CN106645090A - Novel SERS substrate-based method for quantitatively testing pathogenic bacteria - Google Patents
Novel SERS substrate-based method for quantitatively testing pathogenic bacteria Download PDFInfo
- Publication number
- CN106645090A CN106645090A CN201710019969.7A CN201710019969A CN106645090A CN 106645090 A CN106645090 A CN 106645090A CN 201710019969 A CN201710019969 A CN 201710019969A CN 106645090 A CN106645090 A CN 106645090A
- Authority
- CN
- China
- Prior art keywords
- pathogenic bacteria
- quantitative determination
- signal
- raman
- method based
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- 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
Abstract
The invention discloses a novel SERS substrate-based method for quantitatively testing pathogenic bacteria, and belongs to the technical field of biological detection. Silicon dioxide treated by an immunospectral marker is coated with gold nanoparticles for quantitative test of the pathogenic bacteria. Raman spectra signal monitoring data are more stable and reliable and can be greatly repeated. The method is good in test stability, high in sensitivity, high in specificity, the detection process is simple, fast, accurate and quantitative, and a wide detection interval and a low detection limit are obtained. The method is simple in operation, an SERS immune signal probe and a capture probe can be prepared in advance, the capture probe and the signal probe only need to be sequentially added to a to-be-detected sample for reaction during operation and the test is carried out at once through magnetic separation after the reaction is completed: the collection time is 30s, and then the concentration is immediately read from a standard curve, thereby achieving fast quantitatively testing. The method can meet the requirements of food safety and environment monitoring departments, and has wide practicability.
Description
Technical field
The invention belongs to technical field of biological, and in particular to one kind is based on new SERS substrates quantitative determination pathogenic bacteria
Method.
Background technology
In recent years, the food origin disease that microorganism causes has become world today's property public health focus.In food-borne
In pathogenic bacteria, Escherichia coli (Escherichia coli) O157:H7 is most dangerous one, is also generally acknowledged in the world at present
One of three big topmost food-borne pathogens.Ehec infection O157:The clinical symptoms of H7 include diarrhoea, hemorrhagic enteritis
(HC), hemolytic uremic syndrome (HUS) and thrombotic thrombocytopenic purpura etc..Enteropathogenic E. Coli can pass through
The approach such as pollution drinking-water, food cause Disease code, serious harm health.
Escherichia coli O 157 is detected at present:The goldstandard of H7 is that tradition separates identification method, and the method need to increase bacterium, choosing through pre-
Selecting property increase bacterium, be separately cultured, Physiology and biochemistry identification and the step such as Serotype Identification, whole process loaded down with trivial details time-consuming (3～7 days), and
Whole process needs professional to operate, therefore the method is only used for law enforcement agency and the cause of poisoning is analyzed,
And market security status of food is estimated；Immunology especially immune chromatography method, is not required to complex instrument equipment, detects
Time is comparatively fast easy to operate, but current such method totality detection sensitivity is low, and (bacterial concentration need to reach 105～106CFU/mL),
Therefore for authentic sample detection, the longer increasing bacterium time is generally required.Therefore, quick, sensitive, stable detection method is set up
Detection to food-borne pathogens has great importance.
SERS (Surface-enhanced Raman scattering, SERS) labelling technique is one
Novel spectral marker method is planted, he strengthens the mark adsorbed on its surface using the nano-particle of the precious metals such as gold, silver
The Raman signal of molecule, and as mark tracer signal.SERS labelling techniques have the following advantages, Raman spectrum spectral peak
Narrow, Raman scattering is hardly affected by water, SERS signal is strong, low background, by photobleaching do not affected and be not susceptible to substantially
Quenching phenomenon, the sensitivity with superelevation and selectivity etc..The nano-probe grown up based on SERS technologies biology into
The numerous areas such as picture, protein detection, tumour identification show considerable application prospect.
The content of the invention
In order to overcome the shortcoming and deficiency of prior art, it is an object of the invention to provide a kind of be based on new SERS substrates
The method of quantitative determination pathogenic bacteria.The present invention is quick using the integrated immunomagnetic ca pture technology of Silica-coated gold nano microballoon
Quantitative determination pathogenic bacteria are (such as：Escherichia coli O 157:H7(E.coli O157:H7)).
The method of the present invention has that good stability, sensitivity height, high specificity, detection process be simple and quick, accurate quantitative analysis
The features such as.
The purpose of the present invention is achieved through the following technical solutions：
A kind of method based on new SERS substrates quantitative determination pathogenic bacteria, comprises the steps：
(1) Raman signal molecule is attached into gold nano grain surface, subsequently tetraethyl orthosilicate is reduced by ammoniacal liquor
(TEOS) method forms thin layer of silicon dioxide on the gold nano grain surface for combining Raman signal molecule, to ensure that Raman is believed
The stability of number molecule；
(2) on the silicon layer for being prepared sulfydryl modification in step (1) by silane coupler, then will be caused by intermediate
Germ antibody modification, with bovine serum albumin (BSA) closing residual activity site, is prepared into Raman signal spy in the particle surface
Pin；
(3) magnetic bead of carboxyl is modified with using activator activating surface, bacteria antibody is subsequently added and BSA is prepared into magnetic
Property nano particle, i.e. capture probe；
(4) will be resuspended with water after the centrifugation of cultured pathogenic bacteria, and it is diluted to one group of standard sample with concentration gradient
Liquid；
(5) capture probe in the step of taking same volume (3), is separately added into the standard sample of step (4) variable concentrations
Liquid, is incubated in room temperature, and supernatant is removed after Magneto separate；The Raman signal probe in step (2) is added again, forms " capture spy
" sandwich " structure of pin-pathogenic bacteria-signal probe "；After Magneto separate, letter is carried out to remaining Raman signal probe in supernatant
Number collection；
(6) calibration curve is set up with the corresponding relation of Raman signal intensity according to the bacteria concentration that causes a disease, so as to believe using Raman
Number quantitative determination is carried out to pathogenic bacteria；
In the method for above-mentioned detection pathogenic bacteria：
Described pathogenic bacteria are preferably Escherichia coli (E.coli) O157:H7；
Bacteria antibody described in step (2), (3) is preferably Escherichia coli (E.coli) O157:H7 antibody；
Raman signal molecule described in step (1) is preferably 4, and 4 '-bipyridyl, the molecule one end is connected to colloid golden watch
Face, the other end is connected with antibody, is difunctional mark molecule；
The average grain diameter of the gold nano grain described in step (1) is preferably 25nm～35nm；Preferably 30nm；
The method of the ammoniacal liquor reduction tetraethyl orthosilicate described in step (1) is carried out in the aqueous solution of isopropanol；
Silane coupler described in step (2) is (3- mercaptopropyis) trimethoxy silane (MPTMS), is made by being coupled
With by sulfydryl modification on silicon layer；
Intermediate described in step (2) be thio-N- succinimidos 4- (maleimidomehyl) hexamethylenes of 3--
1- carboxylic acid sodium salts (sulfo-SMCC), wherein one end maleimide are connected on the sulfydryl of silicon layer, and other end succinimide is used
To connect bacteria antibody；
The concentration of the bacteria antibody described in step (2) is preferably 0.5～1mg/mL；More preferably 0.5mg/mL.
Bovine serum albumin (BSA) described in step (2) for mass percent 2.5% BSA, it is and resuspended
0.002mol/L borate buffer solutions (BB) volume ratio (pH=8.2) is preferably 1：50；
Surface modification described in step (3) has the magnetic bead of carboxyl, and its particle diameter is preferably 100～200nm；Preferably surface
It is modified with the ferroso-ferric oxide of carboxyl；
Activator described in step (3) is preferably 1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides
And N-hydroxy-succinamide (NHS) (EDC)；
Pathogenic bacteria incubation described in step (4) is as follows：Frozen bacterial classification is placed in 37 DEG C of environment first
Activation 30min, is then added to the bacterial classification for having activated in sterilized LB culture mediums, in being placed on constant incubator
(120rpm, 37 DEG C) vibrates 10h；
Concentration gradient described in step (4) is 0,10,103、105、107、109CFU/mL, wherein 0CFU/mL are blank
Control；
The time being incubated in room temperature described in step (5) is preferably 1～2h；More preferably 1h；
The mixed volume ratio of " sandwich " structure (capture probe-pathogenic bacteria-signal probe) described in step (5) is
3.5～4.5：1：4.5～5.5；Preferably 4：1：5；When using the ratio when using magnitude relation, LDL and background signal
Size is suitable；
Magneto separate described in step (5) to be referred to and separate magnetic conjugate and supernatant using magnetic field physical action, compares
The step of traditional cleaning solid phase substrate makes a return journey removal of residue, the method more gradual change, efficiently；
Signal described in step (5) is measured by micro-Raman spectroscopy：The behaviour of described micro-Raman spectroscopy
As condition be preferably excitation source be wavelength be 632.8nm He-Ne laser instruments, reach sample laser power be 1mW, signal
Acquisition time is 10～60s；
Signal described in step (5) is the Characteristic Raman spectral peak (1612cm for choosing Raman signal molecule-1) as quantitative
Peak, and with pathogenic bacteria concentration to the spectral peak spectral intensity mapping, and calibration curve is drawn with this；
The mechanism of the present invention：In the case where acquisition time is certain, for same Raman signal molecule, signal strength signal intensity with
The concentration of Raman signal molecule is proportionate；Quantitative determination is exactly realized on this basis.
The present invention has the following advantages and effect relative to prior art：
(1) the Silica-coated gold nano grain of immune spectral marker process is used for pathogenic bacteria (such as by the present invention：
E.coli O157:H7 quantitative determination), the concentration of the signal strength signal intensity of Raman molecular and test substance is connected, similar to
Langbobier law, so as to realize to pathogenic bacteria (such as：E.coli O157:H7 quantitative determination)；
(2) the Silica-coated gold nano grain that the present invention is adopted is new Raman substrate, and first by 4,4 '-connection pyrrole
Pyridine is wrapped in silicon layer, to ensure stability of the Raman signal molecule in subsequent reactions；
(3) the immune signal probe that the present invention is prepared based on Silica-coated gold nano grain, it is naked compared to traditional
Au probe, stability is greatly promoted, and stably can preserve at least 50h under the conditions of 4 DEG C；
(4) present invention need not be by pathogenic bacteria (such as：E.coli O157:H7) elute from immunomagnetic beads, improve and catch
Efficiency is obtained, the coefficient of variation is little during quantitative determination；Reduce workload and living contaminants probability；
(5) present invention is by detecting that the raman spectral signal of solution obtains corresponding result, compared to traditional detection
The method of solid phase substrate, the raman spectral signal Monitoring Data in solution is more reliable and more stable and can repeat in a large number；
(6) present invention detection good stability, detection sensitivity is high, has obtained a wide detection interval (10～109CFU/
) and low test limit (10CFU/mL) mL；
(7) present invention is simple to operate, and SERS immune signals probe can be ready in advance with capture probe, during operation only
Capture probe and signal probe need to be added sequentially to be reacted in testing sample, reaction can be entered at once after terminating by Magneto separate
Row detection：Acquisition time 30s, then from calibration curve reads concentration, so as to realize Quantitative detection at once；This can expire
Sufficient food security and the requirement of environmental monitoring department, with wide applicability.
Description of the drawings
Fig. 1 is Quantitative detection E.coli O157:The method flow schematic diagram of H7.
Fig. 2 is the transmission electron microscope picture of the gold nano grain and Silica-coated gold nano grain for preparing.
Fig. 3 is to use variable concentrations E.coli O157:The Raman emission spectrum figure that H7 standard liquids are obtained.
Fig. 4 is E.coli O157 in standard liquid:The curve map of H7 detections；Wherein, abscissa represents E.coli O157:
The concentration (abscissa is the log10 of concentration) of H7, ordinate represents E.coli O157 in water body:The Raman signal intensity of H7.
Fig. 5 is the signal probe prepared based on Silica-coated gold nano grain and the signal probe prepared based on naked gold
The contrast of stability.
Fig. 6 is E.coli O157:The Raman signal intensity figure of H7 specific detections.
Specific embodiment
With reference to embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention are not limited
In this.
In example below, the experimental technique of unreceipted actual conditions and environment, generally according to normal condition, or manufactory
Condition proposed by business.DP is Raman signal 4,4 '-bipyridyl of molecule in the present invention；BSA is bovine serum albumin；BB represents boric acid
Salt buffer；PBS represents phosphate buffer；EDC represents the imido hydrochloride of 1- (3- dimethylamino-propyls) -3- ethyls carbon two；
NHS represents N-hydroxy-succinamide；APTES represents 3- aminopropyl triethoxysilanes；TEOS represents tetraethyl orthosilicate；
MPTMS represents (3- mercaptopropyis) trimethoxy silane；Sulfo-SMCC represents the thio-N- succinimidos 4- (Malaysias of 3-
Acid imide methyl) hexamethylene -1- carboxylic acid sodium salts.
Quantitative detection E.coli O157:The method flow schematic diagram of H7, as shown in Figure 1.
Embodiment 1
(1), the preparation of gold nano grain
It is being stirred continuously the lower gold chloride (HAuCl by 100mL 1mM4) solution be heated to boiling, be subsequently adding 6mL
The trisodium citrate aqueous solution of 38.8mM.Now the color change of solution is：Yellowish-colourless-black-purple-peony, waits molten
Liquid becomes laking and continues to be heated to reflux 15～20min.Normal temperature is finally cooled to, the gold nano grain of 30nm is prepared (such as
Fig. 2 C), Fig. 2A is the gold nano grain obtained under ideal conditions.
(2), the preparation of Silica-coated gold nano grain
1mL gold nano grains (concentration is about 1mmol/L) are separately added into in 6 1.5mL EP pipes, are then sequentially added
The 4 of 30 μ L 0.01mM, 4 '-bipyridyl solution reacts 10min, 8000rpm centrifugation 10min after fully mixing, remove supernatant,
It is resuspended with 1mL tri-distilled waters respectively.Then the colloid in 6 EP pipes is fully transferred in 25mL serum bottles, in the case where being stirred continuously
Add 10 μ L 2.7%NaSiO in serum bottle successively3Solution (reaction 10min) (being purchased from Sigma Reagent Companies) and 12 μ L
1mM APTES solution (reaction 10min) (being purchased from Sigma Reagent Companies), reaction is split in 6 EP pipes after terminating, 8000rpm
Centrifugation 10min, removes supernatant, and tri-distilled water is resuspended；Again the mixed liquor in 6 EP pipes is added dropwise into 6mL isopropanols water-soluble
In liquid (3mL isopropanol+3mL tri-distilled waters), after stirring 5min, add 200 μ L ammoniacal liquor (mixing 5min) (public purchased from Sigma reagents
Department), in finally 300 μ L TEOS (being purchased from Sigma Reagent Companies) being added dropwise into above-mentioned mixed liquor, stirring reaction 3h, reaction
Terminate；6000rpm is centrifuged 15min, and tri-distilled water is resuspended, finally obtains the gold nano grain (such as Fig. 2 D) of Silica-coated, schemes
2B is the gold nano grain of the Silica-coated synthesized under ideal conditions.
(3), the preparation of Raman signal probe
Add 6 μ L 0.1mM MPTMS (public purchased from Sigma reagents in the gold nano grain of 1mL Silica-coateds
Department), reaction 30min is fully mixed, then using centrifuge 15min, rotating speed is 6000rpm.Remove supernatant after centrifugation,
It is resuspended with 1mL BB buffer solutions；3 μ L 0.1mM Sulfo-SMCC are subsequently added, after reaction 30min, 5 μ L 0.5mg/mL is added
rabbit anti-E.coli O157:H7 (i.e. E.coli O157:H7 antibody) (it is purchased from the limited public affairs of Beijing Bo Aosen biotechnologys
Department), 2h is incubated under room temperature, 10min is centrifuged with 8000rpm, remove supernatant, it is resuspended with BB buffer solutions；It is eventually adding 30 μ L
2.5%BSA, reacts 1h, and supernatant is removed in centrifugation (8000rpm, 10min), the letter that finally give resuspended standby with PBS
Number probe is stored under 4 DEG C of environment.
(4), the preparation of capture probe
Taking 100 μ L surface modifications has the ferroso-ferric oxide Fe of carboxyl3O4, purchased from Aladdin reagent (Shanghai) Co., Ltd., use
PBS twice, is finally settled to 1mL with PBS.Then EDC (4mg/mL) and NHS (1mg/mL) is prepared, first to above-mentioned magnetic
Pearl solution adds the EDC solution of 30 μ L, and the NHS solution of same volume is added after 10min, activates 30min.Then PBS is used
Magnetic bead is twice, resuspended with PBS afterwards, is subsequently added 5 μ L 0.5mg/mL rabbit anti-E.coli O157:H7, reacts 2h
Wash removal Excess antibody with PBS afterwards, add the BSA (2.5%) of 30 μ L, close unconjugated site.After 1h, PBS is used
Above-mentioned magnetic bead is twice, resuspended standby, obtains capture probe.
(5), the preparation of standard sample solution
Select the E.coli O157 of 6 concentration:H7 standard liquids, respectively 0,10,103、105、107、109CFU/mL,
Wherein 0CFU/mL is blank.
(6), " sandwich " structure is prepared and E.coli O157:The detection of H7
Take the standard sample solution mixing prepared in the capture probe and 20 μ L steps (5) prepared in 80 μ L steps (4)
1h, is specifically bound using the antibody on capture probe and Escherichia coli, forms the two-layer knot of " capture probe-Escherichia coli "
Structure；The Raman signal probe prepared in 100 μ L steps (3) is subsequently added, after reaction 2h, " capture probe-large intestine bar is defined
" sandwich " structure of bacterium-signal probe "；With E.coli O157:The increase of H7 concentration, " sandwich " structure has difference
The increase of degree, the Raman signal concentration and probe concentration remained in the supernatant after Magneto separate also has different degrees of reduction.
(7), the measurement of Raman signal
Substrate is separated using the magnetic fields of magnet, retains supernatant.With Japanese Nippon Optical System companies
Micro-Raman spectroscopy, carry out signals collecting to the Raman signal probe in supernatant, it is 632.8nm that excitation source is wavelength
He-Ne laser instruments, reach sample laser power be 1mW, the signal collection time be 30s.Signals collecting passes through after finishing
Origin softwares carry out Baseline Survey to data, obtain clear and intuitive SERS spectra figure (such as Fig. 3).
It can be clearly seen that with E.coli O157 in sample from Fig. 4:The raising of H7 concentration, the SERS letters of collection
Number it is gradually lowered, both sides relation meets Y=1489.24-80.74X, shows that in this interval effectively quantitative point can be carried out
Analysis.
Embodiment 2
Gold nano grain solution obtained in 1mL is taken, in being put into EP pipes, the 4 of 30 μ L 0.01mM is added, 4 '-bipyridyl is molten
Liquid, after room temperature reaction 10min, 8000rpm centrifugations (10min) removes supernatant, resuspended with BB buffer solutions, adds 5 μ L
0.5mg/mL rabbit anti-E.coli O157:H7, after incubation 2h, centrifugation (8000rpm, 10min) goes supernatant, BB to delay
Rush liquid resuspended, add 30 μ L 2.5%BSA, closing uncombined site is centrifuged (8000rpm, 10min) and removes supernatant after 1h,
It is resuspended with PBS, prepare the signal probe of naked gold.It is placed into phase with the signal probe in the step of embodiment 1 (3)
The same time, and measure the Raman signal intensity (such as Fig. 5) of each comfortable same time.Learn from Fig. 5, preliminary research shows this
Plant new SERS substrates and there is following advantage：1) silica silicon layer can ensure that gold nano grain not by extraneous electrochemical conditions
Interference；2) 4,4 '-bipyridyl being wrapped in silicon layer can prevent mark molecule from falling off in subsequent reactions；3) dioxy
SiClx silicon layer can prevent antibody that competitive reaction occurs with mark molecule.
Embodiment 3
The water body example of Guangzhou Section of Pearl River is taken, through filtering, being centrifuged, and dilutes 40 times to eliminate matrix impact with tri-distilled water
(E.coli O157 in the water body dilution of the concentration known:The concentration of H7 is respectively 102、104、106、108CFU/mL).According to
The raman spectral signal intensity obtained in the actual water body example, the calibration curve from Fig. 4 reads corresponding E.coli
O157:The concentration of H7, is multiplied by corresponding extension rate and is E.coli O157 in testing sample:The actual concentrations of H7.According to reality
Apply the concentration of the actual water body example that the linear equation in example 1 draws as shown in Table 1 below.
E.coli O157 in the actual water body example of table 1:H7 concentration and the E.coli measured using the present embodiment method
O157:H7 concentration
| Concentration known (CFU/mL) | Measured concentration (CFU/mL) | The rate of recovery (%) | Relative standard deviation (%) |
| 1×102 | 1×102.06 | 114.82 | 3.77 |
| 1×104 | 1×103.97 | 93.33 | 4.54 |
| 1×106 | 1×106.01 | 102.33 | 12.94 |
| 1×108 | 1×108.07 | 117.49 | 3.69 |
Embodiment 4
In order to illustrate the specificity of the present invention, the Escherichia coli from Guangzhou Disease Prevention-Control Center are prepared for respectively
137 (E.137), Escherichia coli 108G (E.108G), Escherichia coli 974 (E.974), Escherichia coli 1006 (E.1006) are golden yellow
Color staphylococcus (Staphylococcus aureus) (SA), salmonella (Salmonella enteriditis) (SE), Lee
This special bacterium (Listeria monocytogenes) (Lm) standard liquid, specific implementation step reference example 1；The signal strength signal intensity for obtaining
Fig. 6 is can refer to, illustrates that the present invention has very strong specificity.
Above-described embodiment is the present invention preferably embodiment, but embodiments of the present invention not by above-described embodiment
Limit, other any Spirit Essences without departing from the present invention and the change, modification, replacement made under principle, combine, simplification,
Equivalent substitute mode is should be, is included within protection scope of the present invention.
Claims (10)
1. a kind of method based on new SERS substrates quantitative determination pathogenic bacteria, it is characterised in that comprise the steps：
(1) Raman signal molecule is attached into gold nano grain surface, the method for subsequently reducing tetraethyl orthosilicate by ammoniacal liquor exists
The gold nano grain surface for combining Raman signal molecule forms thin layer of silicon dioxide；
(2) on the silicon layer for being prepared sulfydryl modification in step (1) by silane coupler, then by intermediate by pathogenic bacteria
Antibody modification, with BSA closing residual activities site, is prepared into Raman signal probe in the particle surface；
(3) magnetic bead of carboxyl is modified with using activator activating surface, bacteria antibody is subsequently added and BSA is prepared into magnetic and receives
Rice grain, i.e. capture probe；
(4) will be resuspended with water after the centrifugation of cultured pathogenic bacteria, and it is diluted to one group of standard sample liquid with concentration gradient；
(5) capture probe in the step of taking same volume (3), is separately added into the standard sample liquid of step (4) variable concentrations, room
Middle benefit gas are incubated, and supernatant is removed after Magneto separate；The Raman signal probe in step (2) is added again, forms " capture probe-cause
" sandwich " structure of germ-signal probe "；After Magneto separate, signal is carried out to remaining Raman signal probe in supernatant and is adopted
Collection；
(6) calibration curve is set up with the corresponding relation of Raman signal intensity according to the bacteria concentration that causes a disease, so as to using Raman signal pair
Pathogenic bacteria carry out quantitative determination.
2. the method based on new SERS substrates quantitative determination pathogenic bacteria according to claim 1, it is characterised in that：
Described pathogenic bacteria are Escherichia coli O 157:H7；
Bacteria antibody described in step (2), (3) is Escherichia coli O 157:H7 antibody.
3. the method based on new SERS substrates quantitative determination pathogenic bacteria according to claim 1, it is characterised in that：
Raman signal molecule described in step (1) is 4,4 '-bipyridyl；
The average grain diameter of the gold nano grain described in step (1) is 25nm～35nm.
4. the method based on new SERS substrates quantitative determination pathogenic bacteria according to claim 1, it is characterised in that：
Silane coupler described in step (2) is (3- mercaptopropyis) trimethoxy silane；
Intermediate described in step (2) is thio-N- succinimidos 4- (maleimidomehyl) hexamethylene -1- carboxylics of 3-
Acid sodium-salt；
The concentration of the bacteria antibody described in step (2) is 0.5～1mg/mL.
5. the method based on new SERS substrates quantitative determination pathogenic bacteria according to claim 1, it is characterised in that：
Surface modification described in step (3) has the magnetic bead of carboxyl, and its particle diameter is 100～200nm；
Activator described in step (3) is 1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides and N- hydroxyl ambers
Amber acid imide.
6. the method based on new SERS substrates quantitative determination pathogenic bacteria according to claim 1, it is characterised in that：
Concentration gradient described in step (4) is 0,10,103、105、107、109CFU/mL, wherein 0CFU/mL are blank.
7. the method based on new SERS substrates quantitative determination pathogenic bacteria according to claim 1, it is characterised in that：
The time being incubated in room temperature described in step (5) is 1～2h.
8. the method based on new SERS substrates quantitative determination pathogenic bacteria according to claim 1, it is characterised in that：
The mixed volume ratio of " sandwich " structure of " capture probe-pathogenic bacteria-signal probe " described in step (5) is
3.5～4.5：1：4.5～5.5.
9. the method based on new SERS substrates quantitative determination pathogenic bacteria according to claim 1, it is characterised in that：
Signal described in step (5) is measured by micro-Raman spectroscopy：The operation bar of described micro-Raman spectroscopy
Part be excitation source be wavelength be 632.8nm He-Ne laser instruments, reach sample laser power be 1mW, the signal collection time
For 10～60s.
10. the method based on new SERS substrates quantitative determination pathogenic bacteria according to claim 1, it is characterised in that：
Signal described in step (5) be the Characteristic Raman spectral peak for choosing Raman signal molecule as quantitative peak, and with pathogenic bacteria
Concentration draws calibration curve to the spectral peak spectral intensity mapping with this.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710019969.7A CN106645090A (en) | 2017-01-11 | 2017-01-11 | Novel SERS substrate-based method for quantitatively testing pathogenic bacteria |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710019969.7A CN106645090A (en) | 2017-01-11 | 2017-01-11 | Novel SERS substrate-based method for quantitatively testing pathogenic bacteria |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN106645090A true CN106645090A (en) | 2017-05-10 |
Family
ID=58842744
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710019969.7A Pending CN106645090A (en) | 2017-01-11 | 2017-01-11 | Novel SERS substrate-based method for quantitatively testing pathogenic bacteria |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106645090A (en) |
Cited By (17)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108680637A (en) * | 2018-05-15 | 2018-10-19 | 苏州大学 | Heterotope double labelling method based on maleimide derivatives combination |
| CN108872194A (en) * | 2018-08-22 | 2018-11-23 | 暨南大学 | A kind of method of sandwich structure SERS detection pathogen |
| CN109253998A (en) * | 2018-10-25 | 2019-01-22 | 珠海彩晶光谱科技有限公司 | Metal-wrappage-antibody composite nanoparticle quantitative detection tumor marker method based on Raman enhancing |
| CN109765361A (en) * | 2018-12-04 | 2019-05-17 | 武汉市农业科学院 | A kind of Detection Method of Pathogenic Microorganism |
| CN110018303A (en) * | 2019-03-19 | 2019-07-16 | 江苏大学 | A kind of food-borne pathogens quantitative detection System structure method based on nanometer enzymatic |
| CN110296972A (en) * | 2019-03-19 | 2019-10-01 | 江苏大学 | A kind of staphylococcus aureus quantitative detecting method based on SERS technology |
| CN110376182A (en) * | 2019-08-15 | 2019-10-25 | 西南交通大学 | SERS substrate and preparation method thereof with inherent Raman photon guidance capability |
| CN110470828A (en) * | 2019-07-30 | 2019-11-19 | 山东第一医科大学(山东省医学科学院) | A kind of preparation method and detection method of cerebral apoplexy marker immunosensor |
| CN110632302A (en) * | 2019-10-30 | 2019-12-31 | 中国农业科学院农产品加工研究所 | Method for simultaneously detecting contents of escherichia coli and salmonella in sample to be detected |
| CN111337470A (en) * | 2020-01-15 | 2020-06-26 | 武汉市农业科学院 | Method for measuring content of escherichia coli in water |
| CN111398241A (en) * | 2020-01-15 | 2020-07-10 | 武汉市农业科学院 | High-flux SERS detection method for food-borne pathogenic bacteria |
| CN111474162A (en) * | 2020-03-18 | 2020-07-31 | 华南师范大学 | SERS (surface enhanced Raman Scattering) labeled nano-probe with gold-hexanethiol-mesoporous silicon structure as well as preparation method and application thereof |
| CN111948386A (en) * | 2020-09-03 | 2020-11-17 | 吉林大学 | Raman homogeneous detection method for recognizing and combining antigen by using antibody |
| CN112098389A (en) * | 2020-08-31 | 2020-12-18 | 华南理工大学 | Detection method of Listeria monocytogenes |
| CN112540072A (en) * | 2020-11-20 | 2021-03-23 | 江苏大学 | Preparation method of surface-enhanced Raman spectrum substrate material for detecting xanthoxylin in hotpot condiment |
| CN113125407A (en) * | 2020-01-16 | 2021-07-16 | 武汉市农业科学院 | Cr (chromium)6+Ion rapid detection method |
| CN114544591A (en) * | 2022-02-25 | 2022-05-27 | 江南大学 | Gram-positive bacterium detection method based on surface enhanced Raman scattering |
Citations (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1444045A (en) * | 2003-04-15 | 2003-09-24 | 吉林大学 | Surface enhancement Raman scattering labelling immunodetection method |
| WO2006096468A2 (en) * | 2005-03-03 | 2006-09-14 | Sword Diagnostics, Inc. | Enhanced detection of biological and bioactive components by resonance raman spectroscopy |
| CN1894421A (en) * | 2003-09-12 | 2007-01-10 | 英特尔公司 | Methods to increase nucleotide signals by raman scattering |
| EP1778010A2 (en) * | 2004-07-30 | 2007-05-02 | Acrymed, Inc. | Antimicrobial silver compositions |
| WO2008016375A3 (en) * | 2005-12-29 | 2008-08-14 | Intel Corp | Modification of metal nanoparticles for improved analyte detection by surface enhanced raman spectroscopy (sers) |
| CN101768625A (en) * | 2010-02-23 | 2010-07-07 | 厦门大学 | Pathogen detection method |
| CN102115778A (en) * | 2010-12-03 | 2011-07-06 | 江南大学 | Method for identifying foodborne pathogen by surface enhanced Raman spectroscopy |
| CN202083634U (en) * | 2010-11-11 | 2011-12-21 | 卢磊磊 | Rapid and sensitive virus and pathogenic bacteria detecting device and data processing system |
| DE102009050287B4 (en) * | 2009-10-20 | 2012-07-05 | Friedrich-Schiller-Universität Jena | SERS substrate |
| CN102559190A (en) * | 2012-01-12 | 2012-07-11 | 东南大学 | Dual-mode optical coding probe and preparation method thereof |
| CN102590176A (en) * | 2012-03-01 | 2012-07-18 | 中国科学院苏州纳米技术与纳米仿生研究所 | Surface-enhanced Raman scattering probe and preparation method thereof |
| CN103990812A (en) * | 2014-06-06 | 2014-08-20 | 厦门大学 | Method for preparing surface enhanced Raman substrate |
| CN104749366A (en) * | 2013-12-31 | 2015-07-01 | 中国科学院上海微***与信息技术研究所 | Method for rapidly detecting pathogenic bacteria |
| CN105004671A (en) * | 2015-07-07 | 2015-10-28 | 中国人民解放军第三军医大学第一附属医院 | Water content-based terahertz spectrum detection cell for rapid and label-free detection of pathogenic bacteria, and method thereof |
| CN105784673A (en) * | 2016-04-12 | 2016-07-20 | 江苏大学 | Quick identifying method for positive and negative food-borne pathogenic bacteria |
-
2017
- 2017-01-11 CN CN201710019969.7A patent/CN106645090A/en active Pending
Patent Citations (16)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1444045A (en) * | 2003-04-15 | 2003-09-24 | 吉林大学 | Surface enhancement Raman scattering labelling immunodetection method |
| CN1894421A (en) * | 2003-09-12 | 2007-01-10 | 英特尔公司 | Methods to increase nucleotide signals by raman scattering |
| EP1778010A2 (en) * | 2004-07-30 | 2007-05-02 | Acrymed, Inc. | Antimicrobial silver compositions |
| WO2006096468A2 (en) * | 2005-03-03 | 2006-09-14 | Sword Diagnostics, Inc. | Enhanced detection of biological and bioactive components by resonance raman spectroscopy |
| WO2008016375A3 (en) * | 2005-12-29 | 2008-08-14 | Intel Corp | Modification of metal nanoparticles for improved analyte detection by surface enhanced raman spectroscopy (sers) |
| DE102009050287B4 (en) * | 2009-10-20 | 2012-07-05 | Friedrich-Schiller-Universität Jena | SERS substrate |
| CN101768625A (en) * | 2010-02-23 | 2010-07-07 | 厦门大学 | Pathogen detection method |
| CN202083634U (en) * | 2010-11-11 | 2011-12-21 | 卢磊磊 | Rapid and sensitive virus and pathogenic bacteria detecting device and data processing system |
| CN102115778A (en) * | 2010-12-03 | 2011-07-06 | 江南大学 | Method for identifying foodborne pathogen by surface enhanced Raman spectroscopy |
| CN102559190A (en) * | 2012-01-12 | 2012-07-11 | 东南大学 | Dual-mode optical coding probe and preparation method thereof |
| CN102590176A (en) * | 2012-03-01 | 2012-07-18 | 中国科学院苏州纳米技术与纳米仿生研究所 | Surface-enhanced Raman scattering probe and preparation method thereof |
| CN104749366A (en) * | 2013-12-31 | 2015-07-01 | 中国科学院上海微***与信息技术研究所 | Method for rapidly detecting pathogenic bacteria |
| CN103990812A (en) * | 2014-06-06 | 2014-08-20 | 厦门大学 | Method for preparing surface enhanced Raman substrate |
| CN105004671A (en) * | 2015-07-07 | 2015-10-28 | 中国人民解放军第三军医大学第一附属医院 | Water content-based terahertz spectrum detection cell for rapid and label-free detection of pathogenic bacteria, and method thereof |
| CN105004671B (en) * | 2015-07-07 | 2017-11-17 | 中国人民解放军第三军医大学第一附属医院 | Tera-hertz spectra detection cell and method based on the quick markless detection pathogenic bacteria of water content |
| CN105784673A (en) * | 2016-04-12 | 2016-07-20 | 江苏大学 | Quick identifying method for positive and negative food-borne pathogenic bacteria |
Non-Patent Citations (2)
| Title |
|---|
| BURCU GUVEN.ETAL: "SERS-based sandwich immunoassay using antibody coated magnetic nanoparticles for Escherichia coli enumeration", 《ANALYST》 * |
| 杨晶等: "基于金纳米聚集体的核壳型SERS 探针及其细胞内应用", 《化学学报》 * |
Cited By (25)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108680637A (en) * | 2018-05-15 | 2018-10-19 | 苏州大学 | Heterotope double labelling method based on maleimide derivatives combination |
| CN108872194B (en) * | 2018-08-22 | 2020-09-04 | 暨南大学 | Method for detecting pathogenic bacteria by sandwich structure SERS |
| CN108872194A (en) * | 2018-08-22 | 2018-11-23 | 暨南大学 | A kind of method of sandwich structure SERS detection pathogen |
| CN109253998A (en) * | 2018-10-25 | 2019-01-22 | 珠海彩晶光谱科技有限公司 | Metal-wrappage-antibody composite nanoparticle quantitative detection tumor marker method based on Raman enhancing |
| CN109765361A (en) * | 2018-12-04 | 2019-05-17 | 武汉市农业科学院 | A kind of Detection Method of Pathogenic Microorganism |
| CN110296972B (en) * | 2019-03-19 | 2022-02-15 | 江苏大学 | Quantitative detection method for staphylococcus aureus based on SERS technology |
| CN110296972A (en) * | 2019-03-19 | 2019-10-01 | 江苏大学 | A kind of staphylococcus aureus quantitative detecting method based on SERS technology |
| CN110018303A (en) * | 2019-03-19 | 2019-07-16 | 江苏大学 | A kind of food-borne pathogens quantitative detection System structure method based on nanometer enzymatic |
| CN110470828A (en) * | 2019-07-30 | 2019-11-19 | 山东第一医科大学(山东省医学科学院) | A kind of preparation method and detection method of cerebral apoplexy marker immunosensor |
| CN110470828B (en) * | 2019-07-30 | 2023-11-10 | 山东第一医科大学(山东省医学科学院) | Preparation method and detection method of cerebral apoplexy marker immunosensor |
| CN110376182B (en) * | 2019-08-15 | 2021-11-30 | 西南交通大学 | SERS substrate with intrinsic Raman photon guiding capability and preparation method thereof |
| CN110376182A (en) * | 2019-08-15 | 2019-10-25 | 西南交通大学 | SERS substrate and preparation method thereof with inherent Raman photon guidance capability |
| CN110632302A (en) * | 2019-10-30 | 2019-12-31 | 中国农业科学院农产品加工研究所 | Method for simultaneously detecting contents of escherichia coli and salmonella in sample to be detected |
| CN110632302B (en) * | 2019-10-30 | 2023-07-18 | 中国农业科学院农产品加工研究所 | Method for simultaneously detecting contents of escherichia coli and salmonella in sample to be detected |
| CN111398241A (en) * | 2020-01-15 | 2020-07-10 | 武汉市农业科学院 | High-flux SERS detection method for food-borne pathogenic bacteria |
| CN111337470A (en) * | 2020-01-15 | 2020-06-26 | 武汉市农业科学院 | Method for measuring content of escherichia coli in water |
| CN113125407A (en) * | 2020-01-16 | 2021-07-16 | 武汉市农业科学院 | Cr (chromium)6+Ion rapid detection method |
| CN113125407B (en) * | 2020-01-16 | 2024-05-03 | 武汉市农业科学院 | Cr (chromium)6+Ion rapid detection method |
| CN111474162A (en) * | 2020-03-18 | 2020-07-31 | 华南师范大学 | SERS (surface enhanced Raman Scattering) labeled nano-probe with gold-hexanethiol-mesoporous silicon structure as well as preparation method and application thereof |
| CN112098389A (en) * | 2020-08-31 | 2020-12-18 | 华南理工大学 | Detection method of Listeria monocytogenes |
| CN112098389B (en) * | 2020-08-31 | 2022-04-22 | 华南理工大学 | Detection method of Listeria monocytogenes |
| CN111948386A (en) * | 2020-09-03 | 2020-11-17 | 吉林大学 | Raman homogeneous detection method for recognizing and combining antigen by using antibody |
| CN111948386B (en) * | 2020-09-03 | 2023-04-07 | 吉林大学 | Raman homogeneous phase non-diagnosis target detection method using antibody to recognize combined antigen |
| CN112540072A (en) * | 2020-11-20 | 2021-03-23 | 江苏大学 | Preparation method of surface-enhanced Raman spectrum substrate material for detecting xanthoxylin in hotpot condiment |
| CN114544591A (en) * | 2022-02-25 | 2022-05-27 | 江南大学 | Gram-positive bacterium detection method based on surface enhanced Raman scattering |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106645090A (en) | Novel SERS substrate-based method for quantitatively testing pathogenic bacteria | |
| Gong et al. | Ag/SiO2 core-shell nanoparticle-based surface-enhanced Raman probes for immunoassay of cancer marker using silica-coated magnetic nanoparticles as separation tools | |
| Bhardwaj et al. | Fluorescent nanobiosensors for the targeted detection of foodborne bacteria | |
| Wu et al. | Magnetic nanobead-based immunoassay for the simultaneous detection of aflatoxin B1 and ochratoxin A using upconversion nanoparticles as multicolor labels | |
| Tallury et al. | Nanobioimaging and sensing of infectious diseases | |
| Jia et al. | Recent advances on immunosensors for mycotoxins in foods and other commodities | |
| Xue et al. | A sensitive immunoassay for simultaneous detection of foodborne pathogens using MnO2 nanoflowers-assisted loading and release of quantum dots | |
| Huang et al. | Application and development of superparamagnetic nanoparticles in sample pretreatment and immunochromatographic assay | |
| Su et al. | Gold nanoparticles as colorimetric sensor: A case study on E. coli O157: H7 as a model for Gram-negative bacteria | |
| Torun et al. | Comparison of sensing strategies in SPR biosensor for rapid and sensitive enumeration of bacteria | |
| Ouyang et al. | Upconversion nanoprobes based on a horseradish peroxidase-regulated dual-mode strategy for the ultrasensitive detection of Staphylococcus aureus in meat | |
| Bu et al. | Applicability of biological dye tracer in strip biosensor for ultrasensitive detection of pathogenic bacteria | |
| Ye et al. | An electrochemical immunoassay for Escherichia coli O157: H7 using double functionalized Au@ Pt/SiO2 nanocomposites and immune magnetic nanoparticles | |
| Du et al. | Quantum dot: Lightning invisible foodborne pathogens | |
| Zhang et al. | One-step colorimetric detection of Staphylococcus aureus based on target-induced shielding against the peroxidase mimicking activity of aptamer-functionalized gold-coated iron oxide nanocomposites | |
| CN108535236A (en) | A method of based on dual amplification SERS signal system super sensitivity detection miRNA | |
| Zhang et al. | Recent advances of fluorescent sensors for bacteria detection-A review | |
| Fei et al. | A sandwich electrochemical immunoassay for Salmonella pullorum and Salmonella gallinarum based on a AuNPs/SiO 2/Fe 3 O 4 adsorbing antibody and 4 channel screen printed carbon electrode electrodeposited gold nanoparticles | |
| Wu et al. | Immunoassay for carcinoembryonic antigen based on the Zn2+-enhanced fluorescence of magnetic-fluorescent nanocomposites | |
| Gabrovska et al. | Immunofluorescent analysis with magnetic nanoparticles for simultaneous determination of antibiotic residues in milk | |
| CN110133262A (en) | A kind of kit for detecting bacterium | |
| Zhang et al. | Rapid and sensitive pathogen detection platform based on a lanthanide-labeled immunochromatographic strip test combined with immunomagnetic separation | |
| Zhang et al. | Au-Fe3O4 heterodimer multifunctional nanoparticles-based platform for ultrasensitive naked-eye detection of Salmonella typhimurium | |
| Wang et al. | Colorimetric determination of Listeria monocytogenes using aptamer and urease dual-labeled magnetic nanoparticles and cucurbit [7] uril-mediated supramolecular assembly of gold nanoparticle | |
| Liu et al. | Rapid detection of Escherichia coli O157: H7 in milk, bread, and jelly by lac dye coloration‐based bidirectional lateral flow immunoassay strip |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20170510 |