CN106940310B - 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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Abstract
The present invention provides the preparation methods that substrate is immunized in a kind of controllable self assembly gold nanorods SERS.It includes the silicon wafer of 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 that substrate, which is immunized, in the SERS.The present invention uses Dual Surfactants method to prepare gold nanorods first, and the gold nanorods are added drop-wise on the burnishing surface of the silicon wafer of hydrophilic treated mistake, and the gold nanorods are arranged in continuous self-assembled structures on the burnishing surface of silicon wafer.During preparation, keep silicon wafer with respect to 35~45 ° of ground inclination, to overcome coffee ring effect, so that self assembly gold nanorods SERS is immunized substrate and has excellent SERS enhancing characteristic and repeatability, applied to highly sensitive immune detection.In addition, the preparation process of the immune substrate of the self assembly gold nanorods SERS is simple, low in cost, time-consuming less, yield is high, and it is easy to spread, convenient for application.
Description
Technical field
The invention belongs to nano material preparation 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 technique
Since Surface enhanced Raman scattering (SERS) phenomenon is found, SERS spectra technology is just because of its highly sensitive and nothing
The advantage of detection is damaged by extensive concern, and is rapidly developed.Noble metal nano grain is synthesized using chemistry or physical method
Son, and be prepared into the active substrate with SERS enhancing ability, then it can be active to SERS is located at using SERS spectra technology
The analyte of substrate surface carries out highly sensitive dactylogram detection.Therefore, the high-performance of very big SERS enhancing ability is prepared
SERS active-substrate is applied to the fields of biomedicine such as bio-sensing, medical imaging, treatment of cancer, drug delivery with important
Meaning.
Generally, being primarily upon it to the performance requirement of the SERS active-substrate of noble metal nano particles composition 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.Currently, being included the following three types using the common method that noble metal nano particles prepare SERS substrate:
The first is that the noble metal nano particles colloidal sol of chemical synthesis preparation is directly added dropwise on substrate (such as silicon wafer or glass) certainly
So form SERS substrate；Second is using the surface of chemical method modification noble metal nano particles and substrate, in electrostatic masterpiece
Substrate surface, which is fixed on, with lower metal nanoparticle forms SERS substrate；The third is to utilize physical etchings or electrochemical deposition etc.
Method prepares the SERS substrate with noble metal nano particles array structure.In fact, the above-mentioned method for preparing SERS substrate
Shortcomings.For example, first method is relatively easy, but the noble metal nano particles distribution in the SERS substrate usually prepared is not
, lead to the less reproducible of SERS signal；The SERS substrate that second method obtains has stronger SERS signal, but stablizes
Property is not good enough；The SERS substrate of the third method preparation can be obtained strong SERS signal, and stability and reproducible, but make
Make complex process, higher cost.
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 substrate 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
The features such as property and biocompatibility, gold nanorods colloidal sol is deposited on the silicon wafer that hydrophilic treated is crossed by solvent evaporated method and is 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 application No. is the preparation methods of the immunocompetence substrate of 201310015549.3 patent disclosure in the prior art, though
Right method preparation process is simple, but prepared gold nanoparticle package assembly small scale, is easy by coffee ring effects,
Application range is restricted.
Summary of the invention
The present invention provides a kind of self assembly gold nanorods SERS and substrate and preparation method thereof is immunized, in the immune substrate
Gold nanorods are continuously arranged self-assembled structures, and the SERS signal for generating the immune substrate is with intensity height and surely
Fixed feature.
The present invention to solve above-mentioned technical problem the following technical schemes are provided:
Material needed for substrate is immunized in a kind of self assembly gold nanorods SERS provided by the invention includes the silicon of single-sided polishing
Piece, the gold nanorods being fixed on the burnishing surface of the single-sided polishing silicon wafer and the antibody for being linked at the gold nanorods surface.
The silicon wafer of the single-sided polishing can use monocrystalline silicon silicon wafer or polysilicon silicon wafer.
Be fixed on the side length of the gold nanorods on the burnishing surface of the single-sided polishing silicon wafer preferred scope be 60~
80nm。
Be fixed on the trans D of the gold nanorods on the burnishing surface of the single-sided polishing silicon wafer preferred scope be 20~
30nm。
The preferred scope for being fixed on the mass concentration of the gold nanorods on the burnishing surface of the single-sided polishing silicon wafer is 0.10
~0.60g/L.
The preferred scope of the mass concentration of the 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 wafer is described from group at self-assembled structures
Assembling structure includes the self-assembled structures of gold nanorods transversely arranged self-assembled structures and gold nanorods longitudinal arrangement；The cross
It is fixed on the burnishing surface of silicon wafer by the side of the gold nanorods 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 the longitudinal arrangement by the gold nanorods end
Face is fixed on the burnishing surface of silicon wafer and what the side of adjacent gold nanorods was connected to form is vertically arranged self-assembled structures.
The link is the combination that the surface of antibody and the gold nanorods is realized in the form of valence link.
The preparation method of substrate is immunized in the self assembly gold nanorods SERS provided by the invention, comprising the following steps:
1) by the cetyl trimethyl bromination of the chlorauric acid solution of 0.45~0.55mmol/L, 0.15~0.25mol/L
Ammonium salt solution, 0.005~0.02mol/L sodium borohydride solution and deionized water according to volume ratio be 10~15:10~15:5.0~
6.0:1 mixing, stands, and obtains golden kind of solution；
2) by 0.0768mol/L cetyl trimethylammonium bromide and 0.0162mol/L sodium oleate solution according to volume ratio
It mixes for 1:1 to being completely dissolved, obtains mixed liquor；4mmol/L silver nitrate solution is sequentially added into the mixed liquor again,
1mmol/L chlorauric acid solution, the hydrochloric acid solution and 10mmol/L ascorbic acid solution of volumetric concentration 37% obtain growth liquid；?
The 4mmol/L silver nitrate solution being added in the mixed liquor, 1mmol/L chlorauric acid solution, 37% hydrochloric acid solution and
The volume ratio of 10mmol/L ascorbic acid solution is 50~80:1:5~8:2~4；
3) it is 1 according to volume ratio by golden kind of the solution that the step 1) obtains and the growth liquid that the step 2) obtains:
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
It is ultrasonically treated, the gold nanorods solution dispersed；
4) by the silicon wafer of single-sided polishing it is hydrophilic treated and dry after obtain pretreated silicon wafer；
5) the gold nanorods solution that the step 3) obtains is added drop-wise on the burnishing surface for the silicon wafer that the step 4) obtains,
When being added drop-wise in the gold nanorods solution on the burnishing surface of the silicon wafer, the silicon wafer is with respect to the horizontal plane kept
Tilt angle is 35~45 °；There is the silicon wafer of the gold nanorods solution to be dried under the conditions of 10~50 DEG C dropwise addition, obtains
Gold nanorods onto the burnishing surface for being fixed on the silicon wafer；The gold nanorods continuous arrangement is at self-assembled structures；
6) antibody-solutions are added drop-wise on gold nanorods described obtained in the step 5), stand, uses phosphorus
Phthalate buffer cleaning silicon chip is dried the silicon wafer after cleaning under the conditions of 35~38 DEG C, and the antibody is linked to
The gold nanorods surface；
7) it is linked in Xiang Suoshu step 6) and bovine serum albumin solution is added dropwise on the gold nanorods for having the antibody, stood about
After 4h, with the slow cleaning of phosphate buffer, it is dried and substrate is immunized to get to self assembly gold nanorods SERS.
Step 1)~3) and step 4) between there is no the limitation of time sequencing.
The range of preferred centrifugal rotational speed is 5000~6000rpm in the step 3)；In the step 3) preferably from
The range of heart time is 15~25min.
The excellent of the gold nanorods liquor capacity on the burnishing surface of the silicon wafer of unit area is added drop-wise in the step 5)
Selecting range is 0.8~2 μ L/cm2。
The preferred scope of the mass concentration for the antibody-solutions being added dropwise in the step 6) is 1~3mg/mL；It is described to be added drop-wise to
The preferred scope of the antibody-solutions volume being fixed on the silicon wafer polishing face of gold nanorods of unit area is 1~4 μ L/
cm2；
The preferred scope of the mass concentration of bovine serum albumin solution described in the step 7) is 5~20mg/mL；It is described
The preferred scope for being added drop-wise to the bovine serum albumin solution volume on the silicon wafer polishing face of unit area is 1~4 μ L/cm2。
In conclusion the preparation method of substrate is immunized in self assembly gold nanorods SERS provided by the invention, by with double tables
Face active agent method prepares the gold nanorods of size uniformity, and the gold nanorods are added dropwise to the polishing for the silicon wafer that hydrophilic treated is crossed
Form controllable self-assembled structures on face, silicon wafer and horizontal plane are tilted a certain angle the shadow for overcoming coffee ring effect when dropwise addition
Ring, ensure that gold nanorods continuous arrangement on the burnishing surface of the silicon wafer forms self-assembled structures, formation from group
Assembling structure significantly enhances SERS signal intensity, and detection is also greatly improved while greatling improve detection sensitivity
Reliability.Self assembly gold nanorods SERS provided by the invention be immunized substrate preparation method, simple process, it is low in cost,
Time-consuming is small, yield is high, easy to spread, convenient for application.
Detailed description of the invention
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 obtains are vertically arranged self-assembled structures；
Fig. 3 is that the scanning electron microscope for the continuously arranged multilayer self-assembled structures of gold nanorods that embodiment 3 obtains is shone
Piece；
Fig. 4 is that the scanning electron microscope for the continuously arranged single layer self-assembled structures of gold nanorods that embodiment 4 obtains is shone
Piece；
Fig. 5 is the Raman labels point linked on the gold nanorods self-assembled structures of Examples 1 to 4 preparation in embodiment 5
The SERS spectra figure of sub- 4MBA；
Fig. 6 is to detect various concentration by immune substrate prepared by the gold nanorods self-assembled structures of embodiment 2 in embodiment 6
The corresponding SERS spectra figure of prostate-specific antigen (PSA)；
Fig. 7 is to detect fixed concentration by immune substrate prepared by the gold nanorods self-assembled structures of embodiment 2 in embodiment 6
When PSA antigen, the 1078cm of different test point acquisitions in substrate-1SERS peak intensity histogram.
Specific embodiment
Material needed for substrate is immunized in a kind of self assembly gold nanorods SERS provided by the invention includes the silicon of single-sided polishing
Piece, the gold nanorods being fixed on the burnishing surface of the single-sided polishing silicon wafer and the antibody for being adsorbed on the gold nanorods surface.
In the present invention, the size of the silicon wafer 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 wafer of the single-sided polishing is not particularly limited, using those skilled in the art
The preparation method of the silicon wafer of known single-sided polishing.In the embodiment of the present invention, the source of the silicon wafer of the single-sided polishing
Purchased from Tianhe Optical Energy Co., Ltd., Changzhou.
In the present invention, the mass concentration of the gold nanorods on the burnishing surface for being fixed on the single-sided polishing silicon wafer
Preferably 0.10~0.60g/L, more preferably 0.25~0.45g/L.
In the present invention, the gold nanorods continuous arrangement on the burnishing surface for being fixed on the single-sided polishing silicon wafer is at certainly
Package assembly, the self-assembled structures include the transversely arranged self-assembled structures of gold nanorods and gold nanorods longitudinal arrangement
Self-assembled structures；The transversely arranged self-assembled structures are fixed on the burnishing surface of silicon wafer by the side of the 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 the longitudinal arrangement by
The end face of the gold nanorods is fixed on the vertical row that on the burnishing surface of silicon wafer and the side of adjacent gold nanorods is connected to form
Column self-assembled structures.
In the present invention, the preferred scope of the side length of the gold nanorods is 60~80nm, and more preferably range is
70~75nm；The preferred scope of the trans D of the gold nanorods is 20~30nm, and more preferably range is 22~27nm,
The trans D of optimization is 25nm.
In the present invention, the preferred scope of the mass concentration of the antibody for being adsorbed on the gold nanorods surface is 1~
3mg/ml, more preferably range are 1.5~2.5mg/ml, and the mass concentration of optimization is 2.0mg/ml.The kind of the antibody
Class includes prostate-specific antigen antibody (anti-PSA), alpha-fetoprotein antibody (anti-AFP) or sugar antigen antibody
(anti-CA19-9) antibody such as.
The present invention provides the self assembly gold nanorods SERS preparation method that substrate is immunized, comprising the following steps:
1) by the chlorauric acid solution of 0.45~0.55mmol/L, the cetyl trimethyl bromination of 0.15~0.25mol/L
Ammonium salt solution, 0.005~0.02 sodium borohydride solution and deionized water are 10~15:10~15:5.0~6.0:1 according to volume ratio
Mixing is stood, and obtains golden kind of solution；
It 2) is that 1:1 is mixed to being completely dissolved according to volume ratio by 0.0768mol/L and 0.0162mol/L sodium oleate solution,
Sequentially add 4mmol/L silver nitrate solution to mixed liquor, 1mmol/L chlorauric acid solution, the hydrochloric acid solution of volumetric concentration 37% and
10mmol/L ascorbic acid solution obtains growth liquid；The 4mmol/L silver nitrate solution that the mixed liquor sequentially adds, 1mmol/L
Chlorauric acid solution, 37% hydrochloric acid solution and the volume ratio of 10mmol/L ascorbic acid solution are 50~80:1:5~8:2~4；
3) golden kind of the solution that the step 1) obtains and the growth liquid that the step 2) obtains is mixed for 1:1 according to volume ratio
It closes, stands, 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
It is ultrasonically treated, the gold nanorods solution dispersed；
4) dry by the silicon wafer hydrophilic treated of single-sided polishing, obtain pretreated silicon wafer；
5) the gold nanorods solution that the step 3) obtains is added drop-wise on the burnishing surface for the silicon wafer that the step 4) obtains,
There are 10~50 DEG C of silicon wafer drying process of gold nanorods solution to dropwise addition；When the dropwise addition by silicon wafer tilt angle be 35~45 °；
6) antibody-solutions are added drop-wise on the silicon wafer polishing face for being fixed with gold nanorods in the step 5), after standing, clearly
Silicon wafer is washed, the silicon wafer after cleaning is dried under the conditions of 35~38 DEG C, obtains the silicon for being fixed with gold nanorods link antibody
Piece；
7) it is fixed in Xiang Suoshu step 6) on the silicon wafer polishing face of gold nanorods link antibody and lock solution is added dropwise, stood
After clean, to after cleaning silicon wafer be dried, obtain self-assembled structures gold nanorods SERS be immunized substrate；
Step 1)~3) and step 4) between there is no the limitation of time sequencing.
In step 1) of the present invention, 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, the preferred scope of the time of repose is 2~6h, the time of repose of optimization
For 4h.
In step 1) of the present invention, 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) and step 3) of the present invention, the hybrid mode of the solution is not particularly limited, and is used
The technical solution of mixed solution well-known to those skilled in the art.
In step 3) of the present invention, the preferred scope of the centrifugal rotational speed is 5000~6000rpm, is more highly preferred to
Range 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, the preferred scope of the time of repose is 10~12h, the standing of optimization
Time is 11h.
In step 3) of the present invention, the solvent that the colloidal sol redissolves is deionized water, the conductance of deionized water
Rate is 18.2M Ω cm.
In step 3) of the present invention, the preferred scope of the ultrasonic cleaning 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.
It is dry by the silicon wafer hydrophilic treated of single-sided polishing in step 4) of the present invention, obtain pretreated silicon
Piece.The method of the silicon wafer hydrophilic treated preferably includes following steps:
I, the silicon wafer of single-sided polishing acid solution is placed in be cleaned by ultrasonic；
II, the silicon wafer after ultrasonic cleaning is rinsed with deionized water；
III, the silicon wafer after rinsing deionized water is placed in alkaline cleaning fluid and is cleaned by ultrasonic, the silicon wafer after ultrasonic cleaning
It is rinsed with deionized water；
IV, the silicon wafer after rinsing the step III is cleaned by ultrasonic with acidic cleaning solution, is finally rinsed, is obtained with deionized water
To the silicon wafer of hydrophilic treated.
In the step of silicon wafer hydrophilic treated of the present invention I, the acid solution includes the H that volumetric concentration is 50%2SO4
The H that solution and volumetric concentration are 20%2O2Solution.
In the step of silicon wafer hydrophilic treated of the present invention III, the alkaline cleaning fluid includes that volumetric concentration is 10%
NH4The H that OH and volumetric concentration are 20%2O2Aqueous solution.
In the step of silicon wafer hydrophilic treated of the present invention IV, the acidic cleaning solution includes that volumetric concentration is 20%
H2O2The aqueous solution for the HCl for being 10% with volumetric concentration.
In the step of silicon wafer hydrophilic treated of the present invention, what the ultrasonic cleaning process used is cleaned by ultrasonic the excellent of power
Selecting range is 100~150w, and the ultrasonic cleaning power of optimization is 120w；It is described ultrasonic cleaning the time preferred scope be 15~
30min, the ultrasonic cleaning time of optimization are 20min.
In step 4) of the present invention, the preferred scope of the silicon wafer drying temperature is 35~38 DEG C, is optimized
Drying temperature be 37 DEG C；The preferred scope of the silicon wafer drying time is 10~14h, and the drying time of optimization is 12h.
In step 5) of the present invention, the gold nanorods solution is added drop-wise to unit plane on the burnishing surface of the silicon wafer
The preferred scope of long-pending volume is 0.8~2 μ L/cm2, more preferably range is 1.2~1.8 μ L/cm2, the volume of optimization is
1.5μL/cm2。
It is described that the preferred model for having the drying temperature of silicon wafer of gold nanorods solution is added dropwise in step 5) of the present invention
Enclosing is 10~20 DEG C, and the drying temperature of optimization is 15 DEG C.The dropwise addition has the drying time of the silicon wafer of gold nanorods solution
Preferred scope is 2~6h, and the drying time of optimization is 4h.
In step 5) of the present invention, the optimization tilt angle of inclination silicon wafer is 40 ° when the dropwise addition.
In step 6) of the present invention, 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 range, 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 type of the antibody is without spy
Different limitation, the antibody studied using those skilled in the art.
In step 6) of the present invention, the preferred scope of the temperature of the standing is 2~8 DEG C, is more highly preferred to range
It is 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, the 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 wafer after TRIS buffer cleaning use again deionized water into
Row rinses.
In step 6) of the present invention, the preferred scope of the drying temperature is 35~38 DEG C, the temperature of optimization
It is 37 DEG C.
In step 7) of the present invention, 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 on the silicon wafer polishing face of gold nanorods is 1~4 μ L/
cm2。
In step 7) of the present invention, the bovine serum albumin solution is added drop-wise to the silicon wafer 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, the cleaning solution is the trihydroxy methyl ammonia containing 0.05%Tween 20
Methylmethane 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 wafer after TRIS buffer cleaning use again deionized water into
Row rinses.
In step 7) of the present invention, the preferred scope of the drying temperature is 35~38 DEG C, the temperature of optimization
It is 37 DEG C.
Substrate and its preparation side are immunized to a kind of self assembly gold nanorods SERS provided by the invention below with reference to embodiment
Method is described in detail, but they cannot be interpreted as limiting the scope of the present invention.
Embodiment 1
Prepare monodispersed uniform gold nanorods solution, detailed process are as follows: a1, at room temperature by the chlorine of 5mL 0.50mM gold
The cetyl trimethylammonium bromide solution of acid solution and 5mL 0.20M stir evenly, and the 0.6mL of frost is added thereto rapidly
0.01M sodium borohydride solution, and 0.4mL deionized water is added, golden kind of solution is obtained, it is stand-by to stand at least 2h at room temperature；a2,
2.5mL 0.0768M cetyl trimethylammonium bromide and 2.5mL 0.0162M sodium oleate solution are mixed to being completely dissolved, to
180 μ L4mM silver nitrate solutions are wherein added, stand 15min, then 2.5mL 1mM chlorauric acid solution is added thereto, stirring
After 15min, after ie in solution becomes colourless, the hydrochloric acid solution of 21 μ L 37% is added, is added followed by 12 μ L 10mM ascorbic acid
Solution obtains growth liquid；A3,8 μ L are added in the growth liquid prepared by a2 step by golden kind of solution prepared by a1 step,
12h is stood at room temperature, finally obtains gold nanorods colloidal sol.The gold nanorods solution injection centrifuge tube being prepared, then to gold
Nanometer rods solution carries out eccentric cleaning, and supernatant is then removed after eccentric cleaning, retains and is deposited in centrifugation bottom of the tube
Simultaneously deionized water is added in gold nanorods colloidal sol, and centrifugation repeatedly, is ultrasonically treated, and the suspension for obtaining monodispersed uniform gold nanorods is molten
Liquid；Here, carrying out the revolving speed of eccentric cleaning to gold nanorods solution is 6000r/min, centrifugation time is 20min, past to be deposited in
Being centrifuged the deionized water being added in the gold nanorods colloidal sol of bottom of the tube is 40mL, and centrifugation number is 2 times.The place being ultrasonically treated
The reason time is 25min.
Prepare the gold nanorods substrate of self assembly, detailed process are as follows: take the single-sided polishing for the square that one piece of side length is 5mm
Silicon wafer, then silicon wafer is cleaned using silicon wafer cleaning process, cleaning process are as follows: first that silicon wafer is clear with Acidic Liquid ultrasound
It washes, is then rinsed with deionized water, then be cleaned by ultrasonic with alkaline cleaning fluid, then surpassed after deionized water is rinsed with acidic cleaning solution
Sound cleaning, is finally rinsed with deionized water；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, the H that volumetric concentration is 20%2O2Water
Solution；The acidic cleaning solution includes the H that volumetric concentration is 20%2O2, the aqueous solution for the HCl that volumetric concentration is 10%.To cleaning
Clean silicon wafer is dried with spare, will be added drop-wise to processing by the 40 μ L of gold nanorods solution of mass concentration 0.60g/L
On the burnishing surface for the silicon wafer crossed, and silicon wafer and horizontal plane is kept to tilt 40 °, is then maintained in 10 DEG C of insulating box and is dried
Processing, has obtained 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 being deposited on the gold nanorods on silicon wafer is uniform, the size of single gold nanorods are as follows: longitudinal long 75 ± 5nm,
Trans D is 25 ± 2nm.
Embodiment 2
Prepare monodispersed uniform gold nanorods solution, detailed process are as follows: a1, at room temperature by the chlorine of 5mL 0.45mM gold
The cetyl trimethylammonium bromide solution of acid solution and 5mL 0.25M stir evenly, and the 0.6mL of frost is added thereto rapidly
0.005M sodium borohydride solution, and 0.4mL deionized water is added, golden kind of solution is obtained, it is stand-by to stand at least 2h at room temperature；
A2,2.5mL 0.0768M cetyl trimethylammonium bromide and 2.5mL 0.0162M sodium oleate solution are mixed to completely molten
180 μ L4mM silver nitrate solutions are added in solution thereto, stand 15min, then 2.5mL 1mM chlorauric acid solution is added thereto, stir
After mixing 15min, after ie in solution becomes colourless, the hydrochloric acid solution of 21 μ L 37% is added, is added followed by 12 μ L 10mM Vitamin Cs
Acid solution obtains growth liquid；A3,8 μ L are added in the growth liquid prepared by a2 step by golden kind of solution prepared by a1 step,
12h is stood at room temperature, finally obtains gold nanorods colloidal sol.The gold nanorods solution injection centrifuge tube being prepared, it is then right
Gold nanorods solution carries out eccentric cleaning, and supernatant is then removed after eccentric cleaning, and reservation is deposited in centrifugation bottom of the tube
Gold nanorods colloidal sol and deionized water is added, centrifugation repeatedly, ultrasonic treatment, obtain the suspension of monodispersed uniform gold nanorods
Solution；Here, carrying out the revolving speed of eccentric cleaning to gold nanorods solution is 6000r/min, centrifugation time is 20min, toward precipitating
The deionized water being added in the gold nanorods colloidal sol of centrifugation bottom of the tube is 40mL, and centrifugation number is 2 times.It is ultrasonically treated
The processing time is 25min.
Prepare the gold nanorods substrate of self assembly, detailed process are as follows: take the single-sided polishing for the square that one piece of side length is 5mm
Silicon wafer, then silicon wafer is cleaned using silicon wafer cleaning process, cleaning process are as follows: first that silicon wafer is clear with Acidic Liquid ultrasound
It washes, is then rinsed with deionized water, then be cleaned by ultrasonic with alkaline cleaning fluid, then surpassed after deionized water is rinsed with acidic cleaning solution
Sound cleaning, is finally rinsed with deionized water；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, the H that volumetric concentration is 20%2O2Water
Solution；The acidic cleaning solution includes the H that volumetric concentration is 20%2O2, the aqueous solution for the HCl that volumetric concentration is 10%.To cleaning
Clean silicon wafer is dried with spare, will be added drop-wise to processing by the 50 μ L of gold nanorods solution of mass concentration 0.45g/L
On the burnishing surface for the silicon wafer crossed, and silicon wafer and horizontal plane tilt 45 °, are then maintained in 15 DEG C of insulating box and place is dried
Reason, obtains 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.Scheming
In 2, the size for being deposited on the gold nanorods on silicon wafer is uniform, the size of single gold nanorods are as follows: longitudinal long 70 ± 5nm, it is horizontal
It is 27 ± 2nm to diameter.
Embodiment 3
Prepare monodispersed uniform gold nanorods solution, detailed process are as follows: a1, at room temperature by the chlorine of 5mL 0.55mM gold
The cetyl trimethylammonium bromide solution of acid solution and 5mL 0.15M stir evenly, and the 0.6mL of frost is added thereto rapidly
0.02M sodium borohydride solution, and 0.4mL deionized water is added, golden kind of solution is obtained, it is stand-by to stand at least 2h at room temperature；a2,
2.5mL 0.0768M cetyl trimethylammonium bromide and 2.5mL 0.0162M sodium oleate solution are mixed to being completely dissolved, to
180 μ L4mM silver nitrate solutions are wherein added, stand 15min, then 2.5mL 1mM chlorauric acid solution is added thereto, stirring
After 15min, after ie in solution becomes colourless, the hydrochloric acid solution of 21 μ L 37% is added, is added followed by 12 μ L 10mM ascorbic acid
Solution obtains growth liquid；A3,8 μ L are added in the growth liquid prepared by a2 step by golden kind of solution prepared by a1 step,
12h is stood at room temperature, finally obtains gold nanorods colloidal sol.The gold nanorods solution injection centrifuge tube being prepared, then to gold
Nanometer rods solution carries out eccentric cleaning, and supernatant is then removed after eccentric cleaning, retains and is deposited in centrifugation bottom of the tube
Simultaneously deionized water is added in gold nanorods colloidal sol, and centrifugation repeatedly, is ultrasonically treated, and the suspension for obtaining monodispersed uniform gold nanorods is molten
Liquid；Here, carrying out the revolving speed of eccentric cleaning to gold nanorods solution is 6000r/min, centrifugation time is 20min, past to be deposited in
Being centrifuged the deionized water being added in the gold nanorods colloidal sol of bottom of the tube is 50mL, and centrifugation number is 3 times.The place being ultrasonically treated
The reason time is 25min.
Prepare the gold nanorods substrate of self assembly, detailed process are as follows: take the single-sided polishing for the square that one piece of side length is 5mm
Silicon wafer, then silicon wafer is cleaned using silicon wafer cleaning process, cleaning process are as follows: first that silicon wafer is clear with Acidic Liquid ultrasound
It washes, is then rinsed with deionized water, then be cleaned by ultrasonic with alkaline cleaning fluid, then surpassed after deionized water is rinsed with acidic cleaning solution
Sound cleaning, is finally rinsed with deionized water；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, the H that volumetric concentration is 20%2O2Water
Solution；The acidic cleaning solution includes the H that volumetric concentration is 20%2O2, the aqueous solution for the HCl that volumetric concentration is 10%.To cleaning
Clean silicon wafer is dried with spare, will be added drop-wise to processing by the 50 μ L of gold nanorods solution of mass concentration 0.25g/L
On the burnishing surface for the silicon wafer crossed, and silicon wafer and horizontal plane tilt 38 °, and then, silicon wafer is maintained in 40 DEG C of insulating box and carries out
It is dried, obtains 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 Fig. 3, the size for being deposited on the gold nanorods on silicon wafer is uniform, the size of single gold nanorods are as follows: and longitudinal direction length 72 ±
5nm, trans D are 25 ± 2nm.
Embodiment 4
Prepare monodispersed uniform gold nanorods solution, detailed process are as follows: a1, at room temperature by the chlorine of 5mL 0.50mM gold
The cetyl trimethylammonium bromide solution of acid solution and 5mL 0.20M stir evenly, and the 0.6mL of frost is added thereto rapidly
0.01M sodium borohydride solution, and 0.4mL deionized water is added, golden kind of solution is obtained, it is stand-by to stand at least 2h at room temperature；a2,
2.5mL 0.0768M cetyl trimethylammonium bromide and 2.5mL 0.0162M sodium oleate solution are mixed to being completely dissolved, to
180 μ L4mM silver nitrate solutions are wherein added, stand 15min, then 2.5mL 1mM chlorauric acid solution is added thereto, stirring
After 15min, after ie in solution becomes colourless, the hydrochloric acid solution of 21 μ L 37% is added, is added followed by 12 μ L 10mM ascorbic acid
Solution obtains growth liquid；A3,8 μ L are added in the growth liquid prepared by a2 step by golden kind of solution prepared by step a1,
12h is stood at room temperature, finally obtains gold nanorods colloidal sol.The gold nanorods solution injection centrifuge tube being prepared, then to gold
Nanometer rods solution carries out eccentric cleaning, then removes supernatant after eccentric cleaning, toward the gold for being deposited in centrifugation bottom of the tube
Nanometer rods colloidal sol is added in 10ml deionized water, and repeatedly, ultrasonic treatment obtains the suspension of monodispersed uniform gold nanorods for centrifugation
Solution；Here, carrying out the revolving speed of eccentric cleaning to gold nanorods solution is 6000r/min, centrifugation time is 20min, toward precipitating
The deionized water being added in the gold nanorods colloidal sol of centrifugation bottom of the tube is 10mL, and centrifugation number is 2 times.It is ultrasonically treated
The processing time is 25min.
Prepare the gold nanorods substrate of self assembly, detailed process are as follows: take the single-sided polishing for the square that one piece of side length is 5mm
Silicon wafer, then silicon wafer is cleaned using silicon wafer cleaning process, cleaning process are as follows: first that silicon wafer is clear with Acidic Liquid ultrasound
It washes, is then rinsed with deionized water, then be cleaned by ultrasonic with alkaline cleaning fluid, then surpassed after deionized water is rinsed with acidic cleaning solution
Sound cleaning, is finally rinsed with deionized water；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, the H that volumetric concentration is 20%2O2Water
Solution；The acidic cleaning solution includes the H that volumetric concentration is 20%2O2, the aqueous solution for the HCl that volumetric concentration is 10%.To cleaning
Clean silicon wafer is dried with spare, silicon wafer will be added dropwise by the 50 μ L of gold nanorods solution of mass concentration 0.10g/L
On burnishing surface, and silicon wafer and horizontal plane tilt 38 °, and then, silicon wafer is maintained in 50 DEG C of insulating box and is dried, i.e.,
Self assembly gold nanorods substrate is made.
It is in continuously arranged single 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 being deposited on the gold nanorods on silicon wafer is uniform, the size of single gold nanorods are as follows: and longitudinal direction length 70 ±
5nm, trans D are 30 ± 2nm.
Embodiment 5
The present embodiment provides a kind of by the link Raman labels point of self assembly gold nanorods substrate 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) Raman labels molecule is used as to mercaptobenzoic acid (4MBA), 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, is cleaned with deionized water
The substrate of 4MBA mark molecule is not linked, after cleaning 2~3 times, keeps 12h dry in 37 DEG C of insulating boxs.
3) SERS of the 4MBA mark molecule of the self assembly gold nanorods substrate link prepared by the step 2) is measured
Spectrum.When detection, make laser emit laser line focus after vertical irradiation in assembling substrate on, labeled point of the laser of irradiation
It is received after the substrate reflection of son by Raman spectrometer, the feature SERS spectra of the 4MBA mark molecule of Raman spectrometer record is as schemed
In 5 shown in curve 1.The time of integration for acquiring SERS spectra is 1~10s, and the power for irradiating laser is 49.55mw, optical maser wavelength
For 785nm.
According to above-mentioned same step, measurement by the embodiment of the present invention 2, embodiment 3 and embodiment 4 be prepared from group
The SERS spectra for filling gold nanorods substrate, as a result respectively as shown in curve 2, curve 3 and curve 4 in Fig. 5.
As seen from Figure 5, corresponding after the 4MBA Raman labels molecule of different self assembly gold nanorods substrate link same concentrations
4MBA SERS spectra feature peak intensity size have it is 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 obtains are most
By force.
Embodiment 6
The present embodiment provides the implementation process for preparing a kind of immune substrate of self assembly gold nanorods SERS.With the system of embodiment 2
For standby self assembly gold nanorods substrate, the specific steps are as follows:
1) the anti-PSA antibody-solutions for being used to carry out PSA antigen detection are added drop-wise to prepared by processed embodiment 2
Self assembly gold nanorods substrate, and 10~12h is stood under conditions of temperature is 2~8 DEG C, 0.05%Tween is successively used later
20 TRIS buffers and deionized water rinse silicon wafer, to remove the unadsorbed anti-PSA on gold nanorods
Antibody is finally dried the self assembly gold nanorods substrate for being adsorbed with anti-PSA antibody, dropwise addition 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 later
TRIS buffer and deionized water silicon wafer is rinsed, to remove the unadsorbed ox on gold nanorods
Seralbumin, then silicon wafer is dried, the drying is to keep 12h in 37 DEG C of insulating boxs, and tool is prepared
There is the SERS of specific selection function that substrate is immunized.
It 2) is the PSA antigenic solution to be measured of 0fg/mL, 2.1fg/mL, 210fg/mL, 21pg/mL and 2.1ng/mL from concentration
It takes 20 μ L to be added drop-wise in sample respectively to be immunized in substrate by 5 self assembly gold nanorods SERS of step 1) preparation, is 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 and is dried, it is dense to obtain the different PSA antigens of 5 correspondences
Substrate is immunized in the SERS of degree.
3) preparation according to the prior art, such as application No. is the gold nanosphere colloidal sol of 201310015549.3 patent disclosure
Method, the gold nanosphere colloidal sol that preparation diameter is 20nm.It is the molten to mercaptobenzoic acid solution (4MBA) of 10mM in concentration
It takes 10 μ L to be added drop-wise in the gold nanosphere colloidal sol of 1mL in liquid, after being sufficiently mixed and standing 4~6h, modification is prepared
There is the gold nanosphere probe solution of the 20nm of 4MBA.After placing 12h at room temperature, centrifugal treating is carried out, with 11000rpm
Revolving speed be centrifuged 30min, remove the supernatant liquor in centrifuge tube later, then deposit is dissolved in the phosphate buffer solution of 1mL
(PBS) in.In the case where being slowly stirred, into the solution obtained after step c processing, 20 μ L is added 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 the revolving speed of 11000rpm,
To remove the unadsorbed anti-PSA antibody on gold nanoparticle surface, the supernatant liquor in centrifuge tube is removed later, then super
Under sonication environment, the deposit in centrifuge tube is dissolved in the phosphate buffer solution of 1mL.Here, the antibody being added is molten
The amount of liquid can control within the scope of 15~30 μ L, and concentration is can be controlled within the scope 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 within the scope of 1~3mL.It is immune that gold nanosphere is prepared as a result,
Probe solution.
4) the anti-PSA antibody-solutions for being used to carry out PSA antigen detection are added drop-wise to prepared by processed embodiment 2
Self assembly gold nanorods substrate, and 10~12h is stood under conditions of temperature is 2~8 DEG C, 0.05%Tween is successively used later
20 TRIS buffers and deionized water rinse silicon wafer, to remove the unadsorbed anti-PSA on gold nanorods
Antibody is finally dried the self assembly gold nanorods substrate for being adsorbed with anti-PSA antibody, dropwise addition 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 later
TRIS buffer and deionized water silicon wafer is rinsed, to remove the unadsorbed ox on gold nanorods
Seralbumin, then silicon wafer is dried, the drying is to keep 12h in 37 DEG C of insulating boxs, and tool is prepared
There is the SERS of specific selection function that substrate is immunized.
5) taking 20 μ L to be added drop-wise to link prepared by step 2) respectively from immunological probe liquid prepared by step 4) has to be measured resist
5 former SERS are immunized in substrate, react 1.5~3h in the case where temperature is 4 DEG C of constant temperature, keep the PSA to be measured being added dropwise anti-
Antigen and SERS in original solution are immunized the anti-PSA antibody in the anti-PSA antibody and immunological probe in substrate and occur
Immune recombination reaction.Later, with the TRIS buffer containing 0.05%Tween 20 and deionized water to every
A SERS is immunized substrate and is rinsed, then substrate is immunized to all SERS and is dried, and keeps 12h in 37 DEG C of insulating boxs
Drying to get to the test sample of 5 " sandwich " structures.
6) SERS is carried out using test sample of the Raman spectrometer to 5 " sandwich " structures obtained after step 4) processing
Spectral measurement detects the chaacteristic fingerprint spectrum of Raman labels object in immunological probe.By the SERS light for 5 samples that the present embodiment obtains
Spectrum is as shown in Figure 6.
From fig. 6 it can be seen that when PSA antigen solution concentration is down to 2.1fg/mL, in Raman shift 1078cm-1Place is still
So there is apparent SERS signal.So substrate, which is immunized, by the self assembly gold nanorods SERS that embodiment 2 is prepared carries out PSA
Antigen concentration detection, has high sensitivity.
To verify the reproducibility and signal that underlying structure is immunized in the self assembly gold nanorods SERS that uses in the present embodiment
Stability carries out repeated detection for the sample that PSA antigen concentration is 2.1ng/mL, as shown in fig. 7,25 differences in substrate
Test point acquires 1078cm-1SERS peak intensity size, as can be seen from the figure the substrate reproducibility, stability are preferable.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Claims (6)
1. the preparation method that substrate is immunized in self assembly gold nanorods SERS, which comprises the following steps:
1) cetyl trimethylammonium bromide of the chlorauric acid solution of 0.45~0.55mmol/L, 0.15~0.25mol/L is molten
Liquid, 0.005~0.02mol/L sodium borohydride solution and deionized water are 10~15:10~15:5.0~6.0:1 according to volume ratio
Mixing is stood, and obtains golden kind of solution；
It 2) is 1:1 according to volume ratio by 0.0768mol/L cetyl trimethylammonium bromide and 0.0162mol/L sodium oleate solution
It mixes to being completely dissolved, obtains mixed liquor；4mmol/L silver nitrate solution, 1mmol/L are sequentially added into the mixed liquor again
Chlorauric acid solution, the hydrochloric acid solution and 10mmol/L ascorbic acid solution of volumetric concentration 37% obtain growth liquid；It is mixed in described
The 4mmol/L silver nitrate solution being added in conjunction liquid, 1mmol/L chlorauric acid solution, 37% hydrochloric acid solution and 10mmol/L are anti-bad
The volume ratio of hematic acid solution is 50~80:1:5~8:2~4；
3) golden kind of the solution that the step 1) obtains and the growth liquid that the step 2) obtains is mixed for 1:2500 according to volume ratio
It closes, stands, 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, the gold nanorods solution dispersed；
4) dry by the silicon wafer hydrophilic treated of single-sided polishing, obtain pretreated silicon wafer；
5) the gold nanorods solution that the step 3) obtains is added drop-wise on the burnishing surface for the silicon wafer that the step 4) obtains, to drop
It is dried under the conditions of 10~50 DEG C added with the silicon wafer of gold nanorods solution, the gold nanorods Jenner being secured on silicon wafer
Rice stick；The gold nanorods continuous arrangement is at self-assembled structures；When gold nanorods solution is added dropwise, the silicon wafer need to be tilted,
Tilt angle is 35~45 °；
6) antibody-solutions are added drop-wise to the gold nanorods surface being fixed on silicon wafer that the step 5) obtains, after standing, cleaning
Silicon wafer is dried the silicon wafer after cleaning under the conditions of 35~38 DEG C, obtains the gold nanorods that chain is connected to antibody；
7) chain that Xiang Suoshu step 6) obtains, which is connected on the gold nanorods of antibody, is added dropwise lock solution, cleans after standing, to cleaning
Silicon wafer afterwards is dried, and obtains self assembly gold nanorods SERS and substrate is immunized；Step 1)~3) and step 4) between do not have
The limitation of having time sequence.
2. preparation method according to claim 1, which is characterized in that the revolving speed being centrifuged in the step 3) is 5000~
6000r/min；The time of the centrifugation is 15~25min；The mass concentration of gold nanorods solution is 0.10~0.60g/L.
3. preparation method according to claim 1, which is characterized in that be added drop-wise to the described of unit area in the step 5)
Gold nanorods liquor capacity on the burnishing surface of silicon wafer is 0.8~2 μ L/cm2。
4. preparation method according to claim 1, which is characterized in that the mass concentration of antibody-solutions is in the step 6)
1~3mg/mL.
5. preparation method according to claim 1, which is characterized in that lock solution is bovine serum albumin in the step 7)
White solution；The mass concentration of the bovine serum albumin solution is 5~20mg/mL.
6. preparation method according to claim 3, which is characterized in that on the silicon wafer polishing face for being added drop-wise to unit area
Bovine serum albumin solution volume be 1~4 μ L/cm2。
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