CN109647555A - Micro-fluidic chip nanowire signal enhancing structure processing method - Google Patents
Micro-fluidic chip nanowire signal enhancing structure processing method Download PDFInfo
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- CN109647555A CN109647555A CN201910046479.5A CN201910046479A CN109647555A CN 109647555 A CN109647555 A CN 109647555A CN 201910046479 A CN201910046479 A CN 201910046479A CN 109647555 A CN109647555 A CN 109647555A
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
- B01L3/502707—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by the manufacture of the container or its components
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
- B01L2200/10—Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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Abstract
The present invention relates to a kind of micro-fluidic chip nanowire signal enhancing structure processing methods, include: step 1: preparing integrated metal nanostructure on solid phase carrier sheet glass using the method for glancing angle deposition, the metal Nano structure is gold nano structure or silver nanostructured;Step 2: DSP solution is added in the glass sheet surface for being integrated with metal Nano structure, reacts at room temperature, obtains the integrated metal nanostructure glass carrier that metallic nanostructured surface is bonded with DSP.Micro-fluidic chip nanowire signal enhancing structure processing method provided by the invention, processing technology is simple, pass through the specified region evaporation metal nanostructure in solid phase carrier, fluorescence enhancement is realized by localized surface plasmons resonance effect, substantially increase the fluorescence detection sensitivity of protein-chip, early detection and diagnosis to disease have important references value, can meet the needs of practical application well.
Description
Technical field
The invention belongs to field of biomedicine technology, and in particular to a kind of micro-fluidic chip nanowire signal enhancing structure processing
Method.
Background technique
Microfluidic chip technology is bases such as biology, chemistry, the sample preparation of medical analysis process, reaction, separation, detections
This operating unit is integrated on the chip of one piece of micro-meter scale, is automatically performed the technology of analysis overall process.Since it is in biology, change
The great potential in the fields such as, medicine has been developed as biology, chemistry, medicine, fluid, electronics, material, a machinery etc.
The brand-new research field of subject crossing.Protein-chip is the lattice array type hybridization hybrid chip that micrometeor is zero, belongs to micro-fluidic core
The specific type of piece (micro-chip).Traditional protein chip is usually integrally to be carried out at special chemistry to solid phase carrier
Reason, then known protein molecular is fixed thereon the testing protein that capture can be specifically bound therewith to realize that biomolecule is examined
It surveys and analyzes, strong technical support is provided for fields such as antigen and antibody and drug screenings.However, traditional protein chip
Fluorescent assay signal is very weak when solid phase carrier protein concentration is lower, and the detection sensitivity of protein-chip is too low, and processes work
Skill is complicated.
Summary of the invention
For above-mentioned problems of the prior art, it can avoid above-mentioned skill occur the purpose of the present invention is to provide one kind
The micro-fluidic chip nanowire signal enhancing structure processing method of art defect.
In order to achieve the above-mentioned object of the invention, technical solution provided by the invention is as follows:
A kind of micro-fluidic chip nanowire signal enhancing structure processing method, comprising:
Step 1: integrated metal nanostructure, institute are prepared on solid phase carrier sheet glass using the method for glancing angle deposition
Stating metal Nano structure is gold nano structure or silver nanostructured;
Step 2: DSP solution is added in the glass sheet surface for being integrated with metal Nano structure.
Further, in the step 2: be integrated with metal Nano structure glass sheet surface be added concentration be 3~
The DSP solution of 5mg/ml reacts 1.8~2.2h at room temperature;After cleaning up, obtains metallic nanostructured surface and be bonded with
The integrated metal nanostructure glass carrier of DSP.
Further, the micro-fluidic chip nanowire signal enhancing structure processing method further include:
Step 3: ELISA experiment is carried out on the glass carrier.
Further, the step 3 includes:
Step (1) is reacted at room temperature in the cTnI monoclonal antibody solution that 18~22 μ g/ml are added in the glass carrier surface
1.8~2.2h realizes cTnI capture;
After step (2) cleans the glass carrier, the bovine serum albumen solution for adding 4~6% reacts 0.8 at room temperature~
1.2h;
After step (3) cleans the glass carrier, the cTnI solution for adding various concentration reacts 1.8~2.2h at room temperature;
After step (4) cleans the glass carrier, the cTnI polyclonal antibody solution that 18~22 μ g/ml are added reacts at room temperature
1.8~2.2h;
After step (5) cleans the glass carrier, the rabbit-anti goat IgG solution of the fluorescent marker of 1.8~2.2 μ g/ml is added
0.8~1.2h is reacted at room temperature;
After step (6) cleans the glass carrier, the corresponding fluorescence intensity of each concentration c TnI is measured under fluorescence microscope, is obtained
It is compared to fluorescence immunoassay curve, and with without chemically treated clean sheet glass.
Further, in the step (3), the concentration values range of cTnI solution is 1pg/ml-1 μ g/ml.
Further, the temperature range of the room temperature is 22 DEG C~26 DEG C.
Further, in said step 1, when using the method evaporation metal nanostructure of glancing angle deposition, solid
One layer of metal die is fixed to realize that metal Nano structure, which is integrated in carrier, specifies region in phase carrier glass piece surface.
Further, in said step 1, the metal Nano structure being prepared be average diameter be 50nm-150nm,
With a thickness of 200nm-300nm, the metal nano array that spacing is 100nm-200nm.
Micro-fluidic chip nanowire signal enhancing structure processing method provided by the invention, processing technology is simple, by solid
The specified region evaporation metal nanostructure of phase carrier realizes fluorescence enhancement by localized surface plasmons resonance effect, greatly
The fluorescence detection sensitivity for improving protein-chip greatly, early detection and diagnosis to disease have important references value, can
To meet the needs of practical application well.
Detailed description of the invention
Fig. 1 is the flow diagram for preparing integrated gold nano structure on the glass sheet using the method for glancing angle deposition;
Fig. 2 show the silver nanostructured SEM figure comparison diagram of different-thickness;
Fig. 3 is the relational graph of silver nanostructured thickness and fluorescence enhancement;
Fig. 4 is fluorescence incident direction and the relational graph for enhancing factor;
Fig. 5 is the relational graph of wavelength and scattering and reflection;
Fig. 6 is silver nanostructured enhancing coefficient analogous diagram;
Fig. 7 is distance and electric-field enhancing relational graph;
Fig. 8 is fluorescence difference incident direction schematic diagram;
When Fig. 9 is using the chip for being integrated with gold nano structure, fluorescein concentration and fluorescence enhancement Relationship of Coefficients figure;
CTnI concentration and fluorescence intensity relational graph when Figure 10 is the chip detection cTnI for being integrated with gold nano structure;
DNA concentration and fluorescence intensity relational graph when Figure 11 is the chip detection DNA for being integrated with gold nano structure;
When Figure 12 is the chip detection DNA for being integrated with gold nano structure, DNA hybridization and fluorescence intensity relational graph at 50 DEG C;
Figure 13 is gold nano structure SEM figure side view;
Figure 14 is gold nano structure SEM figure top view;
Figure 15 is the effect picture that micro-fluidic chip of the invention detects protein.
Specific embodiment
In order to make the objectives, technical solutions, and advantages of the present invention clearer, with reference to the accompanying drawing and specific implementation
The present invention will be further described for example.It should be appreciated that described herein, specific examples are only used to explain the present invention, and does not have to
It is of the invention in limiting.Based on the embodiments of the present invention, those of ordinary skill in the art are not making creative work premise
Under every other embodiment obtained, shall fall within the protection scope of the present invention.
A kind of micro-fluidic chip nanowire signal enhancing structure processing method, comprising:
Step 1: using the method for glancing angle deposition (oblique angle deposition, OAD) in solid phase carrier glass
Glass on piece prepares integrated metal nanostructure, and the metal Nano structure is gold nano structure or silver nanostructured.
OAD is a kind of physical gas-phase deposite method, and principle is to be existed by the diffusion of shadow effect and adatom
Controllable nano array is grown in substrate.Using OAD method vapor deposition gold nano structure or it is silver nanostructured when, in solid phase carrier glass
One layer of metal die (shadow mask) is fixed to realize gold nano structure or silver nanostructured be integrated in carrier in glass piece surface
Specified region is as shown in Figure 1 the flow diagram of the integrated gold nano structure of preparation.
Region, fixed PDMS mold and integrated gold nano structure or silver nanostructured solid phase is specified to carry in the carrier
Body, for specifying region to be chemically treated in solid phase carrier.
The metal Nano structure being prepared in the present invention is that average diameter is 50nm-180nm, with a thickness of 200nm-
3000nm, the metal nano array (gold-nano array or silver nanoparticle array) that spacing is 100nm-200nm, preferred average diameter is
50nm-150nm, with a thickness of 200nm-300nm, spacing be 100nm-200nm metal nano array (gold-nano array or silver are received
Rice array), it is used for fluorescence enhancement, high detection sensitivity can be reached.
Step 2: it is added in the glass sheet surface for being integrated with metal Nano structure (gold nano structure or silver nanostructured) dense
Degree is the DSP solution of 3~5mg/ml (preferably 4mg/ml), reacts 1.8~2.2h (preferably 2h) at room temperature.
3, the 3'- dithiodipropionic acid phenodiazine-being dissolved in dimethyl sulfoxide (dimethyl sulfoxide, DMSO)
Hydroxysuccinimide ester (3,3'-Dithiodipropionic acid di (N-hydroxysuccinimide ester),
DSP) 2h is reacted with the glass sheet surface for being integrated with metal Nano structure (gold nano structure or silver nanostructured) at room temperature.
After cleaning up, the collection that metal Nano structure (gold nano structure or silver nanostructured) surface bond has DSP is obtained
At metal Nano structure glass carrier.
Step 3: ELISA experiment is carried out on the glass carrier;Specifically includes the following steps:
The cTnI of 18~22 μ g/ml (preferably 20 μ g/ml) is added on the glass carrier surface in step (1)
(cTnI) monoclonal antibody solution reacts 1.8~2.2h (preferably 2h) at room temperature, realizes cTnI capture;
After step (2) cleans the glass carrier, bovine serum albumin (BSA) solution room of 4~6% (preferably 5%) is added
Temperature is lower to react 0.8~1.2h (preferably 1h);
After step (3) cleans the glass carrier, add various concentration (concentration values range is 1pg/ml-1 μ g/ml)
CTnI solution reacts 1.8~2.2h (preferably 2h) at room temperature;
After step (4) cleans the glass carrier, the cTnI polyclonal antibody of 18~22 μ g/ml (preferably 20 μ g/ml) is added
(Capra) solution reacts 1.8~2.2h (preferably 2h) at room temperature;
After step (5) cleans the glass carrier, the rabbit-anti of the fluorescent marker of 1.8~2.2 μ g/ml (preferably 2 μ g/ml) is added
Goat IgG solution reacts 0.8~1.2h (preferably 1h) at room temperature;
After step (6) cleans the glass carrier, the corresponding fluorescence intensity of each concentration c TnI is measured under fluorescence microscope, is obtained
It is compared to fluorescence immunoassay curve, and with without chemically treated clean sheet glass.
The room temperature mentioned in the present embodiment refers to 22 DEG C~26 DEG C of temperature range.
The silver nanostructured chip of different parameters is tested, its fluorescence enhancement effect, the Yin Na of different parameters are tested
Rice fabric chip experimental data is as follows:
Experimental result referring to figure 2.~Fig. 8 shown in, wherein Fig. 2 show different-thickness silver nanostructured SEM figure pair
Than figure;Fig. 3 is the relational graph of silver nanostructured thickness and fluorescence enhancement;Fig. 4 is fluorescence incident direction and the pass for enhancing factor
System's figure;Fig. 5 is the relational graph of wavelength and scattering and reflection;Fig. 6 is silver nanostructured enhancing coefficient analogous diagram;Fig. 7 be distance with
Electric-field enhancing relational graph;Fig. 8 is fluorescence difference incident direction schematic diagram.
It is tested using the chip for being integrated with gold nano structure, experiment content and result are with reference to shown in Fig. 9~Figure 15, figure
9 be fluorescein concentration and fluorescence enhancement Relationship of Coefficients figure when using the chip for being integrated with gold nano structure;Figure 10 is to be integrated with gold
CTnI concentration and fluorescence intensity relational graph when the chip of nanostructure detects cTnI;Figure 11 is the chip for being integrated with gold nano structure
DNA concentration and fluorescence intensity relational graph when detecting DNA;When Figure 12 is the chip detection DNA for being integrated with gold nano structure, at 50 DEG C
DNA hybridization and fluorescence intensity relational graph;Figure 13 is gold nano structure SEM figure side view;Figure 14 is that gold nano structure SEM figure is overlooked
Figure;Figure 15 is the effect picture that micro-fluidic chip of the invention detects protein.
Micro-fluidic chip nanowire signal enhancing structure processing method provided by the invention, processing technology is simple, by solid
The specified region evaporation metal nanostructure (gold nano structure or silver nanostructured) of phase carrier, passes through local surfaces plasma
Resonance effects realizes fluorescence enhancement, substantially increases the fluorescence detection sensitivity of protein-chip, to the early detection of disease with
Diagnosis has important references value, can meet the needs of practical application well.
Embodiments of the present invention above described embodiment only expresses, the description thereof is more specific and detailed, but can not
Therefore limitations on the scope of the patent of the present invention are interpreted as.It should be pointed out that for those of ordinary skill in the art,
Without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to protection model of the invention
It encloses.Therefore, the scope of protection of the patent of the invention shall be subject to the appended claims.
Claims (8)
1. a kind of micro-fluidic chip nanowire signal enhancing structure processing method characterized by comprising
Step 1: integrated metal nanostructure, the gold are prepared on solid phase carrier sheet glass using the method for glancing angle deposition
Category nanostructure is gold nano structure or silver nanostructured;
Step 2: DSP solution is added in the glass sheet surface for being integrated with metal Nano structure.
2. micro-fluidic chip signal enhancing structure processing method according to claim 1, which is characterized in that in the step
In two: the DSP solution that concentration is 3~5mg/ml is added in the glass sheet surface for being integrated with metal Nano structure, at room temperature instead
Answer 1.8~2.2h;After cleaning up, the integrated metal nanostructure glass load that metallic nanostructured surface is bonded with DSP is obtained
Body.
3. micro-fluidic chip signal enhancing structure processing method according to claim 1 to 2, which is characterized in that the miniflow
Control chip nanowire signal enhancing structure processing method further include:
Step 3: ELISA experiment is carried out on the glass carrier.
4. micro-fluidic chip signal enhancing structure processing method according to claim 1 to 3, which is characterized in that the step
Three include:
Step (1) reacts 1.8 in the cTnI monoclonal antibody solution that 18~22 μ g/ml are added in the glass carrier surface at room temperature~
2.2h realizes cTnI capture;
After step (2) cleans the glass carrier, the bovine serum albumen solution for adding 4~6% reacts 0.8~1.2h at room temperature;
After step (3) cleans the glass carrier, the cTnI solution for adding various concentration reacts 1.8~2.2h at room temperature;
After step (4) cleans the glass carrier, the cTnI polyclonal antibody solution that 18~22 μ g/ml are added reacts 1.8 at room temperature
~2.2h;
After step (5) cleans the glass carrier, the rabbit-anti goat IgG solution room temperature of the fluorescent marker of 1.8~2.2 μ g/ml is added
0.8~1.2h of lower reaction;
After step (6) cleans the glass carrier, the corresponding fluorescence intensity of each concentration c TnI is measured under fluorescence microscope, is obtained glimmering
Curve is immunized in light, and compares with without chemically treated clean sheet glass.
5. micro-fluidic chip signal enhancing structure processing method described in -4 according to claim 1, which is characterized in that in the step
Suddenly in (3), the concentration values range of cTnI solution is 1pg/ml-1 μ g/ml.
6. micro-fluidic chip signal enhancing structure processing method according to claim 2 or 4, which is characterized in that the room
The temperature range of temperature is 22 DEG C~26 DEG C.
7. micro-fluidic chip signal enhancing structure processing method according to claim 1, which is characterized in that in the step
In one, when using the method evaporation metal nanostructure of glancing angle deposition, one layer of gold is fixed in solid phase carrier glass sheet surface
Belong to mold to realize that metal Nano structure, which is integrated in carrier, specifies region.
8. micro-fluidic chip signal enhancing structure processing method according to claim 1, which is characterized in that in the step
In one, the metal Nano structure being prepared is that average diameter is 50nm-150nm, is with a thickness of 200nm-300nm, spacing
The metal nano array of 100nm-200nm.
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