CN115452796A - High-sensitivity identification method of immunochromatography test strip T line based on surface-enhanced Raman scattering imaging - Google Patents
High-sensitivity identification method of immunochromatography test strip T line based on surface-enhanced Raman scattering imaging Download PDFInfo
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/65—Raman scattering
- G01N21/658—Raman scattering enhancement Raman, e.g. surface plasmons
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54366—Apparatus specially adapted for solid-phase testing
- G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54366—Apparatus specially adapted for solid-phase testing
- G01N33/54386—Analytical elements
- G01N33/54387—Immunochromatographic test strips
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
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Abstract
The invention relates to a high-sensitivity identification method of an immunochromatographic test strip T line based on surface-enhanced Raman scattering imaging. The immunochromatographic strip based on surface enhanced Raman scattering imaging comprises: the sample pad, the gold label pad, the nitrocellulose membrane and the absorbent pad are sequentially overlapped with each other; wherein, the gold nano star marked with Raman reporter molecules and novel coronavirus protein antibodies is used as an SERS probe on a gold-labeled pad of the immunochromatographic test strip; and a detection line T line and a quality control line C line are sequentially arranged on the nitrocellulose membrane from the sample pad to the absorption pad.
Description
Technical Field
The invention relates to the field of lateral flow immunochromatography technology antigen detection and surface enhanced Raman scattering imaging, in particular to a method for identifying a chromatographic test strip T line by combining the lateral flow immunochromatography technology and the surface enhanced Raman scattering imaging so as to detect SARS-CoV-2 negative/positive.
Background
Conventional detection methods, such as nucleic acid detection, are highly sensitive but time consuming (. Gtoreq.2 h) and require specialized equipment, personnel and expensive chemical reagents. Other detection methods such as antibody detection and antigen detection, although capable of screening to some extent, have detection time of 15-30min, but also suffer from disadvantages in use, such as sensitivity two orders of magnitude lower than that of PCR nucleic acid detection, and difficulty in accurate diagnosis in early stage of infection of patients.
Surface Enhanced Raman Spectroscopy (SERS) is a highly sensitive spectroscopic analysis method that can nondestructively detect and identify the chemical structure of a characteristic molecule, with the ability to detect a single biological cell. After capturing nano particles and S protein through immune magnetic beads of new coronavirus SARS-CoV-2S protein by Xiamen university, performing specific immunity, incubating the nano particles and the S protein to construct a sandwich structure, directly measuring an SERS signal through a Raman spectrometer, specifically detecting the new coronavirus SARS-CoV-2 protein within 5 minutes, wherein the sensitivity can reach 5 x 10 5 Tu/mL。
However, in the current SERS-coupled lateral flow immunochromatography method, signals on the T-line of the positive sample are directly detected and compared with signals on the T-line of the negative sample. When the virus load is higher, the method can quickly and accurately identify the positive sample; when viral loads are low, direct comparison of T-line signals for negative and positive samples faces the problem of insufficient accuracy. On one hand, the random agglomeration of gold nanoparticles on a T line may bring about the sharp strengthening of signals at individual positions; on the other hand, there may be a small amount of non-specific binding of the capture antibody on the T-wire to the gold nanoparticles, which may also cause a sudden increase in raman signal, resulting in the appearance of false positives. Furthermore, the position of the T-line cannot be observed by color when the viral load is low, and the position of the T-line needs to be found deliberately, especially when the signal enhancement of the T-line position is not significant, which makes it more difficult to identify the T-line signal. In this way, further improvement of detection sensitivity by combining SERS with the lateral flow immunochromatography is limited.
Disclosure of Invention
Aiming at the problems, the invention provides a method for identifying the T line of the immunochromatographic strip based on the surface enhanced Raman imaging technology, which is used for imaging by scanning Raman signals around the T line of the immunochromatographic strip and judging whether the sample is negative or positive according to the imaging characteristics. The method has the advantages of high accuracy, high sensitivity and high speed, greatly reduces the occurrence of false positives, and has great popularization and application values for signal identification of the chromatographic test strip.
Specifically, in a first aspect, the present invention provides an immunochromatographic strip based on surface enhanced raman scattering imaging, comprising: the sample pad, the gold label pad, the nitrocellulose membrane and the absorbent pad are sequentially overlapped with each other; wherein, the gold nano star marked with Raman reporter molecules and novel coronavirus protein antibodies is used as an SERS probe on a gold-labeled pad of the immunochromatographic test strip; and a detection line T line and a quality control line C line are sequentially arranged on the nitrocellulose membrane from the sample pad to the absorption pad.
Preferably, the structure of the gold nanostars labeled with raman reporter molecules and novel coronavirus protein antibodies comprises from inside to outside: the novel coronavirus protein antibody is characterized by comprising Au nanostars and a novel coronavirus protein antibody which is modified on the surfaces of the Au nanostars and is marked by a Raman reporter molecule;
preferably, the gold nano-star has a tactility structure with the length of 20-50nm and the width of 10-20nm, and the whole grain diameter is 30-100 nm; the Raman reporter molecule is one of 4-mercaptobenzoic acid (4-MBA) or 5,5' -dithiobis (2-nitrobenzoic acid) (DTNB).
Preferably, the UV-Vis absorption peak position of the gold nanosome sol marked with the Raman reporter molecule and the novel coronavirus protein antibody is 750 +/-50 nm.
Preferably, the preparation method of the gold nano star labeled with the raman reporter molecule and the novel coronavirus protein antibody comprises the following steps: adding a novel coronavirus protein antibody marked by a Raman reporter molecule into the gold nano-star solution, and incubating for 6-12 hours at 4-25 ℃; centrifuging and collecting the product, dissolving the product again, adding bovine serum albumin BSA solution, and incubating at 4-25 ℃ for 2-6 hours to obtain the product.
Preferably, the combination pad is dipped in a gold nano star solution marked with Raman reporter molecules and novel coronavirus protein antibodies, and dried to obtain a gold-labeled pad; in the gold nano-star solution marked with the Raman reporter molecule and the novel coronavirus protein antibody, the concentration of gold nano-star particles is 10 10 ～10 12 particles/mL, the concentration of the Raman reporter molecule is 10 -3 ～ 10 -5 M, the concentration of the novel coronavirus protein antibody is 2-10 mug/mL.
Preferably, the detection line T line is obtained by diluting a novel coronavirus protein antibody to 1-5 mg/mL and then scribing, the scribing amount is 0.5-5 mu L/cm, and the scribing speed is 50-100 mm/s; the quality control line C is obtained by diluting goat anti-mouse IgG to 5-10 mg/mL and then scribing, the scribing amount is 0.5-5 mu L/cm, and the scribing speed is 50-100 mm/s.
Preferably, the distance between the detection line T and the quality control line C is 1-2 cm; the width of the C line is 200-500 mu m, and the width of the T line is 200-500 mu m.
In a second aspect, the present invention provides a method for identifying T-line of immunochromatographic strip based on surface enhanced raman scattering imaging for non-diagnostic and therapeutic purposes, comprising:
(1) Respectively configuring novel coronavirus protein solutions with different concentrations in virus lysis solution for immunochromatography test strips based on surface enhanced Raman scattering imaging to perform immunochromatography, and detecting T lines of the immunochromatography test strips and Raman signals around the T lines through portable Raman spectra; mapping imaging is carried out near the T line by using the strongest Raman characteristic peak of the probe molecule;
(2) And judging whether the T line has the virus antigen or not by judging the characteristics of the imaged image and the difference value of the strongest Raman signal value and the weakest Raman signal value in the imaged area.
Preferably, the Mapping imaging area is a rectangular area with X being more than or equal to 1mm and less than or equal to 3mm and Y being more than or equal to 0.2mm and less than or equal to 3mm by taking the geometric center of the T line as the scanning center position; the X direction is the direction of the T line scribing; the Y direction is the liquid flow direction.
Preferably, the scanning step length l in the X direction is controlled x Less than or equal to 200 mu m, preferably less than or equal to 50 mu m x Less than or equal to 200 mu m; scanning step length l in Y direction y Y/3, preferably Y/10. Ltoreq.l y ≤Y/3。
Preferably, the power of the portable Raman spectrum is 20-100 mW, and the single-point integration time is 0.5-2 seconds.
Preferably, the imaging characteristics at line T of the positive test strip include:
(1) Only one color band with the intensity larger than or equal to 2000 and clear boundary exists in the X direction;
(2) The color band pixel point is more than or equal to 2l and the width is more than or equal to 2l x ；
(3) The intensity difference value of the color ribbon in the Y direction is less than or equal to 5000;
(4) Difference value I between the strongest value of Raman signal on the color band and the weakest signal in the imaging area max -I min ≥1500。
Preferably, the imaging characteristics at line T of the negative strip include:
(1) A plurality of color bands with higher intensity than the surrounding color bands exist in the X direction, and the color bands have unclear boundaries;
(2) The color band pixel point is less than 2 and the width is less than 2l x Or no apparent color band;
(3) The difference value I between the strongest value of the Raman signal on the color band and the weakest signal in the imaging area max -I min ≤3000。
Advantageous effects
The invention combines Raman imaging technology and immunochromatography technology, and improves the sensitivity of antigen detection by 1000 times or more; imaging the chromatographic test strip T line through a large number of acquired Raman spectra, visually giving out whether an SERS probe exists on the T line, wherein the relative spectrum reading is a man-machine interface friendly identification mode; the interference caused by some accidental factors, including system errors and non-uniformity of enhanced signals, is avoided, the identification accuracy is improved, and false positive misjudgment is greatly avoided.
Drawings
FIG. 1 is a schematic diagram of immunochromatography and Raman imaging detection based on a gold nano-star SERS probe;
FIG. 2 is a graph showing the results of immunochromatography of solutions of SARS-CoV-2 virus N protein at different concentrations;
FIG. 3 shows the Raman imaging results of the SARS-CoV-2 virus N protein solution at different concentrations after chromatography on a test strip, wherein Mapping images a-g correspond to samples 1-7 in FIG. 2;
FIG. 4 shows the results of immunochromatography of SARS-CoV-2 virus S protein solutions of different concentrations;
FIG. 5 shows the Raman imaging results of the SARS-CoV-2 virus S protein solution at different concentrations after chromatography on the strip, and Mapping images of a-d correspond to samples No. 3-6 in FIG. 4, respectively;
FIG. 6 is a new coronal positive patient C t Immunochromatography results of nasopharyngeal swab samples with a value of 28-38 and four negative human nasopharyngeal swab samples;
FIG. 7 shows a variant C t The Raman imaging results of the SARS-CoV-2 virus positive patient and the negative sample after chromatography on the test strip, a-e are respectively positive samples with Ct value of 30-34, the frame selection area is a color band, f-h are negative samples;
FIG. 8 shows the results of Raman and imaging detection of SARS-CoV-2 virus N protein and negative sample, wherein FIG. a shows 20 spectra collected around the T-line and T-line, respectively, and FIG. b shows the results of Raman imaging of T-line and its surroundings.
Detailed Description
The present invention is further illustrated by the following examples, which are to be understood as merely illustrative, and not restrictive, of the invention.
The invention provides a method for identifying a T line of a SARS-CoV-2 antigen detection side-stream immunochromatographic strip based on a surface enhanced Raman imaging technology, which images by scanning Raman signals around the T line of the immunochromatographic strip and judges whether a sample is negative or positive according to the imaging characteristics. The method has the advantages of high accuracy, high sensitivity and high speed, greatly reduces the occurrence of false positives, and has great popularization and application values for signal identification of the chromatographic test strip.
First, the present invention provides an immunochromatographic strip based on surface enhanced raman scattering imaging, comprising: a sample pad, a gold-labeled pad (i.e., "conjugate pad" shown in fig. 1), a nitrocellulose membrane, and a water absorbent pad, which are sequentially overlapped with each other; wherein, the gold nano star marked with Raman reporter molecules and novel coronavirus N or S protein antibodies is used as an SERS probe on a gold-labeled pad of the immunochromatographic test strip; and a detection line (T line) and a quality control line (C line) are sequentially arranged on the nitrocellulose membrane from the sample pad to the absorbent pad.
Wherein the structure of the gold nanostars marked with the Raman reporter molecule and the novel coronavirus N or S protein antibody comprises from inside to outside: au nanostars and novel coronavirus N or S protein antibodies which are modified on the surfaces of the Au nanostars and labeled by Raman reporter molecules. Preferably, the gold nano-star has a tactility structure with the length of 20-50nm and the width of 10-20nm, and the whole grain diameter is 30-100 nm; the raman reporter molecule may be one of 4-mercaptobenzoic acid (4-MBA), or 5,5' -dithiobis (2-nitrobenzoic acid) (DTNB).
The UV-Vis absorption peak position of the gold nano star sol marked with the Raman reporter molecule and the novel coronavirus N or S protein antibody is 750 +/-50 nm.
In some embodiments, the solution of gold nanostars labeled with raman reporter molecules and novel coronavirus N or S protein antibodies may be prepared by: adding a novel coronavirus N or S protein antibody marked by a Raman reporter molecule into the gold nano-star solution, and incubating for 6-12 hours at 4-25 ℃; centrifuging and collecting the product, dissolving the product again, adding bovine serum albumin BSA solution, and incubating at 4-25 ℃ for 2-6 hours to obtain the product.
And (3) dipping the combination pad into a gold nano star solution marked with Raman reporter molecules and novel coronavirus N or S protein antibodies, and drying to obtain the gold-labeled pad. In the gold nano-star solution marked with the Raman reporter molecule and the novel coronavirus N or S protein antibody, the concentration of gold nano-star particles is 10 10 ～10 12 particles/mL, the concentration of the Raman reporter molecule is 10 -3 ～10 -5 M, the concentration of the novel coronavirus N or S protein antibody is 2-10 mug/mL.
The detection line T line can be obtained by diluting a novel coronavirus N or S protein antibody to 1-5 mg/mL and then scribing, the scribing amount can be 0.5-5 mu L/cm, and the scribing speed can be 50-100 mm/S; the quality control line C can be obtained by diluting goat anti-mouse IgG to 5-10 mg/mL and then scribing, the scribing amount can be 0.5-5 mu L/cm, and the scribing speed can be 50-100 mm/s.
The distance between the detection line (T line) and the quality control line (C line) can be controlled to be 1-2 cm; the width of the C line is 200-500 μm, and the width of the T line is 200-500 μm.
FIG. 1 is a schematic diagram of immunochromatography and Raman imaging detection based on a gold nano star SERS probe. As can be seen from the figure, when the antigen exists (above), the SERS probe is captured by the antibody at the T-line, and the excess or unbound SERS probe is captured by the second antibody at the C-line, and at this time, SERS imaging is performed on the T-line and the surrounding area, and a distinct color band appears; when the antigen is absent (lower), the SERS probe is captured only at the C-line, at which time SERS imaging is performed on the T-line and its surrounding area, with no apparent bands appearing.
The method for identifying T line of SARS-CoV-2 antigen detection immunochromatographic strip based on surface enhanced Raman imaging for non-diagnostic and therapeutic purposes provided by the present invention is illustrated below with reference to FIG. 1, and the identification method mainly comprises the following steps.
S1, immunochromatography based on special colloidal gold. The gold nano star marked with Raman reporter molecules and novel coronavirus N or S protein antibodies is used as an SERS probe on a gold-labeled pad of an immunochromatography test strip, the T line of a nitrocellulose membrane (NC membrane) is marked with another novel coronavirus N or S protein antibody, and the C line is marked with a capture antibody.
Specifically, a novel coronavirus N or S protein antibody marked by a Raman reporter molecule is added into a gold nano-star solution, and the mixture is incubated for 6 to 12 hours at the temperature of between 4 and 25 ℃; centrifuging and collecting the product, adding bovine serum albumin BSA solution after redissolving the product, and incubating for 2-6 hours at 4-25 ℃ to obtain the gold nanostar marked with the Raman reporter molecule and the novel coronavirus N or S protein antibody. In the gold nano-star solution marked with the Raman reporter molecule and the novel coronavirus N or S protein antibody, the concentration of gold nano-star particles is 10 10 ～10 12 particles/mL, the concentration of the Raman reporter molecule is 10 -3 ～10 -5 M, the concentration of the novel coronavirus N or S protein antibody is 2-10 mug/mL.
The colloidal gold used by the immunochromatographic test strip is based on gold nano star (AuNSs) sol marked with Raman reporter molecules, the gold nano star has a touch structure with the length of 20-50nm and the width of 10-20nm, the overall particle size is 30-100nm, and the position of an UV-Vis absorption peak is 750 +/-50 nm. Gold nano star (AuNSs) -Raman reporter molecule-novel coronavirus N or S protein antibody is used as a probe molecule of immunochromatography, and the Raman reporter molecule can be 4MBA or DTNB.
S2, raman signal acquisition and Mapping imaging. Novel coronavirus protein solutions with different concentrations are respectively prepared in virus lysate and used for immunochromatography on an immunochromatography test strip of surface enhanced Raman scattering imaging to perform immunochromatography. Meanwhile, PBS buffer may be additionally used as a control group. Detecting a T line of the chromatographic test strip and a Raman signal around the T line through a portable Raman spectrum; then, mapping imaging is carried out near the T line by using the strongest Raman characteristic peak of the probe molecules, the imaging area is a rectangular area with the geometric center of the T line as the scanning center position, X is more than or equal to 1mm and less than or equal to 3mm, and Y is more than or equal to 0.2mm and less than or equal to 3 mm. Wherein, as shown in fig. 1, the X direction is the T-line scribing direction; the Y direction is the liquid flow direction.
In some embodiments, the scanning step l in the X direction can be controlled x 200 μm or less, and preferably a step size of 50 μm or less l in consideration of detection time and accuracy x Less than or equal to 200 mu m; the scanning step length l in the Y direction can be controlled y Y/3, preferably a step length of Y/10. Ltoreq.l in view of detection time and accuracy y Less than or equal to Y/3. The scanning step length l in the X direction x The distance between the pixels in the X direction; the scanning step length l in the Y direction y The distance between the pixels in the Y direction.
If the scanning step length is too long, the T-line region may be skipped in the X direction, so that the signal cannot be read; in the Y direction, signal interference caused by random particle aggregation is increased because the number of scanned points is too small. If the scanning step length is too small, the time for scanning the whole area is long, and the identification efficiency is influenced finally.
The power of the portable Raman spectrum can be 20-100 mW, and the single-point integration time can be 0.5-2 seconds. The single point integration time is the time required for each pixel point to collect a signal.
And S3, judging whether the sample is negative or positive. And judging whether the T line has the virus antigen or not by judging the characteristics of the imaged image and the difference value of the strongest Raman signal value and the weakest Raman signal value in the imaged area.
Wherein, the imaging of positive test strip T line department has following characteristics simultaneously:
(1) In the X direction, only one color band with high intensity (intensity is more than or equal to 2000) and clear boundary exists. In the raman imaging, which is mapping based on the intensity of a raman signal, when a positive reaction exists at a T-line, the raman signal at the T-line is significantly increased, so that a color band with high intensity and a clear boundary exists in the X-direction;
(2) The color band pixel point is more than or equal to 2l and the width is more than or equal to 2l x . Due to the scanning step l in the X direction x Not more than 200 μm and the width of the T line is about 500 μm, so that the color band appearing at the T line has a certain width in the positive reaction, the pixel point is not less than 2, and the width is not less than 2l x ；
(3) The color bands are close in intensity in the Y direction (the intensity difference is less than or equal to 5000). Generally, the captured antigen is uniformly distributed on a T line after chromatography, so that the Raman intensity of the color band in the Y direction is close;
(4) Difference value I between the strongest value of Raman signal on the color band and the weakest signal in the imaging area max -I min Is more than or equal to 1500. In order to avoid the interference of systematic error and accidental factors, the difference value I between the strongest value of the Raman signal on the color band of the positive test strip and the weakest signal in the imaging area can be controlled max -I min ≥1500。
The imaging at the line T of the negative strip is simultaneously characterized by:
(1) There are several (multiple) color bands in the X direction with higher intensity than the surroundings, and the color band boundaries are not clear. Because the probe of the negative test strip can not be captured on the T line, the probe flows to the C line and the absorbent paper along with the chromatography, or part of the probe randomly remains on the NC membrane, not only one color band with the intensity higher than the surrounding exists in the X direction, but also the color band boundary is unclear;
(2) The color band pixel point is less than 2 and the width is less than 2l x Or no distinct color bands. Because random aggregation of the nanoprobes possibly occurs can cause some isolated strong Raman signals to exist, especially, the randomly aggregated probes cannot be regularly arranged on a T line (Y direction) like a probe of a positive test strip, so that the obtained color band has narrow width, pixel points are less than 2, and the width is less than 2l x (ii) a Or a wide color band but a significant difference in intensity in the Y direction;
(3) The difference value I between the strongest value of the Raman signal on the color band and the weakest signal in the imaging area max -I min Less than or equal to 3000. The difference between the strongest value of the Raman signal in the color band of the negative strip and the weakest signal in the imaging area is generally small due to systematic errors and the signal difference caused by random aggregation of individual probes max -I min ≤3000。
Compared with the prior immunochromatography technology or the technology of combining immunochromatography with Raman spectroscopy, the method can realize high-sensitivity detection and also can intuitively identify the T-line signal as a color development method, thereby improving the accuracy and reliability of signal identification. The method provided by the invention is simple and convenient to operate, high in sensitivity, high in accuracy and high in detection speed, can complete detection within 10min through portable Raman, and has wide application and popularization prospects.
The present invention will be described in detail by way of examples. It should also be understood that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention, and that the insubstantial modifications and adjustments made by those skilled in the art in light of the above disclosure are within the scope of the present invention, and that the specific process parameters and the like described in the following examples are only examples of suitable ranges, i.e., those skilled in the art can select from the suitable ranges described herein, and are not intended to be limited to the specific values listed below.
The following embodimentsIn the examples, unless otherwise specified, the gold nanostars labeled with DTNB and novel coronavirus S protein antibody were used as SERS probes on a gold-labeled pad of an immunochromatographic strip, and the gold-labeled pad was prepared by the following steps: and (3) dipping the combination pad into a gold nano star solution marked with Raman reporter molecules and novel coronavirus S protein antibodies, and drying to obtain the gold-labeled pad. In the gold nano-star solution marked with the Raman reporter molecule and the novel coronavirus S protein antibody, the concentration of gold nano-star particles is 10 11 particles/mL, the concentration of the Raman reporter molecule is 10 -5 M, the concentration of the novel coronavirus S protein antibody is 10 mu g/mL.
Example 1
A method for determining the negative/positive of SARS-CoV-2 virus nucleocapsid protein (N protein) by detecting chromatographic test strip T line based on surface enhanced Raman scattering imaging technology comprises the following steps:
(1) The gold nano star marked with 4MBA and the novel coronavirus N protein antibody is used as an SERS probe on a gold label pad of the immunochromatographic strip. The T line of the nitrocellulose membrane (NC membrane) is marked with another novel coronavirus N protein antibody, and the C line is marked with a capture antibody; the distance between the detection line (T line) and the quality control line (C line) is 1cm, and the width of the T line is 500 mu m;
(2) The virus lysate is respectively prepared with the concentration of 5 multiplied by 10 -7 g/mL～5×10 -12 The protein N solution in g/mL was used for immunochromatography, and PBS buffer was used as a control group. FIG. 2 is a diagram showing the results of immunochromatography of solutions of SARS-CoV-2 virus N protein at different concentrations. As can be seen from the figure, no. 1-6 were each added dropwise at a concentration of 5X 10 -7 g/mL～5×10 -12 g/mL of N protein solution, no. 7 blank control added with PBS buffer solution dropwise, and the color development method can be performed at 5 × 10 -9 Reading new corona positive at g/mL, wherein the subsequent concentration cannot be judged by naked eyes;
(3) The T line is identified by Raman imaging method, and the molecular weight of 4MBA is 1080cm -1 Mapping imaging is carried out on the characteristic peak at the (C-H stretching vibration). The imaging region is a rectangular region having the geometric center of the T line as the center point, where X =2mm, y =0.4mm, the step size in the X direction is 0.1mm, and the step size in the y direction is 0.1mm. Portable Raman spectroscopyThe power of the single-point integration is 50mW, and the single-point integration time is 1 second;
(4) The imaging results are shown in fig. 3. According to the positive identification standard provided by the invention, 3a-e can be accurately judged to be positive; the first three samples of the 3f sample meeting the positive judgment standard are at the critical value of the fourth strip, so that the detection limit of the method can be considered to be reached; and 3g of negative samples obviously do not accord with the positive judgment standard and accord with the negative judgment standard provided by the invention. Compared with a color development method, the Raman imaging method provided by the invention improves the detection limit of the immunochromatographic test strip by 1000 times.
Example 2
A method for determining SARS-CoV-2 virus spike protein (S protein) negative/positive by detecting chromatographic test strip T line based on surface enhanced Raman scattering imaging technology comprises the following steps:
(1) The gold nano star marked with DTNB and the novel coronavirus S protein antibody is used as an SERS probe on a gold-labeled pad of the immunochromatography test strip. The T line of the nitrocellulose membrane (NC membrane) is marked with another novel coronavirus S protein antibody, and the C line is marked with a capture antibody; the distance between the detection line (T line) and the quality control line (C line) is 1cm, and the width of the T line is 450 mu m;
(2) The concentration of each of the virus lysates was 1 × 10 -7 g/mL～1×10 -11 g/mL of the S protein solution was used for immunochromatography, and PBS buffer was used as a control group. As shown in FIG. 4, 1-5 were each added dropwise at a concentration of 1X 10 -7 g/mL-1×10 -11 g/mL S protein solution, no. 6 was added dropwise with PBS blank. As can be seen from the figure, the color development method can be performed at 1X 10 -7 Reading new corona positive at g/mL, wherein the subsequent concentration cannot be judged by naked eyes;
(3) The T line is then identified by Raman imaging with the DTNB molecule at 1326cm -1 Mapping imaging is carried out on the characteristic peak. The imaging region is a rectangular region having the geometric center of the T-line as the center point, where X =1.5mm, y = 0.3mm, the step size in the X direction is 0.075mm, and the step size in the y direction is 0.1mm. The power of the portable Raman spectrum is 50mW, and the single-point integration time is 1 second;
(4) The imaging results are shown in fig. 5. According to the invention1X 10 of the Positive identification criteria -9 g/mL-1×10 -10 The g/mL S protein solution can be accurately judged to be positive; and 1X 10 was added dropwise -11 The test strip and the negative sample of the S protein solution of g/mL obviously do not accord with the positive judgment standard and the negative judgment standard provided by the invention. Therefore, the detection limit of the raman imaging method of the present embodiment is 1 × 10 -10 g/mL. Compared with a color development method, the Raman imaging method provided by the invention improves the detection limit of the immunochromatographic test strip by 1000 times.
Example 3
A method for determining SARS-CoV-2 virus negative/positive of human nasopharyngeal swab based on surface enhanced Raman scattering imaging technology detection chromatography test strip T line comprises the following steps:
(1) The gold nano star marked with 4MBA and the novel coronavirus N protein antibody is used as an SERS probe on a gold label pad of the immunochromatographic strip. The T line of the nitrocellulose membrane (NC membrane) is marked with another novel coronavirus N protein antibody, and the C line is marked with a capture antibody; the distance between the detection line (T line) and the quality control line (C line) is 1cm, and the width of the T line is 400 mu m;
(2) 2mL of each of the new coronal positive patients C were prepared t Nasopharyngeal swab samples with the value of 28-38 (virus quantification adopts major nucleic acid extraction reagent and major nucleic acid detection reagent), live virus is preserved by using phenol red preservation solution, and in addition, four negative human nasopharyngeal swab samples are prepared as a control group, and 15 human samples are totally prepared;
(3) mu.L of virus sample was taken from each human body sample, 100. Mu.L of virus lysate was added thereto and mixed well, and then 80. Mu.L of sample was taken and subjected to immunochromatography. As a result, as shown in FIG. 6, C was read out by the color development method t The value is 28-29, the subsequent concentration of the chromatography result of the new corona positive patient cannot be judged by naked eyes;
(4) The T line is identified by Raman imaging method, and the length of the T line is 1080cm with 4MBA molecules -1 Mapping imaging is carried out on the characteristic peak at the (C-H stretching vibration). The imaging region is a rectangular region having the geometric center of the T line as the center point, where X =2mm, y =0.4mm, the step size in the X direction is 0.1mm, and the step size in the y direction is 0.1mm. The power of the portable Raman spectrum is 50mW, and the single-point integration timeIs 1 second;
(5) The imaging results are shown in fig. 7. Criteria for positive identification proposed according to the invention, C t Positive patients with a value of 30-33 can be accurately judged as new coronas positive; c t The positive patient sample with the value of 34 meets 1, 3 and 4 of the positive judgment criteria, and does not meet the third criterion, so that the detection limit of the method can be considered to be reached; the 7f-h negative sample obviously does not accord with the positive judgment standard and accords with the negative judgment standard provided by the invention. Compared with a color development method, the Raman imaging method provided by the invention improves the detection limit of the immunochromatographic test strip by 10000 times.
Comparative example 1
The result of judging whether SARS-CoV-2 virus N protein is negative/positive by the conventional method is as follows:
(1) The gold nano star marked with 4MBA and the novel coronavirus N protein antibody is used as an SERS probe on a gold-labeled pad of the immunochromatography test strip. The T line of the nitrocellulose membrane (NC membrane) is marked with another novel coronavirus N protein antibody, and the C line is marked with a capture antibody; the distance between the detection line (T line) and the quality control line (C line) is 1cm, and the width of the T line is 500 mu m;
(2) The virus lysate is respectively prepared with the concentration of 5 multiplied by 10 -8 g/mL of an N protein positive solution and PBS buffer were used as negative solutions for the control group, followed by immunochromatography. The chromogenic method can directly read out a new crown positive sample, and then the negative sample is judged by Raman detection;
(3) The samples are first identified by direct raman detection, i.e. without imaging and based on image analysis steps, negative and positive samples are distinguished only by the intensity of the acquired raman spectral signal. As shown in fig. 8a, 20 raman spectra are collected around the T-line and T-line, respectively, and two raman spectra with very high intensity are evident on the T-line, so that the negative strip is likely to be misjudged as a positive sample in actual detection, or a correct judgment cannot be made;
(4) The T line is identified by Raman imaging method, and the length of the T line is 1080cm with 4MBA molecules -1 Mapping imaging is carried out on the characteristic peak at the (C-H stretching vibration). The imaging area is a rectangular area with the geometric center of the T line as the center pointWhere X =2mm, y =0.4mm, the step size in the X direction is 0.1mm, and the step size in the y direction is 0.1mm. The power of the portable Raman spectrum is 50mW, and the single-point integration time is 1 second;
(5) As shown in fig. 8b, according to the negative/positive identification standard provided by the present invention, the color band can be obviously judged as negative, thereby avoiding false positive misjudgment and improving the accuracy of the SERS in combination with the lateral flow immunochromatography.
While the present invention has been described in detail with reference to the preferred embodiments, it should be understood that the above description should not be taken as limiting the invention. Various modifications and alterations to this invention will become apparent to those skilled in the art upon reading the foregoing description. Accordingly, the scope of the invention should be determined from the following claims.
Claims (13)
1. An immunochromatographic strip based on surface-enhanced Raman scattering imaging, comprising: the sample pad, the gold label pad, the nitrocellulose membrane and the absorbent pad are sequentially overlapped with each other; wherein, the gold nano star marked with Raman reporter molecules and novel coronavirus protein antibodies is used as an SERS probe on a gold label pad of the immunochromatographic test strip; and a detection line T line and a quality control line C line are sequentially arranged on the nitrocellulose membrane from the sample pad to the absorption pad.
2. The immunochromatographic strip for surface-enhanced Raman scattering imaging based on claim 1, wherein the structure of the gold nanostars labeled with Raman reporter molecules and novel coronavirus protein antibodies comprises from inside to outside: the novel coronavirus protein antibody is characterized by comprising Au nanostars and a novel coronavirus protein antibody which is modified on the surfaces of the Au nanostars and is marked by a Raman reporter molecule;
preferably, the gold nano-star has a tactility structure with the length of 20-50nm and the width of 10-20nm, and the whole grain diameter is 30-100 nm; the Raman reporter molecule is one of 4-mercaptobenzoic acid (4-MBA) or 5,5' -dithiobis (2-nitrobenzoic acid) (DTNB).
3. The immunochromatographic strip for surface-enhanced Raman scattering imaging-based measurement according to claim 1 or 2, wherein the UV-Vis absorption peak position of the gold nanostar sol labeled with the Raman reporter molecule and the novel coronavirus protein antibody is 750 ± 50nm.
4. The immunochromatographic strip based on surface-enhanced Raman scattering imaging according to any one of claims 1 to 3, wherein the gold nanostars labeled with Raman reporter molecules and novel coronavirus protein antibodies are prepared by the following method: adding a novel coronavirus protein antibody marked by a Raman reporter molecule into the gold nano-star solution, and incubating for 6-12 hours at 4-25 ℃; centrifuging and collecting the product, dissolving the product again, adding bovine serum albumin BSA solution, and incubating at 4-25 ℃ for 2-6 hours to obtain the product.
5. The immunochromatographic strip for surface-enhanced Raman scattering imaging based on any one of claims 1 to 4, wherein the conjugate pad is immersed in a gold nanostar solution labeled with a Raman reporter and a novel coronavirus protein antibody, and dried to obtain a gold-labeled pad; in the gold nano-star solution marked with the Raman reporter molecule and the novel coronavirus protein antibody, the concentration of gold nano-star particles is 10 10 ～10 12 particles/mL, the concentration of the Raman reporter molecule is 10 -3 ～10 -5 M, the concentration of the novel coronavirus protein antibody is 2-10 mug/mL.
6. The immunochromatographic strip based on surface-enhanced Raman scattering imaging according to any one of claims 1 to 5, wherein the detection line T is obtained by streaking a novel coronavirus protein antibody diluted to 1 to 5mg/mL, the streaking amount is 0.5 to 5 μ L/cm, and the streaking speed is 50 to 100mm/s; the quality control line C is obtained by diluting goat anti-mouse IgG to 5-10 mg/mL and then scribing, the scribing amount is 0.5-5 mu L/cm, and the scribing speed is 50-100 mm/s.
7. The immunochromatographic strip based on surface-enhanced Raman scattering imaging according to any one of claims 1 to 6, wherein the detection line T and the quality control line C are spaced from each other by 1 to 2cm; the width of the C line is 200-500 mu m, and the width of the T line is 200-500 mu m.
8. The T-line identification method of the immunochromatographic strip based on surface enhanced Raman scattering imaging according to any one of claims 1 to 7 for non-diagnostic and therapeutic purposes, comprising:
(1) Respectively configuring novel coronavirus protein solutions with different concentrations in virus lysis solution for immunochromatography test strips based on surface enhanced Raman scattering imaging to perform immunochromatography, and detecting T lines of the immunochromatography test strips and Raman signals around the T lines through portable Raman spectra; mapping imaging is carried out near the T line by using the strongest Raman characteristic peak of the probe molecule;
(2) And judging whether the T line has the virus antigen or not by judging the characteristics of the imaged image and the difference value of the strongest Raman signal value and the weakest Raman signal value in the imaged area.
9. The identification method according to claim 8, wherein the Mapping imaging area is a rectangular area with the geometric center of the T line as the scanning center position, and 1mm X3 mm,0.2mm Y3 mm; the X direction is the direction of the T line scribing; the Y direction is the liquid flow direction.
10. Identification method according to claim 8 or 9, characterized in that the scanning step/in the X-direction is controlled x 200 μm or less, preferably 50 μm or less x Less than or equal to 200 mu m; scanning step length l in Y direction y Y/3, preferably Y/10. Ltoreq.l y ≤Y/3。
11. The identification method according to any one of claims 8 to 10, wherein the portable raman spectrum has a power of 20 to 100mW and a single point integration time of 0.5 to 2 seconds.
12. The method of any one of claims 8-11, wherein the imaging characteristic at the T-line of the positive test strip comprises:
(1) Only one color band with the intensity larger than or equal to 2000 and clear boundary exists in the X direction;
(2) The color band pixel point is more than or equal to 2l and the width is more than or equal to 2l x ；
(3) The intensity difference value of the color band in the Y direction is less than or equal to 5000;
(4) Difference value I between the strongest value of Raman signal on the color band and the weakest signal in the imaging area max -I min ≥1500。
13. The method of any one of claims 8-12, wherein the imaging characteristic at the T-line of the negative strip comprises:
(1) A plurality of color bands with the intensity higher than that of the surrounding color bands exist in the X direction, and the color bands have unclear boundaries;
(2) The color band pixel point is less than 2 and the width is less than 2l x Or no distinct color band;
(3) The difference value I between the strongest value of the Raman signal on the color band and the weakest signal in the imaging area max -I min ≤3000。
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