CN104849254B - A kind of micro-fluidic detection chip based on Surface enhanced Raman scattering - Google Patents
A kind of micro-fluidic detection chip based on Surface enhanced Raman scattering Download PDFInfo
- Publication number
- CN104849254B CN104849254B CN201410054113.XA CN201410054113A CN104849254B CN 104849254 B CN104849254 B CN 104849254B CN 201410054113 A CN201410054113 A CN 201410054113A CN 104849254 B CN104849254 B CN 104849254B
- Authority
- CN
- China
- Prior art keywords
- raman
- exogenous
- checked
- chip
- noble metal
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Abstract
The invention discloses a kind of micro-fluidic detection chip based on Surface enhanced Raman scattering.A kind of micro-fluidic detection chip disclosed by the invention, it is that the micro-fluidic detection chip is overlapped to be fixed on carrier successively and formed by material, active substrate, conjugate pad and the sample pad that can produce REFRIGERATION SYSTEM DRIVEN BY CAPILLARY FORCE power according to the order based on made of the principle of Surface enhanced Raman scattering.The advantage of the invention is that：（1）Micro- pointed cone array active substrate is beneficial to uniformly sputter with not staying dead angle or deposited metal layer；（2）Highly sensitive Surface enhanced Raman scattering technology is introduced on the basis of the chromatograph test strip advantage of inheriting tradition, can not only effectively improve sensitivity, can also carry out quantitative analysis；（3）The design of supporting detection device, difficulty of processing are reduced using the self-driven pattern of capillary force, reduces the weight of equipment, enhances the portability of equipment, while also reduce cost.
Description
Technical field
The present invention relates to a kind of micro-fluidic detection chip based on Surface enhanced Raman scattering.
Background technology
One monochromic beam can be scattered when inciding non-uniform medium by medium molecule, frequency, photon such as incident light
When energy size and photon direction change, our inelastic scatterings are referred to as Raman scattering.Raman scattering spectrum can quilt
For characterizing the vibration level of molecule, there is important application in fields such as chemistry, physics, biomedicines.Using drawing
Measuring samples state can be measured without particular/special requirement, liquid phase, solid phase, gas phase sample when graceful scattering spectrum is detected, and had
Have it is non-contact, have to the nondestructive advantage of sample, therefore in biological sample analysis, medicine detection, environmental monitoring etc.
Huge application potential.
However, before laser light source appearance, since the sensitivity of Raman diffused light spectrum analysis is too low, once hindering this
The application of analysis means.From Fleischmann in 1974（Fleischman M,et al,Chem.Phys.Lett.1974,26,
163-166）Deng researcher first since coarse silver electrode surface observes the enhancing Raman scattering signal of Pyridine Molecules,
This new surface photochemical effect-Surface enhanced Raman scattering（Surface Enhanced Raman Scattering,
SERS）Played important in analysis field, the particularly application in the detection of ultrasensitive biological medicine for raman scattering spectrum
Impetus.Surface enhanced Raman scattering refers to coarse noble metal（Such as gold, silver, copper）Or its Nano sol is substrate
The Raman diffused light spectrum signal that surface can be been significantly enhanced when analyzing thing molecule to be checked.Surface enhanced Raman scattering
The enhancing order of magnitude is high, and hollow porous silver-colored microballoon of this laboratory using bacterium as templated synthesis is used as substrate 4-Mercaptopyridine to carry out
Detection, minimum detection limit is up to 10-15M, enhancer reach 1011（Dapeng Yang,et al,Green Chem.2010,
12,2038-2042）.
Immuno-chromatographic assay technology is a kind of bioanalysis means to grow up the eighties in last century, with colloidal gold, glue
It is tracer probe after the labelled antibodies such as body selenium, coloring latex beads or antigen, when liquid sample to be checked is under capillarity driving
Pass sequentially through sample pad, tracer probe pads, cure when having nitrocellulose filter, the water absorption pad of target ligands on nitrocellulose filter
Capture the simultaneously corresponding target molecule of colour generation.The technology is extremely low, easy to operate with manufacturing cost（Only it is loaded an operating procedure）、
It is flexibly and quick to detect flux（Result is obtained in 15 minutes）The advantages of, thus obtained widely in field of biological medicine
Using.But due to above-mentioned common marker material and based on the intrinsic limitation of nitrocellulose filter detection scheme, technology inspection
Survey sensitivity is relatively low, and can not carry out the accurate quantification analysis of target molecule.
So far, existing a variety of different micro-fluidic chip schemes based on Surface Enhanced Raman Scattering Spectrum analysis are used
In analysis field, such as the patent that number of patent application is 201110131032.1《Micro-fluidic Surface enhanced Raman scattering detects device
And preparation method and application》, using the micro-fluidic chip being roughened based on nanometer concave surface prepared by active substrate；Patent
The patent of Application No. 201010117672.2《A kind of micro fluid control detection based on surface-enhanced Raman scattering activity substrate
Part》, using preparing nano-pillar or nanofiber rising structure on substrate and splash-proofing sputtering metal nano-particle layer obtains surface enhanced
Raman scattering active substrate builds micro fluid control detection device；And for example number of patent application is 201110040128.7 patent《One
The analysis system of the kind micro-fluidic Surface enhanced Raman scattering dedicated test chip of array type》, consolidated using multiple dot array active reaction area
Change different tumor markerses, the unmarked swollen of more markers is carried out using the Raman fingerprint databases of various tumor markerses
Tumor markers are analyzed；For another example the patent of Application No. 200610008767.4《With Surface Enhanced Raman Scattering Spectrum active group
The micro-fluidic chip and preparation method at bottom》, coin is prepared using physical evaporation, sputtering or the method for chemical deposition combination mask technique
Race's metal film layer prepares micro-fluidic chip with Surface Enhanced Raman Scattering Spectrum active substrate etc..Using exogenous
Driving force drives the sample introduction of measuring samples, and the designing scheme of such patent must set sample to drive in supporting detector
Module so that the design of detector complicates, while also improves the manufacturing cost of detector.Go out from the angle of practical application
Hair, the increase of function module can also increase the weight of equipment, reduce the portability of equipment.In addition, high-aspect-ratio is vertical poroid
The vertical column construction of structure or draw ratio is also unfavorable for sputtering or deposited metal layer in preparation process is processed, and adds preparation
The complexity of processing.
The content of the invention
The object of the present invention is to provide a kind of micro-fluidic detection chip based on Surface enhanced Raman scattering.
A kind of micro-fluidic detection chip provided by the invention, is based on made of the principle of Surface enhanced Raman scattering, is somebody's turn to do
Micro-fluidic detection chip is by that can produce material, active substrate, conjugate pad and the sample pad of REFRIGERATION SYSTEM DRIVEN BY CAPILLARY FORCE power according to the order
Overlap joint, which is fixed on carrier, successively forms；
The carrier can be any carrier prepared for test strips or prepared by chip, specifically can be such as plastic casing；
The conjugate pad has the polyester film or glass film of exogenous Raman microprobe to cure；
The cured mode combines for non-bonding；
The exogenous Raman microprobe is can be exogenous with the ligand of matter interaction to be checked and with Raman active
The targeting probe that the noble metal nano particles that molecule coats jointly are formed；
The targeting refers to that specificity is directed to material to be checked；
The active substrate is to set test section and matter on the surface of substrate used when Surface enhanced Raman scattering is analyzed
Obtained from control area；
Be connected with the test section it is described can be with the ligand of matter interaction to be checked, being connected with quality control region cannot be with
Matter interaction to be checked but can be with the material of the ligand binding of the matter interaction to be checked；
Side is padded in the test section in the conjugate, and quality control region is in the material side for producing REFRIGERATION SYSTEM DRIVEN BY CAPILLARY FORCE power；
It can be combined, be treated with material to be checked with the ligand of matter interaction to be checked on the test section of the active substrate
Material is examined again with the exogenous Raman microprobe double-antibody sandwich can be passed through with the ligand binding of matter interaction to be checked
Exogenous Raman microprobe is fixed on detection zone by method, by the characteristic for detecting the exogenous molecule on exogenous Raman microprobe
Raman scattering peak intensity reacts the content of material to be checked；
In the quality control region of the active substrate cannot be with matter interaction to be checked but can be with matter interaction to be checked
Ligand binding material can with exogenous Raman microprobe can be with the ligand binding of matter interaction to be checked, it is not necessary to
Exogenous Raman microprobe can be fixed on by quality control region by material to be checked, by detecting the external source on exogenous Raman microprobe
Property molecule characteristic Raman scattering peak intensity to micro-fluidic chip carry out Quality Control.
In said chip, the material for producing REFRIGERATION SYSTEM DRIVEN BY CAPILLARY FORCE power is filter paper, glass film or polymer absorbent material；
The sample pad is glass film；
The noble metal nano particles are nano Au particle or nanometer gold bar；
It is described to be connected with the ligand of matter interaction to be checked by the effect including golden sulfide linkage and nano Au particle
Connect；
The exogenous molecule is connected by the effect including golden sulfide linkage with nano Au particle；
The substrate used when Surface enhanced Raman scattering is analyzed is covered with having for noble metal thin layer for surface and dashes forward upwards
The polymer thick film or plate of the cone-shaped array of structures gone out；
The polymer for can curing molding under certain condition polymer；
It is any enclosed geometric figure at the top of the cone hole of the pointed cone.
The closure geometric figure is specially triangle, trapezoidal, ellipse or circular；
The cone-shaped array of structures is micro- pointed cone array of structures；
Cone height in micro- pointed cone array of structures is 20-500 microns, and cone floor space is micro- for 225-250000 squares
Rice；
The thickness of the noble metal thin layer is 30-300nm；
The thickness of the polymer thick film or plate is 100-3000um；
It is described can the polymer of curing molding under certain condition be polyimides or dimethyl silicone polymer；
Described can be antibody or antigen with the ligand of matter interaction to be checked.
In any of the above-described chip, the exogenous molecule with Raman active is 5,5 ' two thiobis (2- nitre
Yl benzoic acid)；
The material to be checked is ricin (WA)；
The antibody is antiricin polyclonal antibody；
It is described cannot be with matter interaction to be checked but can be sheep with the material of the ligand binding of matter interaction to be checked
Anti-rabbit polyclonal antibody；
What the surface was covered with noble metal thin layer is with cone-shaped the array of structures polymer thick film or plate projected upwards
First pass through the polymer thick film or plate of the polyethylene glycol functionalization of mercapto carboxy difunctionalization；
The polyethylene glycol of mercapto carboxy difunctionalization is connected to the surface by golden sulfide linkage and is covered with noble metal thin layer
With on cone-shaped the array of structures polymer thick film or plate projected upwards；
The antiricin polyclonal antibody and goat-anti rabbit polyclonal antibody are connected to mercapto carboxy pair by amido link
On the polyethylene glycol of functionalization, so be fixed on the surface be covered with noble metal thin layer with the cone-shaped structure projected upwards
On aligned polymer thick film or plate；
The polymer is dimethyl silicone polymer；
The thickness of the polymer thick film or plate is 5000 μm；
The noble metal is gold；
The thickness of the noble metal thin layer is 200nm；
The closure geometric figure is circle；
The pattern diameter is 50 μm, and the distance between circular and circle is 30 μm.
The a length of 3mm of conjugate pad, width 2mm；
The a length of 3mm of sample pad, width 2mm；
The polymer absorbent material a length of 5, width 2mm；
The a length of 4mm of active substrate, width 2mm.
In any of the above-described chip, the preparation method of the exogenous Raman microprobe is as follows：By it is described can with it is to be checked
The ligand of matter interaction is added in the noble metal nano particles solution, is incubated, and supernatant is abandoned in centrifugation to be precipitated；With containing
Sediment is resuspended in the PBS of the 0.1M of the pH7.4 of BSA, adds the exogenous molecule with Raman active, reaction, centrifuges
Precipitation；It is resuspended in containing BSA, PEG20000, Na3N, the PBS of the 10mM pH7.4 of sucrose, trehalose and Tween-20 delay
In fliud flushing, the exogenous Raman microprobe solution is obtained；
The conjugate pad is prepared as follows：The polyester film or glass film are immersed into the exogenous Raman
In probe solution, it is dried overnight to obtain the final product；
The effect of the BSA is that closing can be received with the ligand and exogenous molecule of matter interaction to be checked cladding noble metal
Residual activity site after rice corpuscles；
The active substrate is prepared as follows：It is described in Surface enhanced Raman scattering with the immersion of Piranha solution
Substrate used during analysis, is cleaned, then is dipped in the Aqueous Solutions of Polyethylene Glycol of mercapto carboxy difunctionalization and is reacted, and obtains function
Substrate after change；With 1- ethyls -3- (3- dimethyl aminopropyls)-carbodiimides and n-hydroxysuccinimide can with it is to be checked
Test section is made in the ligand coupling of matter interaction in the substrate of functionalization, equally cannot be with material phase interaction to be checked
With but can be coupled at the material of the ligand binding of matter interaction to be checked in the substrate of functionalization quality control region is made.
In any of the above-described chip, in the preparation method of the exogenous Raman microprobe, the temperature of the incubation is 4
℃；The centrifugation, which abandons after supernatant must precipitate to further include to be suspended again with the PBS buffer of pH7.40.1M, to be precipitated and at 25 DEG C
Stand, the step of supernatant obtains sediment is abandoned in 4 DEG C of centrifugations；The bar for adding the exogenous molecule with Raman active and being reacted
Part be continuously stirred at 4 DEG C 1 it is small when；It is described to be resuspended in containing BSA, PEG20000, Na3N, sucrose, trehalose and
Being further included before in the PBS buffer of the 10mM pH7.4 of Tween-20 must be sunk twice with the PBS centrifuge washings of the 0.1M of pH7.4
The step of shallow lake；
In the preparation method of the active substrate, the temperature of the reaction is 25 DEG C, when the time is 4 small.
A kind of method for preparing any of the above-described chip falls within protection scope of the present invention, including prepares the knot
The method of compound pad and the method for preparing the active substrate；
The method for preparing the conjugate pad, includes the following steps：It is molten that glass film is immersed into exogenous Raman microprobe
In liquid, it is dried overnight to obtain the final product；
The temperature of the drying is 36 DEG C；
The exogenous Raman microprobe solution is prepared as follows：By can be with described in any of the above-described chip
4 DEG C of incubations of the ligand of matter interaction to be checked and noble metal nano solution, 4 DEG C centrifuge and must precipitate, and precipitation is dissolved in containing BSA
PH7.4 0.1M PBS, add the exogenous molecule with Raman active, 4 DEG C of stirrings, 4 DEG C centrifuge and must precipitate, and obtain
The noble metal nano particles that can be coated jointly with the ligand of matter interaction to be checked and the exogenous molecule with Raman active,
It is eventually adding containing BSA, PEG20000, Na3N, the PBS buffer of the 10mM pH7.4 of sucrose, trehalose and Tween-20 are
；
Concentration of the BSA in the exogenous Raman microprobe solution is 1g/100ml, and the PEG20000 is described
Concentration in exogenous Raman microprobe solution is 0.2g/100ml, the Na3N is dense in the exogenous Raman microprobe solution
Spend for 0.02g/100ml, concentration of the sucrose in the exogenous Raman microprobe solution is 10g/100ml, the seaweed
Concentration of the sugar in the exogenous Raman microprobe solution is 2.5g/100ml, and the Tween-20 is visited in the exogenous Raman
Concentration in pin solution is 0.1g/100ml；
The noble metal nano is specially nanogold；
It is described can with the ligand of matter interaction to be checked with the nanogold by including the effect knot including golden sulfide linkage
Close, the exogenous molecule with the nanogold by being combined comprising the effect including golden sulfide linkage, the BSA close can with it is to be checked
Remaining site after ligand and exogenous molecule the cladding nanogold of matter interaction, finally obtains the exogenous Raman and visits
Pin；
The method for preparing the active substrate, includes the following steps：When institute will be analyzed in Surface enhanced Raman scattering
Functionalization is carried out with substrate, obtains the substrate of functionalization；Test section and quality control region are set in the substrate of functionalization, can be with treating
Test section is made in the substrate surface that the ligand of inspection matter interaction is connected to functionalization, it is impossible to material phase interaction to be checked
With but the substrate surface of functionalization can be connected to the material of the ligand binding of matter interaction to be checked quality control region is made；
The substrate used when Surface enhanced Raman scattering is analyzed be specially surface be covered with noble metal thin layer have to
Cone-shaped the array of structures polymer thick film or polymer sheet of upper protrusion；
The functionalization is by the substrate Piranha solution immersion used when Surface enhanced Raman scattering is analyzed, and uses water
25 DEG C of reactions in the Aqueous Solutions of Polyethylene Glycol of mercapto carboxy difunctionalization are dipped in after cleaning again；
The Piranha solution is the aqueous solution of the hydrogen peroxide of the concentrated sulfuric acid and volumn concentration 30% according to volume ratio
3:1 is uniformly mixed so as to obtain；
The time of the immersion is 10-15 minutes；
The surface be covered with noble metal thin layer with the cone-shaped array of structures polymer thick film or polymerization projected upwards
Thing plate is to be formed described with cone-shaped the array of structures polymer thick film or polymer sheet that project upwards sputtering noble metal；
Described with the cone-shaped array of structures polymer thick film projected upwards can be cured described under certain condition
Molding polymer uniformly mixes after-pouring on former with corresponding curing agent, peels off and is solidified into from former after curing
The polymer thick film of type obtains；
The curing agent is the material that can promote or control curing reaction, specific such as silicol；
Or,
It is described with the cone-shaped array of structures polymer sheet projected upwards be by cured polymer sheet and silicon former heat
After pressure bonding, cured polymer sheet is peeled off from former and is obtained；
The former is the former for having cone hole array on hard substrate surface；
The hard substrate is silicon chip or ceramics；
The former is silicon former；
The silicon former is that the silicon chip with the silicon cone hole array being etched is put into corrosion in silicon etch solution to obtain；
The silicon etch solution is by potassium hydroxide：Isopropanol：Water is according to quality 1：2：2 ratio mixes to obtain the final product；
The silicon chip with the silicon cone hole array being etched is to aoxidize silicon chip surface to obtain oxide layer, in oxide layer
Upper polishing one side gets rid of one layer of positive photoetching rubber, the silicon dioxide pattern of the needs that develop etching, by SiO2Corrosive liquid corrosion of silicon is sudden and violent
Reveal silicon dioxide layer, remove positive photoetching rubber, wet etching to obtain the final product；
The SiO2Corrosive liquid is by HF, NH4F and water are mixed according to the ratio of 84ml, 339g, 510ml to obtain the final product；
The former can also use microelectronics and micromechanics（MEMS）It is processed into precision machinery technology with cone hole battle array
The former of row；
The mass ratio of the polymer and the curing agent is specially 10：1；
The oxidated layer thickness is specially 0.1-2 μm；
The thickness of the positive photoetching rubber is specially 5-50 μm.
Whether the method containing desired substance falls within protection scope of the present invention in a kind of qualitative measuring samples of auxiliary, bag
Include following steps：The sample for not containing desired substance is added in the sample pad of any of the above-described chip, mesh not contained
Material sample completely by the chip the polymer absorbent material absorb after, the chip is placed in Raman
On spectrum detection instrument, the characteristic for detecting the exogenous molecule in the Raman scattering peak region wave number of exogenous molecule is drawn
Graceful peak intensity, is averaged plus the value of twice of standard deviation is denoted as Cut-off values；After the same method, measuring samples are carried out
Detection, obtains the characteristic Raman peak intensity of measuring samples, if the characteristic Raman peak intensity of measuring samples is greater than or equal to
Then measuring samples are determined as the sample containing desired substance to Cut-off values, if the characteristic Raman peak intensity of measuring samples is low
In Cut-off values, then measuring samples are judged to not containing the sample of desired substance.
A kind of method for quantitatively detecting desired substance in sample falls within protection scope of the present invention, includes the following steps：
The standard items of the desired substance of various concentrations are added in the sample pad of any of the above-described chip, treat desired substance quilt completely
After the polymer absorbent material on the chip absorbs, chip is placed on Raman spectrum detector, detection is outside
The characteristic Raman peak intensity of the exogenous molecule in the Raman scattering peak region wave number of source property molecule is dense with desired substance
The lg values of degree are abscissa, and characteristic Raman peak intensity is ordinate, make standard curve, obtain calibration curve formula；According to
Same method, is detected measuring samples, the characteristic Raman peak intensity of measuring samples is obtained, by the feature of measuring samples
Property Raman peaks intensity brings calibration curve formula into, obtains the content of desired substance in measuring samples；
The exogenous molecule is specially 5,5 ' two thiobis (2- nitrobenzoic acid)；
The Raman scattering peak region wave number of the exogenous molecule is specially 1330cm-1；
The unit of the desired substance concentration is specially pg/mL；
The desired substance is specially ricin (WA)；
The calibration curve formula is specially y=1395.7x-2077.5.
Application of any of the above-described chip in the product of the desired substance in preparing qualitative or quantitative detection sample
Fall within protection scope of the present invention.
In above application, the desired substance is ricin (WA).
The advantage of the invention is that：（1）Micro- pointed cone array active substrate is beneficial to uniformly not sputter or deposit gold with not staying dead angle
Belong to layer；（2）Highly sensitive Surface enhanced Raman scattering technology is introduced on the basis of the chromatograph test strip advantage of inheriting tradition,
Sensitivity can be not only effectively improved, can also carry out quantitative analysis；（3）Supporting detection is reduced using the self-driven pattern of capillary force to set
Standby design, difficulty of processing, reduce the weight of equipment, enhance the portability of equipment, while also reduce cost.
Brief description of the drawings
Fig. 1 is the standard curve that micro-fluidic detection chip detects ricin (WA).
Embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples, is commercially available unless otherwise specified.
Castor-oil plant (Ricinus communis) seed is purchased from Beijing Xiu He seeds Co., Ltd, and catalog number is castor-oil plant 1
Number.
Equilibrium liquid is 0.01M, the PBS buffer of pH7.2.
Agarose affinity chromatography column is purchased from Amersham companies, catalog number 17-5080-01.
The purebred large ear rabbit of New Zealand is purchased from Test Animal Centre, Academy of Military Medical Sciences, P.L.A.
The preparation method of antiricin polyclonal antibody is as follows in following embodiments：
（One）Prepare the ricin (WA) of detoxification
Slightly carry：100g is weighed after castor seeds are shelled, is soaked in 24h in PBS (0.01M, pH7.2), to seed swelling；
Shell clean, clean seed is placed in 500mL PBS (0.01M, pH7.2) buffer solution, is homogenized in refiner, and in 4
24h overnight is extracted under the conditions of DEG C（Place 24h）, obtain leaching liquor；Next day, after leaching liquor is filtered off residue with absorbent gauze, at 4 DEG C
Under with 12000rpm centrifuge 20min, take supernatant, 0.45 μm of membrane filtration, supernatant tune pH7.2；Slowly add into supernatant
It is 30% to enter saturated ammonium sulfate solution (pH7.4) to saturation degree, after 4 DEG C of magnetic agitation 1h it is static 1 it is small when, 12000rpm centrifugations
20min, abandons and precipitates to obtain supernatant；Supernatant is continued plus saturated ammonium sulfate liquid is to 90% saturation degree, 4 DEG C of magnetic agitation 1h are quiet
24h is only stayed overnight, obtains mixed liquor；Mixed liquor is centrifuged 20min by next day under the conditions of 4 DEG C with 12000rpm, is taken and is precipitated as white；Will
Precipitation is with after PB (10mM, pH7.4) 50mL dissolvings, and dialyse PB under the conditions of 4 DEG C 48h（A not good liquor is changed per 8h）, passed through after dialysis
1200g4 DEG C of centrifugation 20min, takes supernatant, is ricin (WA) crude extract, carries out protein quantification, 12% denaturation, non denatured SDS electricity
Swimming analysis；It is placed on -20 DEG C of stored frozens.
Affinitive layer purification：Affinity chromatography medium is aminophenyl-β-D- galactoside agarose compatible mediums（It is purchased from
Sigma companies, catalog number A-0414）.After taking 5mL affinity media vacuum outgas, slowly load chromatographic column, pay attention to avoiding
Bubble and tomography are produced, cleaning balance is carried out with the PBS of at least 10 times bed volumes.By ricin (WA) crude extract through 0.45 μm
After membrane filtration, with 10 times of volume dilutions of equilibrium liquid, added with 1mL/min flow velocitys in chromatographic column, make ricin (WA) and agglutinin
Fully absorption is on column.After ricin (WA) and agglutinin absorption fully, foreign protein is eluted with equilibrium liquid, flow control is in 1mL/
Min or so, after foreign protein elution completely, uses the PBS buffer one-step elution destination protein containing 0.1M galactolipins instead, collection is washed
De- peak, 12% denaturation, non denatured SDS electrophoretic analysis.
Gel permeation chromatography：Solvent resistant column is SephacrylTMS-100G75 gel prepacked columns（It is public purchased from Amersham
Department）.Sample after affinity chromatography is concentrated loading, loading volume 5mL, concentration 2mg/mL after 0.22 μm of membrane filtration；
Eluted by eluent of PBS, flow velocity 0.5mL/min, the second absworption peak each component is merged after ultraviolet determination, 12% is denatured, is non-
It is ricin (WA) albumen to be denatured SDS electrophoretic analysis；By -20 DEG C of preservations after the dialysis of obtained ricin (WA) albumen, concentration.
Detoxification：The ricin (WA) albumen after gel filtration is diluted to 0.2-0.3mg/ml with PBS (0.1M, pH7.4), is placed
PBS buffer（0.1M, pH8.1）In, 35 DEG C are dialysed 7 days, then place into dialysis 48h in PBS (0.01M, pH7.4),
12000rpm centrifuges 10min, takes supernatant to dispense, -20 DEG C of preservations.
The amino acid sequence of ricin (WA) albumen is as shown in SEQ ID No.1.
（Two）Animal immune
Using the ricin (WA) albumen of detoxification as immunogene, 200 μ g are taken fully to be mixed with isometric Freund's complete adjuvant,
The purebred large ear rabbit of immune New Zealand.Ricin (WA) albumen with 0.5mg detoxifications and isometric incomplete Freund's adjuvant after two weeks
Booster immunization 1 time；Every after three weeks again with the ricin (WA) albumen of 1mg detoxifications and isometric incomplete Freund's adjuvant booster immunization 1
It is secondary；Hereafter ear edge vein exploitating blood, indirect ELISA measure potency weekly.The potency highest of the 7th day serum after booster immunization, is
106, take this serum to carry out more antivenom purifications.
（Three）Antibody purification
More antivenom purifications are carried out with agarose affinity chromatography column.
Comprise the following steps that：By affinity column with level pad（1L level pads are to contain 300mmol NaCl's
1000ml20mM PB buffer solutions, pH7.8）Balance, after column equilibration, by step（Two）Obtained serum is filtered through 0.45 μm of filter membrane
Later with 1mL/min flow velocity upper props, foreign protein is eluted with 1mL/min flow velocitys with level pad, monitoring, treats that foreign protein elution is thorough
Eluent is changed behind bottom（0.1M citrate buffer solutions, pH4.0）Antibody is washed down.The antibody under washing is collected, test tube is collected and adds in advance
The Tris-HCl of suitable 1mol/L, pH9.0, make pH recover to 7.4 or so, in order to avoid lose activity.Purified antibody is with PBS
（5mM, pH7.4）4 DEG C of working solution dialysis 24h, a not good liquor, then 10000r/min centrifugation 30min are changed per 8h, collect supernatant, -70 DEG C
Save backup.
Goat-anti rabbit polyclonal antibody is purchased from Aldrich-Sigma companies of the U.S., catalog number SAB3700883.
SiO2Corrosive liquid：By HF84ml, NH4F339g, the ratio of water 510ml mix to obtain the final product.
Silicon etch solution：By potassium hydroxide：Isopropanol：Water is according to mass ratio 1：2：2 ratio mixes to obtain the final product.
The polyethylene glycol of mercapto carboxy difunctionalization（PEG）, i.e.,（HS-C2H4-CONH-PEG-C3H6-COOH）, purchased from moral
Rapp Polymere companies of state.
The preparation of embodiment 1, micro-fluidic detection chip
First, exogenous Raman microprobe preparation and curing have Raman microprobe glass film preparation
（One）The preparation of nanogold
The preparation scheme of nanogold is reduction of sodium citrate method.Comprise the following steps that：
Take 99mL ultra-pure waters to be heated in maturing vessel, the gold chloride of 1mL0.1g/100ml is added after solution boiling
Aqueous solution simultaneously keeps boiling more than 2min.The trisodium citrate aqueous solution 1mL for the 1g/100ml for being preheated to 80 DEG C is added, keeps boiling
Rise and heat and stir evenly rapidly, react and terminate when reaction solution suddenly becomes claret from black purple, reduce rotating speed, followed by
Stop heating after continuous boiling heating 10min, constant volume is spare to 100mL after being slowly stirred to natural cooling, this solution is nanometer
Gold solution.
（Two）The PBS solution of the antiricin polyclonal antibody of 1ml80 μ g/ml is slowly added to 1ml in stirring to receive
4 DEG C of 8000rpm centrifuge 45min after 1h is incubated in rice gold solution, at 4 DEG C, and abandon its supernatant and retain precipitation.With isometric
The phosphate buffer of pH7.40.1M（PBS）Again suspend and precipitate and stood at 25 DEG C 30min, 8000rpm is centrifuged at 4 DEG C
45min, abandons supernatant and retains precipitation.Then sediment is resuspended with the 0.1M PBS of the pH7.4 of the BSA containing 1g/100ml, adds
5,5 ' two thiobis (2- nitrobenzoic acids) of 20 μ l1mM（DTNB）It is dissolved in the PBS buffer of pH8.5, is continuously stirred at 4 DEG C
8000rpm centrifuges 45min after reacting 1h, is resuspended in 1ml afterwards twice with the 0.1M PBS centrifuge washings of pH7.4 and contains 1g/
100mlBSA, 0.2g/100mlPEG20000,0.02g/100mlNa3N, 10g/100ml sucrose, 2.5g/100ml trehaloses,
In the PBS buffer of 0.1g/100mlTween-20,10mM pH7.4, exogenous Raman microprobe solution is obtained.
（Three）Glass film is immersed into step（Two）In the exogenous Raman microprobe solution prepared, obtaining curing has exogenous drawing
The glass film of graceful probe, 36 DEG C be dried overnight after to cut into 3 × 2mm sizes spare.
2nd, the preparation of micro- pointed cone array active substrate
（One）Silicon chip is once purged, at 1130 DEG C, first wet-oxygen oxidation 5h, then logical dry-oxygen oxidation 2h（At 1130 DEG C of wet oxygen
Superheated vapor aoxidizes, water flow 800ml/min;Dry-oxygen oxidation is that logical purity oxygen aoxidizes in atmosphere, oxygen stream 500ml/min）,
Its Surface Oxygen dissolve a layer thickness be about 2 μm, the oxide layer of even compact, mask layer of the oxide layer as corrosion silicon
（The barrier layer during silicon is etched, i.e., either with or without silicon dioxide layer, there is silica in the place for being not required to etching in the place that need to be etched
Layer is covered）.Polishing one side gets rid of the positive photoetching rubber of one layer of 5-50 μ m-thick on silicon chip after oxidation, after photoetching, 0.5% it is aobvious
The silicon dioxide pattern of the needs that develop in shadow liquid etching（After positive photoetching rubber, graphics field is carried out by litho machine ultraviolet
Line exposing, then develops in developer solution, and the region positive photoetching rubber of exposure is removed, and other local positive photoetching rubbers are protected
Stay）.Figure is 50 μm of circles of diameter, and the distance between circular and circle is 30 μm.After room temperature rises to 50 DEG C, 60min is kept the temperature,
90 DEG C are risen in 30min, keeps the temperature 120 minutes, post bake is completed with furnace cooling.
（Two）SiO2Corrosive liquid corrosion of silicon about 10min exposes silicon dioxide layer.Directly soaked successively with acetone and alcohol
Or directly positive photoetching rubber is removed in erasing.The feature size of silicon is etched on demand（Figure after development is also retained on silicon chip）Control
Wet etching time processed, etching parameters are：Temperature：45 °C, pH value 3, etch rate 400nm/min.Until etching stopping, so that
The silicon cone hole array being etched is left on silicon chip.The silicon chip for having determined zonal corrosion is put into the silicon etch solution prepared, 78
Corrode at DEG C.Since the corrosion of silicon is there are anisotropy, through certain etching time, you can it is required that round cone hole.Separately
Outside, to prevent cone hole bottom from " island " occur, it is necessary to which certain stirring, obtains silicon former in corrosion process.By PDMS（It is poly-
Dimethyl siloxane）With curing agent silicol in mass ratio 10：1 uniformly mixes after-pouring on silicon former, in negative pressure of vacuum
The bubble in mixed liquor is removed under environment, is cured at 75 DEG C, stripping is from silicon chip at room temperature by above-mentioned cured PDMS
Silicon tip array can be left on the PDMS surfaces that silicon female mold surfaces contact.
（Three）By step（Two）The PDMS cut growths with silicon tip array prepared are 4mm, width for after the strip of 2mm
The golden film of 200nm is sputtered thereon.Using LEYBOLD-HERAEUS Z550 sputters, sputtering seed layer process basic parameter：Work(
Rate is 600W, and high-purity argon gas flow is 4.5sccm, and background vacuum is 3.0 × 10-6Mbar, operating air pressure are 5.0 × 10-
3mbar, sputtering time 6min, finally obtain the PDMS bars covered with golden film comprehensively.
（Four）With Piranha solution（The concentrated sulfuric acid：The aqueous hydrogen peroxide solution of volumn concentration 30% is according to volume ratio 3:1
Mixing）The PDMS bars covered with golden film 10-15 minutes comprehensively are soaked, again by the PDMS covered with golden film comprehensively after being cleaned with pure water
Bar immerses the polyethylene glycol for mercapto carboxy difunctionalization that concentration is 1mM（PEG）When 25 DEG C of reactions 4 are small in aqueous solution, pure water is washed
Using conventional 1- ethyls -3- (3- dimethyl aminopropyls)-carbodiimides (EDC) and n-hydroxysuccinimide after net
(NHS) antiricin polyclonal antibody and goat-anti rabbit polyclonal antibody are coupled to micro- pointed cone array active substrate by reagent respectively
Upper test section and quality control region.
3rd, the assembling of micro-fluidic detection chip
There are the glass film of Raman microprobe and polymer absorbent material to cut into respectively curing prepared by glass film, step 1
The coupling prepared after 3 × 2mm, 3 × 2mm and 5 × 2mm sizes with step 2 has antiricin polyclonal antibody and the goat-anti rabbit more
Micro- pointed cone array active substrate of clonal antibody has the glass film of Raman microprobe, micro- pointed cone array activity according to glass film, curing
Substrate and the order of polymer absorbent material overlap successively is pasted on formation micro-fluidic detection chip on plastic casing.Wherein, it is micro-
Goat-anti rabbit polyclonal antibody coupling area is polyclonal in polymer absorbent material direction, antiricin on pointed cone array active substrate
Antibody coupling area has the glass film direction of Raman microprobe curing.
4th, the detection of micro-fluidic detection chip
Take 10 parts of PBS solution for not containing ricin (WA)（Every part of 100ul）It is added to the micro-fluidic chip of step 3 preparation
Glass film side, treat that sample is absorbed by polymer absorbent material completely and finish and be placed on Raman spectrum detector i-Raman（B&W
TekInc, Newark, DE）It is 1330cm to detect respective regions wave number-1DTNB characteristic Raman peak intensities, take average to add twice
Standard deviation is as the cutoff for judging result（Cut-off values）.In unknown sample is detected Van Gogh in the judgement equal to this value
For the positive, the judgement less than this value is feminine gender.
In quantitative detection, the ricin (WA) standard items of various concentrations are prepared, 1330cm is obtained by the above method-1's
DTNB characteristic Raman peak intensities, using the lg values of ricin (WA) concentration as abscissa, Raman signatures peak intensity is ordinate, is made
Standard curve, obtains calibration curve formula, as shown in Figure 1.By the 1330cm of sample of the unknown concentration containing ricin (WA)-1DTNB
Characteristic Raman peak intensity, which substitutes into calibration curve formula, to carry out quantitative analysis to the content of ricin (WA) in unknown sample.
Claims (10)
1. it is the micro-fluidic detection chip based on made of the principle of Surface enhanced Raman scattering a kind of micro-fluidic detection chip
Overlapped and be fixed on successively in the order described above by material, active substrate, conjugate pad and the sample pad that can produce REFRIGERATION SYSTEM DRIVEN BY CAPILLARY FORCE power
Formed on carrier；
The material for producing REFRIGERATION SYSTEM DRIVEN BY CAPILLARY FORCE power is filter paper, glass film or polymer absorbent material；
The conjugate pad has the polyester film or glass film of exogenous Raman microprobe to cure；
The sample pad is glass film；
The exogenous Raman microprobe is can be with the ligand of matter interaction to be checked and the exogenous molecule with Raman active
The targeting probe that the noble metal nano particles coated jointly are formed；
The noble metal nano particles are nano Au particle or nanometer gold bar；
The targeting refers to that specificity is directed to material to be checked；
The active substrate is to set test section and quality control region on the surface of substrate used when Surface enhanced Raman scattering is analyzed
Obtained from；
The substrate used when Surface enhanced Raman scattering is analyzed is covered with having for noble metal thin layer for surface and projects upwards
The polymer thick film or plate of cone-shaped array of structures；
The thickness of the noble metal thin layer is 30-300nm；
The thickness of the polymer thick film or plate is 100-3000um；
Be connected with the test section it is described can with the ligand of matter interaction to be checked, be connected with quality control region cannot with it is to be checked
Matter interaction but can be with the material of the ligand binding of the matter interaction to be checked；
Described can be antibody or antigen with the ligand of matter interaction to be checked；
Side is padded in the test section in the conjugate, and quality control region is in the material side for producing REFRIGERATION SYSTEM DRIVEN BY CAPILLARY FORCE power.
2. chip according to claim 1, it is characterised in that：
The polymer for can curing molding under certain condition polymer；
It is any enclosed geometric figure at the top of the cone hole of the pointed cone；
It is described can the polymer of curing molding under certain condition be polyimides or dimethyl silicone polymer.
3. chip according to claim 2, it is characterised in that：
The exogenous molecule with Raman active is 5,5 ' two thiobis (2- nitrobenzoic acids)；
The material to be checked is ricin (WA)；
The antibody is antiricin polyclonal antibody；
It is described cannot be with matter interaction to be checked but can be goat-anti rabbit with the material of the ligand binding of matter interaction to be checked
Polyclonal antibody；
What the surface was covered with noble metal thin layer is first to pass through with cone-shaped the array of structures polymer thick film or plate projected upwards
Cross the polymer thick film or plate of the polyethylene glycol functionalization of mercapto carboxy difunctionalization；
The polyethylene glycol of mercapto carboxy difunctionalization is connected to the tool that the surface is covered with noble metal thin layer by golden sulfide linkage
Have on cone-shaped the array of structures polymer thick film or plate projected upwards；
It is difunctional that the antiricin polyclonal antibody and goat-anti rabbit polyclonal antibody by amido link are connected to mercapto carboxy
On the polyethylene glycol of change, so be fixed on the surface be covered with noble metal thin layer with the cone-shaped array of structures projected upwards
On polymer thick film or plate；
The polymer is dimethyl silicone polymer；
The noble metal is gold；
The thickness of the noble metal thin layer is 200nm；
The closure geometric figure is circle；
The pattern diameter is 50 μm, and the distance between circular and circle is 30 μm.
4. chip according to claim 1 or 2, it is characterised in that：The preparation method of the exogenous Raman microprobe is as follows：
It can add in the noble metal nano particles solution, incubate, supernatant is abandoned in centrifugation with the ligand of matter interaction to be checked by described
It must precipitate；Sediment is resuspended with the PBS of the 0.1M of the pH 7.4 containing BSA, adds exogenous point with Raman active
Son, reaction, centrifuging to precipitate；It is resuspended in containing BSA, PEG20000, Na3N, sucrose, trehalose and Tween-20
In the PBS buffer of 10mM pH7.4, the exogenous Raman microprobe solution is obtained；
The conjugate pad is prepared as follows：The polyester film or glass film are immersed into the exogenous Raman microprobe
In solution, it is dried overnight to obtain the final product；
The active substrate is prepared as follows：Analyzed with the immersion of Piranha solution is described in Surface enhanced Raman scattering
Shi Suoyong substrates, are cleaned, then are dipped in the Aqueous Solutions of Polyethylene Glycol of mercapto carboxy difunctionalization and are reacted, after obtaining functionalization
Substrate；Can be with material to be checked with 1- ethyls -3- (3- dimethyl aminopropyls)-carbodiimides and n-hydroxysuccinimide
Test section is made in the ligand coupling of interaction in the substrate of functionalization, equally cannot with matter interaction to be checked but
It can be coupled at the material of the ligand binding of matter interaction to be checked in the substrate of functionalization and quality control region is made.
5. chip according to claim 4, it is characterised in that：It is described in the preparation method of the exogenous Raman microprobe
The temperature of incubation is 4 DEG C；The centrifugation abandons after supernatant must precipitate to further include is hanged again with the PBS buffer of 7.4 0.1M of pH
Drift along and form sediment and stood at 25 DEG C, the step of supernatant obtains sediment is abandoned in 4 DEG C of centrifugations；Described add has the exogenous of Raman active
The condition that molecule is reacted be continuously stirred at 4 DEG C 1 it is small when；It is described to be resuspended in containing BSA, PEG20000, Na3N, sugarcane
The PBS centrifugations with the 0.1M of pH 7.4 are further included before in the PBS buffer of the 10mM pH7.4 of sugar, trehalose and Tween-20
Wash twice the step of must precipitating；
In the preparation method of the active substrate, the temperature of the reaction is 25 DEG C, when the time is 4 small.
Prepare the method for any chip in claim 1-5 6. a kind of, including prepares the method for the conjugate pad with
The method for preparing the active substrate；
The method for preparing the conjugate pad, includes the following steps：Glass film is immersed in exogenous Raman microprobe solution,
It is dried overnight to obtain the final product；
The exogenous Raman microprobe solution is prepared as follows：By institute in any chip in claim 1-5
Stating can be with 4 DEG C of incubation of ligand and noble metal nano solution of matter interaction to be checked, and 4 DEG C centrifuge and must precipitate, and precipitation is dissolved in
The PBS of the 0.1M of pH 7.4 containing BSA, adds the exogenous molecule with Raman active, 4 DEG C of stirrings, 4 DEG C centrifuge and must sink
Form sediment, the noble metal for obtaining to coat jointly with the ligand of matter interaction to be checked and the exogenous molecule with Raman active is received
Rice corpuscles, is eventually adding containing BSA, PEG20000, Na3N, the PBS of the 10mM pH7.4 of sucrose, trehalose and Tween-20 delay
Fliud flushing to obtain the final product；
Concentration of the BSA in the exogenous Raman microprobe solution is 1g/100ml, and the PEG20000 is in the external source
Concentration in property Raman microprobe solution is 0.2g/100ml, the Na3Concentration of the N in the exogenous Raman microprobe solution is
0.02g/100ml, concentration of the sucrose in the exogenous Raman microprobe solution is 10g/100ml, and the trehalose exists
Concentration in the exogenous Raman microprobe solution is 2.5g/100ml, and the Tween-20 is molten in the exogenous Raman microprobe
Concentration in liquid is 0.1g/100ml；
The noble metal nano is specially nanogold；
The method for preparing the active substrate, includes the following steps：Will when Surface enhanced Raman scattering is analyzed base used
Bottom carries out functionalization, obtains the substrate of functionalization；Test section and quality control region are set in the substrate of functionalization, can be with thing to be checked
The ligand of matter interaction is connected to the substrate surface of functionalization and test section is made, it is impossible to matter interaction to be checked but
Quality control region is made in the substrate surface that functionalization can be connected to the material of the ligand binding of matter interaction to be checked；
The substrate used when Surface enhanced Raman scattering is analyzed is specially that surface is covered with dashing forward with upward for noble metal thin layer
Cone-shaped the array of structures polymer thick film or polymer sheet gone out；
The functionalization is by the substrate Piranha solution immersion used when Surface enhanced Raman scattering is analyzed, and is eluted with water
It is dipped in 25 DEG C of reactions in the Aqueous Solutions of Polyethylene Glycol of mercapto carboxy difunctionalization again afterwards；
The Piranha solution is the aqueous solution of the hydrogen peroxide of the concentrated sulfuric acid and volumn concentration 30% according to volume ratio 3:1
It is uniformly mixed so as to obtain；
The surface be covered with noble metal thin layer with cone-shaped the array of structures polymer thick film or polymer sheet projected upwards
It is to be formed described with cone-shaped the array of structures polymer thick film or polymer sheet that project upwards sputtering noble metal；
It is described with the cone-shaped array of structures polymer thick film projected upwards be by can curing molding under certain condition it is poly-
Compound uniformly mixes after-pouring on former with corresponding curing agent, is peeled off after curing from former and obtains the polymerization of curing molding
Thing thick film obtains；
The curing agent is the material that can promote or control curing reaction, specific such as silicol；
Or,
Described with the cone-shaped array of structures polymer sheet projected upwards is by cured polymer sheet and silicon former hot pressing key
After conjunction, cured polymer sheet is peeled off from former and is obtained；
The former is the former for having cone hole array on hard substrate surface；
The former is silicon former；
The silicon former is that the silicon chip with the silicon cone hole array being etched is put into corrosion in silicon etch solution to obtain；
The silicon etch solution is by potassium hydroxide：Isopropanol：Water is according to quality 1：2：2 ratio mixes to obtain the final product；
The silicon chip with the silicon cone hole array being etched is to aoxidize silicon chip surface to obtain oxide layer, in oxide layer upthrow
Light one side gets rid of one layer of positive photoetching rubber, the silicon dioxide pattern of the needs that develop etching, by SiO2Corrosive liquid corrosion of silicon exposure two
Silicon oxide layer, removes positive photoetching rubber, wet etching to obtain the final product；
The SiO2Corrosive liquid is by HF, NH4F and water are mixed according to the ratio of 84ml, 339g, 510ml to obtain the final product.
7. in a kind of qualitative measuring samples of auxiliary whether the method containing desired substance, include the following steps：It will be free of purposeful
The sample of material is added in claim 1-5 in the sample pad of any chip, and the sample of desired substance not contained is complete
After the material absorption that REFRIGERATION SYSTEM DRIVEN BY CAPILLARY FORCE power can be produced described on the chip entirely, the chip is placed in Raman spectrum inspection
Survey on instrument, detect the characteristic Raman peak intensity of the exogenous molecule in the Raman scattering peak region wave number of exogenous molecule
Degree, is averaged plus the value of twice of standard deviation is denoted as Cut-off values；After the same method, measuring samples are detected, obtained
To the characteristic Raman peak intensity of measuring samples, if the characteristic Raman peak intensity of measuring samples is greater than or equal to Cut-off
Then measuring samples are determined as the sample containing desired substance to value, if the characteristic Raman peak intensity of measuring samples is less than Cut-
Then measuring samples are judged to not containing the sample of desired substance to off values.
8. a kind of method for quantitatively detecting desired substance in sample, includes the following steps：By the mark of the desired substance of various concentrations
In quasi- product addition claim 1-5 in the sample pad of any chip, treat desired substance completely by the institute on the chip
After stating the material absorption that can produce REFRIGERATION SYSTEM DRIVEN BY CAPILLARY FORCE power, chip is placed on Raman spectrum detector, is detected at exogenous point
The characteristic Raman peak intensity of the exogenous molecule in the Raman scattering peak region wave number of son, with the lg of desired substance concentration
It is ordinate to be worth for abscissa, characteristic Raman peak intensity, makes standard curve, obtains calibration curve formula；According to same
Method, is detected measuring samples, obtains the characteristic Raman peak intensity of measuring samples, by the characteristic Raman of measuring samples
Peak intensity substitutes into calibration curve formula, obtains the content of desired substance in measuring samples.
9. any chips of claim 1-5 are in the product of the desired substance in preparing qualitative or quantitative detection sample
Using.
10. application according to claim 9, it is characterised in that：The desired substance is ricin (WA).
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410054113.XA CN104849254B (en) | 2014-02-18 | 2014-02-18 | A kind of micro-fluidic detection chip based on Surface enhanced Raman scattering |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410054113.XA CN104849254B (en) | 2014-02-18 | 2014-02-18 | A kind of micro-fluidic detection chip based on Surface enhanced Raman scattering |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104849254A CN104849254A (en) | 2015-08-19 |
| CN104849254B true CN104849254B (en) | 2018-04-24 |
Family
ID=53849061
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410054113.XA Active CN104849254B (en) | 2014-02-18 | 2014-02-18 | A kind of micro-fluidic detection chip based on Surface enhanced Raman scattering |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104849254B (en) |
Families Citing this family (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105823768B (en) * | 2016-04-25 | 2020-03-27 | 中国科学院高能物理研究所 | Detection chip based on surface enhanced Raman scattering technology, preparation method and kit |
| CN106338502B (en) * | 2016-08-25 | 2018-12-21 | 济南大学 | The preparation and application of three-dimensional surface enhancing Raman paper chip |
| CN106944164B (en) * | 2017-03-08 | 2019-10-01 | 浙江大学 | A kind of chip automatic filling system of REFRIGERATION SYSTEM DRIVEN BY CAPILLARY FORCE |
| CN109425598A (en) * | 2017-09-05 | 2019-03-05 | 中国人民解放军军事医学科学院微生物流行病研究所 | A kind of capillary micro-fluid self-driven micro-fluidic chip and the preparation method and application thereof |
| CN108103148A (en) * | 2017-12-22 | 2018-06-01 | 惠州清水湾生物材料有限公司 | A kind of preparation and application of the super quick detection probe liquid chips of miRNA |
| CN109060764B (en) * | 2018-08-23 | 2020-12-29 | 安徽中科赛飞尔科技有限公司 | Preparation method of functionalized SERS platform and application of functionalized SERS platform in ATP detection |
| CN109852673B (en) * | 2019-01-17 | 2019-12-03 | 北京市疾病预防控制中心 | A kind of gold/quantum dot nano probe and its application for detecting active ricin (WA) in complex matrices |
| CN110441284B (en) * | 2019-07-23 | 2022-02-15 | 海南大学 | Preparation method of surface-enhanced Raman scattering chip for trace detection, obtained product and application |
| CN110346350B (en) * | 2019-08-08 | 2020-12-25 | 中国科学技术大学 | SERS structure in microfluidic channel and microfluidic SERS detection method |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1444045A (en) * | 2003-04-15 | 2003-09-24 | 吉林大学 | Surface enhancement Raman scattering labelling immunodetection method |
| CN101400976A (en) * | 2005-12-19 | 2009-04-01 | 美国光纳科技 | Chemical detector |
| CN201892678U (en) * | 2010-08-19 | 2011-07-06 | 中国人民解放军军事医学科学院微生物流行病研究所 | Foodborne pathogens detecting test paper tray based on upconversion luminescent ten-channel immunochromatographic |
| CN102180438A (en) * | 2011-03-28 | 2011-09-14 | 中国科学院光电技术研究所 | Manufacturing method of tunable triangular metal nano particle array structure |
| CN102628808A (en) * | 2012-04-13 | 2012-08-08 | 中国科学院光电技术研究所 | Preparation method and application method of high-sensitivity and high-stability surface-enhanced Raman chip |
| CN102914646A (en) * | 2012-11-16 | 2013-02-06 | 湖南大学 | Homogeneous phase multi-component immunoassay method based on surface plasma coupling effect |
| CN103116019A (en) * | 2013-01-16 | 2013-05-22 | 宁波大学 | Preparation method of immune base and antigen or antibody immunoassay method |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US9274105B2 (en) * | 2011-07-13 | 2016-03-01 | Optrotrace (SuZhou) Technologies, Inc. | Analyzing chemical and biological substances using nano-structure based spectral sensing |
| US9134247B2 (en) * | 2011-12-16 | 2015-09-15 | Real-Time Analyzers, Inc. | Method and apparatus for two-step surface-enhanced raman spectroscopy |
-
2014
- 2014-02-18 CN CN201410054113.XA patent/CN104849254B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1444045A (en) * | 2003-04-15 | 2003-09-24 | 吉林大学 | Surface enhancement Raman scattering labelling immunodetection method |
| CN101400976A (en) * | 2005-12-19 | 2009-04-01 | 美国光纳科技 | Chemical detector |
| CN201892678U (en) * | 2010-08-19 | 2011-07-06 | 中国人民解放军军事医学科学院微生物流行病研究所 | Foodborne pathogens detecting test paper tray based on upconversion luminescent ten-channel immunochromatographic |
| CN102180438A (en) * | 2011-03-28 | 2011-09-14 | 中国科学院光电技术研究所 | Manufacturing method of tunable triangular metal nano particle array structure |
| CN102628808A (en) * | 2012-04-13 | 2012-08-08 | 中国科学院光电技术研究所 | Preparation method and application method of high-sensitivity and high-stability surface-enhanced Raman chip |
| CN102914646A (en) * | 2012-11-16 | 2013-02-06 | 湖南大学 | Homogeneous phase multi-component immunoassay method based on surface plasma coupling effect |
| CN103116019A (en) * | 2013-01-16 | 2013-05-22 | 宁波大学 | Preparation method of immune base and antigen or antibody immunoassay method |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104849254A (en) | 2015-08-19 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104849254B (en) | A kind of micro-fluidic detection chip based on Surface enhanced Raman scattering | |
| CN105445449B (en) | Saliva uric acid rapid semi-quantitative detection means and preparation method thereof | |
| CN103335984B (en) | A kind of incorporeity wall micro-array chip based on LSPR and application thereof | |
| CN103694355B (en) | The recombinant antibodies of anti-human cardiac muscle troponin I and construction method thereof and application | |
| CN110292959B (en) | Microfluidic chip and preparation method and application thereof | |
| CN111122679B (en) | DNA biosensor and preparation method and application thereof | |
| CN102105798A (en) | Real-time continuous detection device | |
| CN102778559A (en) | Colloidal gold immunochromatographic detection kit and preparation method thereof | |
| CN111944153B (en) | Molecularly imprinted polymer microsphere for detecting dengue NS1 protein and application thereof | |
| CN105651999A (en) | Molybdenum disulfide-based sensor and preparation method and application thereof | |
| CN106632620A (en) | Dengue virus recombinant antigen as well as preparation method and application thereof | |
| CN101533011A (en) | A Japanese blood fluke ovum antibody magnetic particle EILSA detecting method | |
| CN108051588A (en) | For the antibody fixing means on the micro-fluidic chip of whole blood sample separation detection | |
| Su-Ping et al. | Progress in molecular imprinting electrochemiluminescence analysis | |
| KR20120028914A (en) | Methods of signal generation and signal localization for improvement of signal readability in solid phase based bioassays | |
| CN105601744A (en) | Recombinant antibody resisting influenza a virus nucleoproteins as well as preparation method and application of recombinant antibody | |
| Gao et al. | Bioengineering of silicon nitride | |
| CN109425598A (en) | A kind of capillary micro-fluid self-driven micro-fluidic chip and the preparation method and application thereof | |
| CN108163802B (en) | Antigen detection material and preparation method and application thereof | |
| CN109725149A (en) | A kind of lateral flow biosensor based on golden platinum nano flower | |
| CN108519366A (en) | The method for detecting peptide using the compound substrate based on graphene | |
| CN101738476B (en) | Rapid diagnosis kit for pre-S1 antigens of hepatitis B viruses and method for preparing same | |
| Guan et al. | Surface modification of cellulose paper for quantum dot-based sensing applications | |
| Prusak-Sochaczewski et al. | Detection of human transferrin by the piezoelectric crystal | |
| JPS63315953A (en) | Method for measuring content of trace material in blood or bodily fluid |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| EXSB | Decision made by sipo to initiate substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |