CN109212224A - The method of liquid crystal immunosensor detection HBD-2 based on nano-gold signal amplification - Google Patents

The method of liquid crystal immunosensor detection HBD-2 based on nano-gold signal amplification Download PDF

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CN109212224A
CN109212224A CN201810975899.7A CN201810975899A CN109212224A CN 109212224 A CN109212224 A CN 109212224A CN 201810975899 A CN201810975899 A CN 201810975899A CN 109212224 A CN109212224 A CN 109212224A
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hbd
liquid crystal
slide
lower slide
solution
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苏秀霞
霍文静
徐佳
张婧
张蓉
贺生卓
张海宁
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Shaanxi University of Science and Technology
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    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
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    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
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Abstract

The invention discloses a kind of methods that the liquid crystal immunosensor based on nano-gold signal amplification detects people HBD-2, comprising the following steps: impregnates lower slide with glutaraldehyde solution, the lower slide modified through aldehyde radical is obtained after flushing；AuNPs is added in HBD-2 antibody, then stood, be centrifuged and dispersed, the HBD-2 antibody complex of AuNPs modification is obtained, the modification HBD-2 antibody complex of AuNPs is added drop-wise on the lower slide modified through aldehyde radical, is incubated after blowing open, then dried up after rinsing；Obtained HBD-2-BSA solution is added drop-wise on lower slide, incubates after blowing open, is dried up after flushing；6) upper slide is placed on lower slide, wherein, the Mylar polyester piece with cavity is provided between upper slide and lower slide, make it in isotropic liquid liquid crystal 5CB heating simultaneously, then liquid crystal 5CB is injected into the cavity, then cooled down, obtain liquid crystal pond, HBD-2, the detection mankind beta-defensin -2 that this method can be quick, sensitive, easy are detected further according to the optical image signal in liquid crystal pond.

Description

The method of liquid crystal immunosensor detection HBD-2 based on nano-gold signal amplification
Technical field
The invention belongs to the detection method of mankind's beta-defensin -2, it is related to a kind of liquid crystal based on nano-gold signal amplification and exempts from The method of epidemic disease sensor detection people HBD-2.
Background technique
Since bacterial antibiotic drug resistance continuously emerges, research and development new antibiotic is extremely urgent.Human defense's element is A kind of anti-microbial cationic peptide large family, molecular weight are 4~5k Da, and intramolecular contains the connected cysteine of 6 disulfide bond Residue is a kind of with special space structure, the sun with extensive bactericidal activity, cytotoxicity and immune chemotaxis from Sub- polypeptide is the important component of body natural protection system, and is had closely with the development of some diseases associated with inflammation and tumour Relationship.Mankind's beta-defensin has the activity of broad spectrum antimicrobial, and sphere of action includes gram-negative, positive bacteria, branch bar Bacterium, fungi and conveyor screw etc., human defense's element can also stimulate immune system to participate in immune response, improve the immune level of body It is a kind of antibacterial peptide having wide application prospects, it is expected to resist the microorganism infections such as bacterium, virus to remove pathogenic microorganisms In play a significant role, and the research of these problems needs quick, sensitive, easy detection side mankind's beta-defensin -2 (HBD-2) Method.
Summary of the invention
It is an object of the invention to overcome the above-mentioned prior art, provide it is a kind of based on nano-gold signal amplification The method that liquid crystal immunosensor detects HBD-2, the detection mankind beta-defensin -2 that this method can be quick, sensitive, easy.
In order to achieve the above objectives, the liquid crystal immunosensor of the present invention based on nano-gold signal amplification detects HBD- 2 method the following steps are included:
1) slide is cut into upper slide and lower slide, then upper slide and lower slide is placed in Piranha solution simultaneously It is impregnated at 80 DEG C -100 DEG C, is impregnated after then cleaning with dehydrated alcohol, finally dried up；
2) upper slide is soaked in DMOAP aqueous solution, is dried up after flushing, while lower slide being soaked at 80-100 DEG C In the ethanol solution of APTES and DMOAP, then cleans and dry up；
3) lower slide is impregnated at normal temperature with glutaraldehyde solution, the lower slide modified through aldehyde radical is obtained after flushing； AuNPs is added in HBD-2 antibody, then stood, be centrifuged and dispersed, the HBD-2 antibody complex of AuNPs modification is obtained, The modification HBD-2 antibody complex of AuNPs is added drop-wise on the lower slide modified through aldehyde radical, is placed in 35-40 DEG C after blowing open Lower incubation 2-4h, then dried up after rinsing；
4) HBD-2 solution to be detected is added in the aqueous solution of EDC and NHS, is added drop-wise to after activation in BSA solution, then Concussion reaction obtains HBD-2-BSA solution；
5) the HBD-2-BSA solution that step 4) obtains is added drop-wise on the lower slide obtained through step 3), is placed after blowing open 1-2h is incubated at 35-40 DEG C, is dried up after flushing；
6) the upper slide obtained through step 2) is placed on the lower slide obtained through step 5), wherein upper slide is under It is provided with Mylar polyester piece between slide, cavity is provided in Mylar polyester piece, the side of Mylar polyester piece is provided with the sky The opening of chamber, while making it in isotropic liquid liquid crystal 5CB heating, then liquid crystal 5CB is injected into from the opening In the cavity, and liquid crystal 5CB is made to be covered with entire cavity, be cooled to room temperature, obtain liquid crystal pond, last liquid crystal pond is placed into partially It is detected under light microscope, and HBD-2 is detected according to the optical image signal in liquid crystal pond.
Upper slide and lower slide are placed in Piranha solution in step 1) and impregnated at 80-100 DEG C, then It is cleaned with ultrapure water, is then placed into dehydrated alcohol and impregnates 10-30min, so that upper slide and lower surface of glass slide are fixed with hydroxyl Base finally uses N2Drying；
In step 2) N will be used after upper slide ultrapure water2Drying；Lower slide is successively used into dehydrated alcohol and ultrapure water Cleaning, then uses N2Drying；
Upper slide is soaked in 30-50min in DMOAP aqueous solution in step 2)；
Lower slide is placed in step 2) in the ethanol solution of 80-100 DEG C of APTES and DMOAP and impregnates 2-4h；
In step 3) lower slide is impregnated at normal temperature with glutaraldehyde solution, then is obtained with after ultrapure water through aldehyde radical Change the lower slide of modification；
It is placed in after being blown open in step 3) at 35-40 DEG C and incubates 2-4h, then successively with being used after buffer and ultrapure water N2Drying；
The time of concussion reaction is 2-4h in step 4)；
Successively with using N after buffer and ultrapure water in step 5)2Drying.
The volume fraction of DMOAP aqueous solution is 0.2-0.3% in step 2).
The ethanol solution of the volume fraction 3-5%, APTES and DMOAP of APTES in the ethanol solution of APTES and DMOAP The volume fraction of middle DMOAP is 1-3%.
In step 3), the volume fraction of glutaraldehyde solution is 2-5%, and lower slide is carried out with glutaraldehyde solution at normal temperature It impregnates, and is placed in constant-temperature table and reacts 1-2h, so that lower surface of glass slide aldehyde radical；
The AuNPs of 1mL is added in the HBD-2 antibody of 15-20 μ L in step 3), wherein the concentration of HBD-2 antibody is 0.5mg/mL。
The concentration of the aqueous solution of EDC and NHS is 50-60ng/mL.
Liquid crystal 5CB, which is heated to 40-50 DEG C, makes it in isotropic liquid.
The invention has the following advantages:
The method of liquid crystal immunosensor detection HBD-2 of the present invention based on nano-gold signal amplification is specific When operation, by being added dropwise HBD-2-BSA solution on the lower slide for being fixed with AuNPs modification HBD-2 antibody, temperature after blowing open It educates, so that antigen-antibody reacts, to modify HBD-2 antibody-HBD-2-BSA in lower surface of glass slide assembling AuNPs Interlayer structure to improve the sensitivity of detection, and upsets the vertical orientation of liquid crystal molecule using the interlayer structure, so as to cause light The color and brightness for learning signal change, and to realize the detection to HBD-2, detection speed is fast, and detection sensitivity is high, and grasps Make relatively simple.
Detailed description of the invention
Fig. 1 is the photo figure that embodiment one obtains；
Fig. 2 is the photo figure that embodiment two obtains；
Fig. 3 is the photo figure that embodiment three obtains；
Fig. 4 is the photo figure that example IV obtains；
Fig. 5 is the photo figure that embodiment five obtains；
Fig. 6 is the photo figure that embodiment six obtains；
Fig. 7 is the photo figure that embodiment seven obtains；
Fig. 8 is the photo figure that embodiment eight obtains；
Fig. 9 is the photo figure that embodiment nine obtains；
Figure 10 is the photo figure that embodiment ten obtains.
Specific embodiment
The invention will be described in further detail with reference to the accompanying drawing:
Embodiment one
It is of the present invention based on nano-gold signal amplification liquid crystal immunosensor detection HBD-2 method include with Lower step:
1) slide is cut into upper slide and lower slide, then upper slide and lower slide is placed in Piranha solution simultaneously It is impregnated at 80 DEG C, is then placed into dehydrated alcohol and impregnates 10min, so that upper slide and lower surface of glass slide are fixed with Hydroxyl finally uses N2Drying；
2) upper slide is soaked in 30min in the aqueous solution of DMOAP, then uses N after being cleaned with ultrapure water2Drying；Simultaneously will Lower slide is soaked in 2h in the ethanol solution of APTES and DMOAP at 80 DEG C, then successively clear with dehydrated alcohol and ultrapure water It washes, finally uses N2Drying, wherein the volume fraction of DMOAP aqueous solution is in the ethanol solution of 0.2%, APTES and DMOAP The volume fraction of DMOAP is 1% in the ethanol solution of the volume fraction 3% of APTES, APTES and DMOAP；
3) lower slide is impregnated with glutaraldehyde solution at normal temperature, and is placed in constant-temperature table and reacts 1h, so that Lower surface of glass slide aldehyde radical, then with after ultrapure water the lower slide that be modified through aldehyde radical；The AuNPs of 1mL is added to 15 μ It in the HBD-2 antibody of L 0.5mg/mL, then stood, be centrifuged and dispersed, obtain the HBD-2 antibody complex of AuNPs modification, it will The modification HBD-2 antibody complex of the AuNPs of 75 ng/mL is added drop-wise on the lower slide modified through aldehyde radical, is placed in after blowing open 2h is incubated at 35 DEG C, then successively with using N after buffer and ultrapure water2Drying, wherein the volume fraction of glutaraldehyde solution is 2%；
4) the upper slide obtained through step 2) is placed on the lower slide obtained through step 3), wherein upper slide is under It is provided with Mylar polyester piece between slide, cavity is provided in Mylar polyester piece, the side of Mylar polyester piece is provided with the sky The opening of chamber, while liquid crystal 5CB, which is heated to 40 DEG C, makes it in isotropic liquid, then by liquid crystal 5CB from the opening It is injected into the cavity, and liquid crystal 5CB is made to be covered with entire cavity, be cooled to room temperature, obtain liquid crystal pond, finally by liquid crystal pond It is placed under petrographic microscope and is detected, and HBD-2 is detected according to the optical image signal in liquid crystal pond.
Embodiment two
It is of the present invention based on nano-gold signal amplification liquid crystal immunosensor detection HBD-2 method include with Lower step:
1) slide is cut into upper slide and lower slide, then upper slide and lower slide is placed in Piranha solution simultaneously It is impregnated at 100 DEG C, is then placed into dehydrated alcohol and impregnates 10-30min, so that upper slide and lower surface of glass slide are fixed There is hydroxyl, finally uses N2Drying；
2) upper slide is soaked in 50min in the aqueous solution of DMOAP, then uses N after being cleaned with ultrapure water2Drying；Simultaneously will Lower slide is soaked in 4h in the ethanol solution of APTES and DMOAP at 100 DEG C, then successively clear with dehydrated alcohol and ultrapure water It washes, finally uses N2Drying, wherein the volume fraction of DMOAP aqueous solution is in the ethanol solution of 0.3%, APTES and DMOAP The volume fraction of DMOAP is 3% in the ethanol solution of the volume fraction 5% of APTES, APTES and DMOAP；
3) lower slide is impregnated with glutaraldehyde solution at normal temperature, and is placed in constant-temperature table and reacts 2h, so that Lower surface of glass slide aldehyde radical, then with after ultrapure water the lower slide that be modified through aldehyde radical；The AuNPs of 1mL is added to 20 μ It in the HBD-2 antibody of L 0.5mg/mL, then stood, be centrifuged and dispersed, obtain the HBD-2 antibody complex of AuNPs modification, it will The modification HBD-2 antibody complex of the AuNPs of 100 ng/mL is added drop-wise on the lower slide modified through aldehyde radical, is placed after blowing open 4h is incubated at 40 DEG C, then successively with using N after buffer and ultrapure water2Drying, wherein the volume of glutaraldehyde solution point Number is 5%；
4) the upper slide obtained through step 2) is placed on the lower slide obtained through step 3), wherein upper slide is under It is provided with Mylar polyester piece between slide, cavity is provided in Mylar polyester piece, the side of Mylar polyester piece is provided with the sky The opening of chamber, while liquid crystal 5CB, which is heated to 50 DEG C, makes it in isotropic liquid, then by liquid crystal 5CB from the opening It is injected into the cavity, and liquid crystal 5CB is made to be covered with entire cavity, be cooled to room temperature, obtain liquid crystal pond, finally by liquid crystal pond It is placed under petrographic microscope and is detected, and HBD-2 is detected according to the optical image signal in liquid crystal pond.
Embodiment three
It is of the present invention based on nano-gold signal amplification liquid crystal immunosensor detection HBD-2 method include with Lower step:
1) slide is cut into upper slide and lower slide, then upper slide and lower slide is placed in Piranha solution simultaneously It is impregnated at 90 DEG C, is then placed into dehydrated alcohol and impregnates 20 min, so that upper slide and lower surface of glass slide are fixed with Hydroxyl finally uses N2Drying；
2) upper slide is soaked in 40min in the aqueous solution of DMOAP, then uses N after being cleaned with ultrapure water2Drying；Simultaneously will Lower slide is soaked in 3h in the ethanol solution of APTES and DMOAP at 90 DEG C, then successively clear with dehydrated alcohol and ultrapure water It washes, finally uses N2Drying, wherein the volume fraction of DMOAP aqueous solution is in the ethanol solution of 0.25%, APTES and DMOAP The volume fraction of DMOAP is 2% in the ethanol solution of the volume fraction 4% of APTES, APTES and DMOAP；
3) lower slide is impregnated with glutaraldehyde solution at normal temperature, and is placed in constant-temperature table and reacts 1.5h, made Must descend surface of glass slide aldehyde radical, then with after ultrapure water the lower slide that be modified through aldehyde radical；The AuNPs of 1mL is added to It in the HBD-2 antibody of 18 μ L 0.5mg/mL, then stood, be centrifuged and dispersed, the HBD-2 antibody for obtaining AuNPs modification is compound The modification HBD-2 antibody complex of the AuNPs of 150 ng/mL is added drop-wise on the lower slide modified through aldehyde radical, after blowing open by object It is placed at 38 DEG C and incubates 3h, then successively with using N after buffer and ultrapure water2Drying, wherein the volume of glutaraldehyde solution Score is 3.5%；
4) the upper slide obtained through step 2) is placed on the lower slide obtained through step 3), wherein upper slide is under It is provided with Mylar polyester piece between slide, cavity is provided in Mylar polyester piece, the side of Mylar polyester piece is provided with the sky The opening of chamber, while liquid crystal 5CB, which is heated to 45 DEG C, makes it in isotropic liquid, then by liquid crystal 5CB from the opening It is injected into the cavity, and liquid crystal 5CB is made to be covered with entire cavity, be cooled to room temperature, obtain liquid crystal pond, finally by liquid crystal pond It is placed under petrographic microscope and is detected, and HBD-2 is detected according to the optical image signal in liquid crystal pond.
Example IV
It is of the present invention based on nano-gold signal amplification liquid crystal immunosensor detection HBD-2 method include with Lower step:
1) slide is cut into upper slide and lower slide, then upper slide and lower slide is placed in Piranha solution simultaneously It is impregnated at 85 DEG C, is then placed into dehydrated alcohol and impregnates 15 min, so that upper slide and lower surface of glass slide are fixed with Hydroxyl finally uses N2Drying；
2) upper slide is soaked in 30-50min in the aqueous solution of DMOAP, then uses N after being cleaned with ultrapure water2Drying；Simultaneously Lower slide is soaked in 2.5h in the ethanol solution of APTES and DMOAP at 80-100 DEG C, then successively use dehydrated alcohol and is surpassed Pure water cleaning, finally uses N2Drying, wherein the volume fraction of DMOAP aqueous solution is that the ethyl alcohol of 0.23%, APTES and DMOAP is molten The volume fraction of DMOAP is 1.5% in the ethanol solution of the volume fraction 3.5% of APTES in liquid, APTES and DMOAP；
3) lower slide is impregnated with glutaraldehyde solution at normal temperature, and is placed in constant-temperature table and reacts 1.2h, made Must descend surface of glass slide aldehyde radical, then with after ultrapure water the lower slide that be modified through aldehyde radical；The AuNPs of 1mL is added to It in the HBD-2 antibody of 16 μ L 0.5mg/mL, then stood, be centrifuged and dispersed, the HBD-2 antibody for obtaining AuNPs modification is compound The modification HBD-2 antibody complex of the AuNPs of 200 ng/mL is added drop-wise on the lower slide modified through aldehyde radical, after blowing open by object It is placed at 36 DEG C and incubates 2.5h, then successively with using N after buffer and ultrapure water2Drying, wherein the body of glutaraldehyde solution Fraction is 3%；
4) the upper slide obtained through step 2) is placed on the lower slide obtained through step 5), wherein upper slide is under It is provided with Mylar polyester piece between slide, cavity is provided in Mylar polyester piece, the side of Mylar polyester piece is provided with the sky The opening of chamber, while liquid crystal 5CB, which is heated to 42 DEG C, makes it in isotropic liquid, then by liquid crystal 5CB from the opening It is injected into the cavity, and liquid crystal 5CB is made to be covered with entire cavity, be cooled to room temperature, obtain liquid crystal pond, finally by liquid crystal pond It is placed under petrographic microscope and is detected, and HBD-2 is detected according to the optical image signal in liquid crystal pond.
Embodiment five
It is of the present invention based on nano-gold signal amplification liquid crystal immunosensor detection HBD-2 method include with Lower step:
1) slide is cut into upper slide and lower slide, then upper slide and lower slide is placed in Piranha solution simultaneously It is impregnated at 95 DEG C, is then placed into dehydrated alcohol and impregnates 25min, so that upper slide and lower surface of glass slide are fixed with Hydroxyl finally uses N2Drying；
2) upper slide is soaked in 45min in the aqueous solution of DMOAP, then uses N after being cleaned with ultrapure water2Drying；Simultaneously will Lower slide is soaked in 3.5h in the ethanol solution of APTES and DMOAP at 95 DEG C, then successively clear with dehydrated alcohol and ultrapure water It washes, finally uses N2Drying, wherein the volume fraction of DMOAP aqueous solution is in the ethanol solution of 0.28%, APTES and DMOAP The volume fraction of DMOAP is 2.5% in the ethanol solution of the volume fraction 4.5% of APTES, APTES and DMOAP；
3) lower slide is impregnated with glutaraldehyde solution at normal temperature, and is placed in constant-temperature table and reacts 1.8h, made Must descend surface of glass slide aldehyde radical, then with after ultrapure water the lower slide that be modified through aldehyde radical；The AuNPs of 1mL is added to It in the HBD-2 antibody of 18 μ L 0.5mg/mL, then stood, be centrifuged and dispersed, the HBD-2 antibody for obtaining AuNPs modification is compound The modification HBD-2 antibody complex of the AuNPs of 500 ng/mL is added drop-wise on the lower slide modified through aldehyde radical, after blowing open by object It is placed at 39 DEG C and incubates 3.5h, then successively with using N after buffer and ultrapure water2Drying, wherein the body of glutaraldehyde solution Fraction is 4%；
4) the upper slide obtained through step 3) is placed on the lower slide obtained through step 5), wherein upper slide is under It is provided with Mylar polyester piece between slide, cavity is provided in Mylar polyester piece, the side of Mylar polyester piece is provided with the sky The opening of chamber, while liquid crystal 5CB, which is heated to 48 DEG C, makes it in isotropic liquid, then by liquid crystal 5CB from the opening It is injected into the cavity, and liquid crystal 5CB is made to be covered with entire cavity, be cooled to room temperature, obtain liquid crystal pond, finally by liquid crystal pond It is placed under petrographic microscope and is detected, and HBD-2 is detected according to the optical image signal in liquid crystal pond.
Embodiment six
It is of the present invention based on nano-gold signal amplification liquid crystal immunosensor detection HBD-2 method include with Lower step:
1) slide is cut into upper slide and lower slide, then upper slide and lower slide is placed in Piranha solution simultaneously It is impregnated at 80 DEG C, is then placed into dehydrated alcohol and impregnates 10min, so that upper slide and lower surface of glass slide are fixed with Hydroxyl finally uses N2Drying；
2) upper slide is soaked in 30min in the aqueous solution of DMOAP, then uses N after being cleaned with ultrapure water2Drying；Simultaneously will Lower slide is soaked in 2h in the ethanol solution of APTES and DMOAP at 80 DEG C, then successively clear with dehydrated alcohol and ultrapure water It washes, finally uses N2Drying, wherein the volume fraction of DMOAP aqueous solution is in the ethanol solution of 0.2%, APTES and DMOAP The volume fraction of DMOAP is 1% in the ethanol solution of the volume fraction 3% of APTES, APTES and DMOAP；
3) lower slide is impregnated with glutaraldehyde solution at normal temperature, and is placed in constant-temperature table and reacts 1h, so that Lower surface of glass slide aldehyde radical, then with after ultrapure water the lower slide that be modified through aldehyde radical；The AuNPs of 1mL is added to 15 μ It in the HBD-2 antibody of L 0.5mg/mL, then stood, be centrifuged and dispersed, obtain the HBD-2 antibody complex of AuNPs modification, it will The modification HBD-2 antibody complex of the AuNPs of 150 ng/mL is added drop-wise on the lower slide modified through aldehyde radical, is placed after blowing open 2h is incubated at 35 DEG C, then successively with using N after buffer and ultrapure water2Drying, wherein the volume fraction of glutaraldehyde solution It is 2%；
4) 20 μ L 0.05mg/mL HBD-2 solution to be detected are added in the aqueous solution of EDC and NHS, are added dropwise after activation Into BSA solution, then concussion reaction 2h, obtain HBD-2-BSA solution, wherein the concentration of the aqueous solution of EDC and NHS is 50ng/ mL；
5) the HBD-2-BSA solution that the step 4) of 0.01ng/mL obtains is added drop-wise to the lower slide obtained through step 3) On, it is placed in after blowing open at 35 DEG C and incubates 1h, then successively with using N after buffer and ultrapure water2Drying；
6) the upper slide obtained through step 2) is placed on the lower slide obtained through step 5), wherein upper slide is under It is provided with Mylar polyester piece between slide, cavity is provided in Mylar polyester piece, the side of Mylar polyester piece is provided with the sky The opening of chamber, while liquid crystal 5CB, which is heated to 40 DEG C, makes it in isotropic liquid, then by liquid crystal 5CB from the opening It is injected into the cavity, and liquid crystal 5CB is made to be covered with entire cavity, be cooled to room temperature, obtain liquid crystal pond, finally by liquid crystal pond It is placed under petrographic microscope and is detected, and HBD-2 is detected according to the optical image signal in liquid crystal pond.
Embodiment seven
It is of the present invention based on nano-gold signal amplification liquid crystal immunosensor detection HBD-2 method include with Lower step:
1) slide is cut into upper slide and lower slide, then upper slide and lower slide is placed in Piranha solution simultaneously It is impregnated at 100 DEG C, is then placed into dehydrated alcohol and impregnates 30 min, so that upper slide and lower surface of glass slide are fixed with Hydroxyl finally uses N2Drying；
2) upper slide is soaked in 50min in the aqueous solution of DMOAP, then uses N after being cleaned with ultrapure water2Drying；Simultaneously will Lower slide is soaked in 4h in the ethanol solution of APTES and DMOAP at 100 DEG C, then successively clear with dehydrated alcohol and ultrapure water It washes, finally uses N2Drying, wherein the volume fraction of DMOAP aqueous solution is in the ethanol solution of 0.3%, APTES and DMOAP The volume fraction of DMOAP is 3% in the ethanol solution of the volume fraction 5% of APTES, APTES and DMOAP；
3) lower slide is impregnated with glutaraldehyde solution at normal temperature, and is placed in constant-temperature table and reacts 2h, so that Lower surface of glass slide aldehyde radical, then with after ultrapure water the lower slide that be modified through aldehyde radical；The AuNPs of 1mL is added to 20 μ It in the HBD-2 antibody of L 0.5mg/mL, then stood, be centrifuged and dispersed, obtain the HBD-2 antibody complex of AuNPs modification, it will The modification HBD-2 antibody complex of the AuNPs of 150 ng/mL is added drop-wise on the lower slide modified through aldehyde radical, is placed after blowing open 4h is incubated at 40 DEG C, then successively with using N after buffer and ultrapure water2Drying, wherein the volume fraction of glutaraldehyde solution It is 5%；
4) the HBD-2 solution to be detected of 20 μ L 0.05mg/mL is added in the aqueous solution of EDC and NHS, is dripped after activation It is added in BSA solution, then concussion reaction 4h, obtains HBD-2-BSA solution, wherein the concentration of the aqueous solution of EDC and NHS is 60ng/mL；
5) the HBD-2-BSA solution that the step 4) of 0.1ng/mL obtains is added drop-wise on the lower slide obtained through step 3), It is placed in after blowing open at 40 DEG C and incubates 2h, then successively with using N after buffer and ultrapure water2Drying；
6) the upper slide obtained through step 2) is placed on the lower slide obtained through step 5), wherein upper slide is under It is provided with Mylar polyester piece between slide, cavity is provided in Mylar polyester piece, the side of Mylar polyester piece is provided with the sky The opening of chamber, while liquid crystal 5CB, which is heated to 50 DEG C, makes it in isotropic liquid, then by liquid crystal 5CB from the opening It is injected into the cavity, and liquid crystal 5CB is made to be covered with entire cavity, be cooled to room temperature, obtain liquid crystal pond, finally by liquid crystal pond It is placed under petrographic microscope and is detected, and HBD-2 is detected according to the optical image signal in liquid crystal pond.
Embodiment eight
It is of the present invention based on nano-gold signal amplification liquid crystal immunosensor detection HBD-2 method include with Lower step:
1) slide is cut into upper slide and lower slide, then upper slide and lower slide is placed in Piranha solution simultaneously It is impregnated at 90 DEG C, is then placed into dehydrated alcohol and impregnates 20 min, so that upper slide and lower surface of glass slide are fixed with Hydroxyl finally uses N2Drying；
2) upper slide is soaked in 40min in the aqueous solution of DMOAP, then uses N after being cleaned with ultrapure water2Drying；Simultaneously will Lower slide is soaked in 3h in the ethanol solution of APTES and DMOAP at 90 DEG C, then successively clear with dehydrated alcohol and ultrapure water It washes, finally uses N2Drying, wherein the volume fraction of DMOAP aqueous solution is in the ethanol solution of 0.25%, APTES and DMOAP The volume fraction of DMOAP is 2% in the ethanol solution of the volume fraction 4% of APTES, APTES and DMOAP；
3) lower slide is impregnated with glutaraldehyde solution at normal temperature, and is placed in constant-temperature table and reacts 1.5h, made Must descend surface of glass slide aldehyde radical, then with after ultrapure water the lower slide that be modified through aldehyde radical；The AuNPs of 1mL is added to It in the HBD-2 antibody of 18 μ L 0.5mg/mL, then stood, be centrifuged and dispersed, the HBD-2 antibody for obtaining AuNPs modification is compound The modification HBD-2 antibody complex of the AuNPs of 150 ng/mL is added drop-wise on the lower slide modified through aldehyde radical, after blowing open by object It is placed at 38 DEG C and incubates 3h, then successively with using N after buffer and ultrapure water2Drying, wherein the volume of glutaraldehyde solution Score is 3.5%；
4) the HBD-2 solution to be detected of 20 μ L 0.05mg/mL is added in the aqueous solution of EDC and NHS, is dripped after activation It is added in BSA solution, then concussion reaction 3h, obtains HBD-2-BSA solution, wherein the concentration of the aqueous solution of EDC and NHS is 55ng/mL；
5) the HBD-2-BSA solution that the step 4) of 1ng/mL obtains is added drop-wise on the lower slide obtained through step 3), is blown It is placed in after opening at 38 DEG C and incubates 1.5h, then successively with using N after buffer and ultrapure water2Drying；
6) the upper slide obtained through step 2) is placed on the lower slide obtained through step 5), wherein upper slide is under It is provided with Mylar polyester piece between slide, cavity is provided in Mylar polyester piece, the side of Mylar polyester piece is provided with the sky The opening of chamber, while liquid crystal 5CB, which is heated to 45 DEG C, makes it in isotropic liquid, then by liquid crystal 5CB from the opening It is injected into the cavity, and liquid crystal 5CB is made to be covered with entire cavity, be cooled to room temperature, obtain liquid crystal pond, finally by liquid crystal pond It is placed under petrographic microscope and is detected, and HBD-2 is detected according to the optical image signal in liquid crystal pond.
Embodiment nine
It is of the present invention based on nano-gold signal amplification liquid crystal immunosensor detection HBD-2 method include with Lower step:
1) slide is cut into upper slide and lower slide, then upper slide and lower slide is placed in Piranha solution simultaneously It is impregnated at 85 DEG C, is then placed into dehydrated alcohol and impregnates 15 min, so that upper slide and lower surface of glass slide are fixed with Hydroxyl finally uses N2Drying；
2) upper slide is soaked in 35min in the aqueous solution of DMOAP, then uses N after being cleaned with ultrapure water2Drying；Simultaneously will Lower slide is soaked in 2.5h in the ethanol solution of APTES and DMOAP at 85 DEG C, then successively clear with dehydrated alcohol and ultrapure water It washes, finally uses N2Drying, wherein the volume fraction of DMOAP aqueous solution is in the ethanol solution of 0.22%, APTES and DMOAP The volume fraction of DMOAP is 1.5% in the ethanol solution of the volume fraction 3.5% of APTES, APTES and DMOAP；
3) lower slide is impregnated with glutaraldehyde solution at normal temperature, and is placed in constant-temperature table and reacts 1.5h, made Must descend surface of glass slide aldehyde radical, then with after ultrapure water the lower slide that be modified through aldehyde radical；The AuNPs of 1mL is added to It in the HBD-2 antibody of 16 μ L 0.5mg/mL, then stood, be centrifuged and dispersed, the HBD-2 antibody for obtaining AuNPs modification is compound The modification HBD-2 antibody complex of the AuNPs of 150 ng/mL is added drop-wise on the lower slide modified through aldehyde radical, after blowing open by object It is placed at 36 DEG C and incubates 2.5h, then successively with using N after buffer and ultrapure water2Drying, wherein the body of glutaraldehyde solution Fraction is 2.5%；
4) the HBD-2 solution to be detected of 20 μ L 0.05mg/mL is added in the aqueous solution of EDC and NHS, is dripped after activation It is added in BSA solution, then concussion reaction 2.5h, obtains HBD-2-BSA solution, wherein the concentration of the aqueous solution of EDC and NHS is 53ng/mL；
5) the HBD-2-BSA solution that the step 4) of 10ng/mL obtains is added drop-wise on the lower slide obtained through step 3), It is placed in after blowing open at 36 DEG C and incubates 1.3h, then successively with using N after buffer and ultrapure water2Drying；
6) the upper slide obtained through step 2) is placed on the lower slide obtained through step 5), wherein upper slide is under It is provided with Mylar polyester piece between slide, cavity is provided in Mylar polyester piece, the side of Mylar polyester piece is provided with the sky The opening of chamber, while liquid crystal 5CB, which is heated to 42 DEG C, makes it in isotropic liquid, then by liquid crystal 5CB from the opening It is injected into the cavity, and liquid crystal 5CB is made to be covered with entire cavity, be cooled to room temperature, obtain liquid crystal pond, finally by liquid crystal pond It is placed under petrographic microscope and is detected, and HBD-2 is detected according to the optical image signal in liquid crystal pond.
Embodiment ten
It is of the present invention based on nano-gold signal amplification liquid crystal immunosensor detection HBD-2 method include with Lower step:
1) slide is cut into upper slide and lower slide, then upper slide and lower slide is placed in Piranha solution simultaneously It is impregnated at 95 DEG C, is then placed into dehydrated alcohol and impregnates 25 min, so that upper slide and lower surface of glass slide are fixed with Hydroxyl finally uses N2Drying；
2) upper slide is soaked in 45min in the aqueous solution of DMOAP, then uses N after being cleaned with ultrapure water2Drying；Simultaneously will Lower slide is soaked in 3.5h in the ethanol solution of APTES and DMOAP at 95 DEG C, then successively clear with dehydrated alcohol and ultrapure water It washes, finally uses N2Drying, wherein the volume fraction of DMOAP aqueous solution is in the ethanol solution of 0.29%, APTES and DMOAP The volume fraction of DMOAP is 2.5% in the ethanol solution of the volume fraction 4.5% of APTES, APTES and DMOAP；
3) lower slide is impregnated with glutaraldehyde solution at normal temperature, and is placed in constant-temperature table and reacts 1.9h, made Must descend surface of glass slide aldehyde radical, then with after ultrapure water the lower slide that be modified through aldehyde radical；The AuNPs of 1mL is added to It in the HBD-2 antibody of 19 μ L 0.5mg/mL, then stood, be centrifuged and dispersed, the HBD-2 antibody for obtaining AuNPs modification is compound The modification HBD-2 antibody complex of the AuNPs of 150 ng/mL is added drop-wise on the lower slide modified through aldehyde radical, after blowing open by object It is placed at 39 DEG C and incubates 3.5h, then successively with using N after buffer and ultrapure water2Drying, wherein the body of glutaraldehyde solution Fraction is 4.5%；
4) the HBD-2 solution to be detected of 20 μ L 0.05mg/mL is added in the aqueous solution of EDC and NHS, is dripped after activation It is added in BSA solution, then concussion reaction 3.8h, obtains HBD-2-BSA solution, wherein the concentration of the aqueous solution of EDC and NHS is 58ng/mL；
5) the HBD-2-BSA solution that the step 4) of 100ng/mL obtains is added drop-wise on the lower slide obtained through step 3), It is placed in after blowing open at 39 DEG C and incubates 1.8h, then successively with using N after buffer and ultrapure water2Drying；
6) the upper slide obtained through step 2) is placed on the lower slide obtained through step 5), wherein upper slide is under It is provided with Mylar polyester piece between slide, cavity is provided in Mylar polyester piece, the side of Mylar polyester piece is provided with the sky The opening of chamber, while liquid crystal 5CB, which is heated to 48 DEG C, makes it in isotropic liquid, then by liquid crystal 5CB from the opening It is injected into the cavity, and liquid crystal 5CB is made to be covered with entire cavity, be cooled to room temperature, obtain liquid crystal pond, finally by liquid crystal pond It is placed under petrographic microscope and is detected, and HBD-2 is detected according to the optical image signal in liquid crystal pond.
Wherein, in each embodiment, H in Piranha solution2O2With H2SO4Volume ratio be 3:7.
Liquid crystal pond prepared by embodiment one to five is respectively placed under petrographic microscope and is observed, Fig. 1 to Fig. 5 institute is obtained It is stating as a result, the concentration of HBD-2 antibody is respectively as follows: 75ng/ml, 100ng/ml, 150ng/ml, 200ng/ml and 500ng/ in figure ml；Wherein, when the concentration of HBD-2 antibody is higher than 100ng/ml, there is speck in image, and therefore, HBD-2 antibody content is more than When 100ng/ml, there is significant change in optical signalling.
Liquid crystal pond prepared by embodiment six to ten is respectively placed under petrographic microscope and is observed, obtains Fig. 6 to Figure 10 Shown in as a result, the concentration of HBD-2 is respectively as follows: 0.01ng/ml, 0.1 ng/ml, 1ng/ml, 10ng/ml and 100ng/ in figure ml；Wherein, when the concentration of HBD-2 is higher than 1 ng/ml, there is speck in image, therefore, when HBD-2 content is more than 1ng/ml, optics There is significant change in signal.
The above results show that being substantially reduced using nano-gold signal amplification detection HBD-2 detection limit, in the content of HBD-2 When more than 1ng/ml, there is significant change in optical signalling.

Claims (8)

1. it is a kind of based on nano-gold signal amplification liquid crystal immunosensor detection HBD-2 method, which is characterized in that including with Lower step:
1) slide is cut into upper slide and lower slide, then upper slide and lower slide is placed in Piranha solution and in 80- It is impregnated at 100 DEG C, is impregnated after then cleaning with dehydrated alcohol, finally dried up；
2) upper slide is soaked in DMOAP aqueous solution, is dried up after flushing, while lower slide being soaked at 80-100 DEG C In the ethanol solution of APTES and DMOAP, then cleans and dry up；
3) lower slide is impregnated at normal temperature with glutaraldehyde solution, the lower slide modified through aldehyde radical is obtained after flushing；It will AuNPs is added in HBD-2 antibody, then is stood, is centrifuged and dispersed, and the HBD-2 antibody complex of AuNPs modification is obtained, will The modification HBD-2 antibody complex of AuNPs is added drop-wise on the lower slide modified through aldehyde radical, is placed at 35-40 DEG C after blowing open 2-4h is incubated, then is dried up after rinsing；
4) HBD-2 solution to be detected is added in the aqueous solution of EDC and NHS, is added drop-wise to after activation in BSA solution, then shake Reaction, obtains HBD-2-BSA solution；
5) the HBD-2-BSA solution that step 4) obtains is added drop-wise on the lower slide obtained through step 3), is placed in 35- after blowing open 1-2h is incubated at 40 DEG C, is dried up after flushing；
6) the upper slide obtained through step 2) is placed on the lower slide obtained through step 5), wherein upper slide and lower slide Between be provided with Mylar polyester piece, cavity is provided in Mylar polyester piece, the side of Mylar polyester piece is provided with the cavity Opening, while making it in isotropic liquid liquid crystal 5CB heating, then liquid crystal 5CB is injected into from the opening described In cavity, and liquid crystal 5CB is made to be covered with entire cavity, be cooled to room temperature, obtains liquid crystal pond, it is aobvious that last liquid crystal pond is placed into polarisation It is detected under micro mirror, and HBD-2 is detected according to the optical image signal in liquid crystal pond.
2. the method for the liquid crystal immunosensor detection HBD-2 according to claim 1 based on nano-gold signal amplification, It is characterized in that, upper slide and lower slide are placed in Piranha solution in step 1) and are impregnated at 80-100 DEG C, then It is cleaned with ultrapure water, is then placed into dehydrated alcohol and impregnates 10-30min, so that upper slide and lower surface of glass slide are fixed with hydroxyl Base finally uses N2Drying；
In step 2) N will be used after upper slide ultrapure water2Drying；Lower slide is successively cleaned with dehydrated alcohol and ultrapure water, Then N is used2Drying；
Upper slide is soaked in 30-50min in DMOAP aqueous solution in step 2)；
Lower slide is placed in step 2) in the ethanol solution of 80-100 DEG C of APTES and DMOAP and impregnates 2-4h；
In step 3) lower slide is impregnated at normal temperature with glutaraldehyde solution, then is repaired with being obtained after ultrapure water through aldehyde radical The lower slide of decorations；
It is placed in after being blown open in step 3) at 35-40 DEG C and incubates 2-4h, then successively with using N after buffer and ultrapure water2It blows It is dry；
The time of concussion reaction is 2-4h in step 4)；
Successively with using N after buffer and ultrapure water in step 5)2Drying.
3. the method for the liquid crystal immunosensor detection people HBD-2 according to claim 1 based on nano-gold signal amplification, It is characterized in that, the volume fraction of DMOAP aqueous solution is 0.2%-0.3% in step 2).
4. the method for the liquid crystal immunosensor detection HBD-2 according to claim 1 based on nano-gold signal amplification, It is characterized in that, the ethyl alcohol of the volume fraction 3%-5%, APTES and DMOAP of APTES are molten in the ethanol solution of APTES and DMOAP The volume fraction of DMOAP is 1%-3% in liquid.
5. the method for the liquid crystal immunosensor detection HBD-2 according to claim 1 based on nano-gold signal amplification, It is characterized in that, in step 3), the volume fraction of glutaraldehyde solution is 2%-5%, and lower slide is used glutaraldehyde solution at normal temperature It is impregnated, and is placed in constant-temperature table and reacts 1-2h, so that lower surface of glass slide aldehyde radical.
6. the method for the liquid crystal immunosensor detection HBD-2 according to claim 1 based on nano-gold signal amplification, It is characterized in that, the AuNPs of 1mL is added in the HBD-2 antibody of 15-20 μ L in step 3), wherein the concentration of HBD-2 antibody For 0.5mg/mL.
7. the method for the liquid crystal immunosensor detection HBD-2 according to claim 1 based on nano-gold signal amplification, It is characterized in that, the concentration of the aqueous solution of EDC and NHS is 50-60ng/mL.
8. the method for the liquid crystal immunosensor detection HBD-2 according to claim 1 based on nano-gold signal amplification, It is characterized in that, liquid crystal 5CB, which is heated to 40-50 DEG C, makes it in isotropic liquid.
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