CN110133255A - A kind of quickly SERS- immunochromatography detection method with highly sensitive detection mycoplasma pneumoniae infection - Google Patents
A kind of quickly SERS- immunochromatography detection method with highly sensitive detection mycoplasma pneumoniae infection Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6854—Immunoglobulins
Abstract
The present invention relates to a kind of quickly SERS- immunochromatography detection methods with highly sensitive detection mycoplasma pneumoniae infection, its testing principle is with nitrocellulose filter (Nitrocellulose membranes, it NC) is carrier, the double-deck dyestuff 5, 5 '-two thiobis (2- nitrobenzoic acid) { 5, 5 '-dithiobis- (2-nitrobenzoic acid), DTNB } mark Au@Ag nano material coupling detection antibody as SERS probe, in conjunction with traditional immunochromatographic method (Immunochromatographic assay, ICA the novel immunochromatography technique (SERS-ICA) based on Surface enhanced Raman spectroscopy) is established, it is examined using the technology People IgM and mycoplasma pneumoniae (Mycoplasma pneumonia, MP) specific IgM positive serum sample are surveyed.Detection is divided into sample dilution, the release of SERS probe, the processes such as antigen-antibody reaction, interpretation of result.The detection sensitivity of people IgM and the recall rate of pulmonary branches positive serum sample can be improved through the invention, have great importance for clinical treatment.
Description
Technical field
The present invention relates to technical field of in vitro diagnostic reagents, and in particular to a kind of quick and highly sensitive detection mycoplasma pneumoniae
The SERS- immunochromatography detection method of infection.
Background technique
Mycoplasma pneumoniae is the pathogen of a shortage peptidoglycan cell wall, does not need host cell, can independently replicate；
The all age group being popular in worldwide is the Etiological of pneumonia and respiratory disease.Nearly 6%-30% at
The children of people and 20%-40% suffer from community acquired pneumonia due to pneumonia infection mycoplasma, and every 3-7 is once
The early diagnosis of epidemic peak pulmonary branches is for determining treatment method and guiding suitable Antybody therapy most important.Existing gold
Standard method is still the traditional cell culture diagnostic method most reliable and cheap as pulmonary branches.However, pulmonary branches culture consumes
When, 2-8 weeks is needed, and need special culture dish and technical professional.Another traditional method is serology
Analysis method, since its remolding sensitivity is lower, the diagnosis for pulmonary branches infection is inaccurate.In order to replace traditional detection
Method, the molecular biology method based on antigen and detection of nucleic acids become the main method of identification pulmonary branches, including polymerase connects
React (PCR), DNA sequencing and enzyme linked immune assay (ELISA).These Protocols in Molecular Biologies reach in pulmonary branches detection
Highly sensitive and high selectivity purpose, but there are still some drawbacks, such as need tip device and expensive reagent,
Tediously long and complicated step, there are also false positive results once in a while.
Surface enhanced Raman scattering (Surface-Enhanced Raman Scattering, SERS) technology, surface increase
It is potent Raman signal should to be amplified 1014~1015Times, this high detection sensitivity determines the good application of SERS
Prospect.Colloidal gold immunochromatographimethod (Immunochromatography, IC) technical operation is simple and quick, analysis result understands, easily
In judgement, it has been widely used in fields such as food quality detection, environmental monitoring and medicine.By SERS detection technique
Combine with immuno-chromatographic assay technology, while simple, quick, the easy-operating advantage of immunochromatography and SERS spirit is utilized
The advantages of sensitivity height, high specificity, and the diagnosis infected pulmonary branches in human serum is realized with the method.Operation of the present invention letter
It is single, save time, high sensitivity, high specificity；It is provided quickly for the early diagnosis of mycoplasma pneumoniae infection, it is easy, it is highly sensitive
Detection method.
Summary of the invention
The present invention is directed to the low problem of traditional colloidal gold immunochromatographimethod technology detection sensitivity, discloses SERS- and layer is immunized
The preparation method of analysis test strips, and the method and detection mycoplasma pneumoniae IgM antibody positive serum of detection people IgM are clinical in fact
The experimental method of border sample.Purpose is the detection sensitivity for improving SERS- immunochromatography and the detection of pulmonary branches positive serum sample
Rate.In order to achieve the above object, the present invention includes following detecting step:
1, the double-deck Raman molecular marks the preparation of Au@Ag nano material-antibody complex (SERS probe), comprising: Au@Ag
Nano material, Raman molecular, detection antibody；
2, antibody or antigen are fixed to the control line (C line) and detection line (T line) of nitrocellulose filter with stroke film instrument；
3, Raman molecular first the preparation of SERS- immuno-chromatographic test paper strip: is marked into Au@Ag nano material-antibody complex
(SERS probe) sprays on bonding pad, then nitrocellulose filter, bonding pad, sample pad and blotting paper is successively assembled in poly-
On ethylene PVC bottom plate, after being cut into 6 × 0.35cm specification, become a SERS- immuno-chromatographic test paper strip；
4, the chromatography of sample liquid: being added dropwise the sample liquid comprising detectable substance in the sample pad of SERS- immuno-chromatographic test paper strip,
Detectable substance and SERS probe form immune complex, and by the antigen or antibody capture on T line；
5, testing result is read: being believed using the SERS signal in Raman spectrometer test strip T line region, and according to detection
Number interpretation result.
Au@Ag nano material described in step 1 is to grow Ag shell by Au NPs surface in situ to synthesize Au@Ag core-shell nano
Material, the Raman molecular are 5,5 '-two thiobis (2- nitrobenzoic acid) { 5,5 '-dithiobis- (2-
Nitrobenzoic acid), DTNB }.
The synthesis and preparation of the double-deck Raman molecular label Au@Ag nano material described in step 1 include the following steps:
The sodium citrate solution of 1.5mL 1wt% is added in the chlorauric acid solution of the 0.01wt% of 100mL boiling, prepares the left side 35nm
Right Au NPs.DTNB is added in Au NPs solution to 10 μM of final concentration, and 8000rpm is centrifuged 8 minutes and removes extra DTNB.
0.5mL sodium citrate (1%, w/v) is added to agitating and heating in 50mL Au/DTNB NPs solution, the silver nitrate (1mM) of 1mL
It is added dropwise, stirs and boils 15 minutes.
The double-deck Raman molecular described in step 1 marks Au@Ag nano material-antibody complex (SERS probe) preparation packet
Include the following steps: the EDC/sulfo-NHS solution (1 of 500 μ L Au/DTNB@Ag/DTNB NPs solution and 20 μ L Fresh
Mg/mL it) mixes, mixed liquor shakes 15 minutes.5 μ L antibody (5mg/mL) and 500 μ L BBS buffers (10mM, pH 8.0) add
Enter into above-mentioned solution, mixture room temperature shakes 2 hours with coupled antibody.
The double-deck Raman molecular described in step 1 marks the preparation of Au@Ag nano material-antibody complex (SERS probe)
In, the SERS probe of coupling goat-anti people IgM is for detecting people IgM；The SERS probe of the coupling anti-human IgM of mouse (μ chain) is for examining
Survey the pulmonary branches specific IgM antibodies in human serum.
Double-deck Raman molecular label Au@Ag nano material-antibody complex (SERS probe) described in step 1 is stored in and contains
Have 1% BSA (w/v), 0.1%PVP (w/v), the 50mM Tris-HCl of 0.5%Tween-20 (v/v) and 10% sucrose (w/v)
(pH 8.0) melts in liquid again.
The C line and T line of nitrocellulose filter described in step 2 fix donkey anti-sheep IgG and goat-anti people IgM respectively, for examining
Survey people IgM；The C line and T line of nitrocellulose filter fix sheep anti-mouse igg and MP P1 antigen respectively, for detecting in human serum
Pulmonary branches specific IgM antibodies.
1 × PBST of sample diluting liquid in step 4 is for diluting people IgM, and 2% sodium chloride solution is for diluting clinical serum
Sample.
Sample used liquid product is 70 μ L in step 5.
Signal acquisition described in step 6,10 milliwatt of power the signal acquisition time 5 seconds, are chosen uniform along T line position
5 points of distribution carry out signal acquisition, by software in 1335cm on Raman detection instrument-1Peak value is read when wave band, is being examined
When surveying people IgM, peak value is higher to show that people's IgM concentration is higher；When detecting clinical serum sample, by calculating pulmonary branches feminine gender blood
Clear Raman peak values obtain a critical value, and the serum specimen that peak value is better than critical value is that pulmonary branches is positive, lower than the blood of critical value
Clear sample is that pulmonary branches is negative.
The present invention is the pulmonary branches sense for being used to detect using the double-deck dye marker Au@Ag as SERS probe for the first time in human serum
SERS immune detection probe is fixed on bonding pad by dye by constructing novel SERS- immunochromatography technique, sample liquid warp
Chromatography dissolution SERS probe is crossed, the active principle in sample liquid is captured by SERS probe, forms SERS probe-immune complex
And it chromatographs to T line, the antibody or antigen capture then fixed by T line.It is detected using Raman spectrometer, sample behaviour IgM
When solution, in 1335cm-1Peak value is higher illustrates that people's IgM concentration is higher at place；When sample is clinical serum, peak strength is higher than and faces
It is pulmonary branches positive serum when dividing value, is pulmonary branches negative serum when being lower than critical value.The traditional colloidal gold of remolding sensitivity of the invention
Immunochromatography is high, and detection process is simple, saves the time, can be used for clinical diagnosis.The detection of people IgM can be improved through the invention
The recall rate of sensitivity and mycoplasma pneumoniae IgM positive serum sample solves the problems, such as conventional method positive missing inspection that may be present,
Clinical treatment is aided in the maximum extent.
The characterization of embodiment 1:Au/DTNB@Ag/DTNB SERS probe.
The partial size about 44nm of the partial size of Au NPs about 35nm in the present embodiment, Au@Ag, silver-colored thickness of the shell about 4nm.
The result of embodiment 2:SERS- immuno-chromatographic test paper strip quantitative detection and analysis people IgM.
The detection sensitivity of SERS- immuno-chromatographic test paper strip double-antibody sandwich detection people IgM is 0.1ng/ in the present embodiment
mL。
Embodiment 3:SERS immuno-chromatographic test paper strip detects the result of 20 pulmonary branches IgM positive serum samples.
The recall rate of SERS- immuno-chromatographic test paper strip detection pulmonary branches positive serum sample is 100% in the present embodiment, than
ELISA high 15%.
The specificity experiments result of embodiment 4:SERS- immuno-chromatographic test paper strip detection pulmonary branches positive serum sample.
The serum that the infection of other six kinds of respiratory pathogens is had detected in the present embodiment, it is anti-without intersecting with pulmonary branches positive serum
It answers.
Detailed description of the invention
In Fig. 1, a is the synthetic route chart of Au/DTNB@Ag/DTNB SERS probe；B is that SERS- immunochromatography is quantitatively examined
Survey the schematic illustration of people IgM.
Fig. 2 is the phenogram of Au/DTNB@Ag/DTNB SERS probe in embodiment 1.
A and b is respectively the transmission electron microscope picture of the Au NPs and Au/DTNB@Ag NPs synthesized；It is Au/ that picture is embedded in b
DTNB@Ag NPs high-resolution-ration transmission electric-lens figure (thickness that mark indicates silver-colored shell)；C and d is respectively Au NPs and Au/DTNB@
The dynamic light scattering distribution map of Ag NPs.
Fig. 3 is the result of SERS- immuno-chromatographic test paper strip quantitative detection and analysis people IgM in embodiment 2.
A is the picture for detecting the SERS- immuno-chromatographic test paper strip of various concentration people IgM；B is the SERS light of corresponding detection line
Spectrum, error line represent the standard deviation of five measured values；C is people IgM in 0.1-1000ng mL-11335cm when concentration-1Locate SERS
The calibration curve of signal strength；D is the people IgM of various concentration in 1335cm-1The mapping that SERS signal intensity at displacement is formed
Image.
Fig. 4 is the result that SERS immuno-chromatographic test paper strip detects 20 pulmonary branches IgM positive serum samples in embodiment 3.
A is the picture for detecting the SERS- immuno-chromatographic test paper strip of serum specimen, and the number above test strips represents pulmonary branches spy
Anisotropic IgM male/female serum specimen number；B is the SERS signal in corresponding SERS- immuno-chromatographic test paper strip detection line, accidentally
Poor line represents the standard deviation of five measured values；C is 20 pulmonary branches specific IgM positive serum samples, 10 pulmonary branches specificity
IgM negative serum sample, serum dilution (blank), negative control, positive control and three kinds of standard items (P10, P50 and
P100) ELISA in 96 orifice plates is tested, and indicates that 3 pulmonary branches specific IgM positive serum samples do not cause at circles mark
Solution colour becomes buff；D is absorbance value of all samples at 450nm wavelength.
Fig. 5 is the specificity experiments knot that SERS- immuno-chromatographic test paper strip detects pulmonary branches positive serum sample in embodiment 4
Fruit.
A is the picture that SERS- immuno-chromatographic test paper strip detects pulmonary branches IgM serum and other respiratory disease serum；B is phase
The SERS signal in test strips detection line is answered, error line indicates the standard deviation of five measured values.
Specific embodiment
It is further elucidated above to the present invention below in conjunction with the embodiment in the present invention.Obviously, described embodiment is only
It is only a part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, the common skill in this field
Art personnel all other embodiment obtained without creative efforts belongs to the model that the present invention protects
It encloses.
Embodiment 1, the characterization of Au/DTNB@Ag/DTNB SERS probe
Experimental procedure: this time in experiment, 1.5mL is added in the chlorauric acid solution of the 0.01wt% of 100mL boiling
The sodium citrate solution of 1wt% is persistently boiled 15 minutes, is then cooled to room temperature.The AuNPs partial size that this parameter is prepared exists
35nm or so.10 μ L 10mM DTNB ethanol solutions are added in the AuNPs solution of 10mL, and mixture is stirred at room temperature 4
Hour.8000rpm is centrifuged 8 minutes and removes extra DTNB molecule, and sediment is resuspended in the deionized water of 10mL, 10 mL's
Four times of deionized water of Au NPs/DTNB solution dilute and boil.Then, 0.5mL sodium citrate (1%, w/v) is added to
It is stirred in solution.Finally, the silver nitrate (1mM) of 1mL is added dropwise in above-mentioned solution, stirs and boil 15 minutes.At this
The solution in a stage, Au NPs becomes crocus, shows the formation of Au/DTNB@Ag NPs.
Experimental result: in this experiment, for the Au NPs of average grain diameter 35nm or so as seed, Raman dyestuff DTNB is logical
Cross the surface that disulfide bond can be adsorbed on Au easily.The silver nitrate solution (10mM) of 1mL, which is added in Au seed, forms silver
Shell, the bright Ag thickness of the shell of high-resolution-ration transmission electric-lens chart of Au@Ag NPs is close to 4nm.DYNAMIC DISTRIBUTION scatter diagram also indicates that Au NPs
Average grain diameter increase 44nm or so from 35nm after being coated with Ag shell.It is specifically shown in Fig. 2.
Embodiment 2, the result of SERS- immuno-chromatographic test paper strip quantitative detection and analysis people IgM
Experimental procedure: people IgM is successively diluted to 1000ng mL-1, 100ng mL-1, 10ng mL-1, 1ng mL-1,
0.5ng mL-1With 0.1ng mL-1Totally six concentration, 1 × pBST (1 × PBS, 0.05%Tween-20) are used as blank control；
The sample liquid of 70 μ L is taken to be added sequentially to the loading area of SERS- immuno-chromatographic test paper strip, be examined after 15 minutes with Raman spectrometer
Survey the Raman signal of detection zone.Draw Raman peaks 1335cm-1The SERS signal intensity and people's IgM concentration (0.1-1000ng at place
mL-1) calibration curve between logarithm.With 1335cm-1Locate the SERS in peak value one extended area (250 50 μm of μ m) of drafting
Spectrum, step-length are 5 μm, obtain the homogeneity result of SERS signal in detection line.
Experimental result: when people's IgM concentration increases, due to the formation of many immune complexs, detection zone forms visible
Red lines.The concentration of someone IgM is higher than 10ng mL-1When, detection zone just has visual color.It is examined by detection
The specific Raman signal quantitative analysis person IgM of survey line SERS probe improves detection sensitivity, when people's IgM concentration reduces,
The SERS signal of detection line dies down, when people's IgM concentration drops to 0.1ng mL-1When, 1335cm-1Main peak at displacement is still aobvious
It writes.Therefore, the detection sensitivity of the SERS- immuno-chromatographic test paper strip based on the double-deck dye marker SERS probe is 0.1ng mL-1。
It also, is 0.1ng mL in the concentration of people IgM-1To 1000ng mL-1When have good linear relationship (R2=0.952).It is whole
The SERS signal of a mapping area is in high concentration people IgM (100ng mL-1) when signal be uniform, but in low concentration
(100-500pg mL-1) only the signal of central area be uniform.It is specifically shown in Fig. 3.
Embodiment 3, SERS immuno-chromatographic test paper strip detect the result of 20 pulmonary branches IgM positive serum samples
Experimental procedure: 20 pulmonary branches specific IgM positive clinical serum specimens and 10 pulmonary branches specific IgMs are negative clinical
Serum specimen is detected with SERS- immuno-chromatographic test paper strip.The sodium chloride solution (w/v) of 10 μ L clinical serums and 60 μ L are mixed
It after conjunction, is added dropwise on the loading pad of ready SERS- immuno-chromatographic test paper strip, testing result after 15 minutes.In order to avoid
Based on epidemic disease reaction and chromatography, pulmonary branches specific IgM positive serum sample will will form two macroscopic red detections
Line and control line, pulmonary branches specific IgM negative serum sample only form a macroscopic red control line.It detects and divides
Analysis 1335cm in SERS- immuno-chromatographic test paper strip detection line-1Raman peak intensity at displacement, by calculating 10 lungs
The average value of the SERS signal intensity of branch specific IgM negative serum obtains critical value plus three times standard deviation.SERS signal is strong
The serum specimen that degree is more than or equal to critical value is defined as pulmonary branches specific IgM positive sample, and SERS signal intensity is lower than
The serum specimen of critical value is defined as pulmonary branches specific IgM ' negative ' specimens.Using SeroMPTMIgM kit (SavyonR
Diagnose Co., Ltd, Israel), i.e., enzyme-linked immunosorbent assay (ELISA) detects identical pulmonary branches specific IgM positive blood
Clearly/negative serum sample.In ELISA experiment, MP P1 is coated with 96 orifice plates, for capturing the spy of the pulmonary branches in pulmonary branches positive serum
Anisotropic IgM antibody, the pulmonary branches specific IgM antibodies in serum are by MP P1 antigen capture, and HRP- is coupled anti-human IgM, and (detection is anti-
Body) it is captured again by pulmonary branches specific IgM antibodies, after enzyme linked immunological compound is formed, it is added tetramethyl benzidine (TMB)
Substrate, and with terminate liquid (1M H2SO4) reaction is terminated, the last one step can cause the color of solution in 96 orifice plates by pale yellow
At buff, microplate reader measures the absorbance value at 450nm wavelength and analyzes result discoloration.Positive control, P100 are (high positive
Pulmonary branches human serum), the absorbance value of P50 (middle the positive pulmonary branches human serum) and P10 (low positive pulmonary branches human serum) all answers
In the reasonable scope.According to operating instruction, the serum specimen that absorbance value is higher than P10 absorbance value is considered the pulmonary branches positive, low
It is considered pulmonary branches feminine gender in the serum specimen of P10 absorbance value.
Experimental result: 2,3,5,7,10,11,14 and No. 20 samples of positive group of SERS- immuno-chromatographic test paper strip detection group
Detection line it is high-visible, the detection lines of all negative groups are invisible.The negative SERS organized on all Samples detection lines of statistics
It is 1542.76 that signal value, which obtains critical value,.The result shows that the SERS signal intensity of pulmonary branches specific IgM positive serum sample is high
In the SERS signal intensity of pulmonary branches specific IgM negative serum sample, and it is higher than critical value.The result shows that being designed with us
SERS- immuno-chromatographic test paper strip, the recall rate of pulmonary branches specific IgM positive clinical serum specimen is 100%.ELISA experiment
In, color change caused by most of pulmonary branches specific IgM positive serum sample (except 12,15 and 18) is deeper than 10 pulmonary branches
Color change caused by specific IgM negative serum sample, and absorbance value is higher than the absorbance value of P10 standard value.As a result table
Bright, the recall rate of the pulmonary branches specific IgM positive serum sample of ELISA experiment is 85%.In conclusion SERS- immunochromatography
The recall rate that test strips detect pulmonary branches positive sample is higher than ELISA.It is specifically shown in Fig. 4.
Embodiment 4, SERS- immuno-chromatographic test paper strip detect the specificity experiments result of pulmonary branches positive serum sample
Experimental procedure: detection by other six kinds of respiratory pathogens (chlamydia pneumoniae, adenovirus, Respiratory Syncytial Virus(RSV),
Influenza A virus, influenza B virus and parainfluenza virus) infection serum, and these respiratory disease serum are
Serum reagent box is commercialized to make a definite diagnosis.After the mixing of the sodium chloride solution (w/v) of 10 μ L clinical serums and 60 μ L, it is added dropwise to
On the loading pad of ready SERS- immuno-chromatographic test paper strip, T line Raman signal is detected after 15 minutes.
Experimental result: there is two red tapes, appearance when detecting other six kinds of serum in the test strips of detection pulmonary branches positive serum
One red control line.SERS testing result also indicates that these serum can only generate faint Raman signal in detection line,
And pulmonary branches positive serum generates strong signal.Also, the SERS signal intensity that other six kinds of serum generate is lower than critical value.Also
It is to say, detects the test strips and other six kinds of respiratory disease serum and no cross reaction of pulmonary branches, be pulmonary branches negative serum.
The result shows that our SERS- immuno-chromatographic test paper strip is when detecting pulmonary branches specific IgM positive serum with good special
Property and selectivity.It is specifically shown in Fig. 5.
Claims (9)
1. a kind of be quickly characterized in that with the SERS- immunochromatography detection method of highly sensitive detection mycoplasma pneumoniae infection, include
Following detecting step:
(1) the double-deck Raman molecular marks the preparation of Au@Ag nano material-antibody complex (SERS probe), comprising: Au@Ag receives
Rice material, Raman molecular, detection antibody；
(2) antibody or antigen are fixed to the control line (C line) and detection line (T line) of nitrocellulose filter with stroke film instrument；
(3) Raman molecular first the preparation of SERS- immuno-chromatographic test paper strip: is marked into Au@Ag nano material-antibody complex
(SERS probe) sprays on bonding pad, then nitrocellulose filter, bonding pad, sample pad and blotting paper are successively assembled in poly- second
On alkene PVC bottom plate, after being cut into 6 × 0.35cm specification, become a SERS- immuno-chromatographic test paper strip；
(4) sample liquid comprising detectable substance, inspection the chromatography of sample liquid: are added dropwise in the sample pad of SERS- immuno-chromatographic test paper strip
It surveys object and SERS probe forms immune complex, and by the antigen or antibody capture on T line；
(5) testing result is read: using the SERS signal in Raman spectrometer test strip T line region, and according to detection signal
Interpretation result.
2. a kind of quickly SERS- immunochromatography inspection with highly sensitive detection mycoplasma pneumoniae infection according to claim 1
Survey method, it is characterised in that: Au@Ag nano material described in step (1) is that the synthesis of Ag shell is grown by Au NPs surface in situ
Au@Ag core-shell nano material, the Raman molecular are 5,5 '-two thiobis (2- nitrobenzoic acid) { 5,5 '-dithiobis-
(2-nitrobenzoic acid), DTNB }.
3. a kind of quickly SERS- immunochromatography with highly sensitive detection mycoplasma pneumoniae infection according to claims 1 and 2
Detection method, it is characterised in that: the synthesis of the double-deck Raman molecular label Au@Ag nano material and preparation packet described in step (1)
It includes the following steps: the sodium citrate solution of 1.5mL 1wt% being added in the chlorauric acid solution of the 0.01wt% of 100mL boiling,
Prepare the Au NPs of 35nm or so.DTNB is added in Au NPs solution to 10 μM of final concentration, and 8000rpm is centrifuged 8 minutes and removes
Remove extra DTNB.0.5mL sodium citrate (1%, w/v) is added to agitating and heating in 50mL Au/DTNB NPs solution, 1mL's
Silver nitrate (1mM) is added dropwise, and stirs and boils 15 minutes.
4. a kind of quickly SERS- immunochromatography inspection with highly sensitive detection mycoplasma pneumoniae infection according to claim 1
Survey method, it is characterised in that: the double-deck Raman molecular described in step (1) marks Au@Ag nano material-antibody complex (SERS
Probe) preparation include the following steps: the EDC/ of 500 μ L Au/DTNB@Ag/DTNB NPs solution and 20 μ L Fresh
Sulfo-NHS solution (1mg/mL) mixing, mixed liquor shake 15 minutes.5 μ L antibody (5mg/mL) and 500 μ L BBS buffers
(10mM, pH8.0) is added in above-mentioned solution, and mixture room temperature shakes 2 hours with coupled antibody.
5. SERS- immunochromatography according to claim 1 is quickly built with the method for highly sensitive detection mycoplasma pneumoniae infection
It is vertical, it is characterised in that: the double-deck Raman molecular described in step (1) marks Au@Ag nano material-antibody complex (SERS probe)
Preparation in, the SERS probe of coupling goat-anti people IgM is for detecting people IgM；The SERS probe for being coupled the anti-human IgM of mouse (μ chain) is used
Pulmonary branches specific IgM antibodies in detection human serum.
6. a kind of quickly SERS- immunochromatography inspection with highly sensitive detection mycoplasma pneumoniae infection according to claim 1
Survey method, it is characterised in that: the double-deck Raman molecular described in step (1) marks Au@Ag nano material-antibody complex (SERS
Probe) it is stored in containing 1%BSA (w/v), 0.1%PVP (w/v), 0.5%Tween-20 (v/v) and 10% sucrose (w/v)
50mM Tris-HCl (pH8.0) melts in liquid again.
7. a kind of quickly SERS- immunochromatography inspection with highly sensitive detection mycoplasma pneumoniae infection according to claim 1
Survey method, it is characterised in that: the C line of nitrocellulose filter and T line fix the anti-sheep IgG of donkey and goat-anti people respectively in step (2)
IgM, for the people IgM in test sample；The C line and T line of nitrocellulose filter fix sheep anti-mouse igg and MP P1 antigen respectively,
For detecting the pulmonary branches specific IgM antibodies in human serum.
8. a kind of quickly SERS- immunochromatography inspection with highly sensitive detection mycoplasma pneumoniae infection according to claim 1
Survey method, it is characterised in that: for diluting people IgM, 2% sodium chloride solution is used for 1 × PBST of sample diluting liquid in step (4)
Dilute clinical serum sample.
9. a kind of quickly SERS- immunochromatography inspection with highly sensitive detection mycoplasma pneumoniae infection according to claim 1
Survey method, it is characterised in that: signal acquisition described in step (5), 10 milliwatt of power, the signal acquisition time 5 seconds, along T line
Position chooses equally distributed 5 points and carries out signal acquisition, by software in 1335cm on detecting instrument-1Peak is read when wave band
Value, when detecting people IgM, peak value is higher to show that people's IgM concentration is higher；When detecting clinical serum sample, by calculating pulmonary branches
The Raman peak values of negative serum obtain a critical value, and the serum specimen that peak value is better than critical value is that pulmonary branches is positive, lower than critical
The serum specimen of value is that pulmonary branches is negative.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110531071A (en) * | 2019-09-03 | 2019-12-03 | 上海交通大学 | A kind of preparation and application of highly sensitive Sidestream chromatography immunity test strip |
| CN111175517A (en) * | 2020-01-07 | 2020-05-19 | 中国人民解放军军事科学院军事医学研究院 | Immunochromatography test paper for detecting radiation marker, preparation method and application thereof, and kit |
| CN111537493A (en) * | 2020-05-07 | 2020-08-14 | 中国人民解放军军事科学院军事医学研究院 | Method for detecting rotavirus by combining surface enhanced Raman scattering with immunochromatography |
| CN111650370A (en) * | 2020-08-10 | 2020-09-11 | 苏州微湃医疗科技有限公司 | Method and device for detecting novel coronavirus SARS-CoV-2 |
| CN112505323A (en) * | 2021-02-03 | 2021-03-16 | 南京市产品质量监督检验院 | Raman immunochromatographic test strip for detecting lead ions and preparation method and application thereof |
Citations (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20090082220A1 (en) * | 2005-03-15 | 2009-03-26 | Krause Duncan C | Surface enhanced Raman spectroscopy (SERS) systems for the detection of bacteria and methods of use thereof |
| TW200932913A (en) * | 2007-11-26 | 2009-08-01 | Univ Nat Yang Ming | Method for identifying microorganism or detecting its morphology alteration using surface enhanced raman scattering (SERS) |
| CN102507932A (en) * | 2011-12-02 | 2012-06-20 | 无锡博慧斯生物医药科技有限公司 | IgM (immunoglobulin M) antibody detection test strip |
| CN102928587A (en) * | 2012-11-16 | 2013-02-13 | 南京凯基生物科技发展有限公司 | Colloidal gold method detection test strip and reagent kit for IgM and IgG antibodies of mycoplasma pneumoniae and preparation method of reagent kit |
| US20130157254A1 (en) * | 2011-12-16 | 2013-06-20 | Real-Time Analyzers, Inc. | Method and apparatus for two-step surface-enhanced raman spectroscopy |
| CN104007264A (en) * | 2014-05-27 | 2014-08-27 | 江南大学 | Preparation method and detection method of high-sensitivity paper capable of rapidly detecting algal toxin through Raman visualization |
| CN204666637U (en) * | 2015-05-29 | 2015-09-23 | 珠海丽珠试剂股份有限公司 | Mycoplasma pneumoniae IgM, IgG, IgA tri-test card |
| CN105259345A (en) * | 2015-11-19 | 2016-01-20 | 国家纳米科学中心 | Colloidal gold test strip and test strip card for detecting IgM antibody, and preparation and detection method |
| CN105259158A (en) * | 2015-11-13 | 2016-01-20 | 暨南大学 | Surface enhanced Raman scattering immunochromatography test paper strip and preparation method and application |
| CN105759034A (en) * | 2016-04-01 | 2016-07-13 | 山东德诺生物科技有限公司 | Mycoplasma pneumoniae detection kit |
| CN106053802A (en) * | 2016-06-03 | 2016-10-26 | 无锡市人民医院 | Double-labeled nano time-resolved fluorescence immunochromatographic quantitative test paper for mycoplasma pneumoniae antibodies and preparation method of test paper |
| CN106153928A (en) * | 2016-07-06 | 2016-11-23 | 广东工业大学 | A kind of bisphenol-A immunity test strip containing bisphenol-A immunological probe and application thereof |
| KR20170036630A (en) * | 2015-09-23 | 2017-04-03 | 한양대학교 에리카산학협력단 | High-sensitive lateral flow immunoassay strip based on a surface-enhanced raman scattering and method using the same |
| CN107101991A (en) * | 2017-05-31 | 2017-08-29 | 东南大学 | A kind of high sensitivity multiplex detection chromatograph test strip |
-
2018
- 2018-02-02 CN CN201810104303.6A patent/CN110133255A/en active Pending
Patent Citations (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20090082220A1 (en) * | 2005-03-15 | 2009-03-26 | Krause Duncan C | Surface enhanced Raman spectroscopy (SERS) systems for the detection of bacteria and methods of use thereof |
| TW200932913A (en) * | 2007-11-26 | 2009-08-01 | Univ Nat Yang Ming | Method for identifying microorganism or detecting its morphology alteration using surface enhanced raman scattering (SERS) |
| CN102507932A (en) * | 2011-12-02 | 2012-06-20 | 无锡博慧斯生物医药科技有限公司 | IgM (immunoglobulin M) antibody detection test strip |
| US20130157254A1 (en) * | 2011-12-16 | 2013-06-20 | Real-Time Analyzers, Inc. | Method and apparatus for two-step surface-enhanced raman spectroscopy |
| CN102928587A (en) * | 2012-11-16 | 2013-02-13 | 南京凯基生物科技发展有限公司 | Colloidal gold method detection test strip and reagent kit for IgM and IgG antibodies of mycoplasma pneumoniae and preparation method of reagent kit |
| CN104007264A (en) * | 2014-05-27 | 2014-08-27 | 江南大学 | Preparation method and detection method of high-sensitivity paper capable of rapidly detecting algal toxin through Raman visualization |
| CN204666637U (en) * | 2015-05-29 | 2015-09-23 | 珠海丽珠试剂股份有限公司 | Mycoplasma pneumoniae IgM, IgG, IgA tri-test card |
| KR20170036630A (en) * | 2015-09-23 | 2017-04-03 | 한양대학교 에리카산학협력단 | High-sensitive lateral flow immunoassay strip based on a surface-enhanced raman scattering and method using the same |
| CN105259158A (en) * | 2015-11-13 | 2016-01-20 | 暨南大学 | Surface enhanced Raman scattering immunochromatography test paper strip and preparation method and application |
| CN105259345A (en) * | 2015-11-19 | 2016-01-20 | 国家纳米科学中心 | Colloidal gold test strip and test strip card for detecting IgM antibody, and preparation and detection method |
| CN105759034A (en) * | 2016-04-01 | 2016-07-13 | 山东德诺生物科技有限公司 | Mycoplasma pneumoniae detection kit |
| CN106053802A (en) * | 2016-06-03 | 2016-10-26 | 无锡市人民医院 | Double-labeled nano time-resolved fluorescence immunochromatographic quantitative test paper for mycoplasma pneumoniae antibodies and preparation method of test paper |
| CN106153928A (en) * | 2016-07-06 | 2016-11-23 | 广东工业大学 | A kind of bisphenol-A immunity test strip containing bisphenol-A immunological probe and application thereof |
| CN107101991A (en) * | 2017-05-31 | 2017-08-29 | 东南大学 | A kind of high sensitivity multiplex detection chromatograph test strip |
Non-Patent Citations (3)
| Title |
|---|
| JUNFENG WANG ET AL.: "Magnetically Assisted Surface-Enhanced Raman Spectroscopy for the Detection of Staphylococcus aureus Based on Aptamer Recognition", 《APPLIED MATERIALS & INERFACES》 * |
| 张美玲等: "商品化酶免疫检测试剂盒在支原体肺炎血清学诊断中的应用", 《微生物学免疫学进展》 * |
| 贾潇潇等: "表面增强拉曼光谱技术在微生物鉴定中的应用进展", 《生物工程学报》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110531071A (en) * | 2019-09-03 | 2019-12-03 | 上海交通大学 | A kind of preparation and application of highly sensitive Sidestream chromatography immunity test strip |
| CN110531071B (en) * | 2019-09-03 | 2022-03-15 | 上海交通大学 | Preparation and application of high-sensitivity lateral flow chromatography immunoassay test paper |
| CN111175517A (en) * | 2020-01-07 | 2020-05-19 | 中国人民解放军军事科学院军事医学研究院 | Immunochromatography test paper for detecting radiation marker, preparation method and application thereof, and kit |
| CN111175517B (en) * | 2020-01-07 | 2023-08-08 | 中国人民解放军军事科学院军事医学研究院 | Immunochromatography test paper for detecting radiation markers, preparation method and application thereof and kit |
| CN111537493A (en) * | 2020-05-07 | 2020-08-14 | 中国人民解放军军事科学院军事医学研究院 | Method for detecting rotavirus by combining surface enhanced Raman scattering with immunochromatography |
| CN111650370A (en) * | 2020-08-10 | 2020-09-11 | 苏州微湃医疗科技有限公司 | Method and device for detecting novel coronavirus SARS-CoV-2 |
| CN112505323A (en) * | 2021-02-03 | 2021-03-16 | 南京市产品质量监督检验院 | Raman immunochromatographic test strip for detecting lead ions and preparation method and application thereof |
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