CN106940310A - Substrate and preparation method thereof is immunized in a kind of self assembly gold nanorods SERS - Google Patents
Substrate and preparation method thereof is immunized in a kind of self assembly gold nanorods SERS Download PDFInfo
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- CN106940310A CN106940310A CN201710128016.4A CN201710128016A CN106940310A CN 106940310 A CN106940310 A CN 106940310A CN 201710128016 A CN201710128016 A CN 201710128016A CN 106940310 A CN106940310 A CN 106940310A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/65—Raman scattering
- G01N21/658—Raman scattering enhancement Raman, e.g. surface plasmons
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
Abstract
The invention provides the preparation method that substrate is immunized in a kind of controllable self assembly gold nanorods SERS.Silicon chip of the substrate including single-sided polishing, the gold nanorods being fixed on the silicon wafer polishing face and the antibody for being linked at the gold nanorods surface is immunized in described SERS.The present invention prepares gold nanorods using Dual Surfactants method first, and described gold nanorods are added drop-wise on the burnishing surface of the silicon chip of hydrophilic treated mistake, and described gold nanorods are arranged in continuous self-assembled structures on the burnishing surface of silicon chip.During preparation, silicon chip is kept with respect to 35~45 ° of ground inclination, to overcome coffee ring effect so that substrate, which is immunized, in self assembly gold nanorods SERS has excellent SERS enhancing characteristics and repeatability, applied to highly sensitive immune detection.In addition, substrate is immunized in the self assembly gold nanorods SERS preparation technology it is simple, with low cost, it is time-consuming less, yield it is high, it is easy to promote, be easy to application.
Description
Technical field
Prepared the invention belongs to nano material and field of nanometer technology, and in particular to synthesis and one kind to gold nanorods are certainly
Assemble the preparation method that substrate is immunized in gold nanorods SERS.
Background technology
Since being found from SERS (SERS) phenomenon, SERS spectra technology is just because of its high sensitivity and nothing
Damage the advantage of detection and by extensive concern, and be rapidly developed.Utilize chemistry or physical method synthesis noble metal nano grain
Son, and be prepared into the active substrate with SERS enhancing abilities, then SERS activity can be just pointed to using SERS spectra technology
The analyte of substrate surface carries out highly sensitive dactylogram detection.Therefore, preparing very big SERS strengthens the high-performance of ability
SERS active-substrate, has important applied to biomedical sectors such as bio-sensing, medical imaging, treatment of cancer, drug deliveries
Meaning.
Usually, can the performance requirement for the SERS active-substrate that constituted to noble metal nano particles is primarily upon it provide
Stable, sensitive, repeatable SERS signal.It is known that the performance of SERS active-substrate and its pattern are closely related,
It is closely related with preparation process.At present, the common method for preparing SERS substrates using noble metal nano particles includes following three kinds：
The first is that the noble metal nano particles colloidal sol for preparing chemical synthesis is directly added dropwise on substrate (such as silicon chip or glass) certainly
So form SERS substrates；Second is the surface for being modified using chemical method noble metal nano particles and substrate, in electrostatic masterpiece
Substrate surface formation SERS substrates are fixed on lower metal nanoparticle；The third is to utilize physical etchings or electrochemical deposition etc.
Method, prepares the SERS substrates with noble metal nano particles array structure.In fact, the above-mentioned method for preparing SERS substrates
Shortcomings.For example, first method is relatively easy, but the noble metal nano particles distribution in the SERS substrates generally prepared is not
, the repeated poor of SERS signal is caused；The SERS substrates that second method is obtained have stronger SERS signal, but stably
Property is not good enough；SERS substrates prepared by the third method can obtain strong SERS signal, and stability and reproducible, but system
Make complex process, cost is higher.
Therefore, using the noble metal nano particles for being chemically synthesized special appearance, and in certain physical condition
The controllable preparation of the lower SERS substrates for realizing specific noble metal nano particles self-assembled structures, will be a kind of inexpensive and simple height
The method of effect.We are according to the distinctive anisotropy of gold nanorods, unique surface plasma body resonant vibration characteristic, good stabilization
Property and the features such as biocompatibility, gold nanorods colloidal sol is deposited on the silicon chip that hydrophilic treated is crossed by solvent evaporated method and formed
The continuously arranged self-assembled structures of gold nanorods, so that the nanometer for obtaining being suitable as high-performance SERS active-substrate prepares material
Material.Such as preparation method of immunocompetence substrate disclosed in the patent of Application No. 201310015549.3 in the prior art, though
Right method preparation process is simple, but prepared golden nanometer particle package assembly small scale, easily by coffee ring effects,
Application is restricted.
The content of the invention
The present invention provides a kind of self assembly gold nanorods SERS and substrate and preparation method thereof is immunized, in described immune substrate
Gold nanorods are continuously arranged self-assembled structures, and the SERS signal for producing described immune substrate has intensity high and steady
The characteristics of determining.
The present invention provides following technical scheme to solve above-mentioned technical problem：
Material needed for substrate is immunized in a kind of self assembly gold nanorods SERS that the present invention is provided includes the silicon of single-sided polishing
Piece, the gold nanorods being fixed on the burnishing surface of the single-sided polishing silicon chip and the antibody for being linked at the gold nanorods surface.
The silicon chip of described single-sided polishing can use monocrystalline silicon silicon chip or polysilicon silicon chip.
The preferred scope of the side length for the gold nanorods being fixed on the burnishing surface of the single-sided polishing silicon chip be 60~
80nm。
The preferred scope of the trans D for the gold nanorods being fixed on the burnishing surface of the single-sided polishing silicon chip be 20~
30nm。
The preferred scope of the mass concentration for the gold nanorods being fixed on the burnishing surface of the single-sided polishing silicon chip is 0.10
~0.60g/L.
The preferred scope of the mass concentration of the described antibody for being linked at the gold nanorods surface is 1~3mg/mL.
Gold nanorods continuous arrangement on the burnishing surface of the single-sided polishing silicon chip is described from group into self-assembled structures
Assembling structure includes the self-assembled structures of the transversely arranged self-assembled structures of gold nanorods and gold nanorods longitudinal arrangement；Described horizontal stroke
It is fixed on by the side of described gold nanorods on the burnishing surface of silicon chip to the self-assembled structures of arrangement and adjacent gold nanorods
The self-assembled structures shoulder to shoulder that side is connected to form；The self-assembled structures of described longitudinal arrangement by described gold nanorods end
Face be fixed on the burnishing surface of silicon chip and adjacent gold nanorods side be connected to form be vertically arranged self-assembled structures.
The combination that described link is antibody and the surface of described gold nanorods is realized in the form of valence link.
The preparation method of substrate is immunized in the described self assembly gold nanorods SERS that the present invention is provided, and comprises the following steps：
1) by the cetyl trimethyl bromination of 0.45~0.55mmol/L chlorauric acid solution, 0.15~0.25mol/L
Ammonium salt solution, 0.005~0.02mol/L sodium borohydride solutions and deionized water are 10~15 according to volume ratio：10~15：5.0~
6.0：1 mixing, stands, and obtains gold and plants solution；
2) by 0.0768mol/L cetyl trimethylammonium bromides and 0.0162mol/L sodium oleate solutions according to volume ratio
For 1：1 mixes to being completely dissolved, and obtains mixed liquor；4mmol/L silver nitrate solutiones are sequentially added into described mixed liquor again,
1mmol/L chlorauric acid solutions, the hydrochloric acid solution and 10mmol/L ascorbic acid solutions of volumetric concentration 37% obtain liquid of growing up；
The 4mmol/L silver nitrate solutiones added in described mixed liquor, 1mmol/L chlorauric acid solutions, 37% hydrochloric acid solution and
The volume ratio of 10mmol/L ascorbic acid solutions is 50~80：1：5~8：2~4；
3) by the step 1) obtained gold plants solution and the step 2) obtained growth liquid according to volume ratio is 1：
2500 mixing, stand, obtain colloidal sol；After the colloidal sol is centrifuged 1~2 time, the colloidal sol for being deposited on centrifugation bottom of the tube is redissolved simultaneously
Progress is ultrasonically treated, obtains scattered gold nanorods solution；
4) silicon chip for being pre-processed the silicon chip of single-sided polishing after hydrophilic treated and dry；
5) by the step 3) obtained gold nanorods solution is added drop-wise to the step 4) on the obtained burnishing surface of silicon chip,
When described gold nanorods solution is added drop-wise on the burnishing surface of described silicon chip, described silicon chip is with respect to the horizontal plane kept
Angle of inclination is 35~45 °；There is silicon chip drying process under the conditions of 10~50 DEG C of described gold nanorods solution to dropwise addition, obtain
To the gold nanorods being fixed on the burnishing surface of described silicon chip；The gold nanorods continuous arrangement is into self-assembled structures；
6) described antibody-solutions are added drop-wise to the step 5) on obtained described gold nanorods, stand, use phosphorus
Phthalate buffer cleaning silicon chip, to the drying process under the conditions of 35~38 DEG C of the silicon chip after cleaning, described antibody is linked to
Described gold nanorods surface；
7) to the step 6) in link have the antibody gold nanorods on be added dropwise bovine serum albumin solution, stand about
After 4h, with the slow cleaning of phosphate buffer, through drying process, that is, obtain self assembly gold nanorods SERS and substrate is immunized.
The step 1)~3) and step 4) between there is no the limitation of time sequencing.
The step 3) in the scope of preferred centrifugal rotational speed be 5000~6000rpm；The step 3) in it is preferred from
The scope of heart time is 15~25min.
The step 5) in be added drop-wise to the excellent of gold nanorods liquor capacity on the burnishing surface of the silicon chip of unit area
It is 0.8~2 μ L/cm to select scope2。
The step 6) in be added dropwise antibody-solutions mass concentration preferred scope be 1~3mg/mL；It is described to be added drop-wise to
The preferred scope of the antibody-solutions volume being fixed with the silicon wafer polishing face of gold nanorods of unit area is 1~4 μ L/
cm2；
The step 7) described in bovine serum albumin solution mass concentration preferred scope be 5~20mg/mL；It is described
The preferred scope for the bovine serum albumin solution volume being added drop-wise on the silicon wafer polishing face of unit area is 1~4 μ L/cm2。
In summary, the preparation method of substrate is immunized in the self assembly gold nanorods SERS that the present invention is provided, by using double tables
Face active agent method prepares the gold nanorods of size uniformity, and described gold nanorods are added dropwise to the polishing for the silicon chip that hydrophilic treated is crossed
Controllable self-assembled structures are formed on face, silicon chip overcomes the shadow of coffee ring effect with horizontal plane certain angle during dropwise addition
Ring, it is ensured that described gold nanorods continuous arrangement formation self-assembled structures on the burnishing surface of the silicon chip, formation from group
Assembling structure significantly enhances SERS signal intensity, and detection is also drastically increased while detection sensitivity is greatly enhanced
Reliability.Self assembly gold nanorods SERS that the present invention is provided is immunized the preparation method of substrate, technique is simple, with low cost,
Take small, yield high, it is easy to promote, be easy to application.
Brief description of the drawings
Fig. 1 is the electron scanning micrograph of the obtained gold nanorods of embodiment 1 self-assembled structures shoulder to shoulder；
Fig. 2 is the electron scanning micrograph that the gold nanorods that embodiment 2 is obtained are vertically arranged self-assembled structures；
Fig. 3 is that the SEM for the continuously arranged multilayer self-assembled structures of gold nanorods that embodiment 3 is obtained is shone
Piece；
Fig. 4 is that the SEM for the continuously arranged individual layer self-assembled structures of gold nanorods that embodiment 4 is obtained is shone
Piece；
Fig. 5 divides for the Raman labels linked in embodiment 5 on gold nanorods self-assembled structures prepared by embodiment 1~4
Sub- 4MBA SERS spectra figure；
Fig. 6 detects various concentrations for the immune substrate prepared in embodiment 6 by the gold nanorods self-assembled structures of embodiment 2
The corresponding SERS spectra figure of PSA (PSA)；
Fig. 7 detects fixed concentration for the immune substrate prepared in embodiment 6 by the gold nanorods self-assembled structures of embodiment 2
During PSA antigens, the 1078cm of different test point collections in substrate-1SERS peak intensity block diagrams.
Embodiment
Material needed for substrate is immunized in a kind of self assembly gold nanorods SERS that the present invention is provided includes the silicon of single-sided polishing
The antibody of piece, the gold nanorods being fixed on the burnishing surface of the single-sided polishing silicon chip and absorption on the gold nanorods surface.
In the present invention, the size of the silicon chip of the single-sided polishing is not particularly limited, ripe using those skilled in the art institute
The die size known；The preparation method of the silicon chip of the single-sided polishing is not particularly limited, using those skilled in the art
The preparation method of the silicon chip of known single-sided polishing.In the embodiment of the present invention, the source of the silicon chip of the single-sided polishing
Purchased from Tianhe Optical Energy Co., Ltd., Changzhou.
In the present invention, the mass concentration of described gold nanorods being fixed on the burnishing surface of the single-sided polishing silicon chip
Preferably 0.10~0.60g/L, more preferably 0.25~0.45g/L.
In the present invention, the described gold nanorods continuous arrangement being fixed on the burnishing surface of the single-sided polishing silicon chip is into certainly
Package assembly, described self-assembled structures include the transversely arranged self-assembled structures of gold nanorods and gold nanorods longitudinal arrangement
Self-assembled structures；Described transversely arranged self-assembled structures are fixed on the burnishing surface of silicon chip by the side of described gold nanorods
The self-assembled structures shoulder to shoulder that the side of upper and adjacent gold nanorods is connected to form；The self-assembled structures of described longitudinal arrangement by
The end face of described gold nanorods be fixed on the burnishing surface of silicon chip and adjacent gold nanorods the vertical row that is connected to form of side
Row self-assembled structures.
In the present invention, the preferred scope of the side length of the gold nanorods is 60~80nm, and the scope being more highly preferred to is
70~75nm；The preferred scope of the trans D of the gold nanorods is 20~30nm, and the scope being more highly preferred to is 22~27nm,
The trans D of optimization is 25nm.
In the present invention, the preferred scope of the mass concentration of antibody of the absorption on the gold nanorods surface for 1~
3mg/ml, the scope being more highly preferred to is 1.5~2.5mg/ml, and the mass concentration of optimization is 2.0mg/ml.The kind of the antibody
Class includes PSA antibody (anti-PSA), alpha-fetoprotein antibody (anti-AFP) or sugar antigen antibody
(anti-CA19-9) antibody such as.
The present invention provides the preparation method that substrate is immunized in described self assembly gold nanorods SERS, comprises the following steps：
1) by 0.45~0.55mmol/L chlorauric acid solution, 0.15~0.25mol/L cetyl trimethyl bromination
Ammonium salt solution, 0.005~0.02 sodium borohydride solution and deionized water are 10~15 according to volume ratio：10~15：5.0~6.0：1
Mixing, stands, and obtains gold and plants solution；
2) it is 1 according to volume ratio by 0.0768mol/L and 0.0162mol/L sodium oleate solutions：1 mixes to being completely dissolved,
Sequentially add 4mmol/L silver nitrate solutiones to mixed liquor, 1mmol/L chlorauric acid solutions, the hydrochloric acid solution of volumetric concentration 37% and
10mmol/L ascorbic acid solutions, obtain liquid of growing up；The 4mmol/L silver nitrate solutiones that the mixed liquor is sequentially added, 1mmol/L
Chlorauric acid solution, 37% hydrochloric acid solution and the volume ratio of 10mmol/L ascorbic acid solutions are 50~80：1：5~8：2~4；
3) by the step 1) obtained gold plants solution and the step 2) obtained growth liquid according to volume ratio is 1:1 mixes
Close, stand, the colloidal sol after standing is centrifuged 1~2 time, redissolve the colloidal sol for being deposited on centrifugation bottom of the tube with deionized water after centrifugation
Progress is ultrasonically treated, obtains scattered gold nanorods solution；
4) by the silicon chip hydrophilic treated of single-sided polishing, dry, the silicon chip pre-processed；
5) by the step 3) obtained gold nanorods solution is added drop-wise to the step 4) on the obtained burnishing surface of silicon chip,
There are 10~50 DEG C of drying process of silicon chip of gold nanorods solution to dropwise addition；During the dropwise addition by silicon chip angle of inclination be 35~45 °；
6) antibody-solutions are added drop-wise to the step 5) in be fixed with the silicon wafer polishing face of gold nanorods, after standing, clearly
Silicon chip is washed, to the drying process under the conditions of 35~38 DEG C of the silicon chip after cleaning, the silicon that gold nanorods link antibody is fixed
Piece；
7) to the step 6) in be fixed with gold nanorods link antibody silicon wafer polishing face on lock solution is added dropwise, stand
After clean, to the silicon chip drying process after cleaning, substrate is immunized in the gold nanorods SERS for obtaining self-assembled structures；
The step 1)~3) and step 4) between there is no the limitation of time sequencing.
In step 1 of the present invention) in, the molar concentration of the optimization of the chlorauric acid solution is 0.50mmol/L；Institute
The molar concentration for stating the optimization of cetyl trimethylammonium bromide solution is 0.20mol/L；The sodium borohydride solution is most
The molar concentration of optimization is 0.01mol/L.
In step 1 of the present invention) in, the preferred scope of described time of repose is 2~6h, the time of repose of optimization
For 4h.
In step 1 of the present invention) in, the chlorauric acid solution, cetyl trimethylammonium bromide solution, sodium borohydride
The volume ratio of the optimization of solution and deionized water is 12.5：12：5.5：1.
In step 2 of the present invention) and step 3) in, the hybrid mode of described solution is not particularly limited, and is used
The technical scheme of mixed solution well-known to those skilled in the art.
In step 3 of the present invention) in, the preferred scope of the centrifugal rotational speed is 5000~6000rpm, is more highly preferred to
Scope be 5500rpm；The preferred scope of the centrifugation time is 15~25min, and the centrifugation time of optimization is 20min.
In step 3 of the present invention) in, the preferred scope of described time of repose is 10~12h, the standing of optimization
Time is 11h.
In step 3 of the present invention) in, the solvent that described colloidal sol redissolves is deionized water, the conductance of deionized water
Rate is 18.2M Ω cm.
In step 3 of the present invention) in, the preferred scope for being cleaned by ultrasonic power is 100~150w, optimization
Ultrasonic cleaning power is 120w；The preferred scope of the ultrasonic cleaning time is 15~30min, the ultrasonic cleaning time of optimization
For 20min.
In step 4 of the present invention) in, the silicon chip hydrophilic treated of single-sided polishing is dried, the silicon pre-processed
Piece.The method of the silicon chip hydrophilic treated preferably includes following steps：
Ith, the silicon chip of single-sided polishing is placed in into acid solution to be cleaned by ultrasonic；
IIth, by the silicon chip deionized water rinsing after ultrasonic cleaning；
IIIth, the silicon chip after deionized water rinsing is placed in into alkaline cleaning fluid to be cleaned by ultrasonic, the silicon chip after ultrasonic cleaning
Use deionized water rinsing；
IVth, the silicon chip after the step III is rinsed is cleaned by ultrasonic with acidic cleaning solution, finally with deionized water rinsing, is obtained
To the silicon chip of hydrophilic treated.
In the step of silicon chip hydrophilic treated of the present invention I, the acid solution includes the H that volumetric concentration is 50%2SO4
Solution and the H that volumetric concentration is 20%2O2Solution.
In the step of silicon chip hydrophilic treated of the present invention III, it is 10% that the alkaline cleaning fluid, which includes volumetric concentration,
NH4The OH and H that volumetric concentration is 20%2O2The aqueous solution.
In the step of silicon chip hydrophilic treated of the present invention IV, it is 20% that the acidic cleaning solution, which includes volumetric concentration,
H2O2With the aqueous solution of the volumetric concentration for 10% HCl.
In the step of silicon chip hydrophilic treated of the present invention, what the ultrasonic cleaning process was used is cleaned by ultrasonic the excellent of power
It is 100~150w to select scope, and the ultrasonic cleaning power of optimization is 120w；The preferred scope of the ultrasonic cleaning time be 15~
30min, the ultrasonic cleaning time of optimization is 20min.
In step 4 of the present invention) in, the preferred scope of described silicon chip drying temperature is 35~38 DEG C, is optimized
Drying temperature be 37 DEG C；The preferred scope of described silicon chip drying time is 10~14h, and the drying time of optimization is 12h.
In step 5 of the present invention) in, the gold nanorods solution is added drop-wise to unit plane on the burnishing surface of the silicon chip
The preferred scope of long-pending volume is 0.8~2 μ L/cm2, the scope being more highly preferred to is 1.2~1.8 μ L/cm2, the volume of optimization is
1.5μL/cm2。
In step 5 of the present invention) in, the preferred model of the drying temperature that the silicon chip for having gold nanorods solution is added dropwise
Enclose for 10~20 DEG C, the drying temperature of optimization is 15 DEG C.The drying time that the silicon chip for having gold nanorods solution is added dropwise
Preferred scope is 2~6h, and the drying time of optimization is 4h.
In step 5 of the present invention) in, the optimization angle of inclination that silicon chip is tilted during the dropwise addition is 40 °.
In step 6 of the present invention) in, the preferred scope of the mass concentration of the antibody-solutions is 1~3mg/mL, more
It is 1.5~2.5mg/mL for preferred scope, the mass concentration of optimization is 2mg/mL.The antibody-solutions are added drop-wise to described solid
Surely the preferred scope for having the volume of the unit area on the silicon wafer polishing face of gold nanorods is 1~4 μ L/cm2, the volume of optimization
For 3 μ L/cm2.The antibody-solutions are the mixed solution of a kind of antibody-solutions or Multiple Antibodies.The species of the antibody is without spy
Different limitation, the antibody studied using those skilled in the art.
In step 6 of the present invention) in, the preferred scope of the temperature of the standing is 2~8 DEG C, is more highly preferred to scope
For 3~5 DEG C, the temperature of optimization is 4 DEG C.The time preferred scope of the standing is 10~12h, and the time of optimization is 11h.
In step 6 of the present invention) in, described cleaning agent is the trihydroxy methyl amino containing 0.05%Tween 20
Aminomethane buffer.The preferred scope of the pH value of the TRIS buffer containing 0.05%Tween 20 is
7.5~8.0, the pH of optimization is 7.6.Silicon chip after the TRIS buffer cleaning is entered with deionized water again
Row is rinsed.
In step 6 of the present invention) in, the preferred scope of the drying temperature is 35~38 DEG C, the temperature of optimization
For 37 DEG C.
In step 7 of the present invention) in, the lock solution is bovine serum albumin solution.The bovine serum albumin
The preferred scope of the mass concentration of white solution is 5~20mg/mL, and the mass concentration of optimization is 10mg/mL.It is added drop-wise to unit plane
The preferred scope of the long-pending bovine serum albumin solution volume being fixed with the silicon wafer polishing face of gold nanorods is 1~4 μ L/
cm2。
In step 7 of the present invention) in, the bovine serum albumin solution is added drop-wise to the silicon chip for being fixed with gold nanorods
The preferred scope of dwell temperature after on burnishing surface is 20~27 DEG C, and the temperature of optimization is 25 DEG C.The time of the standing
Preferred scope is 2~4h, and the time of optimization is 3h.
In step 7 of the present invention) in, described cleaning solution is the trihydroxy methyl ammonia containing 0.05%Tween 20
Methylmethane buffer solution.The preferred scope of the pH value of the TRIS buffer containing 0.05%Tween 20 is
7.5~8.0, the pH of optimization is 7.6.Silicon chip after the TRIS buffer cleaning is entered with deionized water again
Row is rinsed.
In step 7 of the present invention) in, the preferred scope of the drying temperature is 35~38 DEG C, the temperature of optimization
For 37 DEG C.
Substrate and its preparation side are immunized to a kind of self assembly gold nanorods SERS that the present invention is provided with reference to embodiment
Method is described in detail, but they can not be interpreted as limiting the scope of the present invention.
Embodiment 1
Monodispersed uniform gold nanorods solution is prepared, detailed process is：A1, at room temperature by 5mL 0.50mM chlorine gold
Acid solution and 5mL 0.20M cetyl trimethylammonium bromide solution are stirred evenly, and add the 0.6mL of frost thereto rapidly
0.01M sodium borohydride solutions, and 0.4mL deionized waters are added, obtain gold and plant solution, at least 2h is stood at room temperature stand-by；a2、
2.5mL 0.0768M cetyl trimethylammonium bromides and 2.5mL 0.0162M sodium oleate solutions are mixed to being completely dissolved, to
180 μ L4mM silver nitrate solutiones are wherein added, 15min is stood, then add 2.5mL 1mM chlorauric acid solutions, stirring thereto
After 15min, after ie in solution becomes colourless, 21 μ L 37% hydrochloric acid solution is added, 12 μ L 10mM ascorbic acid are added followed by
Solution, obtains liquid of growing up；A3, by 8 μ L by a1 steps prepare gold plant solution be added in the growth liquid prepared by a2 steps,
12h is stood at room temperature, finally obtains gold nanorods colloidal sol.The gold nanorods solution injection centrifuge tube prepared, then to gold
Nanometer rods solution carries out eccentric cleaning, and supernatant is then removed after eccentric cleaning terminates, and retains and is deposited in centrifugation bottom of the tube
Gold nanorods colloidal sol simultaneously adds deionized water, and centrifugation is multiple, and ultrasonically treated, the suspension for obtaining monodispersed uniform gold nanorods is molten
Liquid；Here, the rotating speed that eccentric cleaning is carried out to gold nanorods solution is 6000r/min, centrifugation time is 20min, past to be deposited in
The deionized water added in the gold nanorods colloidal sol for centrifuging bottom of the tube is 40mL, and centrifugation number of times is 2 times.Carry out ultrasonically treated place
The reason time is 25min.
The gold nanorods substrate of self assembly is prepared, detailed process is：Take the square single-sided polishing that one piece of length of side is 5mm
Silicon chip, then silicon chip is cleaned using silicon wafer cleaning process, cleaning process is：It is first that silicon chip is clear with Acidic Liquid ultrasound
Wash, then with deionized water rinsing, then be cleaned by ultrasonic with alkaline cleaning fluid, then after deionized water rinsing it is super with acidic cleaning solution
Sound is cleaned, and finally uses deionized water rinsing；The Acidic Liquid is the H that volumetric concentration is 50%2SO4Solution and volumetric concentration are
20% H2O2Solution；The alkaline cleaning fluid includes the NH that volumetric concentration is 10%4OH, volumetric concentration is 20% H2O2Water
Solution；The acidic cleaning solution includes the H that volumetric concentration is 20%2O2, volumetric concentration is the 10% HCl aqueous solution.To cleaning
Processing is dried with standby in clean silicon chip, will be added drop-wise to processing by the mass concentration 0.60g/L μ L of gold nanorods solution 40
On the burnishing surface for the silicon chip crossed, and keep being dried in 40 ° of silicon chip and horizontal plane, the insulating box for being then maintained at 10 DEG C
Processing, that is, be made self assembly gold nanorods substrate.
It is in the self-assembled structures arranged shoulder to shoulder by the gold nanorods in substrate manufactured in the present embodiment, as shown in Figure 1.
In Fig. 1, the size for the gold nanorods being deposited on silicon chip is homogeneous, and the size of its single gold nanorods is：Long 75 ± the 5nm in longitudinal direction,
Trans D is 25 ± 2nm.
Embodiment 2
Monodispersed uniform gold nanorods solution is prepared, detailed process is：A1, at room temperature by 5mL 0.45mM chlorine gold
Acid solution and 5mL 0.25M cetyl trimethylammonium bromide solution are stirred evenly, and add the 0.6mL of frost thereto rapidly
0.005M sodium borohydride solutions, and 0.4mL deionized waters are added, obtain gold and plant solution, at least 2h is stood at room temperature stand-by；
A2, by 2.5mL 0.0768M cetyl trimethylammonium bromides and 2.5mL 0.0162M sodium oleate solutions mix to completely it is molten
Solution, adds 180 μ L4mM silver nitrate solutiones thereto, stands 15min, then adds 2.5mL 1mM chlorauric acid solutions thereto, stirs
Mix after 15min, after ie in solution becomes colourless, add 21 μ L 37% hydrochloric acid solution, be added followed by 12 μ L 10mM Vitamin Cs
Acid solution, obtains liquid of growing up；A3, by 8 μ L by a1 steps prepare gold plant solution be added in the growth liquid prepared by a2 steps,
12h is stood at room temperature, finally obtains gold nanorods colloidal sol.The gold nanorods solution injection centrifuge tube prepared, it is then right
Gold nanorods solution carries out eccentric cleaning, and supernatant is then removed after eccentric cleaning terminates, and reservation is deposited in centrifugation bottom of the tube
Gold nanorods colloidal sol and add deionized water, centrifugation is multiple, ultrasonically treated, obtains the suspension of monodispersed uniform gold nanorods
Solution；Here, the rotating speed that eccentric cleaning is carried out to gold nanorods solution is 6000r/min, centrifugation time is 20min, toward precipitation
The deionized water added in the gold nanorods colloidal sol of centrifugation bottom of the tube is 40mL, and centrifugation number of times is 2 times.Carry out ultrasonically treated
Treatment time is 25min.
The gold nanorods substrate of self assembly is prepared, detailed process is：Take the square single-sided polishing that one piece of length of side is 5mm
Silicon chip, then silicon chip is cleaned using silicon wafer cleaning process, cleaning process is：It is first that silicon chip is clear with Acidic Liquid ultrasound
Wash, then with deionized water rinsing, then be cleaned by ultrasonic with alkaline cleaning fluid, then after deionized water rinsing it is super with acidic cleaning solution
Sound is cleaned, and finally uses deionized water rinsing；The Acidic Liquid is the H that volumetric concentration is 50%2SO4Solution and volumetric concentration are
20% H2O2Solution；The alkaline cleaning fluid includes the NH that volumetric concentration is 10%4OH, volumetric concentration is 20% H2O2Water
Solution；The acidic cleaning solution includes the H that volumetric concentration is 20%2O2, volumetric concentration is the 10% HCl aqueous solution.To cleaning
Processing is dried with standby in clean silicon chip, will be added drop-wise to processing by the mass concentration 0.45g/L μ L of gold nanorods solution 50
On the burnishing surface for the silicon chip crossed, and 45 ° of silicon chip and horizontal plane, place is dried in the insulating box for being then maintained at 15 DEG C
Reason, that is, be made self assembly gold nanorods substrate.
It is in the self-assembled structures being vertically arranged by the gold nanorods in substrate manufactured in the present embodiment, as shown in Figure 2.In figure
In 2, the size for the gold nanorods being deposited on silicon chip is homogeneous, and the size of its single gold nanorods is：Long 70 ± the 5nm in longitudinal direction, it is horizontal
It is 27 ± 2nm to diameter.
Embodiment 3
Monodispersed uniform gold nanorods solution is prepared, detailed process is：A1, at room temperature by 5mL 0.55mM chlorine gold
Acid solution and 5mL 0.15M cetyl trimethylammonium bromide solution are stirred evenly, and add the 0.6mL of frost thereto rapidly
0.02M sodium borohydride solutions, and 0.4mL deionized waters are added, obtain gold and plant solution, at least 2h is stood at room temperature stand-by；a2、
2.5mL 0.0768M cetyl trimethylammonium bromides and 2.5mL 0.0162M sodium oleate solutions are mixed to being completely dissolved, to
180 μ L4mM silver nitrate solutiones are wherein added, 15min is stood, then add 2.5mL 1mM chlorauric acid solutions, stirring thereto
After 15min, after ie in solution becomes colourless, 21 μ L 37% hydrochloric acid solution is added, 12 μ L 10mM ascorbic acid are added followed by
Solution, obtains liquid of growing up；A3, by 8 μ L by a1 steps prepare gold plant solution be added in the growth liquid prepared by a2 steps,
12h is stood at room temperature, finally obtains gold nanorods colloidal sol.The gold nanorods solution injection centrifuge tube prepared, then to gold
Nanometer rods solution carries out eccentric cleaning, and supernatant is then removed after eccentric cleaning terminates, and retains and is deposited in centrifugation bottom of the tube
Gold nanorods colloidal sol simultaneously adds deionized water, and centrifugation is multiple, and ultrasonically treated, the suspension for obtaining monodispersed uniform gold nanorods is molten
Liquid；Here, the rotating speed that eccentric cleaning is carried out to gold nanorods solution is 6000r/min, centrifugation time is 20min, past to be deposited in
The deionized water added in the gold nanorods colloidal sol for centrifuging bottom of the tube is 50mL, and centrifugation number of times is 3 times.Carry out ultrasonically treated place
The reason time is 25min.
The gold nanorods substrate of self assembly is prepared, detailed process is：Take the square single-sided polishing that one piece of length of side is 5mm
Silicon chip, then silicon chip is cleaned using silicon wafer cleaning process, cleaning process is：It is first that silicon chip is clear with Acidic Liquid ultrasound
Wash, then with deionized water rinsing, then be cleaned by ultrasonic with alkaline cleaning fluid, then after deionized water rinsing it is super with acidic cleaning solution
Sound is cleaned, and finally uses deionized water rinsing；The Acidic Liquid is the H that volumetric concentration is 50%2SO4Solution and volumetric concentration are
20% H2O2Solution；The alkaline cleaning fluid includes the NH that volumetric concentration is 10%4OH, volumetric concentration is 20% H2O2Water
Solution；The acidic cleaning solution includes the H that volumetric concentration is 20%2O2, volumetric concentration is the 10% HCl aqueous solution.To cleaning
Processing is dried with standby in clean silicon chip, will be added drop-wise to processing by the mass concentration 0.25g/L μ L of gold nanorods solution 50
On the burnishing surface for the silicon chip crossed, and silicon chip and 38 ° of horizontal plane, then, silicon chip are maintained in 40 DEG C of insulating box and carried out
Drying process, that is, be made self assembly gold nanorods substrate.
It is in continuously arranged multilayer self-assembled structures by the gold nanorods in substrate manufactured in the present embodiment, as shown in Figure 3.
In figure 3, the size for the gold nanorods being deposited on silicon chip is homogeneous, and the size of its single gold nanorods is：Longitudinal direction length 72 ±
5nm, trans D is 25 ± 2nm.
Embodiment 4
Monodispersed uniform gold nanorods solution is prepared, detailed process is：A1, at room temperature by 5mL 0.50mM chlorine gold
Acid solution and 5mL 0.20M cetyl trimethylammonium bromide solution are stirred evenly, and add the 0.6mL of frost thereto rapidly
0.01M sodium borohydride solutions, and 0.4mL deionized waters are added, obtain gold and plant solution, at least 2h is stood at room temperature stand-by；a2、
2.5mL 0.0768M cetyl trimethylammonium bromides and 2.5mL 0.0162M sodium oleate solutions are mixed to being completely dissolved, to
180 μ L4mM silver nitrate solutiones are wherein added, 15min is stood, then add 2.5mL 1mM chlorauric acid solutions, stirring thereto
After 15min, after ie in solution becomes colourless, 21 μ L 37% hydrochloric acid solution is added, 12 μ L 10mM ascorbic acid are added followed by
Solution, obtains liquid of growing up；A3, by 8 μ L by step a1 prepare gold plant solution be added in the growth liquid prepared by a2 steps,
12h is stood at room temperature, finally obtains gold nanorods colloidal sol.The gold nanorods solution injection centrifuge tube prepared, then to gold
Nanometer rods solution carries out eccentric cleaning, then removes supernatant after eccentric cleaning terminates, toward the gold for being deposited in centrifugation bottom of the tube
Nanometer rods colloidal sol is added in 10ml deionized waters, and centrifugation is multiple, ultrasonically treated, obtains the suspension of monodispersed uniform gold nanorods
Solution；Here, the rotating speed that eccentric cleaning is carried out to gold nanorods solution is 6000r/min, centrifugation time is 20min, toward precipitation
The deionized water added in the gold nanorods colloidal sol of centrifugation bottom of the tube is 10mL, and centrifugation number of times is 2 times.Carry out ultrasonically treated
Treatment time is 25min.
The gold nanorods substrate of self assembly is prepared, detailed process is：Take the square single-sided polishing that one piece of length of side is 5mm
Silicon chip, then silicon chip is cleaned using silicon wafer cleaning process, cleaning process is：It is first that silicon chip is clear with Acidic Liquid ultrasound
Wash, then with deionized water rinsing, then be cleaned by ultrasonic with alkaline cleaning fluid, then after deionized water rinsing it is super with acidic cleaning solution
Sound is cleaned, and finally uses deionized water rinsing；The Acidic Liquid is the H that volumetric concentration is 50%2SO4Solution and volumetric concentration are
20% H2O2Solution；The alkaline cleaning fluid includes the NH that volumetric concentration is 10%4OH, volumetric concentration is 20% H2O2Water
Solution；The acidic cleaning solution includes the H that volumetric concentration is 20%2O2, volumetric concentration is the 10% HCl aqueous solution.To cleaning
Processing is dried with standby in clean silicon chip, silicon chip will be added dropwise by the mass concentration 0.10g/L μ L of gold nanorods solution 50
On burnishing surface, and 38 ° of silicon chip and horizontal plane, then, silicon chip, which is maintained in 50 DEG C of insulating box, is dried processing, i.e.,
Self assembly gold nanorods substrate is made.
It is in continuously arranged individual layer self-assembled structures by the gold nanorods in substrate manufactured in the present embodiment, as shown in Figure 4.
In Fig. 4, the size for the gold nanorods being deposited on silicon chip is homogeneous, and the size of its single gold nanorods is：Longitudinal direction length 70 ±
5nm, trans D is 30 ± 2nm.
Embodiment 5
The present embodiment provides a kind of self assembly gold nanorods substrate link Raman labels point by prepared by above-described embodiment
Son and the implementation process for detecting its SERS spectra.By taking self assembly gold nanorods substrate prepared by embodiment 1 as an example, specific steps are such as
Under：
1) to mercaptobenzoic acid (4MBA) as Raman labels molecule, preparation concentration is 10mM to mercaptobenzoic acid solution
(4MBA) solution.
2) self assembly gold nanorods substrate prepared by embodiment 1 is immersed in 4h in 4MBA solution, cleaned with deionized water
The substrate of 4MBA mark molecules is not linked, after cleaning 2~3 times, keeps 12h to dry in 37 DEG C of insulating boxs.
3) measurement by described step 2) prepare self assembly gold nanorods substrate link 4MBA mark molecules SERS
Spectrum.During detection, make laser launch laser line focus after vertical irradiation in assembling substrate on, labeled point of the laser of irradiation
Received after the substrate reflection of son by Raman spectrometer, the feature SERS spectra of the 4MBA mark molecules of Raman spectrometer record is as schemed
In 5 shown in curve 1.The time of integration for gathering SERS spectra is 1~10s, and the power of irradiation laser is 49.55mw, optical maser wavelength
For 785nm.
According to above-mentioned same step, measure by embodiments of the invention 2, embodiment 3 and embodiment 4 prepare from group
The SERS spectra of gold nanorods substrate is filled, as a result respectively as shown in curve 2, curve 3 and curve 4 in Fig. 5.
As seen from Figure 5, correspondence after the 4MBA Raman labels molecules of different self assembly gold nanorods substrate link same concentrations
The 4MBA size of feature peak intensity of SERS spectra have significantly different.So, the SERS of self assembly gold nanorods substrate is special
Property depend on different arrangement modes of the gold nanorods on silicon wafer polishing face, and when gold nanorods on silicon wafer polishing face from group
When dress formation is vertically arranged, the characteristic Raman signals for the 4MBA that the self assembly gold nanorods substrate prepared by embodiment 2 is obtained are most
By force.
Embodiment 6
The present embodiment, which is provided, prepares the implementation process that substrate is immunized in a kind of self assembly gold nanorods SERS.Made with embodiment 2
Exemplified by standby self assembly gold nanorods substrate, comprise the following steps that：
1) it will be added drop-wise to prepared by the embodiment 2 through processing for carrying out the anti-PSA antibody-solutions of PSA antigen detections
Self assembly gold nanorods substrate, and 10~12h is stood under conditions of temperature is 2~8 DEG C, use 0.05%Tween successively afterwards
20 TRIS buffers and deionized water rinsing silicon chip, to remove the unadsorbed anti-PSA on gold nanorods
Antibody, finally the self assembly gold nanorods substrate for being adsorbed with anti-PSA antibody is dried processing, is added dropwise and anti-PSA
The bovine serum albumin solution of antibody-solutions same volume, reacts 2~4h at 25 DEG C, uses contain 0.05%Tween 20 afterwards
TRIS buffer and deionized water silicon chip is rinsed, to remove the unadsorbed ox on gold nanorods
Seralbumin, then processing is dried to silicon chip, described drying is to keep 12h in 37 DEG C of insulating boxs, prepares tool
Substrate is immunized in the SERS for having specific selection function.
2) from the PSA antigenic solutions to be measured that concentration is 0fg/mL, 2.1fg/mL, 210fg/mL, 21pg/mL and 2.1ng/mL
Take 20 μ L to be added drop-wise to by step 1 in sample respectively) prepare 5 self assembly gold nanorods SERS be immunized substrate on, be in temperature
2h is reacted under conditions of 30~38 DEG C；With TRIS buffer and deionized water containing 0.05%Tween 20
Substrate is immunized to SERS to be rinsed, then substrate is immunized to SERS processing is dried, obtain the different PSA antigens of 5 correspondences dense
Substrate is immunized in the SERS of degree.
3) according to prior art, the preparation of gold nanosphere colloidal sol as disclosed in the patent of Application No. 201310015549.3
Method, prepares the gold nanosphere colloidal sol that diameter is 20nm.It is the molten to mercaptobenzoic acid solution (4MBA) of 10mM in concentration
Take 10 μ L to be added drop-wise in 1mL described gold nanosphere colloidal sol in liquid, be sufficiently mixed and stand after 4~6h, prepare modification
There is 4MBA 20nm gold nanosphere probe solution.Place at ambient temperature after 12h, centrifugal treating is carried out, with 11000rpm
Rotating speed centrifugation 30min, the supernatant liquor in centrifuge tube is removed afterwards, then deposit is dissolved in 1mL phosphate buffer solution
(PBS) in.In the case where being slowly stirred, in the solution obtained to after being handled through step c, add 20 μ L and concentration is 2mg/mL
Anti-PSA antibody-solutions, 1.5h, then centrifugal treating are reacted under 4 DEG C of constant temperature, 30min is centrifuged with 11000rpm rotating speed,
To remove the unadsorbed anti-PSA antibody on golden nanometer particle surface, the supernatant liquor in centrifuge tube is removed afterwards, then super
Under sonication environment, the deposit in centrifuge tube is dissolved in 1mL phosphate buffer solution.Here, the antibody added is molten
The amount of liquid can be controlled in the range of 15~30 μ L, and concentration is can be controlled in the range of 1~3mg/mL；The PH of phosphate buffer solution
Value can be 7.0；The amount of phosphate buffer solution is can be controlled in the range of 1~3mL.Thus, gold nanosphere is prepared to be immunized
Probe solution.
4) it will be added drop-wise to prepared by the embodiment 2 through processing for carrying out the anti-PSA antibody-solutions of PSA antigen detections
Self assembly gold nanorods substrate, and 10~12h is stood under conditions of temperature is 2~8 DEG C, use 0.05%Tween successively afterwards
20 TRIS buffers and deionized water rinsing silicon chip, to remove the unadsorbed anti-PSA on gold nanorods
Antibody, finally the self assembly gold nanorods substrate for being adsorbed with anti-PSA antibody is dried processing, is added dropwise and anti-PSA
The bovine serum albumin solution of antibody-solutions same volume, reacts 2~4h at room temperature, uses contain 0.05%Tween 20 afterwards
TRIS buffer and deionized water silicon chip is rinsed, to remove the unadsorbed ox on gold nanorods
Seralbumin, then processing is dried to silicon chip, described drying is to keep 12h in 37 DEG C of insulating boxs, prepares tool
Substrate is immunized in the SERS for having specific selection function.
5) from step 4) take 20 μ L to be added drop-wise to step 2 respectively in the immunological probe liquid for preparing) link for preparing has to be measured anti-
5 former SERS are immunized in substrate, and 1.5~3h is reacted in the case where temperature is 4 DEG C of constant temperature, resist the PSA to be measured being added dropwise
The anti-PSA antibody in substrate is immunized with SERS for antigen in original solution and the anti-PSA antibody in immunological probe occurs
Immune recombination reaction.Afterwards, with TRIS buffer and deionized water containing 0.05%Tween 20 to every
Individual SERS is immunized substrate and is rinsed, then processing is dried to the immune substrates of all SERS, and 12h is kept in 37 DEG C of insulating boxs
Drying, that is, obtain the detection sample of 5 " sandwich " structures.
6) using Raman spectrometer to step 4) the detection sample of 5 " sandwich " structures that obtains after processing carries out SERS
The chaacteristic fingerprint spectrum of Raman labels thing in spectral measurement, detection immunological probe.The SERS light of 5 samples obtained by the present embodiment
Spectrum is as shown in Figure 6.
From fig. 6 it can be seen that in PSA antigen solution concentration as little as 2.1fg/mL, in Raman shift 1078cm-1Place is still
So there is obvious SERS signal.So, the self assembly gold nanorods SERS prepared by embodiment 2 is immunized substrate and carries out PSA
Antigen concentration is detected, with high sensitivity.
The reappearance and signal of underlying structure are immunized for the self assembly gold nanorods SERS that uses in checking the present embodiment
Stability, carries out repeated detection, as shown in fig. 7,25 differences in substrate for PSA antigen concentrations for 2.1ng/mL sample
Test point gathers 1078cm-1SERS peak intensity sizes, as can be seen from the figure the substrate reappearance, stability are preferable.
The above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications also should
It is considered as protection scope of the present invention.
Claims (10)
1. a kind of self assembly gold nanorods SERS is immunized substrate, including single-sided polishing silicon chip, be fixed on the silicon chip list polishing
Gold nanorods and the antibody for being linked at the gold nanorods surface on face.
2. substrate is immunized in SERS according to claim 1, it is characterised in that be fixed on the gold on the silicon chip list burnishing surface
The side length of nanometer rods is 60~80nm, and trans D is 20~30nm.
3. substrate is immunized in SERS according to claim 1, it is characterised in that be fixed on single burnishing surface of the silicon chip
Gold nanorods continuous arrangement formation self-assembled structures.
4. substrate is immunized in the SERS according to claims 1 to 3 any one, it is characterised in that described gold nanorods connect
Continuous to be arranged in self-assembled structures, described self-assembled structures include the transversely arranged self-assembled structures and gold nanorods of gold nanorods
The self-assembled structures of longitudinal arrangement；Described transversely arranged self-assembled structures are fixed on silicon by the side of described gold nanorods
On the burnishing surface of piece and adjacent gold nanorods the self-assembled structures shoulder to shoulder that are connected to form of side；Described longitudinal arrangement from
Package assembly is fixed on the burnishing surface of silicon chip by the end face of described gold nanorods and the side of adjacent gold nanorods is connected shape
Into be vertically arranged self-assembled structures.
5. the preparation method of substrate is immunized in the self assembly gold nanorods SERS described in Claims 1 to 4 any one, its feature exists
In comprising the following steps：
1) it is 0.45~0.55mmol/L chlorauric acid solution, 0.15~0.25mol/L cetyl trimethylammonium bromide is molten
Liquid, 0.005~0.02mol/L sodium borohydride solutions and deionized water are 10~15 according to volume ratio：10~15：5.0~6.0：1
Mixing, stands, and obtains gold and plants solution；
2) it is 1 according to volume ratio by 0.0768mol/L cetyl trimethylammonium bromides and 0.0162mol/L sodium oleate solutions：1
Mix to being completely dissolved, obtain mixed liquor；4mmol/L silver nitrate solutiones, 1mmol/L are sequentially added into described mixed liquor again
Chlorauric acid solution, the hydrochloric acid solution and 10mmol/L ascorbic acid solutions of volumetric concentration 37% obtain liquid of growing up；Mixed in described
The 4mmol/L silver nitrate solutiones added in liquid are closed, 1mmol/L chlorauric acid solutions, 37% hydrochloric acid solution and 10mmol/L is anti-bad
The volume ratio of hematic acid solution is 50~80：1：5~8：2~4；
3) by the step 1) obtained gold plants solution and the step 2) obtained growth liquid according to volume ratio is 1：2500 mix
Close, stand, obtain colloidal sol；After the colloidal sol is centrifuged 1~2 time, the colloidal sol for being deposited on centrifugation bottom of the tube is redissolved and surpassed
Sonication, obtains scattered gold nanorods solution；
4) by the silicon chip hydrophilic treated of single-sided polishing, dry, the silicon chip pre-processed；
5) by the step 3) obtained gold nanorods solution is added drop-wise to the step 4) on the obtained burnishing surface of silicon chip, to drop
Added with silicon chip drying process under the conditions of 10~50 DEG C of gold nanorods solution, the gold nanorods Jenner on silicon chip is secured to
Rice rod；Described gold nanorods continuous arrangement is into self-assembled structures；When gold nanorods solution is added dropwise, the silicon chip need to be tilted,
Angle of inclination is 35~45 °；
6) antibody-solutions are added drop-wise to the step 5) the obtained gold nanorods surface being fixed on silicon chip, after standing, cleaning
Silicon chip, to the drying process under the conditions of 35~38 DEG C of the silicon chip after cleaning, obtains the gold nanorods that chain is connected to antibody；
7) to the step 6) obtained chain is connected on the gold nanorods of antibody and lock solution is added dropwise, cleaned after standing, to cleaning
Silicon chip drying process afterwards, obtains self assembly gold nanorods SERS and substrate is immunized；
The step 1)~3) and step 4) between there is no the limitation of time sequencing.
6. preparation method according to claim 5, it is characterised in that the step 3) in centrifugation rotating speed for 5000~
6000r/min；The time of the centrifugation is 15~25min；The mass concentration of gold nanorods solution is 0.10~0.60g/L.
7. preparation method according to claim 5, it is characterised in that the step 5) in be added drop-wise to the described of unit area
Gold nanorods liquor capacity on the burnishing surface of silicon chip is 0.8~2 μ L/cm2。
8. preparation method according to claim 5, it is characterised in that the step 6) in the mass concentrations of antibody-solutions be
1~3mg/mL.
9. preparation method according to claim 5, it is characterised in that the step 7) in lock solution be bovine serum albumin
White solution；The mass concentration of the bovine serum albumin solution is 5~20mg/mL.
10. preparation method according to claim 5, it is characterised in that the silicon wafer polishing face for being added drop-wise to unit area
On bovine serum albumin solution volume be 1~4 μ L/cm2。
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