CN103954771B - A kind of low cost is caught/is discharged and detects the method for biomolecule - Google Patents
A kind of low cost is caught/is discharged and detects the method for biomolecule Download PDFInfo
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- CN103954771B CN103954771B CN201410174644.2A CN201410174644A CN103954771B CN 103954771 B CN103954771 B CN 103954771B CN 201410174644 A CN201410174644 A CN 201410174644A CN 103954771 B CN103954771 B CN 103954771B
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- 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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54353—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals with ligand attached to the carrier via a chemical coupling agent
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- 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
Abstract
The invention discloses a kind of method that low cost is caught/discharged and detects biomolecule, utilize protein phase in version method, lysozyme is fixed to non-contaminated surface, on this basis, specific binding principle between recycling biomolecule, further fixed target biomolecule, the specificity realizing biomolecule is caught, then, by guanidine solution cleaning substrate surface, substrate surface after cleaning not only still remains initial antifouling property, further can also adsorb lysozyme phase in version product, achieve the reversible transition of between biologically inert and biologically active base material nearly 100%.The present invention is simple to operate, can direct high-sensitivity detection target biological molecules from serum, blood plasma, lymph, interstitial fluid, urine, transudate, cytolysate, juice etc., and the recycling of base material can be realized, reduce cost, achieve Green Chemistry.
Description
Technical field
The invention belongs to the detection technique field of biomolecule, be specifically related to a kind ofly realize the low cost of biomolecule, the method for high-sensitivity detection by the reversible transition between biologically active base material and biologically inert base material.
Background technology
At inert inorganic or organic material surface fixing biological molecules for significant bio-medical material.Fixed cell, protein, the enzyme even bio-carrier of DNA fragmentation are widely used among cell culture medium, Medical rack, protein-chip, biology sensor and genetic chip.And securing the base material of biomolecule, the circulation after realize target analysis detects repeatedly utilizes, and effectively can reduce costs, realize Green Chemistry.
First, in prepared by base material, one of method of fixing biological molecules is exactly that effects on surface carries out modification, produces functional group from the teeth outwards, realizes surface-functionalized.Traditional chip preparing process, because inert base surface chemical structure only has measured response efficiency, it is special to need to carry out surface of solid phase carriers, complicated physical/chemical process, again known protein molecular product is fixed thereon (as enzyme, antigen, antibody, acceptor, part, cell factor etc.), according to the characteristic of these biomolecule, catch and the testing protein of specific binding with it (serum can be present in, blood plasma, lymph, interstitial fluid, urine, transudate, cytolysate, juice etc.), through washing, purifying, carry out again confirming and biochemical analysis.Few people report mild condition, environmental friendliness and efficient preparation method.As Robert S Matteson etc. finds a kind of method of the substrate for the preparation of arrangement biomolecule or cell, method comprises the steps: that (a) provides the solid carrier with surface; And the polymerization deposition polymerization coating on said surface that (b) is mediated by chemical plasma, wherein saidly polymer coatedly comprise at least one side functional group, it can in conjunction with described biomolecule or cell.Visible, said method needs to carry out complicated physical/chemical process to substrate surface, and this is just unfavorable for the integrality keeping base material nature.
Secondly, in various biologic applications field, in the urgent need to the Reversible binding/delivery systme of biomolecule.Such transformation will guide a desirable process effectively, namely promotes the enrichment of in-line purification and low concentration biomolecule and detects further.In addition, intelligent biomolecule of catching/discharge also is widely used at other high-tech areas many such as biological medicine, organizational project and information feed back etc.Actual biomedical method needs the reversible system utilizing biomolecule/cell capture/release, this reversible transition, first separate targets biomolecule/cell, fix from biological fluid and focus on surface, finally discharged, so that the spectrum/fluoroscopic examination done subsequently is separated with cell patch/biological membrane.Although a lot of method is reasonable and desirable, but realize catching and discharge completely reversibility and have very much a challenge, because extensively there is non-specific adsorption on traditional intelligent substrate surface, not easily remove non-specific adsorption, the reversible transition of biologically active and biologically inert 100% can not be reached.Therefore, the premium properties of relevant device is restricted.Therefore explore a kind of new system and catch to realize the reversible of biomolecule/discharge, significant.
Again, for realizing Green Chemistry, making full use of resource and the energy, reducing costs, except adopting nontoxic, harmless raw material, also should reduce refuse to environmental emission; This poses a new problem, what namely how to realize antipollution base material repeats recycling.The non-contaminated surface that it requires secondary to utilize, not only remains the antifouling property that it is original, can also fixing biological molecules in a mild condition further, realizes the recycled for multiple times of base material.Completely bioactivity surface deactivation to be changed into inactive surfaces, because low chemical reaction productive rate and speed are greatly limited, therefore can not realize the transformation completely of nearly 100%.Therefore the effective conversion of biologically active substrate surface to biologically inert substrate surface will be realized, challenging equally.
Summary of the invention
Technical matters to be solved by this invention is to overcome in existing biomolecule detecting method, antipollution base material has to pass through complexity, harsh physical/chemical process, and the base material after detecting cannot reusable shortcoming, there is provided a kind of with low cost, antipollution base material treatment mild condition, base material after process can specificity target acquisition biomolecule, and the method for high-sensitivity detection biomolecule that the base material after detecting can reuse.
Solve the problems of the technologies described above adopted technical scheme to be made up of following step:
1, biologically active base material is prepared
Be that the 4-hydroxyethyl piperazine ethanesulfonic acid aqueous solution of 7.4 is for solvent with 10mmol/LpH value, prepare respectively 50mmol/LpH value be 7.4 three (2-carboxyethyl) phosphine solution and quality-volumetric concentration be the biotin labeled lysozyme soln of 1 ~ 5mg/mL, then by preparation two kinds of solution by volume for 1:1 mixes, with protein point sample instrument immediately point sample to antipollution substrate surface, under moist environment, normal temperature is cultivated 1 ~ 4 hour, then successively by the 4-hydroxyethyl piperazine ethanesulfonic acid aqueous solution that 10mmol/LpH value is 7.4, washed with de-ionized water, be prepared into biologically active base material.
2, target biological molecules is detected
Be that the 4-hydroxyethyl piperazine ethanesulfonic acid aqueous solution of 7.4 is for solvent with 10mmol/LpH value, preparation quality-volumetric concentration is the solution of streptavidin of 0.01 ~ 1mg/mL, then biologically active substrate surface prepared by step 1 is spread into, under moist environment, normal temperature is cultivated 1 ~ 4 hour, then successively by 4-hydroxyethyl piperazine ethanesulfonic acid aqueous solution, washed with de-ionized water that 10mmol/LpH value is 7.4, at antibody or the antigen of the target biological molecules of substrate surface fixed biologically element mark, detect the target biological molecules in testing sample with this antibody or antigen.
3, base material recycling
By the guanidine aqueous solution soaking 1 ~ 2 minute of the base material after having detected in step 2 at 3 ~ 6mol/L, take out, by washed with de-ionized water, naturally dry, be again prepared into biologically active base material according to the method for step 1, reuse.
In above-mentioned preparation biologically active base material step 1, be preferably that the 4-hydroxyethyl piperazine ethanesulfonic acid aqueous solution of 7.4 is for solvent with 10mmol/LpH value, prepare respectively 50mmol/LpH value be 7.4 three (2-carboxyethyl) phosphine solution and quality-volumetric concentration be the biotin labeled lysozyme soln of 2mg/mL, then by preparation two kinds of solution by volume for 1:1 mixes, with protein point sample instrument immediately point sample to antipollution substrate surface, under moist environment, normal temperature cultivates 2 hours, then successively by the 4-hydroxyethyl piperazine ethanesulfonic acid aqueous solution that 10mmol/LpH value is 7.4, washed with de-ionized water, be prepared into biologically active base material.
In above-mentioned detection target biological molecules step 2, quality-volumetric concentration preferably 0.1 ~ 0.5mg/mL of described solution of streptavidin.
In above-mentioned base material recycling step 3, the preferred 3mol/L of concentration of described guanidine aqueous solution.
The invention has the beneficial effects as follows:
1, the present invention's non-confrontational pollution background does any activation and Passivation Treatment, directly adopt Simple temperature and and biomolecule is fixed on antipollution substrate surface by eco-friendly method fast, simple to operate, preparation efficiency is high, and raw materials used nontoxic, wide material sources.
2, the present invention can direct high-sensitivity detection target biological molecules from serum, blood plasma, lymph, interstitial fluid, urine, transudate, cytolysate, juice etc., as tumor markers, cancer cell, and the base material after having detected can realize the reversible transition of between biologically inert and biologically active base material nearly 100% through the cleaning of guanidine solution, the circulation achieving base material repeatedly utilizes, reduce cost, the washing lotion of collection can also be done follow-up spectrum/fluoroscopic examination and be separated with cell patch/biological membrane.
Accompanying drawing explanation
Fig. 1 is the fluorescence photo detecting variable concentrations alpha-fetoprotein in serum.
Fig. 2 is the fluorescence photo detecting variable concentrations green fluorescent protein in damping fluid.
Fig. 3 is the fluorescence spectrum figure detecting the Streptavidin that variable concentrations cyanine type dye Cy-5 marks in damping fluid and serum.
Fig. 4 is the turbidity histogram of different substrate materials.
Fig. 5 is the fluorescence photo that in embodiment 1, the Streptavidin of marked by fluorescein isothiocyanate fixed by heavy freshly prepd biologically active base material.
Fig. 6 is the fluorescence photo of base material after the cleaning of guanidine solution in Fig. 5.
Fig. 7 is the fluorescence photo that the Streptavidin of marked by fluorescein isothiocyanate fixed by base material in Fig. 6.
Embodiment
Below in conjunction with drawings and Examples, the present invention is described in more detail, but protection scope of the present invention is not limited only to these embodiments.
Embodiment 1
1, biologically active base material is prepared
Volume ratio methoxypolyethylene glycol methacrylate being added methyl alcohol and distilled water is in the mixed liquor of 1:1, being mixed with quality-volumetric concentration is 0.01g/mL methoxypolyethylene glycol methacrylic acid ester solution, then clean glass surface is spin-coated on, dry under natural conditions, obtain antipollution base material.Be that the 4-hydroxyethyl piperazine ethanesulfonic acid aqueous solution of 7.4 is for solvent with 10mmol/LpH value, prepare respectively 50mmol/LpH value be 7.4 three (2-carboxyethyl) phosphine solution and quality-volumetric concentration be the biotin labeled lysozyme soln of 2mg/mL, then by preparation two kinds of solution by volume for 1:1 mixes, with protein point sample instrument immediately point sample to the antipollution substrate surface obtained, under moist environment, normal temperature cultivates 2 hours, then successively by the 4-hydroxyethyl piperazine ethanesulfonic acid aqueous solution that 10mmol/LpH value is 7.4, deionized water respectively cleans 2 times, each 15 minutes, be prepared into biologically active base material.
2, the alpha-fetoprotein in serum is detected
With 10mmol/LpH value be the 4-hydroxyethyl piperazine ethanesulfonic acid aqueous solution of 7.4 for solvent, prepare the alpha-fetoprotein antibody solution of solution of streptavidin that quality-volumetric concentration is 0.1mg/mL, the biotin labeled alpha-fetoprotein antibody solution of 0.2mg/mL, 0.01mg/mL marked by fluorescein isothiocyanate respectively.It is the biologically active substrate surface that the solution of streptavidin of 0.1mg/mL spreads into step 1 and prepares by quality-volumetric concentration, under moist environment, normal temperature cultivates 2 hours, be the 4-hydroxyethyl piperazine ethanesulfonic acid aqueous solution of 7.4 successively by 10mmol/LpH value, deionized water respectively cleans 2 times, each 15 minutes, then be that the biotin labeled alpha-fetoprotein antibody solution of 0.2mg/mL spreads into substrate surface by quality-volumetric concentration, under moist environment, normal temperature cultivates 2 hours, be the 4-hydroxyethyl piperazine ethanesulfonic acid aqueous solution of 7.4 successively by 10mmol/LpH value, deionized water respectively cleans 2 times, each 15 minutes, be that the serum of 10 μ g/mL alpha-fetoproteins spreads into substrate surface again by quality-volumetric concentration, under moist environment, normal temperature cultivates 2 hours, be the 4-hydroxyethyl piperazine ethanesulfonic acid aqueous solution of 7.4 successively by 10mmol/LpH value, deionized water respectively cleans 2 times, each 15 minutes, be finally that the alpha-fetoprotein antibody solution of 0.01mg/mL marked by fluorescein isothiocyanate spreads into substrate surface by quality-volumetric concentration, under moist environment, normal temperature cultivates 2 hours, be the 4-hydroxyethyl piperazine ethanesulfonic acid aqueous solution of 7.4 successively by 10mmol/LpH value, deionized water respectively cleans 2 times, each 15 minutes, biochip scanner is adopted to detect fluorescence signal.
3, base material recycling
By the guanidine aqueous solution soaking 1 minute of the base material after having detected in step 2 at 3mol/L, take out, by washed with de-ionized water, naturally dry, be again prepared into biologically active base material according to the method for step 1, reuse.
Embodiment 2
In the step 2 of embodiment 1, be that the 4-hydroxyethyl piperazine ethanesulfonic acid aqueous solution of 7.4 is for solvent with 10mmol/LpH value, prepare the solution of streptavidin that quality-volumetric concentration is 0.01mg/mL respectively, the biotin labeled green fluorescent protein antibody-solutions of 0.01mg/mL, 10 μ g/mL green fluorescent protein solution, it is the biologically active substrate surface that the solution of streptavidin of 0.01mg/mL spreads into step 1 and prepares by quality-volumetric concentration, under moist environment, normal temperature cultivates 2 hours, be the 4-hydroxyethyl piperazine ethanesulfonic acid aqueous solution of 7.4 successively by 10mmol/LpH value, deionized water respectively cleans 2 times, each 15 minutes, then be that the biotin labeled green fluorescent protein antibody-solutions of 0.01mg/mL spreads into substrate surface by quality-volumetric concentration, under moist environment, normal temperature cultivates 2 hours, be the 4-hydroxyethyl piperazine ethanesulfonic acid aqueous solution of 7.4 successively by 10mmol/LpH value, deionized water respectively cleans 2 times, each 15 minutes, to be that 10 μ g/mL green fluorescent protein solution spread into substrate surface containing quality-volumetric concentration again, under moist environment, normal temperature cultivates 2 hours, be the 4-hydroxyethyl piperazine ethanesulfonic acid aqueous solution of 7.4 successively by 10mmol/LpH value, deionized water respectively cleans 2 times, each 15 minutes, inverted fluorescence microscope is adopted to detect fluorescence signal.Other steps are identical with embodiment 1.
In order to prove beneficial effect of the present invention, inventors performed a large amount of laboratory study tests, concrete test situation is as follows:
1, specificity catches/release test
Adopt the method for embodiment 1 to detect the serum containing variable concentrations alpha-fetoprotein, simultaneously using serum as blank, detect fluorescence signal with biochip scanner, test findings is shown in Fig. 1.As seen from Figure 1, time in serum not containing alpha-fetoprotein (namely blank), fluorescence signal do not detected, when the concentration of Serum Alpha Fetoprotein is 10ng/mL, there is faint fluorescence signal, when the concentration of Serum Alpha Fetoprotein is more than 100ng/mL, fluorescence signal obviously strengthens, illustrate that the present embodiment method specificity can catch alpha-fetoprotein in serum, realize the high-sensitivity detection of alpha-fetoprotein, detect and be limited to 1 ~ 10ng/mL.
Adopt the damping fluid of the method for embodiment 1 to the green fluorescent protein containing variable concentrations to detect, simultaneously using damping fluid as blank, detect fluorescence signal with inverted fluorescence microscope, test findings is shown in Fig. 2.As seen from Figure 2, time in damping fluid not containing green fluorescent protein (namely blank), fluorescence signal do not detected, when the concentration of damping fluid Green fluorescin is 0.1 μ g/mL, there is faint fluorescence signal, when the concentration of damping fluid Green fluorescin is 10 μ g/mL, fluorescence signal obviously strengthens, illustrate that the present embodiment method can green fluorescent protein in capture buffer liquid, realize the high-sensitivity detection of green fluorescent protein, detect and be limited to 0.1 μ g/mL.
After adopting the method for embodiment 1 to detect the solution of streptavidin marked containing variable concentrations cyanine type dye Cy-5 in damping fluid and serum, with the guanidine aqueous cleaning base material of 3mol/L, then adopt fluorescence spectrophotometer to test cleaning fluid, test result is shown in Fig. 3.As seen from Figure 3, when in damping fluid or serum, the concentration of the Streptavidin that cyanine type dye Cy-5 marks is 10ng/mL, there is faint fluorescence signal, when in damping fluid or serum, the concentration of the Streptavidin that cyanine type dye Cy-5 marks is 100ng/mL, fluorescence signal obviously strengthens, illustrate the present embodiment method can catch/buffer release liquid or serum in the Streptavidin that marks of cyanine type dye Cy-5, realize the high-sensitivity detection of the Streptavidin that cyanine type dye Cy-5 marks, detect and be limited to 1 ~ 10ng/mL.
2, turbidity test
Inventor adopt ultraviolet spectrophotometer to the antipollution base material in embodiment 1, detected after base material and detected with guanidine solution cleaning after base material carry out turbidity test respectively, to the results are shown in Figure in 3, figure 1 ~ 6 be antipollution base material successively, detected after base material and detected the turbidity that rear guanidine solution cleans 15 seconds, 30 seconds, 45 seconds, 60 seconds.
As seen from Figure 4, the more former antipollution base material of turbidity having detected rear substrate surface obviously increases, and after cleaning 60 seconds with guanidine solution, the turbidity of substrate surface returns to identical with former antipollution base material substantially, illustrate that employing guanidine solution can be clean by substrate surface Rapid Cleaning, the base material after cleaning is reusable.
3, contact angle test
Adopt video contact angle measuring instrument to the antipollution base material in embodiment 1 and detected and clean the base material after 60 seconds with guanidine solution and carry out contact angle test, experimental result shows, the contact angle of antipollution substrate surface is 67 ± 2 °, having detected and having cleaned the substrate surface contact angle after 60 seconds with guanidine solution is 68 ± 2 °, illustrate that substrate surface character is constant, prove that base material is reusable further.
4, base material recycling test
Process in embodiment 1 step 3 according to the method for embodiment 1 step 1 with the base material after the cleaning of guanidine solution, then be that the solution of streptavidin of 0.01mg/mL marked by fluorescein isothiocyanate spreads into the substrate surface after process by quality-volumetric concentration, under moist environment, normal temperature cultivates 2 hours, be the 4-hydroxyethyl piperazine ethanesulfonic acid aqueous solution of 7.4 successively by 10mmol/LpH value, deionized water respectively cleans 2 times, each 15 minutes, inverted fluorescence microscope is adopted to detect fluorescence signal, the results are shown in Figure 5, then by the guanidine aqueous solution soaking 1 minute of base material at 3mol/L, take out, by washed with de-ionized water, naturally dry, continue to detect fluorescence signal, the results are shown in Figure 6, the Streptavidin of marked by fluorescein isothiocyanate is fixed again according to the method described above at substrate surface, detect fluorescence signal, the results are shown in Figure 7.
From Fig. 5 ~ 7, still can being prepared into required biologically active base material with the base material after the cleaning of guanidine solution, for detecting biomolecule, achieving reusing of base material.
Claims (3)
1. low cost is caught/is discharged and detects a method for biomolecule, it is characterized in that it is made up of following step:
(1) biologically active base material is prepared
Be that the 4-hydroxyethyl piperazine ethanesulfonic acid aqueous solution of 7.4 is for solvent with 10mmol/LpH value, prepare respectively 50mmol/LpH value be 7.4 three (2-carboxyethyl) phosphine solution and quality-volumetric concentration be the biotin labeled lysozyme soln of 1 ~ 5mg/mL, then by preparation two kinds of solution by volume for 1:1 mixes, with protein point sample instrument immediately point sample to antipollution substrate surface, under moist environment, normal temperature is cultivated 1 ~ 4 hour, then successively by the 4-hydroxyethyl piperazine ethanesulfonic acid aqueous solution that 10mmol/LpH value is 7.4, washed with de-ionized water, be prepared into biologically active base material,
(2) target biological molecules is detected
Be that the 4-hydroxyethyl piperazine ethanesulfonic acid aqueous solution of 7.4 is for solvent with 10mmol/LpH value, preparation quality-volumetric concentration is the solution of streptavidin of 0.1 ~ 0.5mg/mL, then biologically active substrate surface prepared by step (1) is spread into, under moist environment, normal temperature is cultivated 1 ~ 4 hour, then successively by 4-hydroxyethyl piperazine ethanesulfonic acid aqueous solution, washed with de-ionized water that 10mmol/LpH value is 7.4, at antibody or the antigen of the target biological molecules of substrate surface fixed biologically element mark, detect the target biological molecules in testing sample with this antibody or antigen;
(3) base material recycling
By the guanidine aqueous solution soaking 1 ~ 2 minute of the base material after having detected in step (2) at 3 ~ 6mol/L, taking-up, by washed with de-ionized water, naturally dries, is again prepared into biologically active base material, reuses according to the method for step (1).
2. low cost according to claim 1 is caught/is discharged and detects the method for biomolecule, it is characterized in that: in described preparation biologically active base material step (1), be that the 4-hydroxyethyl piperazine ethanesulfonic acid aqueous solution of 7.4 is for solvent with 10mmol/LpH value, prepare respectively 50mmol/LpH value be 7.4 three (2-carboxyethyl) phosphine solution and quality-volumetric concentration be the biotin labeled lysozyme soln of 2mg/mL, then by preparation two kinds of solution by volume for 1:1 mixes, with protein point sample instrument immediately point sample to antipollution substrate surface, under moist environment, normal temperature cultivates 2 hours, then successively by the 4-hydroxyethyl piperazine ethanesulfonic acid aqueous solution that 10mmol/LpH value is 7.4, washed with de-ionized water, be prepared into biologically active base material.
3. low cost according to claim 1 is caught/is discharged and detects the method for biomolecule, it is characterized in that: in base material recycling step (3), the concentration of described guanidine aqueous solution is 3mol/L.
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