CN107918010A - A kind of method of highly sensitive liquid crystal type Non-labeled Immunosensor detection Human beta-defensin 2 - Google Patents
A kind of method of highly sensitive liquid crystal type Non-labeled Immunosensor detection Human beta-defensin 2 Download PDFInfo
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Abstract
The present invention provides a kind of highly sensitive liquid crystal type Non-labeled Immunosensor detection HBD 2（Human beta-defensin 2）Method, first 2 antibody of HBD is fixed in the substrate surface of self-assembled film modification, reacted by direct immunization by 2 antibody bindings of HBD to sensor base surface, so that substrate surface pattern changes, cause the change of the oriented of liquid crystal molecule, cause optical image signal to change, realize the detection to HBD 2.Liquid crystal biology sensor is introduced into the research of HBD 2 by the present invention, realizes simple and quick, detects HBD 2 high sensitivity.
Description
Technical field
The present invention relates to a kind of detection method of mankind's beta-defensin -2, and in particular to a kind of highly sensitive liquid crystal type non-marked
The method of immunosensor detection mankind beta-defensin -2.
Background technology
Since bacterial antibiotic drug resistance continuously emerges, research and development new antibiotic is extremely urgent.Alexin
(defensins) it is that the one kind being stored in extensively in animal and plant body is free of the alkaline kation polypeptide of sugar chain, there is broad-spectrum antiseptic
Activity, is generally made of (29~45) a amino acid residue, is the important component of body defenses barrier.Human defense's element is
A kind of anti-microbial cationic peptide large family, molecular weight are 4 ~ 5 k Da, and intramolecular contains half Guang that 6 disulfide bond are connected
Histidine residue, be it is a kind of there is special space structure, there is extensive bactericidal activity, cytotoxicity and immune chemotaxis
Cationic polypeptide, is the important component of body natural protection system, and is had with the development of some diseases associated with inflammation and tumour
Close relationship.Mankind's beta-defensin have broad spectrum antimicrobial activity, its sphere of action include gram-negative, positive bacteria,
Mycobacteria, fungi and conveyor screw etc., human defense's element can also stimulating immune system participate in immune response, improve exempting from for body
Epidemic disease level is a kind of antibacterial peptide having wide application prospects to remove pathogenic microorganisms, it is expected to is resisting micro- life such as bacterium, virus
Play a significant role in thing infection.And the research of these problems needs quick, sensitive, special, easy mankind's beta-defensin detection
Method.
The content of the invention
In order to overcome the above problem of the prior art, the object of the present invention is to provide a kind of highly sensitive liquid crystal type non-marked to exempt from
The method that epidemic disease sensor detects HBD-2.The present invention is in detection mankind's beta-defensin -2（HBD-2）Introduced during content
Highly sensitive liquid crystal type Non-labeled Immunosensor, realizes simple and quick, the purpose of efficient detection HBD-2.
In order to achieve the above object, the technical solution adopted by the present invention is：
A kind of method of highly sensitive liquid crystal type Non-labeled Immunosensor detection HBD-2, the first base in self-assembled film modification
Basal surface fixes HBD-2 antibody, is reacted by direct immunization by HBD-2 antibody bindings to sensor base surface, so that
Substrate surface pattern changes, and causes the change of the oriented of liquid crystal molecule, causes optical image signal to change, real
Now to the detection of HBD-2.The present invention comprises the following steps：
1）Slide is cut into the cm of 2 cm × 2, with the Piranha solution ［ V (H newly prepared2O2）: V( H2SO4)=
3 :7] 1 h is soaked in 80 DEG C, is then cleaned three times with ultra-pure water, then 10 min are soaked in absolute ethyl alcohol, through N2
Drying, it is dust-proof spare in 110 DEG C of dry 3 h.
2）The assembling of slide
Upper slide DMOAP (N, N- dimethyl-N-octadecyl base (3- [trimethoxy silane] propyl group) ammonium chloride) solution group
Dress：The slide after toasting will be cleaned to soak in the DMOAP aqueous solutions of 0.2 % (mass fraction), stand 30 at normal temperatures
Min, N clean with ultrapure water2 Drying, it is dust-proof spare in 110 DEG C of dry 1 h.
Lower slide is assembled with APTES (3- aminopropyl triethoxysilanes) and DMOAP mixed solutions：Cleaning is toasted
Slide afterwards immerses（3 % - 5 % ）The ethanol solution of (mass fraction) APTES and 1 % (mass fraction) DMOAP
In, in 80 DEG C of 2 h of constant temperature, first washed twice with absolute ethyl alcohol, then, N clean with ultrapure water2Drying, in 110 DEG C
Dry 1 h, it is dust-proof spare.
3）The fixation of HBD-2 antibody
It is with mass fraction by the lower slide that amination is handled（1 % - 3 %）Glutaraldehyde solution 1 h is soaked under room temperature,
Ultra-pure water is cleaned, and the HBD-2 antibody-solutions of (80-120 nmoL/L) is added on the lower slide through aldehyde radicalization modification, with washing
Ear ball is blown open, is incubated 2 h in 37 DEG C, is rinsed with 10 mM PBS buffer (pH=7.4), ultra-pure water is cleaned, N2 Drying
It is spare.HBD-2 antibody can be fixed on slide by the coupled action of glutaraldehyde.
4）HBD-2 antigen and antibody specifics react
Certain density HBD-2 solution is added in and is fixed with the slide of HBD-2 antibody, ear washing bulb is blown open, in 37 DEG C
1 h is incubated, immune response can be achieved in antigen-antibody.After 10 mM PBS (pH=7.4) wash buffers, then with ultrapure
Water rinses, N2Dry up spare.
5）The making in liquid crystal pond
Liquid crystal pond is assembled face-to-face by upper slide and lower slide, is separated between slide with Mylar polyester pieces, Mylar gathers
Convex cavity is opened among ester piece, in addition to opening direction, other three sides edge is fixed with Small clamp.First liquid crystal 5CB is heated to
40 DEG C or so make it be in isotropic liquid, then inject it from Mylar polyester pieces tapping, liquid crystal is due to capillary
Pipe active force and be covered with whole cavity.Naturally cool to 28 DEG C or so and use polarized light microscope observing.
The step 1）Middle acid treatment slide and with 10 min of soaked in absolute ethyl alcohol, fixes substrate surface enough
Hydroxyl.
The step 2）In upper slide 0.2 % (mass fraction) DMOAP aqueous solution soakings.
The step 2）In upper slide soaking time be 30 min.
The step 2）In lower slide use（3 % - 5 %）(mass fraction) APTES and 1 % (mass fraction)
Handled in the ethanol solution of DMOAP, make substrate surface amination.
The step 2）In lower slide soak 2 h in 80 DEG C.
The step 3）It is middle by amination processing lower slide be with mass fraction（1 % - 3 %）Glutaraldehyde it is water-soluble
Liquid soaks under room temperature, makes substrate surface aldehyde radical.
The step 3）The middle lower slide by amination processing is immersed in glutaraldehyde solution, in 37 in constant-temperature table
DEG C reaction 1 h.
The step 3）The middle HBD-2 antibody-solutions by (80-120 nmoL/L) are added in through under aldehyde radicalization modification
On slide, blown open with ear washing bulb, incubate 2 h in 37 DEG C, substrate surface is fixed HBD-2 antibody.
The buffer solution is the PBS buffer of 10 mM, pH=7.4.
The step 4）Middle be added in certain density HBD-2 solution is fixed with the slide of HBD-2 antibody, is washed
Ear ball is blown open, and 1 h is incubated in 37 DEG C, and immune response can be achieved in antigen-antibody.
The step 5）It is middle that upper slide and lower slide are assembled into liquid crystal cell face-to-face, gathered between slide with Mylar
Ester piece separates.
The step 5）It is middle liquid crystal 5CB is first heated to 40 DEG C or so to make it be in isotropic liquid, then
The Mylar polyester pieces tapping that it is opened convex cavity from centre injects, and liquid crystal is covered with entirely due to capillary force
Cavity.
In the step 5) by the liquid crystal pond made naturally cool to 28 DEG C or so with polarized light microscope observings its
Optical phenomena.
Preferably, the method for the highly sensitive liquid crystal type Non-labeled Immunosensor detection HBD-2 of the present invention includes following several
A step：
In order to achieve the above object, the technical solution adopted by the present invention comprises the following steps：
1）Slide is cut into the cm of 2 cm × 2, with the Piranha solution ［ V (H newly prepared2O2 : V( H2SO4) =
3 :7] 1 h is soaked in 80 DEG C, is then cleaned three times with ultra-pure water, then 10 min are soaked in absolute ethyl alcohol, through N2 Blow
It is dry, it is dust-proof spare in 110 DEG C of dry 3 h.
2）The assembling of slide
Upper slide is assembled with DMOAP solution：The DMOAP water of slide 0.2 % of leaching (mass fraction) after toasting will be cleaned
In solution, 30 min, N clean with ultrapure water are stood at normal temperatures2 Drying, it is dust-proof standby in 110 DEG C of dry 1 h
With.
Lower slide is assembled with APTES and DMOAP mixed solutions：The slide after toasting will be cleaned to immerse（3 % - 5
%）In the ethanol solution of (mass fraction) APTES and 1 % (mass fraction) DMOAP, in 80 DEG C of 2 h of constant temperature, first with nothing
Water-ethanol is washed twice, then clean with ultrapure water, N2 Drying, it is dust-proof spare in 110 DEG C of dry 1 h.
3）The fixation of HBD-2 antibody
It is with mass fraction by the lower slide that amination is handled（1 % -3 %）Glutaraldehyde solution 1 h is soaked under room temperature, surpass
Pure water is cleaned, and the HBD-2 antibody-solutions of (80-120 nmoL/L) is added on the lower slide through aldehyde radicalization modification, with washing ear
Ball is blown open, is incubated 2 h in 37 DEG C, is rinsed with 10 mM PBS (pH=7.4) liquid, ultra-pure water is cleaned, N2 Dry up spare.HBD-
2 antibody can be fixed on slide by the coupled action of glutaraldehyde.
4）HBD-2 antigen and antibody specifics react
Certain density HBD-2 solution is added in and is fixed with the slide of HBD-2 antibody, ear washing bulb is blown open, in 37 DEG C
1 h is incubated, immune response can be achieved in antigen-antibody.After wash buffer, then with ultrapure water, N2 Dry up spare.
5）The making in liquid crystal pond
Liquid crystal pond is assembled face-to-face by upper slide and lower slide, is separated between slide with Mylar polyester pieces, Mylar gathers
Convex cavity is opened among ester piece, in addition to opening direction, other three sides edge is fixed with Small clamp.First liquid crystal 5CB is heated to
40 DEG C or so make it be in isotropic liquid, then inject it from Mylar polyester pieces tapping, liquid crystal is due to capillary
Pipe active force and be covered with whole cavity.Naturally cool to 28 DEG C or so and use polarized light microscope observing.
Preferably, the step 2）In upper slide 0.2 % (mass fraction) DMOAP aqueous solution soakings 30
min。
Preferably, the step 2）In lower slide use（3 % - 5 % ）(mass fraction) APTES and 1 % (matter
Measure fraction) DMOAP ethanol solution in 80 DEG C soak 2 h, make substrate surface amination.
Preferably, the step 3）The mass fraction of middle glutaraldehyde is 1 % -3 %.
Preferably, the step 3）The middle lower slide by amination processing is immersed in glutaraldehyde, in constant-temperature table
1 h is reacted in 37 DEG C.
Preferably, the step 3）In the concentration of HBD-2 antibody on slide be fixed on by glutaraldehyde cross-linking effect be
(80 - 120 nmoL/L)。
Compared with prior art, the beneficial effects of the present invention are：
The present invention introduces highly sensitive liquid crystal type Non-labeled Immunosensor during HBD-2 contents are detected, and realizes
It is simple and quick, the purpose of efficient detection HBD-2.
Brief description of the drawings
Fig. 1 be different volumes than APTES/DMOAP substrates self-assembled film prepare liquid crystal pond optical imagery；
Fig. 2 is that influence of the GA contents to liquid crystal pond optical imagery contrasts photo；
Fig. 3 is the liquid crystal pond optical imagery of the fixation HBD-2 Antibody preparations of various concentrations；
Fig. 4 is liquid crystal pond optical imagery prepared by the HBD-2 to be measured of various concentrations.
Embodiment
With reference to embodiment, the present invention is further elaborated, but the present invention is not limited to following embodiments.
Embodiment one
1) slide is cut into the cm of 2 cm × 2, with the Piranha solution ［ V (H newly prepared2O2 ) : V( H2SO4)
= 3 ;7] 1 h is soaked in 80 DEG C, is then cleaned three times with ultra-pure water, then 10 min are soaked in absolute ethyl alcohol, passed through
N2Drying, it is dust-proof spare in 110 DEG C of dry 3 h.
2）The assembling of slide
Upper slide is assembled with DMOAP solution：The DMOAP water of slide 0.2 % of leaching (mass fraction) after toasting will be cleaned
In solution, 30 min, N clean with ultrapure water are stood at normal temperatures2Drying, it is dust-proof standby in 110 DEG C of dry 1 h
With.
Lower slide is assembled with APTES and DMOAP mixed solutions：The slide after toasting will be cleaned and immerse 3 % (quality
Fraction) APTES and 1 % (mass fraction) DMOAP ethanol solution in, in 80 DEG C of 2 h of constant temperature, first with anhydrous second
Alcohol is washed twice, then clean with ultrapure water, N2 Drying, it is dust-proof spare in 110 DEG C of dry 1 h.
3）The fixation of HBD-2 antibody
The lower slide of amination processing is soaked into 1 h, ultrapure washing with the glutaraldehyde solution that volume fraction is 1 % under room temperature
Only, the HBD-2 antibody-solutions of 80 nmoL/L are added on the lower slide through aldehyde radicalization modification, are blown open with ear washing bulb, in 37
DEG C incubate 2 h, with 10 mM PBS (pH=7.4) wash buffers, ultra-pure water is cleaned, N2Dry up spare.HBD-2 antibody
It can be fixed on by the coupled action of glutaraldehyde on slide.
4）HBD-2 antigen and antibody specifics react
Certain density HBD-2 solution is added in and is fixed with the slide of HBD-2 antibody, ear washing bulb is blown open, in 37 DEG C
1 h is incubated, immune response can be achieved in antigen-antibody.After 10 mM PBS (pH=7.4) wash buffers, then use ultra-pure water
Rinse, N2 Dry up spare.
5）The making in liquid crystal pond
Liquid crystal pond is assembled face-to-face by upper slide and lower slide, is separated between slide with Mylar polyester pieces, Mylar gathers
Convex cavity is opened among ester piece, in addition to opening direction, other three sides edge is fixed with Small clamp.First liquid crystal 5CB is heated to
40 DEG C or so make it be in isotropic liquid, then inject it from Mylar polyester pieces tapping, liquid crystal is due to capillary
Pipe active force and be covered with whole cavity.Naturally cool to 28 DEG C or so and use polarized light microscope observing.
Embodiment two
1）Slide is cut into the cm of 2 cm × 2, with the Piranha solution ［ V (H newly prepared2O2 ) : V( H2SO4)]
= 3 :7] 1 h is soaked in 80 DEG C, is then cleaned three times with ultra-pure water, then 10 min are soaked in absolute ethyl alcohol, passed through
N2Drying, it is dust-proof spare in 110 DEG C of dry 3 h.
2）The assembling of slide
Upper slide is assembled with DMOAP solution：The DMOAP water of slide 0.2 % of leaching (mass fraction) after toasting will be cleaned
In solution, 30 min, N clean with ultrapure water are stood at normal temperatures2 Drying, it is dust-proof standby in 110 DEG C of dry 1 h
With.
Lower slide is assembled with APTES and DMOAP mixed solutions：The slide after toasting will be cleaned and immerse 3.5 % (matter
Measure fraction) in the ethanol solution of APTES and 1 % (mass fraction) DMOAP, in 80 DEG C of 2 h of constant temperature, first with anhydrous
Ethanol is washed twice, then clean with ultrapure water, N2 Drying, it is dust-proof spare in 110 DEG C of dry 1 h.
3）The fixation of HBD-2
The lower slide of amination processing is soaked into 1 h, ultra-pure water with the glutaraldehyde solution that volume fraction is 1.5 % under room temperature
Clean, the HBD-2 antibody-solutions of 90 nmoL/L are added on the lower slide through aldehyde radicalization modification, are blown open with ear washing bulb, in
37 DEG C of 2 h of incubation, with 10 mM PBS (pH=7.4) wash buffers, ultra-pure water is cleaned, N2 Dry up spare.HBD-2 antibody
It can be fixed on by the coupled action of glutaraldehyde on slide.
4）HBD-2 antigen and antibody specifics react
Certain density HBD-2 solution is added in and is fixed with the slide of HBD-2 antibody, ear washing bulb is blown open, in 37 DEG C
1 h is incubated, immune response can be achieved in antigen-antibody.After 10 mM PBS (pH=7.4) wash buffers, then with ultrapure
Water rinses, N2 Dry up spare.
5）The making in liquid crystal pond
Liquid crystal pond is assembled face-to-face by upper slide and lower slide, is separated between slide with Mylar polyester pieces, Mylar gathers
Convex cavity is opened among ester piece, in addition to opening direction, other three sides edge is fixed with Small clamp.First liquid crystal 5CB is heated to
40 DEG C or so make it be in isotropic liquid, then inject it from Mylar polyester pieces tapping, liquid crystal is due to capillary
Pipe active force and be covered with whole cavity.Naturally cool to 28 DEG C or so and use polarized light microscope observing.
Embodiment three
1）Slide is cut into the cm of 2 cm × 2, with the Piranha solution ［ V (H newly prepared2O2 ) : V( H2SO4)
= 3 :7] 1 h is soaked in 80 DEG C, is then cleaned three times with ultra-pure water, then 10 min are soaked in absolute ethyl alcohol, passed through
N2 Drying, it is dust-proof spare in 110 DEG C of dry 3 h.
2）The assembling of slide
Upper slide is assembled with DMOAP solution：The DMOAP water of slide 0.2 % of leaching (mass fraction) after toasting will be cleaned
In solution, 30 min, N clean with ultrapure water are stood at normal temperatures2 Drying, it is dust-proof standby in 110 DEG C of dry 1 h
With.
Lower slide is assembled with APTES and DMOAP mixed solutions：The slide after toasting will be cleaned and immerse 4 % (quality
Fraction) APTES and 1 % (mass fraction) DMOAP ethanol solution in, in 80 DEG C of 2 h of constant temperature, first with anhydrous second
Alcohol is washed twice, then clean with ultrapure water, N2 Drying, it is dust-proof spare in 110 DEG C of dry 1 h.
3）The fixation of HBD-2 antibody
The lower slide of amination processing is soaked into 1 h, ultrapure washing with the glutaraldehyde solution that volume fraction is 2 % under room temperature
Only, the HBD-2 antibody-solutions of 100 nmoL/L are added on the lower slide through aldehyde radicalization modification, are blown open with ear washing bulb, in
37 DEG C of 2 h of incubation, with 10 mM PBS (pH=7.4) wash buffers, ultra-pure water is cleaned, N2 Dry up spare.HBD-2 antibody
It can be fixed on by the coupled action of glutaraldehyde on slide.
4）HBD-2 antigen and antibody specifics react
Certain density HBD-2 solution is added in and is fixed with the slide of HBD-2 antibody, ear washing bulb is blown open, in 37 DEG C
1 h is incubated, immune response can be achieved in antigen-antibody.After 10 mM PBS (pH=7.4) wash buffers, then use ultra-pure water
Rinse, N2 Dry up spare.
5）The making in liquid crystal pond
Liquid crystal pond is assembled face-to-face by upper slide and lower slide, is separated between slide with Mylar polyester pieces, Mylar gathers
Convex cavity is opened among ester piece, in addition to opening direction, other three sides edge is fixed with Small clamp.First liquid crystal 5CB is heated to
40 DEG C or so make it be in isotropic liquid, then inject it from Mylar polyester pieces tapping, liquid crystal is due to capillary
Pipe active force and be covered with whole cavity.Naturally cool to 28 DEG C or so and use polarized light microscope observing.
Example IV
1）Slide is cut into the cm of 2 cm × 2, with the Piranha solution ［ V (H newly prepared2O2 ) ; V( H2SO4)
= 3 :7] 1 h is soaked in 80 DEG C, is then cleaned three times with ultra-pure water, then 10 min are soaked in absolute ethyl alcohol, passed through
N2 Drying, it is dust-proof spare in 110 DEG C of dry 3 h.
2）The assembling of slide
Upper slide is assembled with DMOAP solution：The DMOAP water of slide 0.2 % of leaching (mass fraction) after toasting will be cleaned
In solution, 30 min, N clean with ultrapure water are stood at normal temperatures2 Drying, it is dust-proof standby in 110 DEG C of dry 1 h
With.
Lower slide is assembled with APTES and DMOAP mixed solutions：The slide after toasting will be cleaned and immerse 4.5 % (matter
Measure fraction) in the ethanol solution of APTES and 1 % (mass fraction) DMOAP, in 80 DEG C of 2 h of constant temperature, first with anhydrous
Ethanol is washed twice, then clean with ultrapure water, N2 Drying, it is dust-proof spare in 110 DEG C of dry 1 h.
3）The fixation of HBD-2 antibody
The lower slide of amination processing is soaked into 1 h, ultra-pure water with the glutaraldehyde solution that volume fraction is 2.5 % under room temperature
Clean, the HBD-2 antibody-solutions of 110 nmoL/L are added on the lower slide through aldehyde radicalization modification, are blown open with ear washing bulb, in
37 DEG C of 2 h of incubation, with 10 mM PBS (pH=7.4) wash buffers, ultra-pure water is cleaned, N2 Dry up spare.HBD-2 is
It can be fixed on by the coupled action of glutaraldehyde on slide.
4）HBD-2 antigen and antibody specifics react
Certain density HBD-2 solution is added in and is fixed with the slide of HBD-2 antibody, ear washing bulb is blown open, in 37 DEG C of temperature
1 h is educated, immune response can be achieved in antigen-antibody.After 10 mM PBS (pH=7.4) wash buffers, then use ultra-pure water
Rinse, N2 Dry up spare.
5）The making in liquid crystal pond
Liquid crystal pond is assembled face-to-face by upper slide and lower slide, is separated between slide with Mylar polyester pieces, Mylar gathers
Convex cavity is opened among ester piece, in addition to opening direction, other three sides edge is fixed with Small clamp.First liquid crystal 5CB is heated to
40 DEG C or so make it be in isotropic liquid, then inject it from Mylar polyester pieces tapping, liquid crystal is due to capillary
Pipe active force and be covered with whole cavity.Naturally cool to 28 DEG C or so and use polarized light microscope observing.
Embodiment five
1）Slide is cut into the cm of 2 cm × 2, with the Piranha solution ［ V (H newly prepared2O2 ) : V( H2SO4)
= 3:7] 1 h is soaked in 80 DEG C, is then cleaned three times with ultra-pure water, then 10 min are soaked in absolute ethyl alcohol, through N2
Drying, it is dust-proof spare in 110 DEG C of dry 3 h.
2）The assembling of slide
Upper slide is assembled with DMOAP solution：The DMOAP water of slide 0.2 % of leaching (volume fraction) after toasting will be cleaned
In solution, 30 min, N clean with ultrapure water are stood at normal temperatures2 Drying, it is dust-proof standby in 110 DEG C of dry 1 h
With.
Lower slide is assembled with APTES and DMOAP mixed solutions：The slide after toasting will be cleaned and immerse 5 % (quality
Fraction) APTES and 1 % (mass fraction) DMOAP ethanol solution in, in 80 DEG C of 2 h of constant temperature, first with anhydrous second
Alcohol is washed twice, then clean with ultrapure water, N2 Drying, it is dust-proof spare in 110 DEG C of dry 1 h.
3）The fixation of HBD-2 antibody
The lower slide of amination processing is soaked into 1 h, ultrapure washing with the glutaraldehyde solution that mass fraction is 3 % under room temperature
Only, the HBD-2 antibody-solutions of 120 nmoL/L are added on the lower slide through aldehyde radicalization modification, are blown open with ear washing bulb, in 37
DEG C incubate 2 h, with 10 mM PBS (pH=7.4) wash buffers, ultra-pure water is cleaned, N2 Dry up spare.HBD-2
It is fixed on by the coupled action of glutaraldehyde on slide.
4）HBD-2 antigen and antibody specifics react
Certain density HBD-2 solution is added in and is fixed with the slide of HBD-2 antibody, ear washing bulb is blown open, in 37 DEG C
1 h is incubated, immune response can be achieved in antigen-antibody.After 10 mM PBS (pH=7.4) wash buffers, then with ultrapure
Water rinses, and N2 dryings are spare.
5）The making in liquid crystal pond
Liquid crystal pond is assembled face-to-face by upper slide and lower slide, is separated between slide with Mylar polyester pieces, Mylar
Convex cavity is opened among polyester piece, in addition to opening direction, other three sides edge is fixed with Small clamp.First liquid crystal 5CB is heated
Make it be in isotropic liquid to 40 DEG C or so, then inject it from Mylar polyester pieces tapping, liquid crystal is due to hair
Capillary action power and be covered with whole cavity.Naturally cool to 28 DEG C or so and use polarized light microscope observing.
Embodiment six
1）The selection of APTES/DMOAP volume ratios
By step 2 in embodiment five）Described method, respectively by lower slide APTES and DMOAP mixed solution groups
Dress, is made liquid crystal pond, the liquid crystal pond optical imagery under polarized light microscopy Microscopic observation, the results are shown in Figure 1 by the slide after assembling.
In figure, APTES/DMOAP volume ratios are respectively (a) 25:1；(b) 10:1；(c) 5:1； (d) 3:1；(f) 1:1.
It will be seen from figure 1 that when APTES/DMOAP ratios are higher (25:1, figure a), due to DMOAP contents compared with
It is few, it is impossible to which that effectively induction liquid crystal molecule is arranged vertically, and speck is big and more in optical imagery, as APTES/DMOAP ratios subtract
Small, speck gradually decreases in optical imagery, when both are than equal to or less than 5:When 1, optical imagery is homogeneous black pattern.For
It is fixed with substrate not only to can induce liquid crystal vertical arrangement and enough amino fixing biological molecules can be provided, our selections
APTES/DMOAP ratios are 5:1 - 3:Between 1, this example is to 5:1 elaborates.
2）The selection of glutaraldehyde concentration
With APTES/DMOAP volume ratios 5:1 preparation APTES and DMOAP mixed solution groups hold slide, by embodiment
Step 3 in five）Described method, is placed in the glutaraldehyde that volume fraction is 3% ~ 5% by lower slide respectively（GA）In fixer, fill
After the modification modification of point aldehyde radicalization, liquid crystal pond is made in the slide of aldehyde radical, in polarized light microscopy Microscopic observation liquid crystal pond optical imagery,
The results are shown in Figure 2.In figure, (a) APTES/DMOAP is 5:1, GA content is 5% (v/v)；(b) APTES/DMOAP is
5:1, GA content is 4% (v/v)；(c) APTES/DMOAP is 5:1, GA content is 3% (v/v).
GA contents determine substrate surface aldehyde radical density, Fig. 2 the results show that when GA contents be 5% when, APTES/DMOAP
Ratio is 5:Occurs equally distributed larger colored speck in 1 optical imagery.It is bright in image when GA contents are reduced to 4%
Spot is reduced；When GA contents are reduced to 3 %, completely black background is presented in pattern, does not disturb biomolecule detection, therefore select herein
APTES/DMOAP ratios are selected as 5:1, GA content is 3% (v/v).
3）The selection of immobilization HBD-2 antibody concentrations
APTES/DMOAP volume ratios are prepared as 5 using five the method for embodiment:1, GA content is the lower slide of 3% (v/v),
According to five step 3 of embodiment）Described in method, the HBD-2 antibody-solutions of 500,200,120,80 nmoL/L are added in respectively
On lower slide through aldehyde radicalization modification.HBD-2 antibody is fixed on after being fixed on by the coupled action of glutaraldehyde on slide,
Liquid crystal pond is made into, in polarized light microscopy Microscopic observation liquid crystal pond optical imagery, the results are shown in Figure 3.In figure, HBD-2 antibody
Solution is respectively (a) 500 nmoL/L；(b)200 nmoL/L；(c)120 nmoL/L；(d)80 nmoL/L.
With the reduction of immobilization HBD-2 antibody concentrations, liquid crystal molecular orientation is upset and is reduced, liquid crystal pond optical imagery
Gradually dimmed, the results show that when immobilization HBD-2 antibody concentrations are 120,80 nmoL/ L, optical imagery presents full Fig. 3
Black (c, d in figure), due to needing enough antibody and antigen-reactive, is chosen so that optical imagery background keeps dark
120 nmoL/L of highest immobilization HBD-2 antibody concentrations is fixed on substrate surface.
4）HBD-2 antigen and antibody specific reaction monitoring effects
According to step 4 in embodiment five）The HBD-2 solution of various concentrations, is added dropwise is being fixed with respectively by the method for record
On the slide of HBD-2 antibody, after immune response is complete, liquid crystal pond is made into, in polarized light microscopy Microscopic observation liquid crystal pond optics
Imaging, the results are shown in Figure 4.
Fig. 4 is shown, with the increase of HBD-2 concentration, liquid crystal pond optical imagery gradually brightens, when concentration reaches 10
During nmoL/L, occur speck in image, show detectable as low as mankind's beta-defensin -2 of 10 nmoL/L.
The above results show that the present invention using direct immunization method detection mankind beta-defensin -2 can as low as 10 nmoL/L, from
And realize the purpose of simple and quick high sensitivity detection mankind beta-defensin -2.
Claims (10)
- A kind of 1. method of highly sensitive liquid crystal type Non-labeled Immunosensor detection HBD-2, it is characterised in that including following step Suddenly：1）The pretreatment of slideSlide is cut to required size, with the Piranha solution newly prepared in 80 DEG C of fully immersions, then uses ultra-pure water Cleaning, then fully soaked in absolute ethyl alcohol, through N2Drying, it is fully dry, it is dust-proof spare；2）The assembling of slide2-1）Upper slide is assembled with DMOAP solution：By step 1）The slide of pretreatment is dipped in DMOAP aqueous solutions, in room temperature Lower fully immersion, with ultrapure water, N2Drying, it is fully dry, it is dust-proof spare；2-2）Lower slide is assembled with APTES and DMOAP mixed solutions：By step 1）The slide of pretreatment immerses APTES: DMOAP mass ratioes are 3-5:In 1 ethanol solution, in 80 DEG C of fully immersions, first with washes of absolute alcohol, then with surpassing Pure water cleans, N2Drying, it is fully dry, it is dust-proof spare；3）The fixation of HBD-2 antibodyBy step 2-2）The lower slide of amination processing is fully soaked with glutaraldehyde solution under room temperature, is cleaned, obtained with ultra-pure water To the lower slide modified through aldehyde radicalization；Certain density HBD-2 antibody-solutions are added on the lower slide through aldehyde radicalization modification, Blow open, incubate 2 h in 37 DEG C, cleaned with wash buffer, then with ultra-pure water, N2Dry up spare, HBD-2 antibody is It can be fixed on by the coupled action of glutaraldehyde on slide；4）HBD-2 antigen and antibody specifics reactCertain density HBD-2 solution is added dropwise in step 3）It is fixed with the slide of HBD-2 antibody, blows open, in 37 DEG C 1 h is incubated, antigen-antibody can react；With wash buffer, then with ultrapure water, N2 Dry up spare；5）The making in liquid crystal pondLiquid crystal pond is by step 2-1）Obtained upper slide and step 4）Obtained lower slide assembles, and is equipped between slide Mylar polyester pieces, Mylar polyester piece while be equipped with opening, the three side edges that upper and lower slide is not open by being arranged on Clip is fixed；Liquid crystal 5CB first is heated to 40 DEG C or so makes it be in isotropic liquid, then gathers it from Mylar The opening injection of ester piece, liquid crystal are covered with whole cavity due to capillary force；28 DEG C or so are naturally cooled to, with inclined Light microscope is observed.
- 2. the method for highly sensitive liquid crystal type Non-labeled Immunosensor detection HBD-2 according to claim 1 a kind of, its It is characterized in that：The step 1）10 min of soaked in absolute ethyl alcohol is used after middle acid treatment slide, substrate surface is fixed enough Hydroxyl.
- 3. the method for highly sensitive liquid crystal type Non-labeled Immunosensor detection HBD-2 according to claim 1 a kind of, its It is characterized in that：The step 2-1）In upper slide mass fraction be 0.2 % 30 min of DMOAP aqueous solution soakings.
- 4. the method for highly sensitive liquid crystal type Non-labeled Immunosensor detection HBD-2 according to claim 1 a kind of, its It is characterized in that：The step 2-2）In lower slide with APTES mass fractions be that 3%-5% and DMOAP mass fractions are Handled in 1% ethanol solution, make substrate surface amination.
- 5. the method for highly sensitive liquid crystal type Non-labeled Immunosensor detection HBD-2 according to claim 1 a kind of, its It is characterized in that：The step 2-2）In lower slide soak 2 h in 80 DEG C.
- 6. the method for highly sensitive liquid crystal type Non-labeled Immunosensor detection HBD-2 according to claim 1 a kind of, its It is characterized in that：The step 3）It is middle by amination processing lower slide with the glutaraldehyde solution that mass fraction is 1%-3% in Soaked under room temperature, make substrate surface aldehyde radical.
- 7. the method for highly sensitive liquid crystal type Non-labeled Immunosensor detection HBD-2 according to claim 1 a kind of, its It is characterized in that：The step 3）The middle lower slide by amination processing is immersed in glutaraldehyde, and 1 is reacted in constant-temperature table h。
- 8. the method for highly sensitive liquid crystal type Non-labeled Immunosensor detection HBD-2 according to claim 1 a kind of, its It is characterized in that：The step 3）It is middle that the HBD-2 antibody-solutions of 80-120 nmol/L are added in through under aldehyde radicalization modification On slide, blown open with ear washing bulb, 2 h are incubated in 37 DEG C, substrate surface is fixed HBD-2 by the coupled action of glutaraldehyde Antibody.
- 9. the method for highly sensitive liquid crystal type Non-labeled Immunosensor detection HBD-2 according to claim 1 a kind of, its It is characterized in that：The step 4）It is middle certain density HBD-2 solution to be added in the slide for being fixed with HBD-2 antibody On, ear washing bulb is blown open, and 1 h is incubated in 37 DEG C, and immune response can be achieved in antigen-antibody.
- 10. the method for highly sensitive liquid crystal type Non-labeled Immunosensor detection HBD-2 according to claim 1 a kind of, It is characterized in that：The step 5）It is middle liquid crystal 5CB is first heated to 40 DEG C or so to make it be in isotropic liquid, Then the Mylar polyester pieces tapping for it being opened to convex cavity from centre injects, and liquid crystal is covered with due to capillary force Whole cavity.
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