CN105861295A - Biosensor for detecting salmonella typhimurium and preparation and detection methods - Google Patents
Biosensor for detecting salmonella typhimurium and preparation and detection methods Download PDFInfo
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Abstract
The invention provides a biosensor for detecting salmonella typhimurium. The biosensor is established through a nucleic acid probe (FAM-P) labeled by non-covalent coupling carboxyl fluorescein (FAM) on the surface of graphene oxide based on non-covalent coupling nucleic acid molecules and strong fluorescence quenching characteristics of the graphene oxide (GO), is referred to as an FAM-P/GO biosensor. After a target is added for the FAM-P/GO biosensor, a target sequence forms double strands with a probe sequence through competition and complementary pairing, so that the probe is separated from the surface of the graphene oxide and system fluorescence is recovered. The higher the target sequence concentration is, the higher the fluorescence value is. The invention further provides preparation and usage methods of the FAM-P/GO biosensor. The FAM-P/GO biosensor is simple in operation, accurate in detection, high in sensitivity and good in specificity and can quantitatively detect the concentration of the SSeC gene containing salmonella typhimurium.
Description
Technical field
The present invention relates to biosensor technology field, more specifically, relate to a kind of Salmonella typhimurium that detects
Biosensor and preparation, detection method.
Background technology
Salmonella typhimurium (S.typhimurium) is a kind of common infecting both domestic animals and human cause of disease bacterium, its mechanism of causing a disease and bacterium
The toxin of the pathogenicity island gene expression secretion of body is relevant with virulence protein.The pathogenicity island gene of Salmonella typhimurium is SSeC
Gene, SSeC gene is positioned on pathogenicity island-2 (SPI-2), and sequence is the most conservative, the toxin that SSeC gene code is expressed
Albumen can cause the symptoms such as body generation organ dysfunction, and therefore SSeC gene can be as detection Salmonella typhimurium
One important marker of bacterium.
At present, the detection method of Salmonella typhimurium has traditional microorganism culturing detection method, biochemical identification method, with point
Method for quick based on sub-biology and the detection technique based on immunology, wherein, with molecular biology
Based on method for quick include polymerase chain reaction (PCR), biochip technology etc., based on immunology
Detection technique include elisa (ELISA), isotope marked antibodies, immunofluorescence, immune magnetic
Isolation technics, automatic enzyme-labeled immunity detector etc., above-mentioned detection method contain microorganism culturing, foranalysis of nucleic acids, antigen-
The technology such as antibody response.But, the traditional detection method such as microorganism culturing detection method, biochemical identification method exist time-consuming long,
The shortcoming that sensitivity is low, accuracy in detection is low etc.；Although the method detection speed such as ELISA and PCR are fast, but there is operation
Loaded down with trivial details, the most contaminated, high to equipment requirements shortcoming, and the specificity detected is not highly desirable, it is impossible to meet
The requirement of the quickly detection of modern clinic and food.
For reply disadvantages mentioned above, in recent years, the method for detection Salmonella typhimurium occurs in that biosensor detection method.
Nano biological sensor is by having bioactive identification original paper and signal adapter forms, and bioactive substance generally includes
Enzyme, Ag-Ab, aptamers etc..Due to nano biological sensor have increasingly automated, miniaturization, can be complicated
System carries out the function of real-time monitoring etc., thus at aspects such as pharmacy, food, chemical industry, biomedicine, Clinical Laboratorys
Bring huge economic benefit and be widely applied prospect.But most nano biological sensor is sensitive owing to existing
Spend the shortcoming low, poor specificity, detection range are narrow, limit the use on a large scale of nano biological sensor.
Summary of the invention
It is an object of the invention to provide a kind of biosensor detecting Salmonella typhimurium and preparation, detection method, profit
With nano graphene oxide, absorption and the fluorescent quenching of single stranded DNA are built fluorescent probe, and then realize containing SSeC base
Because of the quick detection of Salmonella typhimurium and highly sensitive, specificity good, detection range width.
In order to solve above-mentioned technical problem, the present invention provides following technical scheme:
A kind of biosensor detecting Salmonella typhimurium, described biosensor includes graphene oxide and fluorescence mark
The nucleic probe of note, wherein, described graphene oxide is carrier, described graphene oxide and described fluorescently-labeled nucleic acid
Probe passes through non-covalent bond coupling, and the gene order of described fluorescently-labeled nucleic probe is the mutual of described SSeC gene order
Mend chain-ordering.
Preferably, the gene order of described fluorescently-labeled nucleic probe is 6-FAM-GCCTCCTCTG CCATCTCATTCG.
Preferably, the concentration of described fluorescently-labeled nucleic probe is 50nM, and the concentration of described graphene oxide is
0.02-0.09mg/mL。
Preferably, the concentration of described graphene oxide is 0.05mg/mL.
Preferably, described non-covalent bond is π-π covalent bond.
A kind of preparation method of the biosensor detecting Salmonella typhimurium, described preparation method includes:
Compound concentration is the graphene oxide solution of 1mg/ml；
By fluorescently-labeled nucleic probe degenerative treatments 5min under conditions of temperature is 95 DEG C；
Described graphene oxide solution being joined in the described fluorescently-labeled nucleic probe after degenerative treatments, stirring is all
Even, stand 15min, form the biosensor of detection Salmonella typhimurium.
Preferably, described compound concentration is that the graphene oxide solution of 1mg/ml includes:
Weigh 0.1g graphene oxide to be positioned in 100ml ultra-pure water, be placed in ultrasonic disperse 120min in ice bath and obtain
Described graphene oxide solution.
Preferably, described in stir, standing includes: it is glimmering that described graphene oxide solution adds after described degenerative treatments
After the nucleic probe of signal, use phosphate buffer dilution, and make both be sufficiently mixed, be 37 DEG C of conditions in temperature
Lower standing 15min.
A kind of using method of the biosensor detecting Salmonella typhimurium, described using method includes:
Build the standard curve of fluorescent value and Salmonella typhimurium concentration；
The DNA of detected sample is joined in biosensor formation mixed liquor, by described mixed liquor the temperature of 37 DEG C
Under hatch 30min；
Detect the fluorescent value of described mixed liquor after hatching end, and obtain Mus in described detection sample according to described standard curve
The concentration of salmonella typhi.
Preferably, described structure fluorescent value includes with the standard curve of Salmonella typhimurium concentration:
Choose graphene oxide and nucleic probe matches the concentration of optimum, prepare biosensor；
Prepare variable concentrations gradient containing SSeC DNA rat salmonella typhi solution；
The DNA rat salmonella typhi solution containing SSeC of described variable concentrations gradient is added separately to described bio-sensing
In device, and form detection system；
Described detection system is hatched at a temperature of 37 DEG C 30min, and detects described biosensor and described inspection respectively
The fluorescent value of survey system；
According to detected fluorescent value and described variable concentrations gradient containing SSeC DNA rat salmonella typhi solution
Concentration draw curve, obtain the standard curve of fluorescent value and Salmonella typhimurium concentration.
The invention provides a kind of biosensor detecting Salmonella typhimurium, described biosensor includes aoxidizing stone
Ink alkene and fluorescently-labeled nucleic probe, wherein, graphene oxide is carrier, and described graphene oxide and described nucleic acid are visited
Pin passes through non-covalent bond coupling, and the gene order of described nucleic probe is the complementary strand sequence of described SSeC gene order.This
The biosensor of the detection Salmonella typhimurium that invention provides is by with graphene oxide as carrier, based on graphite oxide
The non-covalent associations nucleic acid molecules of alkene and the characteristic of quencher fluorescence, on the surface of graphene oxide, non-covalent bond coupling is the most glimmering
The nucleic probe of signal, the biosensor of preparation detection Salmonella typhimurium.The biosensor that the present invention provides
When being in non-detection status, owing to the distance between graphene oxide and fluorescently-labeled nucleic probe is the least, therefore, raw
Thing sensor occurs resonance energy transfer, and then fluorescent quenching；When biosensor is in detection state, i.e. biological
When adding the single stranded DNA of detected sample in sensor, single stranded DNA can be complementarily shaped to double with fluorescently-labeled nucleic probe
Chain DNA, due to non-more than between graphene oxide and fluorescently-labeled nucleic probe of adhesion when forming double-stranded DNA
Covalent bonding forces, therefore, double-stranded DNA dissociates to get off from the surface of graphene oxide, and then recovers fluorescence, glimmering by detection
Light value just can learn the concentration in detected sample containing SSeC DNA rat salmonella typhi.The detection Mus that the present invention provides
The biosensor of salmonella typhi have simple to operate, detection accurately, Monitoring lower-cut is low, highly sensitive, specificity
Good feature, and can the detection by quantitative concentration containing SSeC DNA rat salmonella typhi.
Accompanying drawing explanation
For the technical scheme being illustrated more clearly that in the embodiment of the present invention, required use in embodiment being described below
Accompanying drawing be briefly described, it should be apparent that, for those of ordinary skills, do not paying creative labor
On the premise of Dong, it is also possible to obtain other accompanying drawing according to these accompanying drawings.
Fig. 1 is the preparation flow figure of the biosensor of the detection Salmonella typhimurium that the embodiment of the present invention provides；
Fig. 2 is graphene oxide and the fluorescence of the biosensor of the detection Salmonella typhimurium that the embodiment of the present invention provides
The concentration optimization figure of the nucleic probe of labelling；
Fig. 3 is the using method flow chart of the biosensor of the detection Salmonella typhimurium that the embodiment of the present invention provides；
Fig. 4 is target DNA and fluorescent value in the biosensor detecting Salmonella typhimurium that the embodiment of the present invention provides
Canonical plotting；
Fig. 5 is the biosensor specific detection figure of the detection Salmonella typhimurium that the embodiment of the present invention provides；
Fig. 6 is the biosensor detection testing sample sensitivity of the detection Salmonella typhimurium that the embodiment of the present invention provides
Linear diagram；
Fig. 7 is special when being the biosensor detection testing sample of the detection Salmonella typhimurium that the embodiment of the present invention provides
The collection of illustrative plates of property.
Detailed description of the invention
The biosensor of the detection Salmonella typhimurium that the embodiment of the present invention provides and preparation, detection method, utilization is received
Rice graphene oxide builds fluorescent probe to absorption and the fluorescent quenching of single stranded DNA, and then realizes containing SSeC DNA rat
The quick detection of salmonella typhi.
For the technical scheme making those skilled in the art be more fully understood that in the embodiment of the present invention, and the present invention is made to implement
The above-mentioned purpose of example, feature and advantage can become apparent from understandable, below in conjunction with the accompanying drawings to the technology in the embodiment of the present invention
Scheme is described in further detail.
The invention provides a kind of biosensor detecting Salmonella typhimurium, described biosensor includes aoxidizing stone
Ink alkene (graphene oxide, GO) and fluorescently-labeled nucleic probe (FAM-P), wherein, graphene oxide is for carrying
Body, graphene oxide and fluorescently-labeled nucleic probe pass through non-covalent bond coupling, the gene of fluorescently-labeled nucleic probe
Sequence is the complementary strand sequence of described SSeC gene order.
Concrete, the biosensor of the detection Salmonella typhimurium that the present invention provides is referred to as FAM-P/GO biology and passes
Sensor.GO is nano material, and FAM-P is the nucleic acid of CF 5(6)-Carboxyfluorescein (carboxy-fluorescein, FAM) labelling,
The gene order of FAM-P is the complementary strand sequence of SSeC gene order, and wherein, SSeC gene order is 5 '
-CGAATGAGATGGCAGAGGAGGC-3 ', then its complementary strand sequence i.e. gene order of FAM-P is
6-FAM-GCCTCCTCTG CCATCTCATTCG；Non-covalent bond between GO and FAM-P is combined into π-π covalent bond and combines,
The concentration of FAM-P be the concentration of 50nM, GO be 0.02-0.09mg/L.
The Cleaning Principle of the FAM-P/GO biosensor that the present invention provides is: non-covalent associations nucleic acid molecules based on GO
With the characteristic of quencher fluorescence, introduce FAM-P on the surface of GO.Particularly as follows: when FAM-P/GO biosensor is in non-
During detection state, when the most not adding target DNA, FAM-P is free rolled state, between GO and FAM-P
Distance is the least, therefore, resonance energy transfer, and then fluorescent quenching occurs in FAM-P/GO biosensor, can't detect
Fluorescent value；When FAM-P/GO biosensor is in detection state, i.e. FAM-P/GO biosensor add to be checked
During single stranded DNA (single-stranded DNA, the ssDNA) of test sample product, when i.e. adding target DNA, target DNA's
SsDNA can be complementarily shaped to double-stranded DNA (double-stranded DNA, dsDNA) with FAM-P, during owing to forming dsDNA
Adhesion more than the non-covalent bonding force between GO and FAM-P, therefore, dsDNA can dissociate to get off from the surface of GO,
And then recovery fluorescence, just can learn the DNA rat Salmonella typhimurium Han SSeC in detected sample finally by detection fluorescent value
The concentration of bacterium, detection is accurately, simple to operate.
Based on above-mentioned principle, the target DNA of addition is the most, and the double-strand of formation is the most, and fluorescence is replied the strongest,
And then can detection by quantitative containing the concentration of SSeC DNA rat salmonella typhi, meanwhile, the FAM-P/GO that the present invention provides
Biosensor also has the feature that Monitoring lower-cut is low, highly sensitive.
The embodiment of the present invention additionally provides the preparation method of FAM-P/GO biosensor, specifically refer to accompanying drawing 1.
The preparation method of FAM-P/GO biosensor includes:
S101: compound concentration is the GO solution of 1mg/ml；
S102: by FAM-P degenerative treatments 5min under conditions of temperature is 95 DEG C；
S103: join in FAM-P by GO solution, stir, stands 15min, forms FAM-P biosensor.
Wherein, compound concentration is that the GO solution concrete operations of 1mg/ml are: weighing 0.1g GO, to be positioned over 100ml ultrapure
In water, it is placed in ultrasonic disperse 120min in ice bath, and then GO solution can be obtained；Stirring, standing includes:
After described GO solution adds the FAM-P after described degenerative treatments, use phosphate buffer (phosphate buffer
Saline, PBS) dilution, and make both be sufficiently mixed, under the conditions of temperature is 37 DEG C, stand 15min.
The preparation method of FAM-P/GO biosensor that the embodiment of the present invention provides is simple, easily operated, will not be dirty
Dye, the requirement to equipment is the highest, it is adaptable to laboratory, unit or the preparation of factory on a large scale.In the preparation present invention
During the FAM-P/GO biosensor that embodiment provides, the SSeC gene order of Salmonella typhimurium and FAM-P base
Because of sequence it is all various nucleotide sequences used in outsourcing, and the description below and various thalline is outsourcing, the present invention
This is not limited.
Owing to FRET (fluorescence resonance energy transfer) is affected very big, meanwhile, for making this by nano material GO and FAM-P ratio
Bright embodiment provide FAM-P/GO biosensor have sensitive detection performance and detect extremely low concentration containing SSeC base
Because of the performance of Salmonella typhimurium, embodiments provide FAM-P/GO biosensor for being ground by ratio optimization
Studying carefully, concrete research contents is:
Set in FAM-P/GO biosensor system the constant concentration of FAM-P solution as 50nM, investigate GO solution concentration
Be respectively 0.02mg/mL, 0.04mg/mL, 0.05mg/mL, 0.06mg/mL, 0.07mg/mL, 0.08mg/mL,
Fluorescent value during 0.09mg/mL.Specific implementation process is: be separately added into ultrasonic place in the FAM-P solution of certain volume
The GO solution of the variable concentrations after reason forms mixed liquor, and above-mentioned mixed liquor is degenerative treatments under conditions of temperature is 95 DEG C
5min, and it is diluted to 100ul formation composite solution with PBS, this composite solution is sufficiently mixed and stands 15min at 37 DEG C,
Detect each fluorescent value.When carrying out fluorescent value detection, arranging excitation wavelength is 480nm, launches wavelength 517nm, measures multiple
Close solution fluorescence value F and Raw fluorescence value F0, by GO solution concentration and fluorescence relative value F/F0Relation between-1 is learnt
When the concentration of FAM-P solution is 50nM, the optium concentration of the GO solution matched with FAM-P, specifically refer to accompanying drawing 2.
Accompanying drawing 2 shows the concentration of FAM-P Yu GO in the FAM-P/GO biosensor system that the embodiment of the present invention provides
Optimize figure.It can be seen that along with the continuous increase of GO concentration from accompanying drawing 2, FAM-P/GO biosensor system
Fluorescence relative value F/F0-1 constantly raises, and this shows to be notable downward trend along with the increase fluorescent value of GO concentration, when
GO concentration reach 0.05mg/mL and above time, fluorescence relative value F/F0-1 is almost unchanged and close to 1.0, and exceedes
The fluorescence of the FAM-P of 95%, by GO quencher, it is possible to show that in FAM-P/GO biosensor system, FAM-P is the completeest
Entirely being attached to GO surface, there is not free FAM-P in solution, background value is low, and now, the concentration of FAM-P solution is
50nM, the optium concentration of the GO solution matched with FAM-P is 0.05mg/mL.
The embodiment of the present invention additionally provides the using method of FAM-P/GO biosensor, specifically refer to accompanying drawing 3.
The using method of FAM-P/GO biosensor includes:
S201: build the standard curve of fluorescent value and Salmonella typhimurium concentration；
S202: the ssDNA of detected sample is joined formation mixed liquor in FAM-P/GO biosensor, by described
Mixed liquor hatches 30min at a temperature of 37 DEG C；
S203: detect the fluorescent value of described mixed liquor after hatching end, and obtain described detection sample according to described standard curve
The concentration of Salmonella typhimurium in product.
Wherein, the standard curve building fluorescent value and Salmonella typhimurium concentration in step S201 includes again:
S2011: choose GO and FAM-P match optimum concentration, prepare FAM-P/GO biosensor；
S2012: prepare variable concentrations gradient containing SSeC DNA rat salmonella typhi solution；
S2013: the DNA rat salmonella typhi solution containing SSeC of described variable concentrations gradient is added separately to
In FAM-P/GO biosensor, and form detection system；
S2014: described detection system is hatched at a temperature of 37 DEG C 30min, and detect respectively described biosensor and
The fluorescent value of described detection system；
S2015: according to detected fluorescent value and described variable concentrations gradient containing SSeC DNA rat Salmonella typhimurium
The concentration of bacterium solution draws curve, obtains the standard curve of fluorescent value and Salmonella typhimurium concentration.
Build the fluorescent value standard curve with Salmonella typhimurium concentration particularly as follows: according to the concentration set be
The FAM-P solution of 50nM, and the GO solution allocation usefulness that optium concentration is 0.05mg/L matched with FAM-P is excellent
Good FAM-P/GO biosensor complex liquid system, arrange variable concentrations gradient containing SSeC DNA rat Salmonella typhimurium
Bacterium solution, this SSeC gene is target DNA, and the concentration of Salmonella typhimurium solution be set to 0.05 μm/L,
0.1μm/L、0.2μm/L、0.3μm/L、0.4μm/L、0.5μm/L、0.6μm/L、0.7μm/L、0.8μm/L、0.9μm/L、
With 1.0 μm/L.The target DNA of above-mentioned 11 variable concentrations is added separately to FAM-P/GO biosensor complex liquid
In system, under conditions of temperature is 37 DEG C, hatch 30min, measure fluorescent value the most respectively.Carrying out fluorescent value detection
Time, arranging excitation wavelength is 480nm, launches wavelength 517nm, measures composite solution fluorescent value F, dense with target DNA
Degree is abscissa, and fluorescent value is that vertical coordinate utilizes software Origin8.0 to draw standard curve, obtains the linear of standard curve
Regression equation, specifically refer to accompanying drawing 4.
Accompanying drawing 4 shows target DNA and the mark of fluorescent value in the FAM-P/GO biosensor that the embodiment of the present invention provides
Directrix curve figure.Can be learnt by the curve in accompanying drawing 4, when the concentration of target DNA is in the range of 0.05-1.0 μm/L, target
Mark DNA presents good linear relationship with fluorescent value, and target DNA with the equation of linear regression of fluorescent value is
Y=23.37X-174.915, linearly dependent coefficient is 0.9921, it is possible to explanation, when the concentration of target DNA is relatively low,
The FAM-P/GO biosensor that the embodiment of the present invention provides remains able to target DNA be detected, further relates to the present invention real
The FAM-P/GO biosensor that executing example provides has relatively low Monitoring lower-cut.
The embodiment of the present invention additionally provides the detection specific experimentation of FAM-P/GO biosensor system, specifically real
Testing content is: configures three kinds of concentration and is the base mispairing DNA of 1.0 μm/L, wherein, mismatches base number and be respectively 1,4
With 5, the base sequence that mismatches of its correspondence is: CGAATGAGATGGCAGAGGAGTC、CGGACGAGATGGAAGAGGAGTC
And CGGGCGAGATGGAAGAGGAGTC, with the base of underscore for mismatching base；Do not add target DNA and complete complementary
Target sequence DNA as a control group, and the concentration of matched group is also 1.0 μm/L, by the DNA of above-mentioned three kinds of base mispairings
And matched group DNA is added separately in the FAM-P/GO biosensor detection system prepared of same volume, and all exist
Temperature is to hatch 30min at 37 DEG C, measures the fluorescent value of FAM-P/GO biosensor system, and concrete outcome refer to attached
Fig. 5.
Accompanying drawing 5 shows the detection FAM-P/GO biosensor system specific detection figure that the embodiment of the present invention provides,
Wherein a is the detection fluorescent value not adding target DNA, and b is the detection fluorescent value of 5 mismatched dnas mismatching base number, c
Being the detection fluorescent value of 4 mismatched dnas mismatching base number, d is that the detection of 1 mismatched dna mismatching base number is glimmering
Light value, e is the detection fluorescent value of target sequence DNA of complete complementary.From accompanying drawing 5 it can be seen that the fluorescent value of e
Height, target sequence DNA of this explanation complete complementary can make the detection fluorescent value of FAM-P/GO biosensor system notable
Restore, thus illustrate, target sequence DNA of complete complementary be combined with FAM-P complementary pairing after from the surface dissociation of GO
Come.And after not adding target DNA, mismatching the mismatched dna of base number, the detection of FAM-P/GO biosensor system
Fluorescent value is extremely low, even if only 1 base there are differences, the detection fluorescent value of target sequence DNA of complete complementary is 1
More than 4 times of the detection fluorescent value of the individual mismatched dna mismatching base number, this explanation base mismatch sequence can not make FAM-P
Splitting away off from GO, thus illustrate, the FAM-P/GO biosensor that the embodiment of the present invention provides has the highest special
Property and selectivity, it is possible to well distinguish target sequence and mismatch sequence, and then same Pseudomonas different serotypes can be differentiated
Salmonella, and during difference reaction interference less, speed fast.
The embodiment of the present invention additionally provides the experimentation of FAM-P/GO biosensor detection testing sample sensitivity, tool
Internal appearance is: Salmonella typhimurium is cultivated under conditions of temperature is 37 DEG C 24h, after plate count, prepares dense
Degree is respectively 103CFU/mL、104CFU/mL、105CFU/mL、106CFU/mL、107CFU/mL and 108The thalline of CFU/mL,
The thalline of above-mentioned variable concentrations is positioned over 15min in the water-bath of 95 DEG C, then places 10min on ice corresponding to obtain
SsDNA, is separately added into the ssDNA obtained in the FAM-P/GO biosensor system configured, is 37 DEG C in temperature
Under conditions of hatch 30min, detect the fluorescent value of each system the most respectively, and according to the fluorescent value of measured each system
Drawing linearity curve with the logarithm of cell concentration, linearity curve result specifically refer to accompanying drawing 6.
Accompanying drawing 6 shows that the FAM-P/GO biosensor that the embodiment of the present invention provides detects the line of testing sample sensitivity
Linearity curve figure.Can learn from accompanying drawing 6, along with the continuous increase of cell concentration, fluorescent value significantly increases, and upper
Stating the logarithm of fluorescent value and cell concentration in concentration range is good linear relationship, the logarithm of fluorescent value and cell concentration
Linear equation is Y=29.192X-45.239, and linearly dependent coefficient is 0.9987, it is possible to explanation, and FAM-P/GO is raw
Thing sensor systems is able to detect that the lower limit of thalline is 103CFU/mL, further relates to what the embodiment of the present invention provided
FAM-P/GO biosensor has the highest sensitivity when detecting measuring samples, and is able to detect that the bacterium of low concentration
Body.
Further, the embodiment of the present invention additionally provides the detection specific experiment of testing sample of FAM-P/GO biosensor
Research, concrete research contents is: by the ssDNA of 20 kinds of different thalline, and being each configured to concentration is 5 × 107CFU/ml's is molten
Liquid, wherein, 20 kinds of different thalline are 1-Salmonella typhimurium；2-Salmonella paratyphi A；3-Salmonella enteritidis；
4-Salmonella choleraesuls；5-shigella；6-staphylococcus aureus；7-E.coli K88；8-Bacillus proteus；
9-vibrio parahaemolyticus；10-hemolytic vibrio；11-Listerella；12-campylobacter jejuni；13-streptococcus thermophilus；14-
Bacillus subtilis；15-Bacillus licheniformis；16-bifidobacterium thermophilum；17-bifidobacterium longum；18-bifidobacterium breve 19-
Bacillus acidophilus, 20-lactobacillus casei, above-mentioned thalline all can use outsourcing thalline.Setting is not added with the ssDNA of thalline
Blank group, is 21, the ssDNA of blank group from above-mentioned 20 kinds of different thalline is separately added into the FAM-P/GO configured
In biosensor system, under conditions of temperature is 37 DEG C, hatch 30min, measure fluorescent value the most respectively, and with respectively
Thalline be arranged as abscissa, fluorescent value be vertical coordinate draw specific manner, concrete outcome refer to accompanying drawing 7.
Accompanying drawing 7 shows that the FAM-P/GO biosensor that the embodiment of the present invention provides detects during testing sample specific
Collection of illustrative plates.Can learn from accompanying drawing 7, the fluorescent value of No. 1 thalline dramatically increases and glimmering apparently higher than remaining 20 groups of thalline
Light value, the Salmonella of remaining kind of No. 2-4 produces part fluorescence owing to its sequence exists certain homology, but
Its fluorescent value is well below the fluorescent value of No. 1 thalline, and the fluorescent value of other kinds of thalline and blank group is lower, thus can
Enough explanations, the FAM-P/GO biosensor that the embodiment of the present invention provides has the most special when detecting measuring samples
Property.
Can be learnt by above-mentioned detection content and description, the FAM-P/GO biosensor that the embodiment of the present invention provides has
The feature that Monitoring lower-cut is low, highly sensitive, specificity is good, and can detection by quantitative DNA rat salmonella typhi Han SSeC
Concentration.
Invention described above embodiment, is not intended that limiting the scope of the present invention.Any the present invention's
Amendment, equivalent and the improvement etc. made within spirit and principle, should be included within the scope of the present invention.
Claims (10)
1. the biosensor detecting Salmonella typhimurium, it is characterised in that described biosensor includes oxidation
Graphene and fluorescently-labeled nucleic probe, wherein, described graphene oxide is carrier, described graphene oxide and described
Fluorescently-labeled nucleic probe passes through non-covalent bond coupling, and the gene order of described fluorescently-labeled nucleic probe is described
The complementary strand sequence of SSeC gene order.
The biosensor of detection Salmonella typhimurium the most according to claim 1, it is characterised in that described glimmering
The gene order of the nucleic probe of signal is 6-FAM-GCCTCCTCTG CCATCTCATTCG.
The biosensor of detection Salmonella typhimurium the most according to claim 1, it is characterised in that described glimmering
The concentration of the nucleic probe of signal is 50nM, and the concentration of described graphene oxide is 0.02-0.09mg/mL.
The biosensor of detection Salmonella typhimurium the most according to claim 3, it is characterised in that described oxygen
The concentration of functionalized graphene is 0.05mg/mL.
The biosensor of detection Salmonella typhimurium the most according to claim 1, it is characterised in that described non-
Covalent bond is π-π covalent bond.
6. a preparation method for the biosensor of detection Salmonella typhimurium as claimed in claim 1, its feature
Being, described preparation method includes:
Compound concentration is the graphene oxide solution of 1mg/ml；
By fluorescently-labeled nucleic probe degenerative treatments 5min under conditions of temperature is 95 DEG C；
Described fluorescently-labeled nucleic probe after described graphene oxide solution is joined degenerative treatments joins described oxygen
In functionalized graphene solution, stir, stand 15min, form the biosensor of detection Salmonella typhimurium.
The preparation method of the biosensor of detection Salmonella typhimurium the most according to claim 6, its feature exists
In, described compound concentration is that the graphene oxide solution of 1mg/ml includes:
Weigh 0.1g graphene oxide to be positioned in 100ml ultra-pure water, be placed in ultrasonic disperse 120min in ice bath and obtain
Described graphene oxide solution.
The most according to claim 6 based on graphene oxide detection DNA rat typhoid fever Salmonella mouse typhus sramana Han SSeC
The preparation method of the biosensor of Salmonella, it is characterised in that described in stir, standing includes:
After described graphene oxide solution adds the fluorescently-labeled nucleic probe after described degenerative treatments, phosphate is used to delay
Rush liquid dilution, and make both be sufficiently mixed, under the conditions of temperature is 37 DEG C, stands 15min.
9. a using method for the biosensor of the detection Salmonella typhimurium as described in claim 1 or 6, its
Being characterised by, described using method includes:
Build the standard curve of fluorescent value and Salmonella typhimurium concentration；
The DNA of detected sample is joined in biosensor formation mixed liquor, by described mixed liquor the temperature of 37 DEG C
Under hatch 30min；
Detect the fluorescent value of described mixed liquor after hatching end, and obtain Mus in described detection sample according to described standard curve
The concentration of salmonella typhi.
The using method of the biosensor of detection Salmonella typhimurium the most according to claim 9, its feature
Being, described structure fluorescent value includes with the standard curve of Salmonella typhimurium concentration:
Choose graphene oxide and fluorescently-labeled nucleic probe matches the concentration of optimum, prepare biosensor；
Prepare variable concentrations gradient containing SSeC DNA rat salmonella typhi solution；
The DNA rat salmonella typhi solution containing SSeC of described variable concentrations gradient is added separately to described biosensor
In, and form detection system；
Described detection system is hatched at a temperature of 37 DEG C 30min, and detects described biosensor and described inspection respectively
The fluorescent value of survey system；
According to detected fluorescent value and described variable concentrations gradient containing SSeC DNA rat salmonella typhi solution
Concentration draws curve, obtains the standard curve of fluorescent value and Salmonella typhimurium concentration.
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