the other hand also result in silver-colored single
Matter is individually nucleated in detecting liquid, and the silver for only depositing to sensitive film surface can just generate the ordered arrangement of liquid crystal molecule and disturb
Disorderly, and then optical signalling is generated.Therefore we select the Ag of 5 mmol/L+As Ag in detection liquid+Concentration.
The pH for detecting buffer solution in liquid can generate very big influence to the activity of alkaline phosphatase, to influence to detect
Signal.Therefore AA-2P and Ag in our fixed test liquid+A concentration of 5 mmol/L, a concentration of the 1 × 10 of ALP-6 U/mL changes
Become the pH of buffer solution, liquid crystal pond polarized light microscope observing testing result is assembled after reacting 1.5 h.Testing result such as Fig. 6 C institutes
Show, when it is 9.8 to detect liquid pH, detection signal reaches maximum, as shown in Fig. 6 C c.
In addition we have also investigated influence of the enzymatic metallization reaction time to detection signal, we utilize 1 × 10-6 U/
ML ALP contain 5 mm Ag in pH9.8+It is reacted the different time in 5 mm AA-2P detection liquid, obtained testing result
As shown in Figure 6 D, with the increase in reaction time, optical signalling gradually increases, we select 1.5 h to metallize as enzymatic
Reaction time, as shown in Fig. 6 D c.
6, the detection of alkaline phosphatase
The alkaline phosphatase of various concentration is detected under optimum reaction condition using the liquid crystal biosensor, experiment knot
Fruit as shown in fig. 7, with alkaline phosphatase enzyme activity increase, the white hot spot appeared in petrographic microscope grey black background is gradual
Increase, when alkaline phosphatase activity is more than 5 × 10-6U/mL （Fig. 7 E）Afterwards, hot spot start to be linked to be piece start to show color it is rich
Rich texture image, color gradually become orange by white（Fig. 7 F）, purple（Fig. 7 G）, it is red（Fig. 7 H）, blue（Fig. 7 I）
Eventually become green（Fig. 7 J）, the main reason is that the presence due to alkaline phosphatase can induce silver-colored simple substance in decorated by nano-gold
Surface of glass slide deposition, so that sensitive environmental microbes is changed, generated double to upset the ordered arrangement of 5CB liquid crystal molecules
Refraction effect.With the increase of ALP concentration, the nano silver size for depositing to surface of glass slide becomes larger, and upsets more 5CB liquid crystal
The ordered arrangement of molecule.It can pass through inspection after generation birefringent phenomenon when mixed white light is irradiated to by petrographic microscope on liquid crystal pond
Polariscope detects speck, becomes larger with the nano silver size of sensitive membrane Surface Creation, liquid crystal molecule that polarised light passes through upset
Optical length also gradually increase, different face is presented because the interference of different levels occurs after polarised light is emitted from liquid crystal pond
Color.As shown in fig. 7, we can utilize the variation pair 5 of the quantity and color of white hot spot on polarized light microscopy optical imagery ×
10-9ALP within the scope of the U/mL of U/mL ~ 0.5 carries out quantitative and semi-quantitative detection, and obtained testing result is bright in luster, face
Color is abundant, is very suitable for Visual retrieval, and the detection of this method is limited down to 5 × 10-9U/mL, the visualization with other ALP are examined
Survey method is compared, and the detection sensitivity of this method improves the 5-7 order of magnitude.
7, the effect of sensitive membrane nano surface gold
In order to inquire into modification to sensitive membrane nano surface gold signal amplification, we also using only modified DMOAP with
The slide of APS is detected the ALP of various concentration as substrate, using identical inspection policies.Testing result such as Fig. 8 institutes
Show, when being detected using the substrate of unmodified nanogold its detection be limited to 5 × 10-6 U/mL（It is detected substantially not less than the concentration
To optical signalling, such as Fig. 8 B, C, D）.Fig. 7 and Fig. 8 are compared, although the slide substrate of unmodified nanogold can also be made
Liquid crystal biosensor substrate to be metallized based on enzymatic obtains detection signal, but its detection sensitivity ratio has modified nanometer
At least low 3 orders of magnitude of sensitive film surface of gold, and cannot get Fig. 7 interference colours coloury like that, it is unfavorable for visual
Change detection.The main reason is that the nanogold of surface of glass slide acts not only as the reduction reaction that catalyst accelerates silver ion, and
And being also used as crystal seed makes the silver of reduction all deposit to surface of glass slide, and the silver being reduced is avoided individually to be nucleated in the solution.
Therefore liquid crystal Visual retrieval signal can not only be amplified by being detected using the liquid crystal sensitive membrane of decorated by nano-gold, but also be also had
More large-sized silver nano-grain is formed conducive in sensitive film surface, to form coloury interference colours, being conducive to can
It is detected depending on changing.
8, specificity is investigated
In order to investigate the specificity of this method, under the same conditions, the interference that concentration is significantly larger than alkaline phosphatase is respectively adopted
Substance, such as 50 μ g/mL horseradish peroxidases, 1.5 U/mL pepsins, 1.5 U/mL carbonic anhydrases, 50 μ g/mL paddy Guangs
As a contrast, experimental result is as shown in figure 9,1 × 10 for sweet peptide, 1.5 U/mL trypsase-6The optical imagery of U/mL ALP（Figure
9F）On there is large stretch of hot spot, and the testing result of other interfering substances（Fig. 9 A-E）It is mainly grey black, only generates pole
The background signal of a small number of white bright spots.Therefore the liquid crystal biosensor has good selection for the detection of alkaline phosphatase
Property.
9, the detection of human serum sample's alkaline phosphatase
10 μ L fresh serum samples are taken, using diethanol amine buffer solution（0.1 mol/L pH 9.8）Dilute different multiples
（1000 times, 10000 times, 50000 times）, the ALP in human serum sample is detected according to the detecting step of ALP, simultaneously will
A certain amount of ALP, which is added in the blood serum sample, measures its recovery of standard addition.
As shown in Figure 10, with the reduction of extension rate, optical signalling gradually increases, when human serum sample dilutes 1000 times
For the texture image of yellow（Figure 10 E）, sample alkaline phosphatase when can obtain 1000 times of dilution is compared with Fig. 8
Content is about 5 × 10-5 U/mL.By the blood serum sample after high-temperature process, the alkaline phosphatase inactivation in serum, in same batten
The background image that can only obtain grey black is detected under part（Figure 10 E）, it was demonstrated that the complex material in serum will not interfere ALP's
It measures.We also carry out mark-on experiment to diluted blood serum sample simultaneously, we are by 1 × 10-6The ALP of U/mL is added to above-mentioned
In diluted blood serum sample, the detection signal after mark-on obviously becomes larger, and testing result shows large stretch of hot spot, these are detected
As a result semi-quantitative analysis can be carried out by being compared with Fig. 8, and testing result can be coincide very well with mark-on amount, it was demonstrated that this method energy
It is enough in the detection of ALP in human serum.
Pass through above every detection, it was demonstrated that the method for the present invention can be applied to the detection of alkaline phosphatase, and method is simple, clever
Sensitivity is high, and Monitoring lower-cut is down to 5 × 10-9U/mL, it is wide to the detection range of ALP, pass through hot spot on the optical imagery that detects
Quantity and the color of liquid crystal texture image are realized to 5 × 10-9ALP in U/mL-0.5 U/mL concentration ranges realizes sxemiquantitative
Detection, and this method does not need complicated instrument, and highly sensitive, the visualization inspection of complex sample alkaline phosphatase may be implemented
It surveys, there is good application prospect in biomedicine is examined.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all essences in the present invention
With within principle, any modification, equivalent replacement, improvement and so on should all be included in the protection scope of the present invention god.
Claims (8)
1. a kind of liquid crystal biosensor of detection of alkaline phosphatase, it is characterised in that：Including liquid crystal sensitive membrane, it is added dropwise described
The upper slide of 5CB liquid crystal molecules and DMOAP modifications in liquid crystal sensitive membrane, the liquid crystal sensitive membrane are to first pass through silanization to repair
Decorations recycle glutaraldehyde that decorated by nano-gold is prepared to surface of glass slide, utilize the Yin Na for depositing to liquid crystal sensitivity film surface
Rice grain, which generates 5-CB liquid crystal molecules, to be upset to realize the detection to alkaline phosphatase.
2. a kind of preparation method of liquid crystal biosensor, it is characterised in that：Include the following steps：
Step（1）, prepare glutathione be ligand nanogold；
Step（2）, slide cleaning；
Step（3）, DMOAP modification upper slide and amido modified lower slide preparation；
Step（4）, liquid crystal sensitive membrane preparation
By step（3）Lower slide immerse in the glutaraldehyde water solution of a concentration of 0.5-1%, react at room temperature 1-2h, it is clear with ultra-pure water
Wash clean, N2Drying, obtains the slide substrate of aldehyde radical function；
Then 200 μ L steps are added dropwise in the slide substrate surface of the aldehyde radical function（1）Nanogold, room temperature reaction 1 h or so,
Unreacted nanogold, N are washed away with ultra-pure water2Drying, obtains liquid crystal sensitive membrane；
Step（5）, liquid crystal biosensor preparation
The alkaline phosphatase of various concentration to be detected is added in detection liquid, it is quick to be added drop-wise to above-mentioned liquid crystal after mixing rapidly
Feel film surface, be protected from light, ultrapure water, N are used after enzymatic metallization reaction2Drying, it is spare；
1 ~ 2 μ L 5CB liquid crystal molecules are added dropwise in liquid crystal sensitive membrane after above-mentioned enzymatic metallization reaction, cover step
（3）DMOAP modifications upper slide to get to the liquid crystal biosensor for detection of alkaline phosphatase.
3. preparation method according to claim 1, it is characterised in that：Step（1）In, a concentration of the 1.41 × 10 of nanogold-8 mol/L。
4. preparation method according to claim 1, it is characterised in that：Step（2）In, it is molten that slide is immersed in Piranha
In liquid, it is placed in 1 ~ 2h of standing in 80 DEG C of water-bath, is rinsed with a large amount of ultra-pure water and absolute ethyl alcohol, is then used after reaction
N2Drying, is positioned over 110 DEG C of dry 2 ~ 4h in exsiccator, is positioned over drying in exsiccator, obtains clean slide.
5. preparation method according to claim 1, it is characterised in that：Step（3）In, the system of the upper slide of DMOAP modifications
Preparation Method is：
Clean slide is immersed in the DMOAP aqueous solutions containing 0.5-1%, reaction 20-30min is stood under room temperature, with nothing
Water-ethanol cleans unreacted DMOAP, then clean, the N with a large amount of ultrapure water2Drying is positioned over drying in baking oven, obtains
The upper slide substrate of DMOAP modifications；
The preparation method of amido modified lower slide is：
Clean slide is immersed in the Acetic acid-sodium acetate buffer solution containing 1%DMOAP and 4-6%APS, 80 DEG C in water-bath
Isothermal reaction 1.5h cleans unreacted DMOAP and APS with ethyl alcohol, then is cleaned up with a large amount of ultra-pure waters, N2Drying is placed
It dries in an oven, makes the upper chain alkyl of surface of glass slide modification and amido functional group.
6. preparation method according to claim 1, it is characterised in that：Step（5）In, detection liquid is to contain 2-8mmol/L
The diethanol amine that 0.1-0.2mol/L, pH of ascorbic acid 2- phosphates and 2-8mmol/L silver nitrates are 9.5-10-nitric acid buffering
Solution；1-2 μ L5CB liquid crystal molecules are added dropwise in liquid crystal sensitivity film surface.
7. preparation method according to claim 1, it is characterised in that：Step（1）Prepare the nanometer that glutathione is ligand
Gold is specific as follows：
0.10mL glutathione (GSH, 0.24 mol/L) is added into the gold chloride of 1mL 1%, at room temperature stirring 2 ~ 5 minutes, so
1 mol/L NaOH to the pH of solution are slowly added dropwise in backward solution and occur largely light yellow sink in 2.5 ~ 3.0, solution
It forms sediment, then utilizes centrifuge to be centrifuged 1 minute using 6000 ~ 8000 revs/min of rotating speed, discard supernatant liquid and obtain Au (I)-GSH
Precipitation, then precipitation 5mmol/L sodium hydroxides are dissolved, 11 mL ultra-pure waters are added, and then adjust solution ph to 7.0
Left and right, is slowly added dropwise into reaction solution into 0.1 mg/mL sodium borohydrides, 100 μ L, until solution becomes brown color, is stirred in magnetic force
The rotating speed using 600 revs/min on device is mixed, reacts 4 h at room temperature, uses molecular cut off to be purified for the super filter tube of 30kD, most
Obtained solution is dispersed in 4 mL ultra-pure waters afterwards, 4 DEG C save backup.
8. the application of the liquid crystal biosensor of the detection of alkaline phosphatase of one of claim 1-7, it is characterised in that：Including such as
Lower step：
Step（1）, the alkaline phosphatase of various concentration is added in detection liquid, is added drop-wise to claim after mixing rapidly
The liquid crystal sensitivity film surface of the decorated by nano-gold of one of 1-6, is protected from light；The detection liquid is to contain ascorbic acid 2-
The diethanol amine of phosphate and silver nitrate-nitric acid buffer solution；
Step（2）, by step（1）Liquid crystal sensitive membrane ultrapure water, N2Then 1-2 μ L5CB liquid crystal point is added dropwise in drying
Son covers the upper slide of DMOAP modifications, obtains liquid crystal biosensor；
Step（3）, then above-mentioned liquid crystal biosensor is placed on 25 DEG C ~ 27 DEG C of heated at constant temperature platform, it is aobvious using polarisation
Micro mirror is observed under cross-polarized light pattern, is taken pictures, and obtains the optic response image of various concentration alkaline phosphatase, utilizes this
The variation of the quantity and color of white hot spot carries out quantitative and semi-quantitative detection to alkaline phosphatase on optical imagery.
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| CN111982818A (en) * | 2020-07-30 | 2020-11-24 | 山东省分析测试中心 | Device for detecting liquid crystal droplet optical digital signal and application thereof |
| CN111982902A (en) * | 2020-08-11 | 2020-11-24 | 南方科技大学 | Liquid crystal-based detection probe and preparation method and application thereof |
| CN112033938A (en) * | 2020-09-11 | 2020-12-04 | 华南师范大学 | Visual sensor based on liquid crystal molecule self-assembly structure and construction method and application thereof |
| CN114414481A (en) * | 2022-01-07 | 2022-04-29 | 大连理工大学 | Liquid crystal sensor for visually detecting organophosphorus pesticide and detection method thereof |
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