CN108088994B - Hollow core-shell nanoparticle, preparation method, test strip and test method - Google Patents
Hollow core-shell nanoparticle, preparation method, test strip and test method Download PDFInfo
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- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6887—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids from muscle, cartilage or connective tissue
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- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/65—Raman scattering
- G01N2021/653—Coherent methods [CARS]
- G01N2021/656—Raman microprobe
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- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
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- G01N2333/4712—Muscle proteins, e.g. myosin, actin, protein
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- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
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- G01N2333/4727—Calcium binding proteins, e.g. calmodulin
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- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/32—Cardiovascular disorders
- G01N2800/325—Heart failure or cardiac arrest, e.g. cardiomyopathy, congestive heart failure
Abstract
The invention discloses a hollow core-shell nanoparticle, a preparation method, a test strip and a test method, which can be used for quantitatively detecting the concentration of an object to be detected in a sample solution and improving the detection sensitivity. The hollow core-shell nanoparticle comprises a core body and a shell, wherein the shell is positioned outside the core body, a space is reserved between the shell and the core body, Raman molecules are respectively marked on the outer surface and the inner surface of the shell, and Raman molecules are marked on the outer surface of the core body; the shell is made of gold-silver alloy, and the core body is made of gold.
Description
Technical Field
The invention relates to the field of biomedical detection, in particular to a hollow core-shell nanoparticle, a preparation method, a test strip and a test method.
Background
Immunochromatography detection technology based on gold nanoparticles (Au NPs) is a membrane-based in-vitro detection and diagnosis method developed on the basis of monoclonal antibody technology and novel nano-material technology, and is widely applied to various fields, including sensitiveness, early pregnancy, myocardial infarction markers, infectious diseases, environmental pollutants, drugs and the like. Taking the early pregnancy test as an example only, the dosage of the kit/test strip required by each year reaches 107And (4) respectively. The principle of immunochromatography detection based on Au NPs is as follows: fixing a specific capture antibody on a Nitrocellulose (NC) membrane to form a strip; loading the Au NPs compound combined with the recognition antibody on a glass fiber pad at the sample end of the NC membrane to prepare a gold-labeled pad; moving the sample solution from one end of the test strip to the other end by using the capillary action of the micropores of the NC membrane; the solution to be measured dripped on the sample pad flows through the gold-labeled padWhen the complex flows through the test area, the complex is captured by the capture antibody fixed on the nitrocellulose membrane to form a double-antibody sandwich structure. As the structure gradually accumulated in the test zone, red bands appeared on the membrane at the test line T, and the quality control line C was used to detect the activity of the protein coated on the immuno-probe Au NPs. The immunochromatography detection technology based on Au NPs is simple and rapid, the result can be judged only by naked eyes, and the position of the technology in point-of-care testing (POCT) is increasingly established in the last 10 years. However, there are still two challenges with this technology: firstly, how to realize high-sensitivity detection, the current immunochromatography detection sensitivity is far less than that of methods such as enzyme-linked immunosorbent assay, immunofluorescence and the like; secondly, the color development of the bands is only a qualitative result through naked eye observation, and the eyes of different people have larger sensitivity difference to the color and low sensitivity.
For the diagnosis of Acute Myocardial Injury (AMI), an electrocardiographic examination must be combined with the detection of the content of cardiac troponin I (cTnI) in the peripheral blood to give a definitive diagnosis. cTnI is one of the three subunits constituting the troponin complex, has high myocardial specificity, and has a blood level content not affected by other diseases such as skeletal muscle injury, strenuous exercise, renal disease, and the like. The content of cTnI in peripheral blood of healthy people is generally lower than 0.06ng/mL, and for patients with acute myocardial infarction, different kinds of proteins in myocardial cells are released into the blood after the myocardial cells are damaged, the concentration of the cTnI in the peripheral blood rapidly rises within 2.2-6.8 h after AMI occurs, the cTnI reaches the peak 195.9ng/mL after 11.2h, and the cTnI can last for 4-10 days. The time when the concentration of cTnI rises from the normal value to the peak is the best time for detecting myocardial infarction. Of the patients who have suffered a myocardial infarction in the clinic, about 25% of the patients do not have obvious symptoms in the early stage of the myocardial infarction, and nearly half of the patients do not have the characteristics of myocardial necrosis in the electrocardiographic examination. Therefore, after myocardial necrosis, the detection of the myocardial marker is of great importance for the early diagnosis of myocardial infarction. At present, although the traditional enzyme-linked immunosorbent assay (ELISA) can realize high-sensitivity detection, the detection steps are more complicated, and the requirement of quick detection cannot be realized. The immunochromatography based on the gold nanoparticles can realize rapid in-vitro diagnosis and is widely applied to cTnI detection, the detection range is 5 ng/mL-1 mu g/mL, and the lowest detectable concentration is 5 ng/mL. On the basis of immunochromatography, Xu et al report that superparamagnetic nanoparticles are used for replacing gold nanoparticles for cTnI immunochromatography rapid detection, a magnetic analyzer is used for detecting magnetic signals on a detection line, the cTnI quantitative detection is realized, and the lowest detection concentration can reach 0.01 ng/mL. The Cui group selects GoldMag gold magnetic particles as carriers, and also realizes the rapid qualitative or quantitative detection of cTnI. However, magnetic immunochromatography is susceptible to interference from the earth and the magnetic field in the surrounding environment during detection.
Disclosure of Invention
The technical problem is as follows: the technical problem to be solved by the invention is as follows: the hollow core-shell nano-particles, the preparation method, the test strip and the test method are provided, the concentration of an object to be detected in a sample solution can be quantitatively detected, and the detection sensitivity is improved.
The technical scheme is as follows: in order to solve the technical problem, the embodiment of the invention adopts the following technical scheme:
the hollow core-shell nanoparticle comprises a core body and a shell, wherein the shell is positioned outside the core body, a space is reserved between the shell and the core body, Raman molecules are respectively marked on the outer surface and the inner surface of the shell, and Raman molecules are marked on the outer surface of the core body; the shell is made of gold-silver alloy, and the core body is made of gold.
As a preferred example, the hollow core-shell nanoparticle is used for a surface-enhanced raman scattering immunochromatography test strip.
A method of making a hollow core-shell nanoparticle, the method comprising:
step 10), preparing gold seeds by a hydrothermal method:
step 20) preparing gold particles by a seed growth-ripening method:
step 30) preparing Au @ Ag gold-silver core-shell nanoparticles by a seed growth method:
and step 40) preparing Au @ Au-Ag hollow core-shell nanoparticles by taking the Au @ Ag gold-silver core-shell nanoparticles prepared in the step 30) as sacrificial templates.
As a preferred example, the step 10) includes: heating water to boil, and sequentially adding chloroauric acid solution and sodium citrate solution to prepare gold seeds; the size of the gold seeds is 10-15 nm.
As a preferred example, the step 20) includes: heating water to boil, and then sequentially adding a sodium citrate solution, a chloroauric acid solution, a hydroquinone solution and the gold seeds prepared in the step 10) to prepare gold particles; the size of the gold particles is 30-100 nm; adding a Nile blue solution into the solution, and modifying a layer of Raman molecules on the surface of the gold particles by using an electrostatic adsorption principle.
As a preferred example, the step 30) includes: taking the gold particles prepared in the step 20) as seeds, sequentially adding an ascorbic acid solution and a silver nitrate solution under stirring at normal temperature, growing a silver shell on the surface of the gold particles, adding an NBA solution under stirring, and performing electrostatic adsorption on Raman molecules modified on the silver surface to prepare the Au @ Ag gold-silver core-shell nanoparticles.
As a preferred example, the step 40) includes: heating the Au @ Ag gold-silver core-shell nanoparticle solution prepared in the step 30) to boiling, and dropwise adding AuCl2 -And after the reaction of the solution is finished, filtering to obtain Au @ Au-Ag nano particle solution, adding NBA solution, and modifying a layer of Raman molecules on the surface of the particles to obtain the Au @ Au-Ag hollow core-shell nano particles.
The test strip containing the hollow core-shell nanoparticles comprises a bottom lining, a sample pad, a nanoparticle combination pad, a chromatographic membrane and a water absorption pad; the chromatographic membrane is provided with a detection line and a quality control line which are parallel to each other; the sample pad, the nanoparticle combination pad, the chromatographic membrane and the water absorption pad are sequentially and tightly connected and attached to the bottom lining; wherein, the hollow core-shell nano-particles marked by the antibody are attached to the nano-particle bonding pad; the capture antibody is attached to the detection line.
As a preferred example, the hollow core-shell nanoparticle comprises a core body and a shell, wherein the shell is positioned outside the core body, a gap is formed between the shell and the core body, raman molecules are respectively labeled on the outer surface and the inner surface of the shell, and raman molecules are labeled on the outer surface of the core body; the shell is made of gold-silver alloy, and the core body is made of gold.
A method for detecting a clinical marker by using the test strip comprises the following steps: and dropwise adding a sample solution containing the object to be detected on the sample pad through the sample adding hole, standing, collecting the Raman spectrum of the detection line position of the test strip by using a Raman spectrometer, and obtaining the concentration of the object to be detected according to the relative intensity of the Raman spectrum.
Has the advantages that: compared with the prior SERS immunochromatography technology using spherical gold nanoparticles or Au @ Ag gold-silver core-shell nanoparticles, the invention using Au @ Au-Ag hollow core-shell nanoparticles has the following beneficial effects:
1. compared with spherical Au or Au @ Ag gold-silver core-shell nanoparticles used by the traditional test strip, the Au @ Au-Ag hollow core-shell nanoparticles have stronger SERS activity, and the test sensitivity of the test strip is greatly improved.
And 2, the distance between the Au @ Au-Ag hollow core-shell nano particles and the core-shell is adjustable, and the Au @ Au-Ag hollow core-shell nano particles have stronger SERS activity. When the distance between Au @ Au-Ag core shells is reduced, the SERS activity of the particles is increased exponentially, and when the distance is less than 2nm, the increase is particularly remarkable. By changing the thickness of the Ag shell layer of the Au @ Ag nano particles, the distance between the Au @ Au-Ag core shells is adjustable within the range of 2nm, and the immunochromatography detection probe with the most excellent SERS activity can be obtained.
3. For spherical gold nanoparticles, Raman molecules are modified on the particle surface, and the particles are easy to fall off from the particle surface in the subsequent process of preparing the test strip. In the embodiment, Raman molecules can be modified on the inner surface and the outer surface of the core shell of the Au @ Au-Ag hollow core-shell nanoparticle and the distance between the inner surface and the outer surface of the core shell of the Au @ Au-Ag hollow core-shell nanoparticle, are protected by the shell layer and are prevented from being interfered by the outside, and are always stable in the subsequent test strip preparation process. The Raman molecule is kept stable, and the preparation and the detection of the test strip have high repeatability.
The Au @ Ag core-shell nanoparticles and the Au @ Au-Ag hollow core-shell nanoparticles have strong SERS activity due to the fact that silver elements are contained in the shell layers, but the Au @ Ag shell layers are made of pure silver, need to be loaded on a polyester fiber film and dried in the process of preparing the test strip, and are prone to oxidative failure in subsequent storage, and the Au @ Au-Ag shell layers are gold-silver alloy shell layers and can still keep stable in severe environments such as oxidative corrosion and the like, and the prepared test strip is easy to store.
5. In the prior art, only Au-Ag alloy nano-particles coated with a surfactant (cetyl trimethyl ammonium bromide) or polymer molecules (polyvinylpyrrolidone) and the like can be obtained by the preparation method, and in subsequent application, other molecules such as antibodies and the like are difficult to modify on the surfaces of the particles. The Au @ Au-Ag nano-particle prepared by the method is also stable by sodium citrate and easy to surface finish, the preparation process of the subsequent test strip is consistent with that of the traditional immunochromatography test strip taking the Au nano-particle with stable sodium citrate as a probe, the method has a ready-made preparation process, no additional improvement is needed, and the production cost is reduced.
The Au @ Au-Ag hollow core-shell nano-particles are used as materials, so that the surface enhanced Raman scattering immunochromatography test strip which is more stable and has stronger signals can be prepared, and the rapid, high-sensitivity and semi-quantitative detection of clinical markers can be realized.
Drawings
FIG. 1 is a schematic structural diagram of Au @ Au-Ag hollow core-shell nanoparticles according to an embodiment of the invention;
FIG. 2 is a schematic structural diagram of a test strip according to an embodiment of the present invention;
FIG. 3 is a graph comparing SERS performance of gold nanoparticles (Au), gold and silver core-shell nanoparticles (Au @ Ag) and Au @ Au-Ag hollow core-shell nanoparticles;
FIG. 4 is an optical photograph of test strips tested for various concentrations of cTnI in an example of the present invention;
FIG. 5 is a Raman spectrum obtained by detecting cTnI with different concentrations by a test strip in an embodiment of the present invention;
FIG. 6 shows that in the present invention, the main peak of the Raman spectrum in FIG. 5 is 592cm-1Standard curve of established intensity (log transformed).
The figure shows that: the kit comprises hollow core-shell nanoparticles 1, a recognition antibody 2, an object to be detected 3, a capture antibody 4, a bottom liner 5, a sample pad 6, a nanoparticle combination pad 7, a chromatographic membrane 8, a detection line 9, a quality control line 10, a water absorption pad 11, a core body 101, a shell 102 and Raman molecules 103.
Detailed Description
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Furthermore, it should be understood that various changes and modifications can be made by those skilled in the art after reading the teachings of the present invention, and such equivalents also fall within the scope of the appended claims.
As shown in fig. 1, embodiments of the present invention provide a hollow core-shell nanoparticle comprising a core and a shell. The shell is located on the outside of the core body with a space between the shell and the core body. The outer surface and the inner surface of the shell are respectively marked with Raman molecules. The outer surface of the nucleus is labeled with raman molecules. The shell is made of gold-silver alloy, and the core body is made of gold. The hollow core-shell nano-particles are used for a surface-enhanced Raman scattering immunochromatography test strip.
In the hollow core-shell nanoparticle in the embodiment, the distance is reserved between the core body and the shell and is adjustable, and the gold-silver alloy beams and columns with small irregular sizes are bridged between the core bodies, so that the core bodies can be kept relatively fixed.
Raman molecules are respectively marked on the outer surface and the inner surface of the shell, and Raman molecules are marked on the outer surface of the core body. The individual particles modify the raman molecules as much as possible, thereby increasing the detection signal intensity. The casing is made of gold-silver alloy, and the core body is made of gold, so that the chemical property is stable, the test strip is not easy to oxidize, and the test strip is favorable for storage.
The preparation method of the hollow core-shell nanoparticle of the embodiment includes:
step 10), preparing gold seeds by a hydrothermal method: heating water to boil, and sequentially adding chloroauric acid solution and sodium citrate solution to prepare gold seeds.
Step 20) preparing gold particles by a seed growth-ripening method: heating water to boil, and then sequentially adding a sodium citrate solution, a chloroauric acid solution, a hydroquinone solution and the gold seeds prepared in the step 10) to prepare gold particles; adding Nile blue (which is fully called Nile blue A, and is abbreviated as NBA) solution into the solution, and modifying a layer of Raman molecules on the surface of the gold particles by the electrostatic adsorption principle.
Step 30) preparing Au @ Ag gold-silver core-shell nanoparticles by a seed growth method: taking the gold particles prepared in the step 20) as seeds, sequentially adding an ascorbic acid solution and a silver nitrate solution under stirring at normal temperature, growing a silver shell on the surface of the gold particles, adding an NBA solution under stirring, and performing electrostatic adsorption on Raman molecules modified on the silver surface to prepare the Au @ Ag gold-silver core-shell nanoparticles.
Step 40), preparing Au @ Au-Ag hollow core-shell nanoparticles by taking the Au @ Ag gold-silver core-shell nanoparticles prepared in the step 30) as sacrificial templates: heating the Au @ Ag gold-silver core-shell nanoparticle solution prepared in the step 30) to boiling, and dropwise adding AuCl2 -And after the reaction of the solution is finished, filtering to obtain Au @ Au-Ag nano particle solution, adding NBA solution, and modifying a layer of Raman molecules on the surface of the particles to obtain the Au @ Au-Ag hollow core-shell nano particles.
In the above embodiment, in the step 10), the size of the gold seed is 10-15 nm; in the step 20), the size of the gold particles is 30-100 nm.
As shown in fig. 2, an embodiment of the present invention further provides a test strip containing the hollow core-shell nanoparticles, which includes a bottom liner 5, a sample pad 6, a nanoparticle binding pad 7, a chromatographic membrane 8, and a water absorption pad 11. The chromatographic membrane 8 is provided with a detection line 9 and a quality control line 10 which are parallel to each other; the sample pad 6, the nano-particle combination pad 7, the chromatographic membrane 8 and the water absorption pad 11 are sequentially and tightly connected and attached to the bottom lining 5; wherein, the antibody and the hollow core-shell nano-particles are attached to the nano-particle combination pad 7; the detection line 9 is attached with a capture antibody.
In this example, the hollow core-shell nanoparticle 1 includes a core body 101 and a shell body 102. The shell 102 is positioned on the outside of the core body 101 with a space between the shell 102 and the core body 101. The outer and inner surfaces of the housing 102 are labeled with raman molecules 103, respectively. The outer surface of the nucleus 101 is labelled with raman molecules 103. The shell 102 is made of gold-silver alloy, and the core body 101 is made of gold.
The method for detecting the clinical marker by using the test strip comprises the following steps: and dropwise adding a sample solution containing the object to be detected 3 on the sample pad 6 through the sample adding hole, standing, collecting the Raman spectrum of the position of the test strip detection line 9 by using a Raman spectrometer, and obtaining the concentration of the object to be detected 3 according to the relative intensity of the Raman spectrum. The standing time may be 10-20 min, for example 15 min.
The nanoparticle conjugate pad 7 is provided with a nanoparticle-labeled recognition antibody 2. The nanoparticle conjugate pad 7 is made of a polyester fiber film. The sample pad 6 is made of a glass fiber membrane. The chromatographic carrier 8 is made of a nitrocellulose membrane. The detection line 9 antibody is the capture antibody 4, and the nanoparticle-labeled antibody is the recognition antibody. The capture antibody 4 and the recognition antibody 2 are 2 monoclonal antibodies which can be matched with each other to form a sandwich structure.
In the embodiment of the invention, the immunochromatographic test strip is prepared from Au @ Au-Ag hollow core-shell nanoparticles, and the rapid, high-sensitivity and semi-quantitative detection of clinical markers is realized through Raman spectrum detection. The Raman spectrum technology is utilized to detect and identify the sample, so that the advantages of non-contact, no damage and the like are achieved, a special sample preparation step is not needed, and the Raman spectrum can be widely applied to the detection of the sample with biological activity in a biological system because the influence of water on the Raman spectrum is very small. The intensity of the Raman scattering spectrum is only 10 of the incident light intensity-10The signal is too weak to collect a detection. The detection sensitivity of the Surface Enhanced Raman Scattering (SERS) technology is greatly improved due to the appearance of the SERS technology. When molecules with Raman activity contact with the metal nano structure, the Raman spectrum of the molecules can be greatly enhanced through the electromagnetic enhancement and chemical enhancement effect action of the metal nano structure, and the enhancement capability reaches 104～107. Under the condition that the concentration of an object to be detected is low, high-sensitivity detection is realized, the gold and silver nanoparticles can greatly enhance the signal intensity of surface Raman molecules of the gold and silver nanoparticles, the intensity of a Raman spectrum has positive correlation with the quantity of the gold and silver nanoparticles, and therefore the SERS characteristic of the gold and silver nanoparticles is applied to immunochromatography detection, so that rapid, high-sensitivity and quantitative detection can be realizedAnd (4) requiring.
One specific example is illustrated below:
aiming at the fast detection of cTnI, preparing an Au @ Au-Ag hollow core-shell nanoparticle labeled SERS immunochromatographic test strip, and analyzing the result:
(1) preparing Au @ Au-Ag hollow core-shell nanoparticles marked by Raman molecular Nile Blue (NBA): a) firstly, preparing 15nm gold seeds by using a traditional hydrothermal method: heating 96mL of water to boil, and sequentially adding 1mL of chloroauric acid solution with the concentration of 25mM and 3mL of sodium citrate solution with the concentration of 1%; b) uniform 50nm gold particles were prepared by a seed growth-ripening method: heating 93mL of water to boil, sequentially adding 1mL of 15mM sodium citrate solution, 1mL of 25mM chloroauric acid solution, 1mL of 25mM hydroquinone solution and 4mL of the 15nm gold seed to obtain 50nm gold particle solution, and adding 0.5mL of 10mM gold particle solution-4Modifying a layer of Raman molecules on the surface of the NBA solution of M by an electrostatic adsorption principle; c) preparing Au @ Ag gold-silver core-shell nanoparticles by a seed growth method again: taking 50mL of the prepared 50nm gold particles as seeds, sequentially adding 0.25mL of ascorbic acid solution with the concentration of 0.1M and 1.25mL of silver nitrate solution with the concentration of 10mM under stirring at normal temperature, growing silver shells on the surfaces of the gold nanoparticles, and adding 0.5mL of 10mM under stirring-4NBA solution of M, which is adsorbed on the surface of silver by static electricity to modify Raman molecules; d) preparing Au @ Au-Ag hollow core-shell nanoparticles by taking Au @ Ag as a sacrificial template: 50mL of the Au @ Ag nanoparticle solution was heated to boiling, and 24mL of 1mM AuCl was added2 -The solution is filtered after the reaction is finished to obtain Au @ Au-Ag nano particle solution, and then 0.5mL of 10-concentration Au-Ag nano particle solution is added-4M NBA solution, a layer of Raman molecules is modified on the surface of the particles.
(2) The products of the three different stages involved in the preparation process of the Au @ Au-Ag hollow core-shell nanoparticle are 50nm gold nanoparticles, Au @ Ag core-shell nanoparticles and Au @ Au-Ag hollow core-shell nanoparticles, one layer, two layers and three layers of NBA Raman molecules are respectively modified due to the respective structures, the Raman spectra of three nanoparticle solutions under the same particle concentration are detected by a Raman spectrometer, and the result is shown in FIG. 3. As can be seen from fig. 3: the Au @ Au-Ag hollow core-shell nanoparticles have the strongest SERS activity, which is far greater than two times and three times of the strength of the Au nanoparticles and the Au @ Ag core-shell nanoparticles.
(3) And adjusting the pH value of the Au @ Au-Ag hollow core-shell nanoparticle solution modified with the NBA molecules to 8.5, adding an identification antibody (a strain of anti-human H-cTnI monoclonal antibody A) to react with the Au @ Au-Ag hollow core-shell nanoparticle solution, centrifuging to obtain a precipitate, and re-suspending the precipitate to obtain the Au @ Au-Ag hollow core-shell nanoparticle solution marked by the identification antibody.
(4) The polyester fiber membrane was treated with 0.05M Tris-HCl buffer (containing 1% by mass of bovine serum albumin, 0.02% by mass of NaN) at pH9.030.1% of PVA, 0.9% of NaCl, 10% of sucrose and 0.1% of TritonX-100), drying, spray-printing the solution of the Au @ Au-Ag hollow core-shell nano-particles marked by the recognition antibody, and placing the solution in a drying box at 37 ℃ for drying and later use.
(5) Spraying and scratching a detection line on the activated nitrocellulose membrane by using a capture antibody (a strain of anti-human H-cTnI monoclonal antibody B), and drying in vacuum; then, spraying goat anti-mouse IgG on the activated nitrocellulose membrane to mark a quality control line, and drying in vacuum;
(6) sticking the sample pad, the Au @ Au-Ag nanoparticle marking pad, the nitrocellulose membrane and the water absorption pad on a plastic plate with a single-sided pressure-sensitive adhesive in sequence, cutting, and filling into a plastic card;
(7) the test strip is used: dropwise adding the sample solution on the sample pad through the sample adding hole, and standing for 15 min;
(8) and (3) reading a detection result: a) observing the color of the detection line by naked eyes, wherein the detection line can develop color, and then qualitatively judging that the sample solution contains the substance to be detected; b) and detecting a Raman signal at the detection line position of the test strip by using a Raman spectrometer, and quantitatively analyzing the concentration of the object to be detected in the sample solution according to the strength of the Raman signal.
Fig. 4 is an optical photograph obtained after test strips tested for various concentrations of cTnI. Consistent with the traditional method, the detection line on the test strip with the concentration of 5ng/mL or more can only be distinguished by observing the immunochromatography detection result of cTnI with different concentrations by naked eyes.
Fig. 5 is a raman spectrum obtained by detecting the test strip detection line using a raman spectrometer, and it can be seen that the intensity of the raman spectrum gradually increases as the concentration of cTnI increases. Main peak 592cm in Raman molecular NBA spectrum-1Intensity is plotted on the ordinate and the concentration of cTnI on the abscissa, and a standard curve is plotted after logarithmic conversion, as shown in fig. 6. As can be seen in fig. 6: the intensity of the NBA spectrum and the cTnI concentration are in a linear relation, the detection limit can reach 0.09ng/mL, and the detection result is improved by 50 times compared with the result observed by naked eyes.
According to the invention, the Au @ Au-Ag hollow core-shell nanoparticles are used for replacing the spherical gold nanoparticles and gold-silver core-shell nanoparticles which are commonly used at present, so that the detection sensitivity and the storage stability of the surface-enhanced Raman scattering immunochromatography test strip are improved, and the simple, rapid, high-sensitivity and semi-quantitative detection of the human serum marker is realized.
The foregoing illustrates and describes the principles, general features, and advantages of the present invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are intended to further illustrate the principles of the invention, and that various changes and modifications may be made without departing from the spirit and scope of the invention, which is also intended to be covered by the appended claims. The scope of the invention is defined by the claims and their equivalents.
Claims (6)
1. A method of making a hollow core-shell nanoparticle, the method comprising:
step 10), preparing gold seeds by a hydrothermal method;
step 20) preparing gold particles by a seed growth-curing method; the step 20) comprises: heating water to boil, and then sequentially adding a sodium citrate solution, a chloroauric acid solution, a hydroquinone solution and the gold seeds prepared in the step 10) to prepare gold particles; in the step 20), the size of the gold particles is 30-100 nm; adding a Nile blue solution into the gold particle solution, and modifying a layer of Raman molecules on the surface of the gold particles by using an electrostatic adsorption principle;
step 30) preparing Au @ Ag gold-silver core-shell nanoparticles by a seed growth method; the step 30) includes: taking the gold particles prepared in the step 20) as seeds, sequentially adding an ascorbic acid solution and a silver nitrate solution under stirring at normal temperature, growing a silver shell on the surface of the gold particles, adding an NBA solution under stirring, and performing electrostatic adsorption on Raman molecules modified on the silver surface to prepare Au @ Ag gold-silver core-shell nanoparticles;
step 40) preparing Au @ Au-Ag hollow core-shell nanoparticles by taking the Au @ Ag gold-silver core-shell nanoparticles prepared in the step 30) as sacrificial templates; by changing the thickness of the Ag shell layer of the Au @ Ag nano particles, the distance between the Au @ Au-Ag core shells is adjustable within the range of 2nm, and the distance between the Au @ Au-Ag core shells is smaller than 2 nm.
2. The method of preparing hollow core-shell nanoparticles according to claim 1, wherein the step 10) comprises: heating water to boil, and sequentially adding chloroauric acid solution and sodium citrate solution to prepare gold seeds; in the step 10), the size of the gold seeds is 10-15 nm.
3. The method of preparing hollow core-shell nanoparticles according to claim 1, wherein the step 40) comprises: heating the Au @ Ag gold-silver core-shell nanoparticle solution prepared in the step 30) to boiling, and dropwise adding AuCl2 -And after the reaction of the solution is finished, filtering to obtain Au @ Au-Ag nano particle solution, adding NBA solution, and modifying a layer of Raman molecules on the surface of the particles to obtain the Au @ Au-Ag hollow core-shell nano particles.
4.A strip containing hollow core-shell nanoparticles prepared by the method of claim 1, wherein the strip comprises a substrate (5), a sample pad (6), a nanoparticle binding pad (7), a chromatographic membrane (8) and a water absorbent pad (11); the chromatographic membrane (8) is provided with a detection line (9) and a quality control line (10) which are parallel to each other; the sample pad (6), the nano-particle combination pad (7), the chromatographic membrane (8) and the water absorption pad (11) are sequentially and tightly connected and attached to the bottom liner (5); wherein, the nanoparticle combination pad (7) is attached with hollow core-shell nanoparticles marked by antibodies; the capture antibody is attached to the detection line (9).
5. The test strip of claim 4, wherein the hollow core-shell nanoparticle comprises a core and a shell, the shell is located outside the core, the shell and the core are spaced apart from each other, the outer surface and the inner surface of the shell are respectively labeled with Raman molecules, and the outer surface of the core is labeled with Raman molecules; the shell is made of gold-silver alloy, and the core body is made of gold.
6. A method of using the test strip of claim 4, the method comprising:
and dropwise adding a sample solution containing the object to be detected (3) on the sample pad (6) through the sample adding hole, standing, collecting the Raman spectrum at the position of the test strip detection line (9) by using a Raman spectrometer, and obtaining the concentration of the object to be detected (3) according to the relative intensity of the Raman spectrum.
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| CN113390852B (en) * | 2021-07-14 | 2023-03-24 | 东南大学 | High-stability surface-enhanced Raman scattering nano-label and preparation method thereof |
| CN114131037B (en) * | 2021-12-07 | 2022-06-21 | 南京医科大学第二附属医院 | Preparation method of gold-silver alloy nanoshell with high SERS activity |
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