CN103713138A - Adenosine detecting method based on micro-fluidic chip and nucleic acid adapter technology - Google Patents
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Abstract
The invention discloses an adenosine detecting method based on a micro-fluidic chip and a nucleic adapter technology. Biotin-modified adenosine nucleotide adapter and biotin-modified casein are fixed on the surface of an avidin-modified microsphere to form a functional microsphere through a biotin-avidin combining method; capture probes modified with horseradish peroxidase and sulfydryl are modified on nanometer particles as signal marks; functional nanometer particles flow to a micro-fluidic microsphere array to detect a detection area of a chip and hybrid with a microsphere array; after elution, an adenosine solution flows to be incubated with hybridized microspheres; a peroxidase substrate solution flows in; biotinylated tyramine is combined with the casein on the surface of the microsphere; the avidin-marked quantum dot flows in after elution; qualification and quantification are carried out on the adenosine by calculating a ratio of a fluorescence amount of the microsphere surface after adenosine inflows and a fluorescence amount of the microsphere surface before the adenosine inflows. The adenosine detecting method provided by the invention can be used for detecting and analyzing a target at high sensitivity, so that the specific and accurate quantitative analysis of the adenosine in a serum sample is realized.
Description
Technical field
The invention belongs to the Biological Detection technical field of micro-total analysis system, relate to a kind of adenosine detection method based on micro-fluidic chip and aptamer recognition technology.
Background technology
Adenosine is a kind of endogenous nucleosides, because of its vital role in nervous centralis and peripheral nervous system signal representation, is subject to extensive concern and research.Adenosine is as the effect substrate of adenosine deaminase simultaneously, its content also can indirect reaction adenosine deaminase internal metabolism level, and the detection of activity of adenosine deaminase all has important references for the diagnosis of clinical numerous disease and is worth, the novel detection method that therefore develops adenosine is significant for medical clinic applications and organism metabolism level.Tradition adenosine detection method is mainly the technology such as HPLC, ultraviolet detection, flow cytometer detection method and fluorometry, and this class technology for detection sensitivity is limited, and needs the detection solution of larger volume, makes to be limited in the application in highly sensitive microanalysis field.Micro-fluidic micropearl array chip is a kind of novel chip pattern developing in micro-fluidic chip research field, the development of this technology has better solved a difficult problem for high flux, highly sensitive detection under micro-example condition, testing process is simple simultaneously, do not need expensive detecting instrument, for developing following robotization high-performance biomolecule detection platform technology, provide new direction.
Aptamer refers to the energy specific bond albumen that screens from random single strain oligonucleotide library by index concentration Fas lignand system evolution technology (SELEX) or the single strain oligonucleotide of other small-molecule substances, and length is generally 25-60 nucleotide.Compare with antibody, aptamer have easily synthetic, easily modify, easily fixing, can Reusability, can preserve for a long time and not have or the minimum features such as immunogenicity, and reveal efficient, single-minded characteristic with target molecule associative list, thereby aptamer biology sensor is used widely at aspects such as protein research, drug test, medical diagnosiss.Enzyme functionalized nano particle, as a kind of certification mark material, owing to carrying a plurality of enzyme molecules on a nano particle, has become a kind of method for amplifying signal that has application prospect in signal amplification technique field.
Summary of the invention
In order to overcome the defect existing in prior art, the invention provides a kind of adenosine detection method based on micro-fluidic chip and aptamer recognition technology, micro-fluidic chip, aptamer identification, multienzyme nanowire signal amplifying technique are combined, developed a kind of high-performance biosensor technique detecting for adenosine, will have broad application prospects.Its technical scheme is as follows:
An adenosine detection method based on micro-fluidic chip and aptamer recognition technology, comprises the following steps:
(1) the adenosine aptamer of biotin modification and the casein of biotin modification are fixed on by biotin-avidin associated methods the surface formation functionalized microsphere that affinity element is modified microballoon, described microballoon is fixed in the corresponding miniature cell of micro-fluidic chip detection zone, forms micro-fluidic micropearl array detection chip;
(2) capture probe of horseradish peroxidase and sulfydryl modification is modified on nano particle as signal mark;
(3) functionalized nano grain flow step (2) being formed enters detection zone and the micropearl array hybridization of micro-fluidic micropearl array detection chip, after wash-out, flowing into adenosine solution hatches with hybridization microballoon, due to adenosine aptamer, can be combined with adenosine and form specific steric configuration, can cause functionalized nano particle to depart from from microsphere surface;
(4) flow into peroxidase substrate solution, by multienzyme nanowire signal amplifying technique, biotinylated tyrasamine is attached on the casein of microsphere surface, the quantum dot that flows into affinity element mark after wash-out, described quantum dot can be identified and fix with the biotin of combination on microsphere surface casein;
(5) by calculating, flow into microsphere surface fluorescence after adenosine, with the ratio that does not flow into adenosine microsphere surface fluorescence, adenosine is carried out to quantitative and qualitative analysis.
Further preferably, the adenosine aptamer of the biotin modification of the functionalized microsphere finishing described in step (1) and the casein of biotin modification, its volumetric molar concentration ratio is 1: 100～1: 120.
Further preferably, the microballoon described in step (1) is any one in silicon dioxide microsphere, polystyrene type organic polymer microballoon, magnetic microsphere and biomacromolecule polymer microballoon.
Further preferably, the nano particle described in step (2) is any one in gold nano grain, nano SiO 2 particle and polystyrene nanoparticles.
Further preferably, the adenosine solution described in step (3) is hatched with hybridization microballoon, and incubation time is 20～25min.
Further preferably, the peroxidase substrate solution described in step (4) comprises 0.007%～0.009%H
2o
2, the biotinylated tyrasamine of 250～350 μ M, 50～100mM NaCl.
Further preferably, the quantum dot described in step (4) is a kind of in CdSe, CdTe, CdS and compound quantum dot, and its transmitting boundary is 400nm～700nm.
Compared with prior art, beneficial effect of the present invention:
Micro-fluidic chip of the present invention has the dual nature of trace detection and high pass Sensitive Detection, can solve preferably the performances such as trace detection in conventional method of analysis, highly sensitive detection and high throughput analysis and be difficult to compatible problem, can realize the High Sensitive Analysis of micro-example; The present invention adopts multienzyme nanowire signal to amplify and quantum dot fluorescence technology, can highly sensitive detection evaluating objects, and can detect the adenosine of 0.1pM, and realize the special and quantitative test accurately of adenosine in blood serum sample; Trace and the highly sensitive detection that is established as adenosine of the present invention provides a kind of strong detection means.
Accompanying drawing explanation
Fig. 1 is that the adenosine based on microballon sensing and aptamer recognition technology of the present invention detects principle schematic;
Fig. 2 is the inside and outside Sensitivity comparison curve map that detects adenosine of the micro-fluidic chip of the embodiment of the present invention;
Fig. 3 is the blood serum sample testing result of the embodiment of the present invention.
Embodiment
Below in conjunction with the drawings and specific embodiments, further illustrate technical scheme of the present invention.
With reference to Fig. 1, it is the principle schematic of the inventive method.Concrete principle shown in Fig. 1 is as follows:
First, the aptamer 3 of biotin modification and the casein 2 of biotin modification are modified the microsphere surface 1 that Avidin is modified according to a certain percentage, prepare and can be used for the functionalized microsphere 4 that adenosine detects; The function nano particle 5 of horseradish peroxidase (HRP) and capture probe has been modified in inflow, and with the functionalized microsphere hybridization in chip, capture probe and aptamer combine by hybridization, and functionalized nano particle is fixed on to microsphere surface 6; Flow into target molecule adenosine 7, adenosine is combined with aptamer and is formed particular space structure and function nano particle is disintegrated down to 8 from microsphere surface; Then flow into substrate for enzymatic activity solution, comprise biotinylated tyrasamine 9 and hydrogen peroxide, the tyrasamine of horseradish peroxidase energy catalysis biological elementization under hydrogen peroxide exists forms bioactive molecule 10, the oxybenzene group generation covalent cross-linking that this biotinylated bioactive molecule can carry with casein functionalized microsphere surface, thereby the microsphere surface 11 of formation biotin group functionalization; Flow into the quantum dot 12 that Avidin is modified, quantum dot utilizes the binding characteristic of Avidin and biotin to be fixed on microsphere surface and has formed identification microballoon 13; Finally according to identification microsphere surface fluorescence signal acquisition, detect information.
The specific implementation process of the embodiment of the present invention below:
(1) caseic preparation: the NaHCO that 0.3-0.5g casein is dissolved in to 10mL150-200mM
3in solution, the dimethyl formamide that is dissolved with 2-4mg hydroxyphenylpropionic acid N-hydroxy-succinamide ester and the biotinylated succinimide ester of 0.5-1.0mg is dropwise joined in casein solution under stirring.Mixed solution at room temperature stirs 1h continuously, the 12-24h that then dialyses in deionized water, then the 12-24h that dialyses in phosphate buffer (20mM, pH7.6), and solution centrifugal is removed slight turbid solution, and precipitation freeze-drying is standby.
(2) preparation of the functional polystyrene microballon detecting for adenosine: adopt 10-30 μ m Avidin to modify polystyrene microsphere as the fixing solid phase interface of aptamer, get the Avidin that 100 μ L concentration are 1%-3% and modify microballoon in centrifuge tube, with 100 μ L affinity elution liquid (20mM Tris pH7.5,1M NaCl, 1mM EDTA, 0.0005%Triton X-100) washed twice, centrifugal condition is 3500rpm, 5min, removes supernatant; Add respectively the affinity elution liquid of 44 μ L, the biotinylated casein of 3 μ L10-15 μ M and 3 μ L0.1-0.3 μ M aptamer as shown in table 1, normal temperature is hatched 10-15h; By centrifuge washing method, remove unconjugated molecule and functionalized microsphere is suspended in to 100 μ L affinity elution liquid.Aptamer and casein are by the biotin of modification and the Avidin specific binding of microsphere surface and be fixed on microsphere surface, thereby have formed the functional polystyrene microballoon with target molecule detectability.
(3) preparation of micro-fluidic micropearl array chip: first the chip structure pattern of design is drawn out by mapping software (CorelDRAW 9.0), and prepared the photomask of chip with the resolution printing of 2400dpi on the film film of Kodak; Then the pattern of photomask is transferred on the pcb board that is coated with photoresist by the method for uv-exposure, and on exposure pcb board, prepared the force plate of chip with chemical etching method; After finally aggressiveness before dimethyl silicone polymer being mixed in 10: 1 ratios with hardening agent, in vacuum pump, remove bubble, be then laid in (thick about 1mm) on chip force plate.Be placed in 65 ℃ of baking oven 3h, take out after cured, dimethyl silicone polymer (PDMS) sheet base is stripped down from force plate.Functionalized microsphere is fixed on and is take miniature cell in the array that unit forms by microballon carrier sheet base; After functionalization microballon is fixing, peel off microballon carrier sheet base and by microballoon fixedly array chip base and reagent transmission sheet base laminating structure detection chip.
(4) preparation of functionalization gold nano grain probe: get gold nano grain centrifugal 15min under 10000rpm prepared by 600 μ L and remove supernatant and be concentrated into 150 μ L, use 0.1M K
2cO
3adjusting pH is 8.0 left and right, adds subsequently 9.0 μ L horseradish peroxidase solution (5 μ g/ μ L), reacts 30 minutes under room temperature, adds capture probe as shown in table 1 60 μ L (10 μ M), 200rpm reaction 20h in shaking table at 10 ℃.Add damping fluid to make in system containing 10mM PBS, 0.02%Tween-20,0.15MNaCl (final concentration), 200rpm reaction 20h in shaking table at 10 ℃.The last centrifugal supernatant that goes of 10000rpm15min, and
The oligonucleotide probe (5 '-3 ' end) that table 1 is synthetic
With cleansing solution washing 5 times, Eddy diffusion is (cleansing solution composition: 0.01M PBS, 0.15M NaCl, 0.025%Tween20,0.1%BSA) in 150 μ L cleansing solutions, in 4 ℃ of refrigerators, preserves.
(5) (a) the interior adenosine of chip detects: according to the operating process shown in Fig. 1, the gold nano grain probe solution that is modified with capture probe and HRP 10 μ L prepared by 2nM step (4), comprise: 25mM Tris Acetate (pH8.2), 200mM NaCl, 5mM MgSO
40.025%Tween20, under pressure-driven, flow into prepared by step (3) the micro-fluidic micropearl array chip detection district that comprises functionalization micropearl array, hatch 30min for 37 ℃, with eluent, ((25mM Tris acetate (pH8.2), 150mM NaCl) washs 5min; Flow under 10 μ L10fM-10nM adenosine solution (comprising: 25mM Tris acetate (pH8.2), 150mM NaCl) normal temperature and hatch 25min; After 5min wash-out, flow into 10 μ L horseradish peroxidase substrate solutions, comprising: 0.008%H
2o
2, 300 μ M biotin-tyramine, 50mM NaCl, 50mM borate (pH8.8), hatch 60min in 37 ℃, after passage wash-out, the affinity element of dilution in 1: 20 is modified to quantum dot solution and passed into detection zone and react 30min with microballoon, finally use TE solution washing 5min.Reacted chip is placed under the CCD imaging system of fluorescence inverted microscope, particle is observed to shooting, and analyze with fluoroscopic image analysis software, by calculating, flow into after adenosine microsphere surface fluorescence (F) and do not flow into adenosine microsphere surface fluorescence (F
0) ratio (F/F
0) adenosine is carried out quantitatively; By obtaining curve 1 as shown in Figure 2 to the chip detection of variable concentrations adenosine.
(b) chip extracellular adenosine detects: in the Eppendorf of 0.2ml pipe, build reaction system outside chip, comprise: 1-2 μ L functionalization microballon, the gold nano grain probe that is modified with capture probe and HRP prepared by 2nM step (4), 25mM Tris Acetate (pH8.2), 200mM NaCl, 5mM MgSO
4, 0.025%Tween20, hatches 30min for 37 ℃; With eluent ((25mM Tris acetate (pH8.2), 150mM NaCl) washing 2 times, the centrifugal supernatant that goes; Add under 10 μ L10fM-10nM adenosine solution (comprising: 25mM Tris acetate (pH8.2), 150mM NaCl) normal temperature and hatch 25min; After washing, add 10 μ L horseradish peroxidase substrate solutions, comprising: 0.008%H
2o
2, 300 μ M biotin-tyramine, 50mM NaCl, 50mM borate (pH8.8), hatches 60min in 37 ℃, washs after centrifugal and removes supernatant, add the affinity element of dilution in 1: 20 to modify quantum dot solution normal-temperature reaction 30min, finally use TE solution washing 2 times.Reacted chip is placed under the CCD imaging system of fluorescence inverted microscope, particle is observed to shooting, and analyze with fluoroscopic image analysis software, by calculating, flow into after adenosine microsphere surface fluorescence (F) and do not flow into adenosine microsphere surface fluorescence (F
0) ratio (F/F
0) adenosine is carried out quantitatively; By obtaining curve 2 as shown in Figure 2 to the chip detection of variable concentrations adenosine.Result shows: adopt the minimum adenosine that 0.1pM can be detected of micro-fluidic control chip sensor building, and adopt the outer minimum adenosine molecule that 50pM can only be detected of detection method of chip.Adenosine sensing technology based on chip has improved 500 times compared with the outer detection system sensitivity of chip, the material Transfer of microfluid is strengthened the property and has been caused the highly sensitive sensitivity for analysis outside chip that in chip, adenosine is analyzed as can be seen here, and this has fully proved the applicability that the method is analyzed for highly sensitive adenosine.
(6) analytical performance of blood serum sample: the gold nano grain probe solution that is modified with capture probe and HRP 10 μ L prepared by 2nM step (4), comprising: 25mM Tris Acetate (pH8.2), 200mM NaCl, 5mM MgSO
40.025%Tween20, under pressure-driven, flow into prepared by step (3) the micro-fluidic micropearl array chip detection district that comprises functionalization micropearl array, hatch 30min for 37 ℃, with eluent, ((25mM Tris acetate (pH8.2), 150mM NaCl) washs 5min; 10% serum that inflow comprises variable concentrations adenosine, hatches 25min under normal temperature; After 5min wash-out, flow into 10 μ L horseradish peroxidase substrate solutions, comprising: 0.008%H
2o
2, 300 μ M biotin-tyramine, 50mM NaCl, 50mM borate (pH8.8), hatch 60min in 37 ℃, after passage wash-out, the affinity element of dilution in 1: 20 is modified to quantum dot solution and passed into detection zone and react 30min with microballoon, finally use TE solution washing 5min.Reacted chip is placed under the CCD imaging system of fluorescence inverted microscope, particle is observed to shooting, and analyze with fluoroscopic image analysis software, by calculating, flow into after adenosine microsphere surface fluorescence (F) and do not flow into adenosine microsphere surface fluorescence (F
0) ratio (F/F
0) adenosine is carried out quantitatively; By the chip detection to the blood serum sample that comprises variable concentrations adenosine, and compare with solution example testing result.Result is as shown in Figure 3: blood serum sample can not disturb the detection of adenosine in chip, and testing result is close with solution example testing result.
The above, be only best mode for carrying out the invention, is anyly familiar with those skilled in the art in the technical scope that the present invention discloses, and the simple change of the technical scheme that can obtain apparently or equivalence are replaced and all fallen within the scope of protection of the present invention.
Claims (7)
1. the adenosine detection method based on micro-fluidic chip and aptamer recognition technology, is characterized in that, comprises the following steps:
(1) the adenosine aptamer of biotin modification and the casein of biotin modification are fixed on by biotin-avidin associated methods the surface formation functionalized microsphere that affinity element is modified microballoon, described microballoon is fixed in the corresponding miniature cell of micro-fluidic chip detection zone, forms micro-fluidic micropearl array detection chip;
(2) capture probe of horseradish peroxidase and sulfydryl modification is modified on nano particle as signal mark;
(3) functionalized nano grain flow step (2) being formed enters detection zone and the micropearl array hybridization of micro-fluidic micropearl array detection chip, flows into adenosine solution and hatch with hybridization microballoon after wash-out;
(4) flow into peroxidase substrate solution, by multienzyme nanowire signal amplifying technique, biotinylated tyrasamine is attached on the casein of microsphere surface, the quantum dot that flows into affinity element mark after wash-out, described quantum dot can be identified and fix with the biotin of combination on microsphere surface casein;
(5) by calculating, flow into microsphere surface fluorescence after adenosine, with the ratio that does not flow into adenosine microsphere surface fluorescence, adenosine is carried out to quantitative and qualitative analysis.
2. the adenosine detection method based on micro-fluidic chip and aptamer recognition technology according to claim 1, it is characterized in that: the adenosine aptamer of the biotin modification of the functionalized microsphere finishing described in step (1) and the casein of biotin modification, its volumetric molar concentration ratio is 1: 100～1: 120.
3. the adenosine detection method based on micro-fluidic chip and aptamer recognition technology according to claim 1, is characterized in that: the microballoon described in step (1) is any one in silicon dioxide microsphere, polystyrene type organic polymer microballoon, magnetic microsphere and biomacromolecule polymer microballoon.
4. the adenosine detection method based on micro-fluidic chip and aptamer recognition technology according to claim 1, is characterized in that: the nano particle described in step (2) is any one in gold nano grain, nano SiO 2 particle and polystyrene nanoparticles.
5. the adenosine detection method based on micro-fluidic chip and aptamer recognition technology according to claim 1, is characterized in that: the adenosine solution described in step (3) is hatched with hybridization microballoon, and incubation time is 20～25min.
6. the adenosine detection method based on micro-fluidic chip and aptamer recognition technology according to claim 1, is characterized in that: the peroxidase substrate solution described in step (4) comprises 0.007%～0.009%H
2o
2, the biotinylated tyrasamine of 250～350 μ M, 50～100mM NaCl.
7. the adenosine detection method based on micro-fluidic chip and aptamer recognition technology according to claim 1, it is characterized in that: the quantum dot described in step (4) is a kind of in CdSe, CdTe, CdS and compound quantum dot, and its transmitting boundary is 400nm～700nm.
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