CN104555913A - Production method of silver-clad gold nano-rods and their application - Google Patents
Production method of silver-clad gold nano-rods and their application Download PDFInfo
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Abstract
The invention discloses a production method of silver-clad gold nano-rods and their application and belongs to the technical field of food quality detection and analysis. Au@ag nano-rods are produced, and with the surface enhanced fluorescence of the nano-rods, the nano-rods are successfully applied to the detection of the gene O157:H7easeA of E. coli. Compared with the traditional methods, the method has the advantages that detection limit is greatly lowered, sensitivity is improved, and good stability is achieved. According to the principle of fluorescence enhancement-weakening, a standard curve for gene O157:H7ease detection of E. coli is built, a linear range is from 10<-17> M to 10<-11> M, correlation coefficient R2 is 0.9947, and the detection limit is 3.33*10<-18> M (S/N = 3). The method has the advantages that specificity is good, labelling experiments show that eaeA gene recovery rate is 98.36% to 101.67%, with good stability, and the nano-rods are applicable to the detection of the gene O157:H7ease of E. coli and have a promising application prospect.
Description
Technical field
The present invention relates to a kind of preparation method and application of silver-colored gold-covered nano rod, belong to food quality and detect analysis technical field.
Background technology
Escherichia coli (Escherichia coli, E.coli) represent bacterium for Escherichia (Escherichia), and be main in people and many animal intestinals and a kind of bacterium that quantity is maximum, main parasitic is in large intestine.When it invades some positions of human body, infection can be caused, as peritonitis, cholecystitis, cystitis and diarrhoea etc.The symptom of people after ehec infection is stomachache, vomits, suffers from diarrhoea and heating.Infection may be fatefulue, especially to child and old man.
Colibacillary morbid substance is mainly: (1) invasiveness: comprise K antigen and colonizing factor; (2) endotoxin: there is activity of endotoxin containing in endotoxic colibacillary cell membrane, the lipid A of lipopolysaccharides layer is toxicity position; (3) enterotoxin: enterotoxin is different to heat endurance, is divided into heat-resisting and thermo-labile two kinds.Heat-labile toxin is to thermally labile, and within when 65 DEG C 30 minutes, can lose activity, and Heat stable Enterotoxin is to thermally-stabilised, 100 DEG C of heating are not still destroyed for 20 minutes, molecular weight, and immunogenicity is weak.Associated diseases comprises: (1) extra intestinal infection: mostly be autogenous infection, based on urinary system infection contamination, as urethritis, cystitis, pyelonephritis.Also peritonitis, cholecystitis, appendicitis etc. can be caused; (2) acute diarrhea, wherein by EHEC (EnterohemorrhagicE coli, EHEC) refer to can causing bleeding property enteritis, hemolytic uremic syndrome a paracolon, can shiga-like toxin be produced.The main bacterial type of EHEC is O157:H7, also comprises 026:H11,0111 etc.
Traditional detection detects the method for large enteric bacterial pathogens E.coli O157:H7: 1) common detection methods: bed board cultivation, counting method, biochemistry detection, microscope, flow cytometer and luminescence method.2) immunological detection method: Western blot and enzyme-linked immunosorbent assay; 3) molecular biology for detection: PCR (PCR) and DNA microarray.
But the method for traditional detection bacterium has been come by Morphological characterization or biochemical analysis, they can not provide conclusion to such an extent as to the discriminating that can not realize on strain level and the Bacteria Detection be separated from environment accurately on bacterial species; Test simultaneously carries out and data processing is all very consuming time, can not meet the Real-Time Monitoring fast and accurately required for Bacteria Detection.PCR method is larger by sample effects, especially in food source property harmful microorganism checks, composition in food materials such as () carbohydrate, acids, greases can normally the carrying out of disturbance reponse, and the environment detected, intermediate treatment link also can be with and serve PCR reaction suppressor, thus makes PCR present higher false positive and false negative rate.Although ELISA has very high sensitivity and repeatability, also there is pathogenic bacteria sample size large, complex operation, kit is expensive, and cost is high, poor accuracy and complicated operation and need the defects such as professional training.Although its good stability of method for biosensor, precision are high, the shortcoming of ubiquity instrument price costliness, can not meet the detection demand of pathogenic bacteria in extensive sample, business-like progress is very slow.
Shiga toxin producing escherichia coli (Shiga toxin-producing Escherichia coli, STEC) mankind can not only be caused to suffer from diarrhoea, also can cause serious hemorrhagic enteritis (hemorrhagic colitis, and hemolytic uremia (haemolytic uraemic syndrome, HUS) HC).Especially EHEC (enterohemorrhage Escherichia coli, EHEC) O157:H7 bacterial strain is occupied an leading position in the infection of report.EaeA, stx2 and hlyA gene is the major virulence gene that STEC causes a disease, E.coli O157:H7's sticks the important embodiment that colonization ability is EHEC (EHEC O157:H7), and it is relevant with the eaeA gene on chromosome to epithelial adhesion.For one of virulence gene that large enteric bacterial pathogens O157:H7, eaeA gene is pathogenic.If can fast, Sensitive Detection goes out virulence gene eaeA in large enteric bacterial pathogens O157:H7, so just significant to diseases such as prevention hemorrhagic enteritis.
The present invention has prepared AuAg nanometer rods, and utilizes the effect of its surface-enhanced fluorescence, is successfully applied to and detects large enteric bacterial pathogens E.coli O157:H7eaeA gene.
Summary of the invention
For the deficiency of existing detection large enteric bacterial pathogens E.coli O157:H7 method, and the toxicity action impact of eaeA gene pairs enterohemorrhagic disease, the invention provides a kind of new method for detecting this gene.First AuAg nanometer rods is prepared, then at its finishing oligonucleotide probe, then the partial nucleotide acid hybridization be connected with fluorescent dye cy3, construct the biology sensor detecting large enteric bacterial pathogens E.coli O157:H7eaeA gene.The Modulatory character of the monodispersity that the present invention has had in conjunction with AuAg nanometer rods and length, wide plasmon absorption is had in ultraviolet and near infrared region, and have point and the features such as strong longitudinal plasma vibrational band than Au nanometer rods, utilize the effect of surface-enhanced fluorescence, establish the biology sensor that AuAg nanometer rods detects large enteric bacterial pathogens eaeA gene first, there is the features such as low detectability, high sensitivity, good stability.
First object of the present invention is to provide a kind of preparation method of silver-colored gold-covered nano rod, and it is characterized in that, described method is: after the gold nanorods solution prepared utilizing seed mediated growth method and CTAB solution mix, add AgNO successively wherein
3solution, ascorbic acid solution, NaOH solution are spent the night at 25-30 DEG C.
Described method comprises: be in the CTAB solution of 0.02-0.08mol/L in every 20ml concentration, adds the gold nanorods solution that 10-20mL concentration is 15-30nmol/L, stirs, and adding 1000-4000 μ L concentration is successively the AgNO of 2-8mmol/L
3solution, 500-800 μ L concentration are the ascorbic acid solution of 0.1-0.5mol/L, 1000-3000 μ L concentration is the NaOH solution of 0.1-0.5mol/L, stir, leave standstill, and namely synthesis obtains AuAg nanometer rods.
The AuAg nanometer rods of described method synthesis, in one embodiment of the invention, have selected the gold nanorods of different length respectively, thus composition length is the AuAg nanometer rods of 30nm, 40nm, 50nm respectively.
Described method synthesizes the AgNO that AuAg nanometer rods that the length obtained is respectively 30nm, 40nm, 50nm uses
3the volume of solution is 1250 μ L respectively, 2500 μ L and 3750 μ L.
Second object of the present invention is to provide a kind of AuAg nanometer rods obtained as stated above.
3rd object of the present invention is to provide a kind of application process of described AuAg nanometer rods.
Described application process is for detecting eaeA gene in one embodiment of the invention.
Described method is modified oligonucleotide probe in AuAg nanometer rods, and after the partial nucleotide acid hybridization that oligonucleotide probe is connected with fluorescent dye cy3, fluorescence signal strengthens.When target dna occurs, probe is combined with target dna, now fluorescent weakening, thus according to Fluorescence Increasing-weaken the detection that principle is applied to large enteric bacterial pathogens E.coli O157:H7eaeA gene.
Described method comprises: (1) is modified oligonucleotide probe in AuAg nanorod surfaces, then with add the nucleotides that fluorescent dye cy3 modifies and hybridize, obtain the AuAg – DNA hybridization solution modified; (2) the fluorescence intensity F of hybridization solution supernatant that obtains of determination step 1
ag; (3) target gene sequence of variable concentrations is joined in AuAg – DNA hybridization supernatant prepared by step 1, reaction, centrifugal, the fluorescence intensity F that measures supernatant
s, finally according to formula F
ag-F
s/ F
agcalculate the change of fluorescence intensity and obtain the changing value F that variable concentrations target gene adds rear fluorescence intensity
ag-F
s/ F
ag; (4) calibration curve of target gene concentration and fluorescence intensity change value is drawn; (5) testing sample is added in hybridization solution prepared by step 1, measure fluorescence intensity, then according to formula F
ag-F
s/ F
agcalculate the changing value of fluorescence intensity, finally according to the calibration curve that step 4 obtains, calculate the concentration of target gene in testing sample.
AuAg nanometer rods in described step (1) in one embodiment of the invention, is adopt length to be the AuAg nanometer rods of 50nm.
Oligonucleotide probe in described step (1) in one embodiment of the invention, is 5 '-H
2n-C
6-GCGAGGAACGCCGATACCATTACTTATACCGCGACGCCTCGC-3 ' (probe sequence is SEQ ID NO.1), can be used for the eaeA gene detecting large enteric bacterial pathogens E.coli O157:H7.
Described step (1), in one embodiment of the invention, comprise: first at the finishing Mercaptoundecanoic acid of AuAg nanometer rods, then EDC/NHS activated carboxyl group is used, then with amido modified oligonucleotides and the Mercaptoundecanoic acid coupling activated, last at 37 DEG C, under PBS condition, the nucleotide hybridization be connected with fluorescent dye cy3.
Described step (1), in one embodiment of the invention, the base number of the nucleotides that fluorescent dye cy3 modifies is optimized, be that the nucleotides of the Different Alkali radix first selecting fluorescent dye cy3 to modify is hybridized, choose the nucleotides modified through fluorescent dye cy3 as final utilization that Fluorescence Increasing effect is the strongest.
Described step (1), in one embodiment of the invention, the nucleotides that the fluorescent dye cy3 selected modifies is: 5 '-cy3-GGTATAAGTAATGGTATCGGCGTT-3 ' (namely nucleotide sequence is as shown in SEQ ID NO.4).
Fluorescence intensity in described step (2), with do not have to hybridize before the partial nucleotide fluorescence intensity that is connected of cy3 variant, this determines according to the distance of fluorescent dye cy3 and AuAg nanorod surfaces, after the fluorescent dye cy3 that Different Alkali radix connects and AuAg nanometer rods are hybridized, there is the effect of Fluorescence Increasing or fluorescent weakening.In one embodiment of the invention, select maximum that nucleotide base chain of Fluorescence Increasing (distance is 10nm), for the Fluorescence Increasing signal in testing.
In described step (3), AuAg – DNA hybridization supernatant is combined with target sequence, at 37 DEG C, under PBS condition, the oligonucleotides that probe and original cy3 of AuAg finishing modify is separated, so the oligonucleotides of fluorescent dye cy3 modification and the distance of AuAg nanorod surfaces increase, namely more than 10nm, and now fluorescent weakening.According to formula F
ag-F
s/ F
agcalculate the change of fluorescence intensity, the concentration of its variable quantity and target gene sequence is proportional, finally draws target gene concentration and fluorescence intensity change value calibration curve.Wherein F
agrepresent fluorescence intensity during AuAg nanometer rods Fluorescence Increasing, F
sthe oligonucleotides that expression fluorescent dye cy3 modifies and target sequence leave fluorescence intensity during AuAg nanorod surfaces fluorescent weakening after competing.
Described calibration curve, in one embodiment of the invention, for detecting the eaeA gene of large enteric bacterial pathogens E.coli O157:H7, be y=95.97x+1992.49, target DNA concentration is 10
-17-10
-11have good linear relationship between M, coefficient correlation is R
2=0.9947, detect and be limited to 3.33 × 10
-18m (S/N=3).
Described method, in one embodiment of the invention, for detecting the eaeA gene of large enteric bacterial pathogens E.coli O157:H7, comprising and is: the preparation of (1) AuAg nanometer rods; (2) AuAg nanorod surfaces modifies carboxylic group; (3) with AuAg nanometer rods and the coupling of amidized stem ring oligonucleotides of carboxylic group; (4) modified the nucleotides sequence that the AuAg nanometer rods of oligonucleotides and fluorescent dye cy3 modify and be listed in 37 DEG C, hybridized under PBS condition, utilize XRF F-7000 to measure hybridization supernatant fluorescence intensity; (5) add complementary target sequence and eaeA gene order in the supernatant of having hybridized to previous step, measure fluorescence intensity, according to formula F
ag-F
s/ F
agcalculate the change of fluorescence intensity, the concentration of its variable quantity and target dna is proportional; (6) testing sample is added in the hybridization solution of step 4, according to the proportional relation that fluorescence intensity change and step 5 obtain, calculate the concentration of target sequence in testing sample.
In described step (5) testing sample can be following any one: the directly DNA of the thalline of extracting from food or his material, or thallus DNA is through the amplified production of PCR.The latter due to fragment length shorter, its Detection results is better than the former.
Beneficial effect of the present invention:
(1) found out a set of novel method preparing AuAg nanometer rods, compared with existing preparation method, the AuAg nanometer rods that the method prepares, its aspect ratio distribution is even, and output is high, and homogeneity is good, good dispersion.The AuAg nanometer rods simultaneously obtaining different draw ratio needs the AgNO adding different volumes
3solution.Adopt the AuAg nanometer rods Fluorescence Increasing effect of 50nm best, the simultaneously Modulatory character of the monodispersity that had than Au nanometer rods of AuAg nanometer rods and size, for genetic test, there is stability high, repeatable high, highly sensitive, the advantage such as the range of linearity is wide of detection.
(2) phenomenon is strengthened greatly owing to only having molecule could produce near metal surface or its, the oligonucleotides that the present invention selects the fluorescent dye cy3 containing 24 base numbers shown in 5 '-cy3-GGTATAAGTAATGGTATCGGCGTT-3 ' to modify, the distance of adjustment fluorescent dye cy3 and AuAg nanometer rods, thus make Fluorescence Increasing or the effect that weakens the most obvious, ensure that the sensitivity detecting large enteric bacterial pathogens E.coli O157:H7.
(3) AuAg nanometer rods of the present invention is for detecting the eaeA gene of E.coli O157:H7, and its detectability can reach 3.33 × 10
-18m, have highly sensitive, favorable reproducibility, fast, high specificity, the feature such as convenient, the use of bonding probes, selective good.
Accompanying drawing explanation
Fig. 1: Au nanometer rods and AuAg nanometer rods TEM (transmission electron microscope picture); Wherein A, B, C are the Au nanometer rods of different length, and D, E, F are the AuAg nanometer rods of different length;
Fig. 2: AuAg nanometer rods detects large enteric bacterial pathogens E.coli O157:H7eaeA gene schematic diagram;
Fig. 3: AuAg nanometer rods UV absorption figure before and after modified oligonucleotide; Note: a represents the UV absorption figure of AuAg nanometer rods; B represents the UV absorption figure after the upper Mercaptoundecanoic acid of AuAg nanometer rods modification; C represents the UV absorption figure after the upper Mercaptoundecanoic acid of AuAg nanometer rods modification and oligonucleotides;
Fig. 4: change in fluorescence figure after cy3-DNA and the AuAg surface oligonucleotide hybridization of Different Alkali radix;
Fig. 5: large enteric bacterial pathogens E.coli O157:H7eaeA genetic test canonical plotting;
Fig. 6: AuAg nanometer rods detects large enteric bacterial pathogens E.coli O157:H7 biology sensor specific test; Wherein a: complete complementary target sequence; B: single base mismatch sequence; C: mismatch completely;
Fig. 7: the AuAg nanometer rods Fluorescence Increasing design sketch of different length.
Detailed description of the invention
In order to more clearly understand technology contents of the present invention, describe in detail especially exemplified by following examples, its object is only better understand content of the present invention but not limit the scope of the invention.
The synthesis of embodiment 1 AuAg nanometer rods
First, take 0.364g CTAB, it is fully dissolved in 20mL water, then, under 27 DEG C of states stirred, in the CTAB aqueous solution, add 10mL gold nanorods solution, after Keep agitation 2min, in reaction system, add 3mM AgNO in the following order respectively
3, 650 μ L concentration are the VC of 0.1M, and 1200 μ L concentration are the NaOH of 0.1M, hold over night after Keep agitation 5-10min, and reaction temperature is 27 DEG C.
Note: according to the difference of synthesized AuAg nanometer rods size, selects AgNO
3the volume of solution is also different, namely selects AgNO
3the volume of solution is 1250 μ L respectively, 2500 μ L and 3750 μ L.
Fig. 1 is Au nanometer rods and AuAg nanometer rods TEM (transmission electron microscope picture).The Au nanometer rods (figure A, B, C) of the present invention to different length has carried out contracted payment experiment, obtain the AuAg nanometer rods (D, E, F) of different length, according to the engineer's scale of figure below, test through contracted payment, the length of AuAg nanometer rods is respectively 30nm, 40nm and 50nm.As seen from Figure 1, the AuAg nanometer rods aspect ratio distribution obtained is even, and output is high, and homogeneity is good, good dispersion.
Embodiment 2 AuAg nanometer rods is used for eaeA genetic test
AuAg nanometer rods detects large enteric bacterial pathogens E.coli O157:H7eaeA gene schematic diagram as shown in Figure 2.Modified oligonucleotide probe in AuAg nanometer rods, after the partial nucleotide acid hybridization that oligonucleotide probe is connected with fluorescent dye cy3, fluorescence signal strengthens.When target dna occurs, probe is combined with target dna, now fluorescent weakening, thus according to Fluorescence Increasing-weaken the detection that principle is applied to large enteric bacterial pathogens E.coli O157:H7eaeA gene.
Concrete grammar is as follows:
(1) AuAg nanorod surfaces modifies MUDA
First, be centrifugal 25 DEG C of the AuAg nanometer rods 8500rpm of 50nm by the length of synthesis, then, according to ligand exchange reaction principle, Mercaptoundecanoic acid (MUDA) in its finishing, adds 33 μ L in the AuAg nanometer rods solution after 3mL purifying per hour, 0.1M MUDA, continue after 3 hours, draw the unnecessary MUDA do not connected by after the centrifugal 25min of AuAg nanometer rods 8500rpm of upper for modification MUDA, finally precipitation is dissolved in 1mL deionized water.
(2) MUDA-AuAg finishing stem ring probe
First, 1-(3-the dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride (EDC HCl) of 1mL MUDA-AuAg nanometer rods and 1mL 0.1M is stirred 3h, for activated carboxyl group under pH4.5.Then, in molar ratio 1:2 respectively to adding 0.1MN-hydroxysuccinimide (NHS) and amidized stem ring probe 5 '-H in system
2n-C
6-GCGAGGAACGCCGATACCATTACTTATACCGCGACGCCTCGC-3 ' (probe sequence is as shown in SEQ ID NO.1) room temperature with constant stirs and spends the night.Finally, by centrifugal for solution 12000rpm 10min, be dissolved in after Aspirate supernatant in TE buffer solution (pH7.4).Before and after modified oligonucleotide, AuAg nanometer rods UV absorption situation as shown in Figure 3, can find out that before and after modified oligonucleotide, AuAg nanometer rods can also keep original form and character.
(3) preparation of AuAg-MUDA-DNA surface fluorescence enhancing
Respectively to the single stranded DNA (as shown in Figure 4) that the fluorescent dye cy3 adding Different Alkali radix in MUDA-AuAg-DNA modifies, final concentration is 10
-7m.This mixture is at PBS buffer solution (pH7.4), and the centrifugal 10min of hybridization reaction 2h at 37 DEG C, last 12000rpm, now supernatant is used for measuring fluorescence intensity.
As seen from Figure 4, when the strongest containing Fluorescence Increasing effect time 24 base (sequence is as shown in SEQ ID NO.4), due to three about 1nm of base, and this sequence and stem ring probe (sequence is as shown in SEQ ID NO.1) partial complementarity, the linear fraction of stem ring probe has 6 bases, illustrates that the distance of fluorescent dye and AuAg nanometer rods is the strongest in about 10nm Fluorescence Increasing effect.
Finally select the 5 '-cy3-GGTATAAGTAATGGTATCGGCGTT-3 ' of Fluorescence Increasing to carry out next step experiment.
(4) preparation of object large enteric bacterial pathogens E.coli O157:H7eaeA gene is detected under Fluorescence Increasing-weaken pattern
Use the primer of sequence as shown in SEQ ID NO.2, SEQ ID NO.3 to carry out pcr amplification, (concentration range is 10 then to obtain the eaeA gene of variable concentrations
-20m-10
-8between M) join in the solution after AuAg – DNA hybridization respectively, at 37 DEG C, with cy3-DNA competitive reaction 2h.The centrifugal 10min of last 12000rpm obtains supernatant.The cy3-DNA amount that competition is fallen depends on the concentration of target eaeA gene, now, measures supernatant fluorescence intensity, due to the increase of distance, and fluorescent weakening, thus according to above-mentioned formula, F
ag-F
s/ F
agcalculate the change of fluorescence intensity, the concentration of its variable quantity and large enteric bacterial pathogens E.coli O157:H7eaeA gene is proportional, F
agrepresent fluorescence intensity during AuAg nanometer rods Fluorescence Increasing, F
srepresent fluorescence intensity when to leave fluorescent weakening after AuAg nanorod surfaces after cy3-DNA and target dna are competed.
Therefore, according to above principle, with the concentration of eaeA gene for abscissa, fluorescence intensity change amount is ordinate, establishes large enteric bacterial pathogens E.coli O157:H7eaeA genetic test calibration curve (as shown in Figure 5).Calibration curve equation is: y=95.97x+1992.49, and the range of linearity is 10
-17to 10
-11between M, coefficient correlation is R
2=0.9947, detect and be limited to 3.33 × 10
-18m (S/N=3).
Added to by testing sample in the supernatant solution after (3) step hybridization reaction, competitive reaction, the fluorescence intensity of centrifugal mensuration supernatant, then according to formula F
ag-F
s/ F
agcalculate the changing value of fluorescence intensity, finally according to calibration curve, calculate the concentration getting final product target eaeA gene in testing sample.Testing sample can be the DNA of the directly thalline of extracting from food, water body or his material, or thallus DNA is through the amplified production of PCR.
Method of the present invention detects gene, has detectability low, highly sensitive, the advantages such as the range of linearity is wide.
The performance evaluation of embodiment 3 AuAg nanometer rods
(1) stability
The AuAg nanometer rods of the different batches prepared with the present invention, detects the eaeA gene of large enteric bacterial pathogens E.coli O157:H7.Result is as shown in table 1.Found that the gene rate of recovery is at 98.36-101.67%, error is little, and AuAg nanometer rods of the present invention and detection method are described, stability, repeatability are all fine.
Table 1 detects the large enteric bacterial pathogens E.coli O157:H7eaeA gene rate of recovery and error result
Note: this experiment is rate of recovery experiment, and background concn is consistent, and variable is spiked levels.
(2) specificity
The AuAg nanometer rods prepared with the present invention, detect containing different gene orders, comprising with the target sequence of oligonucleotide probe part complete complementary (5 '-CGTCGCGGTATAAGTAATGGTATCGGCGTT-3 ', namely as shown in SEQ ID NO.5), single base mismatch sequence (5 '-CGTCGCGGTATAAGTCATGGTATCGGCGTT-3 ', namely as shown in SEQ ID NO.6), complete mismatched target sequence (5 '-GTTCGACTGCTGATGATTGTA AGGTAACA-3 ', namely as shown in SEQ ID NO.7).Result as shown in Figure 6.As we can see from the figure, after adding complete complementary target sequence, fluorescence intensity is minimum, namely fluorescence intensity weakens maximum (variable quantity is maximum), after adding complete mismatched target sequence, fluorescence intensity weakens minimum, can illustrate, the AuAg nanometer rods biology sensor specificity of structure is good.
The Fluorescence Increasing effect of the AuAg nanometer rods of embodiment 4 different length
At AuAg nanometer rods (30nm, 40nm, 50nm) the finishing oligonucleotides of the different length of synthesis, and the oligonucleotide hybridization modified with fluorescent dye cy3, mensuration fluorescence intensity, result is as shown in Figure 7.Found that, length is that the AuAg nanometer rods Fluorescence Increasing effect of 50nm is best.Therefore, this experiment just make use of the AuAg nanometer rods of this length to detect large enteric bacterial pathogens O157; H7, effectively can improve the sensitivity of detection.
The synthesis of embodiment 5 AuAg nanometer rods
Be in the CTAB aqueous solution of 0.02mol/L in every 20ml concentration, under 25 DEG C of conditions, add the gold nanorods solution that 20mL concentration is 15nmol/L, stir, adding 1000 μ L concentration is successively the AgNO of 2mmol/L
3solution, 500 μ L concentration are the ascorbic acid solution of 0.3mol/L, 1000 μ L concentration are the NaOH solution of 0.2mol/L, stir, leave standstill, and namely synthesis obtains AuAg nanometer rods.
The synthesis of embodiment 6 AuAg nanometer rods
Be in the CTAB solution of 0.08mol/L in every 20ml concentration, under 30 DEG C of stirring conditions, add the gold nanorods solution that 15mL concentration is 30nmol/L, stir, adding 4000 μ L concentration is successively the AgNO of 8mmol/L
3solution, 800 μ L concentration are the ascorbic acid solution of 0.5mol/L, 3000 μ L concentration are the NaOH solution of 0.5mol/L, stir, hold over night, and namely synthesis obtains AuAg nanometer rods.
Although the present invention with preferred embodiment openly as above; but it is also not used to limit the present invention, any person skilled in the art, without departing from the spirit and scope of the present invention; all can do various changes and modification, what therefore protection scope of the present invention should define with claims is as the criterion.
Claims (10)
1. a preparation method for silver-colored gold-covered nano rod, is characterized in that, described method is: after the gold nanorods solution prepared utilizing seed mediated growth method and CTAB solution mix, add AgNO successively wherein
3solution, ascorbic acid solution, NaOH solution are spent the night at 25-30 DEG C.
2. method according to claim 1, it is characterized in that, described method comprises: be in the CTAB solution of 0.02-0.08mol/L in every 20ml concentration, add the gold nanorods solution that 10-20mL concentration is 15-30nmol/L, stir, adding 1000-4000 μ L concentration is successively the AgNO of 2-8mmol/L
3solution, 500-800 μ L concentration are the ascorbic acid solution of 0.1-0.5mol/L, 1000-3000 μ L concentration is the NaOH solution of 0.1-0.5mol/L, stir, leave standstill, and namely synthesis obtains AuAg nanometer rods.
3. method according to claim 2, is characterized in that, use the gold nanorods of different length to synthesize in described method respectively AuAg nanometer rods that the length obtained is respectively 30nm, 40nm, 50nm, the AgNO used
3the volume of solution is 1250 μ L respectively, 2500 μ L and 3750 μ L.
4. according to the AuAg nanometer rods that the arbitrary described method of claim 1-3 obtains.
5. AuAg nanometer rods described in claim 4 is in the application of food or water body context of detection.
6. the application of AuAg nanometer rods in genetic test described in claim 4.
7. the method utilizing AuAg nanometer rods described in claim 4 to detect gene, it is characterized in that, described method comprises: (1) is modified oligonucleotide probe in AuAg nanometer rods, then the nucleotides adding fluorescent dye cy3 modification is hybridized, and obtains the AuAg – DNA hybridization solution modified; (2) fluorescence intensity of hybridization solution supernatant that obtains of determination step 1; (3) joined by the target gene of variable concentrations in AuAg – DNA hybridization solution prepared by step 1, reaction, centrifugal, the fluorescence intensity that measures supernatant, calculate the variable quantity that variable concentrations target gene adds rear fluorescence intensity; (4) calibration curve of target gene concentration and fluorescence intensity change amount is drawn; (5) testing sample is added in hybridization solution prepared by step 1, measure fluorescence intensity, calculate fluorescence intensity change amount, then according to the calibration curve that step 4 obtains, calculate the concentration of target gene in testing sample.
8. method according to claim 7, it is characterized in that, described step (1) is specifically: first at the finishing Mercaptoundecanoic acid of AuAg nanometer rods, then EDC/NHS activated carboxyl group is used, then with amido modified oligonucleotides and the Mercaptoundecanoic acid coupling activated, last under PBS condition, the partial nucleotide acid hybridization be connected with fluorescent dye cy3.
9. method according to claim 7, is characterized in that, in described step (1), the sequence of oligonucleotide probe is the sequence shown in SEQ ID NO.1.
10. method according to claim 7, is characterized in that, described step (1) uses length to be the AuAg nanometer rods of 50nm, and the sequence of the nucleotides that the fluorescent dye cy3 of use modifies is the sequence shown in SEQ ID NO.4.
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| CN106404765A (en) * | 2016-08-30 | 2017-02-15 | 中南林业科技大学 | Preparation method of silver-coated gold nano-rod colorimetric probe, and method using probe to detect copper ions |
| CN107824782A (en) * | 2017-10-31 | 2018-03-23 | 上海纳米技术及应用国家工程研究中心有限公司 | Preparation method of gold nanorods of top supported palladium and products thereof and application |
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| CN108254347A (en) * | 2018-01-25 | 2018-07-06 | 青岛大学 | A kind of method of the Fluorescence Increasing detection DNA based on metal nanoparticle coupling |
| CN108941608A (en) * | 2018-08-23 | 2018-12-07 | 安徽中科赛飞尔科技有限公司 | A kind of regulatable silver-colored/golden cavity nanometer rods construction method of shell thickness and its application |
| CN112263490A (en) * | 2020-11-24 | 2021-01-26 | 上海健康医学院 | Toothpaste for developing oral helicobacter pylori and preparation method thereof |
| CN113804665A (en) * | 2021-09-16 | 2021-12-17 | 南京师范大学 | Near-infrared fluorescence sensor with plasma enhanced fluorescence and preparation method and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106404765A (en) * | 2016-08-30 | 2017-02-15 | 中南林业科技大学 | Preparation method of silver-coated gold nano-rod colorimetric probe, and method using probe to detect copper ions |
| CN106404765B (en) * | 2016-08-30 | 2019-08-20 | 中南林业科技大学 | The preparation method of silver-colored gold-covered nano stick colorimetric probe and its method for detecting copper ion |
| CN108085370A (en) * | 2017-10-27 | 2018-05-29 | 南京邮电大学 | A kind of construction method of individual particle bioprobe and application thereof |
| CN107824782A (en) * | 2017-10-31 | 2018-03-23 | 上海纳米技术及应用国家工程研究中心有限公司 | Preparation method of gold nanorods of top supported palladium and products thereof and application |
| CN108254347A (en) * | 2018-01-25 | 2018-07-06 | 青岛大学 | A kind of method of the Fluorescence Increasing detection DNA based on metal nanoparticle coupling |
| CN108254347B (en) * | 2018-01-25 | 2021-01-26 | 青岛大学 | Fluorescence enhancement DNA detection method based on metal nanoparticle coupling |
| CN108941608A (en) * | 2018-08-23 | 2018-12-07 | 安徽中科赛飞尔科技有限公司 | A kind of regulatable silver-colored/golden cavity nanometer rods construction method of shell thickness and its application |
| CN112263490A (en) * | 2020-11-24 | 2021-01-26 | 上海健康医学院 | Toothpaste for developing oral helicobacter pylori and preparation method thereof |
| CN113804665A (en) * | 2021-09-16 | 2021-12-17 | 南京师范大学 | Near-infrared fluorescence sensor with plasma enhanced fluorescence and preparation method and application thereof |
| CN113804665B (en) * | 2021-09-16 | 2024-05-07 | 南京师范大学 | Near infrared fluorescence sensor for plasma enhanced fluorescence and preparation method and application thereof |
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