CN107643400B - The preparation method and applications of cancer markers detection architecture based on silver-colored aggregation-polymer and Crystal structure - Google Patents
The preparation method and applications of cancer markers detection architecture based on silver-colored aggregation-polymer and Crystal structure Download PDFInfo
- Publication number
- CN107643400B CN107643400B CN201710804383.1A CN201710804383A CN107643400B CN 107643400 B CN107643400 B CN 107643400B CN 201710804383 A CN201710804383 A CN 201710804383A CN 107643400 B CN107643400 B CN 107643400B
- Authority
- CN
- China
- Prior art keywords
- silver
- polymer
- crystal structure
- concentration
- buffer solution
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Landscapes
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
Abstract
The invention discloses the preparation method and applications based on silver-colored aggregation-polymer and the cancer markers detection architecture of Crystal structure, feature is the preparation the following steps are included: (1) silver aggregation-polymer SERS immunological probe；(2) preparation of substrate is immunized in Crystal structure；(3) cancer markers detection architecture assembles；Its application is to utilize Raman spectrometer to carry out spectral measurement the cancer markers detection architecture based on silver-colored aggregation-polymer and Crystal structure, according to the quantitative relationship between spectral intensity and antigen concentration, the concentration for obtaining antigen in the phosphate buffer solution containing determined antigen can be calculated, advantage is that the preparation method and applications of the cancer markers detection architecture based on silver-colored aggregation-polymer and Crystal structure of specificity and repeatability of testing result are improved while obtaining lower detectable limit.
Description
Technical field
The present invention relates to a kind of detection methods of cancer markers, more particularly, to one kind based on silver-colored aggregation-polymer
With the preparation method and applications of the cancer markers detection architecture of Crystal structure.
Background technique
In recent years, with the continuous quickening of human production life rhythm, global warming, bio-diversity falls sharply, area
Disease problems caused by the environmental disruptions such as domain property atmosphere combined pollution are more serious.Research shows that 70% or more carcinogenic and rush
Cancer factor and environmental pollution close relation.With the progress of photoelectron science and technology, especially by micro-nano preparation skill
Art, advanced photoelectric detecting technology are combined with modern biotechnology, and people have been developed based on the Gao Te between antigen-antibody
The biochip of specific immunological reaction detection cancer-specific antigen.Currently, the immune inspection of Surface enhanced Raman scattering (SERS) base
One research hotspot and difficult point in survey field are how to obtain further to obtain the high signal output of intensity, and then obtain lower
The immune detection limit.It, need to there are some in immune substrate studies have shown that obtain the lower SERS base immune detection limit
Hot spot region with especially high electromagnetic field intensity.Therefore, advanced micro Nano material technology of preparing, controllable and letter how to be used
Just synthesize the hot spot with high SERS enhancement factor, further promote SERS base immunoassay technology mature still need to compared with
Big effort.The uniformity is higher especially by designing and producing, and signal strength is larger and the immunological probe and substrate pair that last a long time
The clinical serum sample of cancer patient carries out immunoassays and improves the spy of testing result while obtaining lower detectable limit
Anisotropic and repeatability, to improve cancer sufferer rehabilitation and chances of survival be still SERS base field of immunodetection difficult point
And major tasks.
Summary of the invention
Technical problem to be solved by the invention is to provide one kind while obtaining lower detectable limit, improves detection knot
The cancer markers detection architecture based on silver-colored aggregation-polymer and Crystal structure of the specificity and repeatability of fruit
Preparation method and applications.
The technical scheme of the invention to solve the technical problem is: a kind of based on silver-colored aggregation-polymer and gold
The preparation method of the cancer markers detection architecture of nano-wire array, comprising the following steps:
(1) preparation of silver-colored aggregation-polymer SERS immunological probe
A. 1.47-4.41 milligrams of sodium citrates and 1.97-5.91 milligrams of gold chlorides are dissolved in 20-60 milliliters of water and mixing is made
Solution, the concentration that 0.6-1.8 milliliters of Fresh are added when being vigorously stirred at room temperature is the sodium borohydride of 4 mg/mls
Aqueous solution obtains gold seeds solution；
B. 3-9 milliliters of gold seeds solution is taken to be added in the 50-150 milliliters of aqueous solutions containing silver nitrate and sodium citrate,
The aqueous ascorbic acid that 10-30 milliliters of concentration are 2 mg/mls is added dropwise thereto later, reaction obtains Yin Na in 1 hour
Rice grain aqueous solution；
C. taking 1-3 milliliters of silver nano-grain aqueous solution centrifugal concentratings is 10-30 microlitres, and 820-2460 is gradually added thereto
Microlitre dimethylformamide, the dimethyl formamide solution and 20-60 microlitres of water of the 5-15 microlitres of thionaphthol containing 2- are subsequently placed in 60
After being cultivated 3 hours in DEG C baking oven, it is added 80-240 microlitres thereto and contains polystyrene polyacrylic acid diblock copolymer
Dimethyl formamide solution and 180-540 microlitres of water are subsequently placed in after cultivating 2 hours in 110 DEG C of baking ovens and are centrifuged, obtained precipitating
Object is silver-colored aggregation-polymer, will obtain the slow of silver-colored aggregation-polymer in phosphate buffer solution that sediment is dissolved in again
Rush solution；
D. the buffer solution for taking 1-3 milliliters of silver-colored aggregation-polymer, is added the 20-60 microlitres of buffer solution containing antibody, in
It is reacted 2 hours at 4 DEG C, after centrifugation removes extra unreacted antibody, adds the 10-30 microlitres of buffering containing bovine serum albumin(BSA)
Solution reacts 1 hour at room temperature, and to close the site that silver-colored aggregation-polymer is not adhered to by antibody, centrifugation removal is extra not
To get to silver-colored aggregation-polymer SERS immunological probe after the bovine serum albumin(BSA) of reaction, it is placed at 4 DEG C and stores for use；
(2) preparation of substrate is immunized in Crystal structure
A. it is water-soluble the silicon wafer of hydrophiling to be placed in the 3- aminopropyl triethoxysilane that concentration is 1.1-3.3 mg/ml
Make amino in its surface modification within 1 hour in liquid；The silicon wafer for being modified with amino is placed in the gold nano grain water that partial size is 3-5 nanometers
2 hours in solution, make gold nano grain on its adsorption；The silicon wafer for being adsorbed with gold nano grain is placed in containing 4- sulfydryl benzene
After reacting 15 minutes in the aqueous solution of formic acid, gold chloride and ascorbic acid, i.e., Crystal structure is obtained in silicon chip surface；
B. be added dropwise on Crystal structure the 20-60 microlitres of buffer solution (concentration be 2-6 mg/ml) containing antibody in
It is reacted 2 hours at 4 DEG C, after cleaning removes extra unreacted antibody, then the 10-30 microlitres of buffering containing bovine serum albumin(BSA) is added dropwise
Solution reacts 1 hour at room temperature, and to close the site that do not adhered to by antibody on Crystal structure, cleaning removal is not extra anti-
Crystal structure is obtained after the bovine serum albumin(BSA) answered, substrate is immunized, be placed at 4 DEG C and store for use；
(3) cancer markers detection architecture assembles
Phosphate buffer solution containing determined antigen is added drop-wise to Crystal structure to be immunized in substrate, places it in 37 DEG C
It is lower to stand 2 hours, carry out the immune response between antigen and antibody sufficiently, cleaning removes extra unreacted determined antigen；
15 microlitres of silver-colored aggregation-polymer SERS immunological probes are added drop-wise to again and is adsorbed with the Crystal structure of determined antigen and is immunized
In substrate, reacted 2 hours at 37 DEG C, the extra unreacted silver-colored aggregation-polymer SERS immunological probe of cleaning removal to get
To the cancer markers detection architecture based on silver-colored aggregation-polymer and Crystal structure.
Silver nitrate concentration is 0.2 milligram/milli in aqueous solution containing silver nitrate and sodium citrate described in step (1) B
It rises and sodium citrate concentration is 0.6 mg/ml；
In the dimethyl formamide solution of the thionaphthol containing 2- described in step (1) C the concentration of 2- thionaphthol be 10 milligrams/
Milliliter；Polystyrene polypropylene in the dimethyl formamide solution containing polystyrene polyacrylic acid diblock copolymer
The concentration of sour diblock copolymer is 8 mg/mls；
The concentration of antibody is 2 mg/mls in buffer solution containing antibody described in step (1) D, and buffer solution is phosphorus
Hydrochlorate buffer solution；The mass concentration of bovine serum albumin(BSA) is 3% in the buffer solution containing bovine serum albumin(BSA), buffering
Solution is phosphate buffer solution；
Centrifugal speed is 5000-15000 revs/min in step (1) C and D, and centrifugation time is 5-30 minutes.
4- sulfydryl benzene first in aqueous solution containing 4- mercaptobenzoic acid, gold chloride and ascorbic acid described in step (2) A
The concentration of acid is 0.85-2.55 mg/ml, and the concentration of gold chloride is the concentration of 0.07-0.21 mg/ml and ascorbic acid
For 0.7-2.1 mg/ml.
The concentration of antibody is 2-6 mg/ml in buffer solution containing antibody described in step (2) B, and buffer solution is
Phosphate buffer solution；The mass concentration of bovine serum albumin(BSA) is 3% in the buffer solution containing bovine serum albumin(BSA), is delayed
Rushing solution is phosphate buffer solution.
The application of the above-mentioned cancer markers detection architecture based on silver-colored aggregation-polymer and Crystal structure, by institute
The cancer markers detection architecture based on silver-colored aggregation-polymer and Crystal structure stated carries out light using Raman spectrometer
It is slow can to calculate phosphate of the acquisition containing determined antigen according to the quantitative relationship between spectral intensity and antigen concentration for spectrometry
Rush the concentration of antigen in solution.
Compared with the prior art, the advantages of the present invention are as follows: present invention firstly discloses based on silver-colored aggregation-polymer
The SERS base to cancer markers with lower detectable limit that substrate is immunized in SERS immunological probe and Crystal structure is immune
Detection architecture and its application form silver-colored aggregation-polymerization by the high specific immune response between antigen and corresponding antibody
Substrate three-decker is immunized in object SERS immunological probe/determined antigen/Crystal structure, is imitated using Surface enhanced Raman scattering
It answers, the chaacteristic fingerprint spectrum of detection immunological probe surface Raman marker realizes the detection to cancer markers antigen to be measured, has
Following advantage:
(1) the silver nano-grain number reunited in silver-colored aggregation in its silver-colored aggregation-polymer SERS immunological probe is reachable
10 or more, there is the gap taken measurements greatly less than 10 nanometers between each other.Under the excitation of ambient light, in these nano gaps
It will form a large amount of Raman hot spot region, compared to the identical size silver nano-grain of individualism, can produce very high strength
The output of SERS signal.And the present invention makes silver by way of using polymer wrapped in the appropriate time after aggregation occurs
The aggregation of nano particle terminates, and is a kind of method of silver-colored aggregation of controllable preparation, of Argent grain in obtained aggregation
Number essentially 10 or so (as shown in Figure 1), make silver-colored aggregation polymeric inner numbers of particles than more uniform.Obtaining intensity
While high SERS signal exports, it is obviously improved the stability of signal.In addition, this method is by Raman labels molecule packet
It is wrapped within polymer, there is no molecule attachments for the outer surface of polymer.In the immune response after progress, compared to before
The surface markers of report have a gold and silver aggregation of Raman molecular, antigen-antibody be more easily largely adsorbed in the present invention obtain it is clean
On outer surface, the immune detection limit obtained after can also promoting is greatly lowered.
(2) the nanowires of gold length in the present invention in Crystal structure is bordering on parallel between each other up to 1 microns
It is vertically arranged, since nanowire alignment is close, will form a large amount of nano gap in array surface.Meanwhile nano wire have compared with
Big surface area/volume ratio, and the lightning rod effect that its tip has.Under the action of external exciting light, nanowires of gold is generated
Surface plasma cognition propagated and coupled on nano-wire array surface, and generate in nano gap a large amount of Raman heat
Point region can equally obtain the SERS signal output of higher-strength.After being especially immunoreacted, silver-colored aggregation-polymerization
Object SERS immunological probe can be attached to Crystal structure and substrate surface is immunized, and plasma coupling can equally occur therebetween
It closes, further enhances SERS signal, be conducive to obtain the detection limit extremely low to determined antigen in immune detection.Further, since
The length of nanowires of gold is very unified, so that the surface of Crystal structure is very smooth, ensure that after being immunoreacted
Uniform coating of the silver-colored aggregation-polymer SERS immunological probe to its surface.This point is the same as silver-colored aggregation polymeric inner particle
The homogeneity of number together so that SERS signal obtained have high consistency.Therefore, it is immunized using this programme
Detection has high repeatability, and (Raman signal that 15 retests obtain is as shown in figure 3, its 1064 and 1381cm-1It is strong
Degree standard deviation is only 3.8% and 3.2%) (reaches to the detection limit of prostate specific antigen (PSA) with extremely low detectable limit
1 winged gram every milliliter).
(3) technical process of the present invention is simple, and the period is short, at low cost, the clinic easy to spread applied to cancer
Among detection.
Detailed description of the invention
Fig. 1 is the silver-colored aggregation-polymer SERS immunological probe transmission electron microscope prepared in the embodiment of the present invention 1
Photo；
Fig. 2 is that the transmission electron microscope photo top of substrate is immunized in the Crystal structure prepared in the embodiment of the present invention 1
View one；
Fig. 3 is that the transmission electron microscope photo top of substrate is immunized in the Crystal structure prepared in the embodiment of the present invention 1
View two；
Fig. 4 is that the silver-colored aggregation-polymer SERS immunological probe prepared in the embodiment of the present invention 1 and Crystal structure are exempted from
Epidemic disease substrate is by carrying out the Raman spectrogram that Raman detection obtains to substrate after being immunoreacted with determined antigen；
Fig. 5 be the cancer markers detection architecture that is prepared in the embodiment of the present invention 1 containing determined antigen (concentration is 1 milligram every
Milliliter) He Weihan determined antigen comparison Raman spectrogram；
Fig. 6 is that the silver-colored aggregation-polymer SERS immunological probe prepared in the embodiment of the present invention 1 and Crystal structure are exempted from
Epidemic disease substrate passes through and the determined antigen of various concentration (concentration is 1 milligram every milliliter to 1 winged gram every milliliter) carries out being immunoreacted it
The Raman spectrogram that Raman detection obtains is carried out to substrate afterwards；
Fig. 7 is that frequency displacement is 1381cm in the cancer markers detection architecture Raman spectrum prepared in the embodiment of the present invention 1-1Spy
Peak intensity is levied with the variation diagram of determined antigen concentration.
Specific embodiment
The present invention will be described in further detail below with reference to the embodiments of the drawings.
The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..Unless otherwise specified, embodiment
Used in the conventional means that are well known to those skilled in the art of technological means, raw materials used is commercial goods.Embodiment
Used in Raman spectrum detector BWS415 be purchased from U.S. Bi Da Imtech (B&W Tek Inc.).Cancer markers refer to
The substance for directly being generated by tumour cell or being generated by non-tumor cell through tumor cell induction, following embodiment is with prostate spy
Hapten (PSA), alpha-fetoprotein antigen and Carbohydrate Antigen CA125 are illustrated for example.
Embodiment 1
The preparation of prostate specific antigen (PSA) detection architecture based on silver-colored aggregation-polymer and Crystal structure
Method, comprising the following steps:
1, the preparation of silver-colored aggregation-polymer SERS immunological probe
1.47 milligrams of sodium citrates and 1.97 milligrams of gold chlorides are dissolved in 20 milliliters of water, mixed solution is made, at room temperature side
It is vigorously stirred the sodium borohydride aqueous solution (concentration is 4 mg/mls) that 0.6 milliliter of Fresh is added in side, it is molten to obtain gold seeds
Liquid；
3 milliliters of gold seeds solution are taken to be added to 50 milliliters containing silver nitrate (concentration is 0.2 mg/ml) and sodium citrate
In the aqueous solution of (concentration is 0.6 mg/ml), 10 milliliters of aqueous ascorbic acid (concentration 2 are then added dropwise thereto
Mg/ml), reaction obtains silver nano-grain aqueous solution in 1 hour；Taking 1 milliliter of silver nano-grain aqueous solution centrifugal concentrating is 10
Microlitre, be gradually added thereto 820 microlitres of dimethylformamides, 5 microlitres of thionaphthols containing 2- dimethyl formamide solution (wherein
2- thionaphthol concentration is 10 mg/mls) and 20 microlitres of water, it is subsequently placed in 60 DEG C of baking ovens after cultivating 3 hours and adds thereto
Enter 80 microlitres of dimethyl formamide solutions containing polystyrene polyacrylic acid diblock copolymer (concentration is 8 mg/mls)
With 180 microlitres of water, then be placed in 110 DEG C of baking ovens cultivate 2 hours after be centrifuged, obtained sediment is silver-colored aggregation-polymer,
The buffer solution of silver-colored aggregation-polymer will be obtained in phosphate buffer solution that sediment is dissolved in again；Take 1 milliliter of silver aggregation
20 microlitres of buffer solutions containing prostate-specific antibody are added thereto and react 2 at 4 DEG C for body-polymer buffer solution
Hour.After centrifugation removes extra unreacted antibody, 10 microlitres of buffer solutions containing bovine serum albumin(BSA) are added thereto, in
It reacts 1 hour at room temperature, to close the site that silver-colored aggregation-polymer is not adhered to by antibody.Centrifugation removal is extra unreacted
Silver-colored aggregation-polymer SERS immunological probe is obtained after bovine serum albumin(BSA), is placed at 4 DEG C and stores for use.Wherein containing anti-
The concentration of antibody is 2 mg/mls in the buffer solution of body, and buffer solution is phosphate buffer solution；Containing bovine serum albumin(BSA)
Buffer solution in bovine serum albumin(BSA) mass concentration be 3%, buffer solution is phosphate buffer solution；Centrifugal speed is
5000 revs/min, centrifugation time is 30 minutes.
Fig. 1 shows the silver-colored aggregation-polymer SERS immunological probe transmission electron microscope prepared in the present embodiment
Photo.From figure 1 it appears that being core-shell structure in prepared immunological probe, center is the aggregation of a large amount of silver nano-grains
Body, outer layer are polymeric shell layer.
2, the preparation of substrate is immunized in Crystal structure
Hydrophilicity-imparting treatment is done by the silicon chip surface that plasma clean is 1 square centimeter to size.By the silicon of hydrophiling
Piece is placed in APTES aqueous solution (concentration is 1.1 mg/mls) 1 hour, makes amino in its surface modification.Amino will be modified with
Silicon wafer to be placed in partial size be to make gold nano grain on its adsorption 2 hours in 3-5 nanometers of gold nano grain aqueous solution.It will
The silicon wafer for being adsorbed with gold nano grain is placed in that (concentration is containing 4- mercaptobenzoic acid (concentration be 0.85 mg/ml), gold chloride
0.07 mg/ml) and the aqueous solution of ascorbic acid (concentration be 0.7 mg/ml) in react 15 minutes after be in silicon wafer
Surface obtains Crystal structure.The buffer solution of 20 microlitres of antibody containing prostate-specific is added dropwise on Crystal structure in 4
It is reacted 2 hours at DEG C.After cleaning removes extra unreacted antibody, to 10 microlitres of bufferings containing bovine serum albumin(BSA) of dropwise addition thereon
Solution reacts 1 hour at room temperature, to close the site that do not adhered to by antibody on Crystal structure.Cleaning removal is not extra anti-
Crystal structure is obtained after the bovine serum albumin(BSA) answered, substrate is immunized, be placed at 4 DEG C and store for use.Wherein containing antibody
The concentration of antibody is 2 mg/mls in buffer solution, and buffer solution is phosphate buffer solution；It is slow containing bovine serum albumin(BSA)
The mass concentration for rushing bovine serum albumin(BSA) in solution is 3%, and buffer solution is phosphate buffer solution.
Fig. 2 shows that the transmission electron microscope photo top view of substrate is immunized in the Crystal structure prepared in the present embodiment
Figure, figure it is seen that a large amount of nano gaps are contained on prepared Crystal structure surface.
Fig. 3 shows that the transmission electron microscope photo top view of substrate is immunized in the Crystal structure prepared in the present embodiment
Figure, from figure 3, it can be seen that prepared Crystal structure is approximately perpendicular to silicon wafer, arrangement is fine and close.
3, cancer markers detection architecture assembles
Phosphate buffer solution containing prostate specific antigen to be measured is added drop-wise to Crystal structure to be immunized in substrate, it will
It is placed at 37 DEG C and stands 2 hours, carries out the immune response between antigen and antibody sufficiently, and cleaning removal is extra unreacted
Determined antigen；15 microlitres of silver-colored aggregation-polymer SERS immunological probes are added drop-wise to the nanowires of gold for being adsorbed with determined antigen again
It in array immunization substrate, is reacted 2 hours at 37 DEG C, it is immune that cleaning removes extra unreacted silver aggregation-polymer SERS
Probe to get arrive the cancer markers detection architecture based on silver-colored aggregation-polymer and Crystal structure.
4, spectrum survey is carried out to the cancer markers detection architecture obtained after above-mentioned steps are handled using Raman spectrometer
Measure detectable determined antigen.
Fig. 4 is to be exempted from using the silver-colored aggregation-polymer SERS immunological probe and Crystal structure that prepare in the present embodiment
Epidemic disease substrate is obtained by carrying out Raman detection to substrate after being immunoreacted with determined antigen (concentration is 10 micrograms per millilitres)
The Raman signal that 15 retests obtain, 1064 and 1381cm can be calculated in the Raman spectrogram arrived from Fig. 4-1
Intensity standard deviation be only 3.8% and 3.2%.
Fig. 5 is to be exempted from using the silver-colored aggregation-polymer SERS immunological probe and Crystal structure that prepare in the present embodiment
Epidemic disease substrate is obtained by carrying out Raman detection to substrate after being immunoreacted with determined antigen (concentration is 1 milligram every milliliter)
Raman spectrogram.From fig. 5, it can be seen that can detecte the relatively strong of mark molecule 2- thionaphthol when containing determined antigen
Raman signatures spectrum, in 1382cm-1The Raman signal intensity at place reaches 19957.The detection pole of prostate specific antigen (PSA)
Limit can reach 1 winged gram every milliliter.And when not containing determined antigen in solution to be measured, since silver-colored aggregation-polymer SERS exempts from
Epidemic disease probe and Crystal structure are immunized substrate and cannot be combined by specific immune response, and only occur a small amount of non-specific
Property absorption, therefore, only measure very weak Raman spectrum.
Fig. 6 is to be exempted from using the silver-colored aggregation-polymer SERS immunological probe and Crystal structure that prepare in the present embodiment
Epidemic disease substrate passes through and the determined antigen of various concentration (concentration is 1 milligram every milliliter to 1 winged gram every milliliter) carries out being immunoreacted it
The Raman spectrogram that Raman detection obtains is carried out to substrate afterwards.From fig. 6, it can be seen that when the reduction with determined antigen concentration,
The Raman signatures spectral intensity of mark molecule 2- thionaphthol gradually decreases, until the concentration of determined antigen is reduced to 1 winged gram every milliliter
When, the raman characteristic peak of mark molecule is relative to background signal still it is obvious that this concentration is this programme to determined antigen
Detectable limit.
Fig. 7 is that frequency displacement is 1381cm in Raman spectrum-1Feature peak intensity with the variation diagram of determined antigen concentration, pass through fitting
As it can be seen that Raman signatures peak intensity is with concentration when the concentration of determined antigen is from when changing to 1 milligram every milliliter for 1 winged gram every milliliter
Linear change.Fitting result shows that this variation tendency meets this linear equation of Y=2245.42+32865.44*LogX,
Standard deviation is 0.985, and wherein X is the concentration of antigen, and Y is Raman signatures peak intensity.
Embodiment 2
The preparation method of alpha-fetoprotein antigen detection architecture based on silver-colored aggregation-polymer and Crystal structure, packet
Include following steps:
1, the preparation of silver-colored aggregation-polymer SERS immunological probe: by 2.94 milligrams of sodium citrates and 3.94 milligrams of chlorine gold
Acid is dissolved in 40 milliliters of water and mixed solution is made, and the sodium borohydride of 1.2 milliliters of Fresh is added when being vigorously stirred at room temperature
Aqueous solution (concentration is 4 mg/mls), obtains gold seeds solution；It takes 6 milliliters of gold seeds solution to be added to 100 milliliters and contains nitre
In the aqueous solution of sour silver (concentration is 0.2 mg/ml) and sodium citrate (concentration is 0.6 mg/ml), later thereto by
It is added dropwise to 20 milliliters of aqueous ascorbic acids (concentration is 2 mg/mls), reaction obtains silver nano-grain aqueous solution in 1 hour；
Taking 2 milliliters of silver nano-grain aqueous solution centrifugal concentratings is 20 microlitres, and 1640 microlitres of dimethyl are gradually added thereto
Formamide, the dimethyl formamide solution and 40 microlitres of water of 10 microlitres of thionaphthols containing 2- (concentration is 10 mg/mls), then
It is placed in 60 DEG C of baking ovens after cultivating 3 hours 160 microlitres of addition thereto and contains polystyrene polyacrylic acid diblock copolymer
The dimethyl formamide solution and 360 microlitres of water of (concentration is 8 mg/mls), are subsequently placed in 110 DEG C of baking ovens and cultivate 2 hours
It is centrifuged in the phosphate buffer solution for being dissolved in sediment (i.e. silver-colored aggregation-polymer) again afterwards；Take 2 milliliters of silver-colored aggregations-poly-
The buffer solution of object is closed, 40 microlitres of buffer solutions containing alpha-fetoprotein antibody (concentration is 2 mg/mls) are added thereto in 4
It is reacted 2 hours at DEG C, after centrifugation removes extra unreacted antibody, the buffering that 20 microlitres of bovine serum albumin(BSA)s are added thereto is molten
Liquid (mass percent 3%) reacts 1 hour at room temperature, to close the position that silver-colored aggregation-polymer is not adhered to by antibody
Point, centrifugation obtain silver-colored aggregation-polymer SERS immunological probe after removing extra unreacted bovine serum albumin(BSA), are placed in
It is stored at 4 DEG C stand-by.Wherein centrifugal speed is 10000 revs/min, and centrifugation time is 20 minutes.
2, the preparation of substrate is immunized in Crystal structure
Hydrophilicity-imparting treatment is done by the silicon chip surface that plasma clean is 1 square centimeter to size.By the silicon of hydrophiling
Piece is placed in APTES aqueous solution (concentration is 1.1-3.3 mg/ml) 1 hour, makes amino in its surface modification.It will be modified with
The silicon wafer of amino is placed in the gold nano grain aqueous solution having a size of 3-5 nanometers 2 hours, makes gold nano on its adsorption
Grain.The silicon wafer for being adsorbed with gold nano grain is placed in (dense containing 4- mercaptobenzoic acid (concentration is 1.7 mg/mls), gold chloride
Degree be 0.14 mg/ml) and the aqueous solution of ascorbic acid (concentration is 0.14 mg/ml) in react 15 minutes later be
Crystal structure is obtained in silicon chip surface, 40 microlitres are added dropwise on Crystal structure, and (concentration is 4 millis containing alpha-fetoprotein antibody
Grams per milliliter) buffer solution reacted 2 hours at 4 DEG C, it is micro- to 20 are added dropwise thereon after cleaning removes extra unreacted antibody
The buffer solution (mass percent 3%) for rising bovine serum albumin(BSA) reacts 1 hour at room temperature, to close Crystal structure
On the site do not adhered to by antibody, cleaning removes that obtain Crystal structure after extra unreacted bovine serum albumin(BSA) immune
Substrate is placed at 4 DEG C and stores for use.
3, cancer markers detection architecture assembles
Phosphate buffer solution containing alpha-fetoprotein antigen to be measured is added drop-wise to Crystal structure to be immunized in substrate, by it
Be placed at 37 DEG C and stand 2 hours, carry out the immune response between antigen and antibody sufficiently, cleaning removal it is extra it is unreacted to
Survey antigen；15 microlitres of silver-colored aggregation-polymer SERS immunological probes are added drop-wise to the gold nano linear array for being adsorbed with determined antigen again
It arranges in immune substrate, is reacted 2 hours at 37 DEG C, the extra unreacted silver aggregation-polymer SERS of cleaning removal is immune to be visited
Needle to get arrive the cancer markers detection architecture based on silver-colored aggregation-polymer and Crystal structure.
4, spectrum survey is carried out to the cancer markers detection architecture obtained after above-mentioned steps are handled using Raman spectrometer
Measure detectable determined antigen.
Substrate is immunized using the silver-colored aggregation-polymer SERS immunological probe and Crystal structure prepared in the present embodiment
By carrying out the Raman that Raman detection obtains to substrate after being immunoreacted with determined antigen (concentration is 1 milligram every milliliter)
Spectrogram can be seen that when containing determined antigen, and can detecte mark molecule 2- thionaphthol relatively hales graceful characteristic spectrum,
In 1382cm-1The Raman signal intensity at place reaches 17363.The detectable limit of alpha-fetoprotein antigen can reach 2 winged grams every milliliter.
And when not containing determined antigen in solution to be measured, due to silver-colored aggregation-polymer SERS immunological probe and Crystal structure
Immune substrate cannot be combined by specific immune response, and a small amount of non-specific adsorption only occurs, and therefore, only be measured very
Weak Raman spectrum.
Embodiment 3
The preparation method to Carbohydrate Antigen CA125 detection architecture based on silver-colored aggregation-polymer and Crystal structure,
The following steps are included:
1, the preparation of silver-colored aggregation-polymer SERS immunological probe
4.41 milligrams of sodium citrates and 5.91 milligrams of gold chlorides are dissolved in 60 milliliters of water, mixed solution is made, at room temperature side
It is vigorously stirred the sodium borohydride aqueous solution (concentration is 4 mg/mls) that 1.8 milliliters of Fresh are added in side, it is molten to obtain gold seeds
Liquid；
9 milliliters of gold seeds solution are taken to be added to 150 milliliters containing silver nitrate (concentration is 0.2 mg/ml) and citric acid
In the aqueous solution of sodium (concentration is 0.6 mg/ml), 30 milliliters of aqueous ascorbic acid (concentration are added dropwise thereto later
For 2 mg/mls), reaction obtains silver nano-grain aqueous solution in 1 hour；3 milliliters of silver nano-grain aqueous solution centrifugal concentratings are taken to be
30 microlitres, 2460 microlitres of dimethylformamides are gradually added thereto, (concentration is 10 milligrams/milli to 15 microlitres of thionaphthols containing 2-
Rise) dimethyl formamide solution and 60 microlitres of water, be subsequently placed in 60 DEG C of baking ovens cultivate 3 hours and be added 240 thereto later
The dimethyl formamide solution of microlitre polystyrene polyacrylic acid diblock copolymer (concentration is 8 mg/mls) and 540 micro-
Rise water, then be placed in 110 DEG C of baking ovens cultivate 2 hours after the centrifugation phosphoric acid that is dissolved in sediment (i.e. silver aggregation-polymer) again
In salt buffer solution；Take the buffer solution of 3 milliliters of silver-colored aggregation-polymer, be added thereto 60 microlitres it is (dense containing carbohydrate antibodies
Degree be 2 mg/mls) buffer solution reacted 2 hours at 4 DEG C.After centrifugation removes extra unreacted antibody, thereto plus
Enter the buffer solution (mass percent 3%) of 30 microlitres of bovine serum albumin(BSA)s, reacts 1 hour at room temperature, it is poly- with closing silver
The site that collective-polymer is not adhered to by antibody.Centrifugation obtains silver-colored aggregation after removing extra unreacted bovine serum albumin(BSA)
Body-polymer SERS immunological probe is placed at 4 DEG C and stores for use.Wherein centrifugal speed is 15000 revs/min, centrifugation time
It is 5 minutes.
2, the preparation of substrate is immunized in Crystal structure
Hydrophilicity-imparting treatment is done by the silicon chip surface that plasma clean is 1 square centimeter to size.By the silicon of hydrophiling
Piece is placed in APTES aqueous solution (concentration is 3.3 mg/mls) 1 hour, makes amino in its surface modification.Amino will be modified with
Silicon wafer be placed in the gold nano grain aqueous solution having a size of 3-5 nanometers 2 hours, make gold nano grain on its adsorption.It will
The silicon wafer for being adsorbed with gold nano grain is placed in containing 4- mercaptobenzoic acid (concentration be 2.55 mg/mls), and (concentration is gold chloride
0.21 mg/ml) and the aqueous solution of ascorbic acid (concentration be 2.1 mg/mls) in react 15 minutes after be in silicon wafer
Surface obtains Crystal structure.60 microlitres are added dropwise on Crystal structure containing carbohydrate antibodies (concentration is 6 mg/mls)
Buffer solution reacted 2 hours at 4 DEG C.After cleaning removes extra unreacted antibody, to 30 microlitres of cow's serums of dropwise addition thereon
The buffer solution (mass percent 3%) of albumin reacts 1 hour at room temperature, is not resisted on Crystal structure with closing
The site of body attachment.Cleaning obtains the immune substrate of Crystal structure after removing extra unreacted bovine serum albumin(BSA), and
It is placed at 4 DEG C and stores for use.
3, cancer markers detection architecture assembles
Phosphate buffer solution containing sugar antigen to be measured is added drop-wise to Crystal structure to be immunized in substrate, is placed it in
2 hours are stood at 37 DEG C, carries out the immune response between antigen and antibody sufficiently, cleaning removal is extra unreacted to be measured anti-
It is former；15 microlitres of silver-colored aggregation-polymer SERS immunological probes are added drop-wise to again and is adsorbed with the Crystal structure of determined antigen and exempts from
It in epidemic disease substrate, is reacted 2 hours at 37 DEG C, the extra unreacted silver-colored aggregation-polymer SERS immunological probe of cleaning removal, i.e.,
Obtain the cancer markers detection architecture based on silver-colored aggregation-polymer and Crystal structure.
4, using Raman spectrometer to the immune substrate that is obtained after above-mentioned steps are handled carry out spectral measurement it is detectable to
Survey antigen.
Substrate is immunized using the silver-colored aggregation-polymer SERS immunological probe and Crystal structure prepared in the present embodiment
By carrying out the Raman that Raman detection obtains to substrate after being immunoreacted with determined antigen (concentration is 1 milligram every milliliter)
Spectrogram can be seen that when containing determined antigen, and can detecte mark molecule 2- thionaphthol relatively hales graceful characteristic spectrum,
In 1382cm-1The Raman signal intensity at place reaches 15221.The detectable limit of Carbohydrate Antigen CA125 reaches 2 winged grams every milliliter.And
When not containing determined antigen in solution to be measured, since silver-colored aggregation-polymer SERS immunological probe and Crystal structure are exempted from
Epidemic disease substrate cannot be combined by specific immune response, and a small amount of non-specific adsorption only occurs, and therefore, only be measured very weak
Raman spectrum.
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, fall within the scope of the claimed invention without departing from theon the basis of the spirit of the present invention.
Claims (4)
1. a kind of preparation method of the cancer markers detection architecture based on silver-colored aggregation-polymer and Crystal structure,
Be characterized in that the following steps are included:
(1) preparation of silver-colored aggregation-polymer SERS immunological probe
A. by 1.47-4.41 milligrams of sodium citrates and 1.97-5.91 milligrams of gold chlorides be dissolved in 20-60 milliliters of water be made mixing it is molten
Liquid, the concentration that 0.6-1.8 milliliters of Fresh are added when being vigorously stirred at room temperature is the sodium borohydride water of 4 mg/mls
Solution obtains gold seeds solution；
B. 3-9 milliliters of gold seeds solution are taken to be added in the 50-150 milliliters of aqueous solutions containing silver nitrate and sodium citrate, later
The aqueous ascorbic acid that 10-30 milliliters of concentration are 2 mg/mls is added dropwise thereto, reaction obtains silver nanoparticle in 1 hour
Grain aqueous solution；
C. taking 1-3 milliliters of silver nano-grain aqueous solution centrifugal concentratings is 10-30 microlitres, and 820-2460 microlitres is gradually added thereto
Dimethylformamide, the dimethyl formamide solution and 20-60 microlitres of water of the 5-15 microlitres of thionaphthol containing 2- are subsequently placed in 60 DEG C of bakings
After cultivating 3 hours in case, the 80-240 microlitres of diformazan containing polystyrene polyacrylic acid diblock copolymer is added thereto
Base formamide solution and 180-540 microlitres of water are subsequently placed in after cultivating 2 hours in 110 DEG C of baking ovens and are centrifuged, and obtained sediment is i.e.
For silver-colored aggregation-polymer, sediment is dissolved in again obtained in phosphate buffer solution silver-colored aggregation-polymer buffering it is molten
Liquid；
D. the buffer solution for taking 1-3 milliliters of silver-colored aggregation-polymer, is added the 20-60 microlitres of buffer solution containing antibody, in 4 DEG C
Lower reaction 2 hours, after centrifugation removes extra unreacted antibody, it is molten to add the 10-30 microlitres of buffering containing bovine serum albumin(BSA)
Liquid reacts 1 hour at room temperature, and to close the site that silver-colored aggregation-polymer is not adhered to by antibody, centrifugation removal is not extra anti-
To get to silver-colored aggregation-polymer SERS immunological probe after the bovine serum albumin(BSA) answered, it is placed at 4 DEG C and stores for use；
(2) preparation of substrate is immunized in Crystal structure
A. the silicon wafer of hydrophiling is placed in 1 in the 3- aminopropyl triethoxysilane aqueous solution that concentration is 1.1-3.3 mg/ml
Hour makes amino in its surface modification；The silicon wafer for being modified with amino is placed in the gold nano grain aqueous solution that partial size is 3-5 nanometers
In 2 hours, make gold nano grain on its adsorption；The silicon wafer for being adsorbed with gold nano grain is placed in containing 4- sulfydryl benzene first
After reacting 15 minutes in the aqueous solution of acid, gold chloride and ascorbic acid, i.e., Crystal structure is obtained in silicon chip surface；
B. the 20-60 microlitres of buffer solution containing antibody is added dropwise on Crystal structure to react 2 hours at 4 DEG C, cleaning removal
After extra unreacted antibody, then the 10-30 microlitres of buffer solution containing bovine serum albumin(BSA) is added dropwise and reacts at room temperature 1 hour,
To close the site that do not adhered to by antibody on Crystal structure, clean after removing extra unreacted bovine serum albumin(BSA) to obtain the final product
Substrate is immunized to Crystal structure, is placed at 4 DEG C and stores for use；
(3) cancer markers detection architecture assembles
Phosphate buffer solution containing determined antigen is added drop-wise to Crystal structure to be immunized in substrate, is placed it in quiet at 37 DEG C
It sets 2 hours, carries out the immune response between antigen and antibody sufficiently, cleaning removes extra unreacted determined antigen；Again will
15 microlitres of silver-colored aggregation-polymer SERS immunological probes are added drop-wise to the immune substrate of Crystal structure for being adsorbed with determined antigen
On, it is reacted 2 hours at 37 DEG C, cleaning removes extra unreacted silver-colored aggregation-polymer SERS immunological probe to get base is arrived
In the cancer markers detection architecture of silver-colored aggregation-polymer and Crystal structure.
2. according to claim 1 detect body based on the cancer markers of silver-colored aggregation-polymer and Crystal structure
The preparation method of system, it is characterised in that:
In aqueous solution containing silver nitrate and sodium citrate described in step (1) B silver nitrate concentration be 0.2 mg/ml with
And sodium citrate concentration is 0.6 mg/ml；
The concentration of 2- thionaphthol is 10 milligrams/milli in the dimethyl formamide solution of the thionaphthol containing 2- described in step (1) C
It rises；Polystyrene polyacrylic acid in the dimethyl formamide solution containing polystyrene polyacrylic acid diblock copolymer
The concentration of diblock copolymer is 8 mg/mls；
The concentration of antibody is 2 mg/mls in buffer solution containing antibody described in step (1) D, and buffer solution is phosphate
Buffer solution；The mass concentration of bovine serum albumin(BSA) is 3% in the buffer solution containing bovine serum albumin(BSA), buffer solution
For phosphate buffer solution.
3. according to claim 1 detect body based on the cancer markers of silver-colored aggregation-polymer and Crystal structure
The preparation method of system, it is characterised in that: centrifugal speed is 5000-15000 revs/min in step (1) C and D, centrifugation time 5-
30 minutes.
4. according to claim 1 detect body based on the cancer markers of silver-colored aggregation-polymer and Crystal structure
The preparation method of system, it is characterised in that: the water containing 4- mercaptobenzoic acid, gold chloride and ascorbic acid described in step (2) A
The concentration of 4- mercaptobenzoic acid is 0.85-2.55 mg/ml in solution, and the concentration of gold chloride is 0.07-0.21 mg/ml
Concentration with ascorbic acid is 0.7-2.1 mg/ml；Antibody is dense in buffer solution containing antibody described in step (2) B
Degree is 2-6 mg/ml, and buffer solution is phosphate buffer solution；Ox in the buffer solution containing bovine serum albumin(BSA)
Sero-abluminous mass concentration is 3%, and buffer solution is phosphate buffer solution.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710804383.1A CN107643400B (en) | 2017-09-08 | 2017-09-08 | The preparation method and applications of cancer markers detection architecture based on silver-colored aggregation-polymer and Crystal structure |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710804383.1A CN107643400B (en) | 2017-09-08 | 2017-09-08 | The preparation method and applications of cancer markers detection architecture based on silver-colored aggregation-polymer and Crystal structure |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN107643400A CN107643400A (en) | 2018-01-30 |
| CN107643400B true CN107643400B (en) | 2019-05-03 |
Family
ID=61110347
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710804383.1A Active CN107643400B (en) | 2017-09-08 | 2017-09-08 | The preparation method and applications of cancer markers detection architecture based on silver-colored aggregation-polymer and Crystal structure |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107643400B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110530839B (en) * | 2019-07-11 | 2021-11-30 | 宁波大学 | Preparation method of molybdenum disulfide/silver nano immune substrate material and repeatable immunodetection application thereof |
| CN112730836B (en) * | 2020-12-24 | 2023-05-05 | 北京信息科技大学 | Non-diagnostic detection method for multiple tumor markers based on SERS (surface enhanced Raman scattering) sensing substrate |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101566571B (en) * | 2009-06-03 | 2010-09-08 | 哈尔滨工业大学 | Surface enhanced raman spectroscopy substrate of continuous three dimensional structural nano silver and preparation method thereof |
| CN102323412A (en) * | 2011-08-09 | 2012-01-18 | 中国科学院合肥物质科学研究院 | A kind of purposes of Raman coding microball and utilize the Raman coding microball to detect the method for tumor markers |
| CN106137970A (en) * | 2015-04-10 | 2016-11-23 | 中国科学院宁波材料技术与工程研究所 | A kind of composite nano materials with surface enhanced raman spectroscopy function and preparation method thereof |
| CN106939161A (en) * | 2017-01-20 | 2017-07-11 | 广东工业大学 | A kind of orderly nano silver wire and preparation method and application |
| CN107470427A (en) * | 2017-08-01 | 2017-12-15 | 清华大学 | A kind of preparation method of metal and its alloy micro nano structure or nano-wire array |
-
2017
- 2017-09-08 CN CN201710804383.1A patent/CN107643400B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101566571B (en) * | 2009-06-03 | 2010-09-08 | 哈尔滨工业大学 | Surface enhanced raman spectroscopy substrate of continuous three dimensional structural nano silver and preparation method thereof |
| CN102323412A (en) * | 2011-08-09 | 2012-01-18 | 中国科学院合肥物质科学研究院 | A kind of purposes of Raman coding microball and utilize the Raman coding microball to detect the method for tumor markers |
| CN106137970A (en) * | 2015-04-10 | 2016-11-23 | 中国科学院宁波材料技术与工程研究所 | A kind of composite nano materials with surface enhanced raman spectroscopy function and preparation method thereof |
| CN106939161A (en) * | 2017-01-20 | 2017-07-11 | 广东工业大学 | A kind of orderly nano silver wire and preparation method and application |
| CN107470427A (en) * | 2017-08-01 | 2017-12-15 | 清华大学 | A kind of preparation method of metal and its alloy micro nano structure or nano-wire array |
Non-Patent Citations (2)
| Title |
|---|
| Gold Nanowire Forests for SERS Detection;Andrea La Porta,et al.;《ChemistryOpen》;20141231;第3卷;第146-151页 |
| Magnetic gold nanoparticles in SERS-based sandwich immunoassay for antigen detection by well oriented antibodies;Julija Baniukevic，et al.;《Biosensors and Bioelectronics》;20121214;第43卷;第281–288页 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN107643400A (en) | 2018-01-30 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Iarossi et al. | Colorimetric immunosensor by aggregation of photochemically functionalized gold nanoparticles | |
| Liu et al. | SERS substrate fabrication for biochemical sensing: Towards point-of-care diagnostics | |
| Ravalli et al. | Gold and magnetic nanoparticles-based electrochemical biosensors for cancer biomarker determination | |
| Mustafaoglu et al. | Site-specific conjugation of an antibody on a gold nanoparticle surface for one-step diagnosis of prostate specific antigen with dynamic light scattering | |
| Wu et al. | Ultrasensitive ebola virus detection based on electroluminescent nanospheres and immunomagnetic separation | |
| CN101305280A (en) | Diagnostic-nanosensor and its use in medicine | |
| Ye et al. | An electrochemical immunoassay for Escherichia coli O157: H7 using double functionalized Au@ Pt/SiO2 nanocomposites and immune magnetic nanoparticles | |
| CN109991207A (en) | A kind of SERS sensor and its preparation and detection method for detecting the sandwich structure of tyrosinase | |
| Song et al. | Synthesis of novel gold mesoflowers as SERS tags for immunoassay with improved sensitivity | |
| Shen et al. | Ultrasensitive aptasensor for isolation and detection of circulating tumor cells based on CeO2@ Ir nanorods and DNA walker | |
| Ma et al. | Improved lateral flow strip based on hydrophilic− hydrophobic SERS substrate for ultra− sensitive and quantitative immunoassay | |
| CN107478641A (en) | Liquid phase SERS sensor, its preparation method and its purposes for detection of nucleic acids | |
| CN107643400B (en) | The preparation method and applications of cancer markers detection architecture based on silver-colored aggregation-polymer and Crystal structure | |
| CN103837676A (en) | Metal nano island carrier and preparation method thereof as well as application of metal nano island carrier in immunodetection | |
| CN106692991A (en) | Targeting magnetic nanoparticles having double-heating and imaging functions, and preparation method and use thereof | |
| Li et al. | Dual protecting encapsulation synthesis of ultrastable quantum-dot nanobeads for sensitive and accurate detection of cardiac biomarkers | |
| CN109085158B (en) | Used for cancer cell and H2O2Construction of paper-based sensors for detection | |
| CN106940310A (en) | Substrate and preparation method thereof is immunized in a kind of self assembly gold nanorods SERS | |
| Zhang et al. | Spontaneous implantation of gold nanoparticles on graphene oxide for salivary SERS sensing | |
| Huang et al. | Label-free optical biosensor based on localized surface plasmon resonance of immobilized gold nanorods | |
| CN109884029A (en) | Silver/graphite alkene quantum dot nano enzyme, SERS detection kit and application | |
| Tang et al. | LHRH-targeting surface-enhanced Raman scattering tags for the rapid detection of circulating tumor cells | |
| KR20210019767A (en) | Magnetic-Optical Composite Nanoparticles | |
| Lu et al. | Detection of squamous cell carcinoma antigen in cervical cancer by surface-enhanced Raman scattering-based immunoassay | |
| CN110455769A (en) | Shell is interior target core-shell nano Surface enhanced Raman spectroscopy quantitative analysis method |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20221223 Address after: Floor 3, Building 3, Pansheng Biological Industrial Park, Gangyuan 8th Road, Jinan Area, China (Shandong) Pilot Free Trade Zone, 250000, Jinan, Shandong Patentee after: Saier Medical Technology (Shandong) Co.,Ltd. Address before: Room 2202, 22 / F, Wantong building, No. 3002, Sungang East Road, Sungang street, Luohu District, Shenzhen City, Guangdong Province Patentee before: Shenzhen dragon totem technology achievement transformation Co.,Ltd. Effective date of registration: 20221223 Address after: Room 2202, 22 / F, Wantong building, No. 3002, Sungang East Road, Sungang street, Luohu District, Shenzhen City, Guangdong Province Patentee after: Shenzhen dragon totem technology achievement transformation Co.,Ltd. Address before: 315211, Fenghua Road, Jiangbei District, Zhejiang, Ningbo 818 Patentee before: Ningbo University |
|
| TR01 | Transfer of patent right |