CN103901210A - Listeria monocytogenes detecting method based on optical fiber evanescent wave biosensor - Google Patents
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Abstract
The invention discloses a listeria monocytogenes detecting method based on an optical fiber evanescent wave biosensor. The method comprises the following steps: coupling a CdTe quantum dot with a listeria monocytogenes polyclonal antibody; covering an optical fiber with a listeria monocytogenes monoclonal antibody; inserting an optical fiber probe into a solution of bacteria to be detected and fixing an antigen on the optical fiber by specific reaction between the antigen and a captured antibody; inserting the optical fiber probe into a solution containing a detection antibody to combine the nano quantum dot on the antibody on the surface of the optical fiber; and detecting the listeria monocytogenes by using the optical fiber evanescent wave biosensor. The method has an important application value to the detection rate of pathogenic microorganisms in foods and has the important meaning on promoting the human health and the economic development in China.
Description
Technical field
The invention belongs to immunological technique and health detection technique field, be specifically related to a kind of method that detects single increasing listeria spp based on biologic sensor for fast travelling waves of optical fibre.
Background technology
The detection of Listeria Monocytogenes at present has had multiple detection method, as traditional cultivation detection method, immunological detection method, round pcr and nucleic acid probe etc.These traditional detection methods comprise front increasing bacterium, selective enrichment, the checking of microscopy and serology, and experiment that its method relates to is loaded down with trivial details, complicated operation, need to consume the long period, and be difficult to accurate judgement.
Summary of the invention
The object of this invention is to provide a kind of single method that increases listeria spp in safe, quick, sensitive detection food, a kind of method that detects single increasing listeria spp based on biologic sensor for fast travelling waves of optical fibre.
Detect a single method that increases listeria spp based on biologic sensor for fast travelling waves of optical fibre, it comprises:
1) CdTe quantum dot and single coupling that increases listeria spp polyclonal antibody
Getting water-soluble CdTe quantum dots mixes with PBS damping fluid, single listeria spp polyclonal antibody that increases joins in above-mentioned mixed liquor, then will add freshly prepd EDC solution, biased sample lucifuge in 37 ℃ of incubators is reacted 2 hours, then 4 ℃ are spent the night, and reaction mixture is centrifugal, and supernatant carries out ultrafiltration repeatedly with ultra filtration membrane, remove small-molecule substance and unreacted CdTe quantum dot, collect the CdTe-polyclonal antibody conjugate that ultra filtration membrane is held back;
2) preparation of capture antibody optical fiber probe
By polystyrene optical fiber surface, first clean with hydrochloric acid and alcohol mixeding liquid, then use sulfuric acid cleaned, next in ultrapure water, boil, make optical fiber surface there is hydrophilic polarization layer; Then the optical fiber of handling well is put into single solution that increases listeria spp monoclonal antibody and soaked, take out with deionized water and clean, obtain being coated with capture antibody optical fiber probe;
3) single detection that increases listeria spp
CdTe-polyclonal antibody conjugate is made to aqueous solution, capture antibody optical fiber probe is inserted in solution to be measured, and then capture antibody optical fiber probe is put into CdTe-polyclonal antibody conjugate and make aqueous solution, detect with biologic sensor for fast travelling waves of optical fibre;
Biased sample described in step 1), comprises 1.0mL quantum dot, 1.0mL PBS, 400uL antibody, 100uL EDC;
Step 2) described optical fiber is polystyrene optical fiber;
Step 2) solution of described monoclonal antibody is 15 μ g/ml;
It is that 100 μ g/ml detect polyclonal antibody that CdTe-polyclonal antibody conjugate described in step 3) is made aqueous solution.
This method has been set up a kind of method of the quanta point optical fiber probe biology sensor detection Listeria monocytogenes based on evanescent wave field excitation.Quantum dot fluorescence optical fiber probe biology sensor based on evanescent wave field excitation is the one of biological sensor.It mainly combines the high specific and the highly sensitive feature that when light is propagated in optical fiber, produce evanescent wave field and the reaction of antibody-antigen immune.The principle of evanescent wave field excitation is: in the time that light transmits in total reflection mode in optical fiber, produce a kind of ripple that traverses optical fiber, spread out of optical fiber by the intersection of fibre core and covering, this ripple is exponential damping with propagation distance, is called evanescent wave.Because the evanescent wave depth of field being sent in microbial solution to be measured only has wavelength magnitude, so biologic sensor for fast travelling waves of optical fibre can only detect the fluorescence that the fluorescent dye that is incorporated within the scope of evanescent wave field sends, and in solution free fluorescent dye on measurement result without impact.Therefore compared with other biological detection means, biologic sensor for fast travelling waves of optical fibre tool has the following advantages: 1) highly sensitive, biologic specificity is strong; 2) simple to operate, measuring speed is fast; 3) can carry out dynamic monitoring to bioprocesses; 4) can make instrument miniaturization.
And quantum dot has exciting light spectrum width, and continuous distribution, emission spectrum be symmetric and width narrow, color is adjustable with particle size, the quantum dot of different emission wavelengths can be with the optical excitation of Same Wavelength, and photochemical stability is high, is difficult for decomposing.Quantum dot replaces the pathogenic microorganism in traditional detection of fluorescent dyes food, it has obtained stronger fluorescence intensity, longer fluorescent lifetime and the signal having amplified, thus make to have reduced greatly the detectability of testing sample.
The invention provides a kind of method that detects single increasing listeria spp based on biologic sensor for fast travelling waves of optical fibre, it is with CdTe quantum dot and single coupling that increases listeria spp polyclonal antibody, single coated optical fiber of listeria spp monoclonal antibody that increases, optical fiber probe inserts in the solution that contains bacterium to be measured, thereby the idiosyncrasy that antigen and capture antibody are occurred is fixed on antigen on optical fiber, and then this optical fiber probe insertion is sent out and contained in the solution that detects antibody, the nano-quantum point that makes to detect on antibody is also coupled to optical fiber surface, recycling biologic sensor for fast travelling waves of optical fibre detects single listeria spp that increases, the method has improved detection efficiency, the recall rate that improves pathogenic microorganism in food is had to significant application value, for promoting that the economic development of human health and China is significant.
Accompanying drawing explanation
The detection sensitivity experimental result of the mono-increasing of Fig. 1 listeria spp;
Fig. 2. single specific detection result that increases listeria spp.
Embodiment
preparation and the BALB/c mouse immunity of embodiment 1 Listeria monocytogenes antigen bacteria liquid
Get ℃ preservation of these chamber-80 Listeria monocytogenes (
aTCC19111), method of scoring is inoculated in the upper cultivation of trypticase soy yeast extract agar (TSA) nutrient culture media (pH7.3 ± 0.1), and conventional method increases bacterium cultivates, the centrifugal 10min of 6000r/min, collect bacterial sediment, repeat to carry out colony counting (2.4 × 10 after washing three times with sterile saline
9cFU/mL), adding final concentration is that 4 ℃, 0.3% formaldehyde spends the night and makes thalline deactivation.Next day bacterium liquid good wash-out is carried out to ultrasound wave under the condition of ice bath of 20KHZ, 150W and make somatic cells fragmentation, each broken 10s, interval 10s, total used time 20min.Adopt BCA protein determination kit to measure mycoprotein content, result is 2.548 mg/mL.
Get 8 week age female BALB/c mouse and carry out immunity.Head exempts to adopt above-mentioned inactivated bacterial liquid (2 × 10
8cFU/mL) inject 50 μ L with equivalent Freund's complete adjuvant mixing pneumoretroperitoneum, hypodermic injection 50 μ L every immunity in 2 weeks 1 time, use incomplete Freund's adjuvant instead later, and dosage is the same, exempts from altogether 3 times.
3 exempt from after, mouse docking blood sampling, with conventional indirect ELISA method detection serum titer.Wherein, envelope antigen is the prepared Listeria monocytogenes bacterium liquid of embodiment 1, dilutability is 1:40, positive serum dilutability 1:1600, and ELIAS secondary antibody is the anti-mouse IgG of rabbit (dilutability is 1:15000) of HRP (horseradish peroxidase) mark.Measurement result shows, antibody titer is 1:12800.
the coupling of embodiment 2 CdTe quantum dots and Listeria Monocytogenes polyclonal antibody
Get the water-soluble CdTe quantum dots and 1mL PBS(pH=7.4 of 1mL) damping fluid mixes, the Listeria Monocytogenes polyclonal antibody (1mg/ml) of 500uL is joined in above-mentioned mixed liquor, then freshly prepd 100uL EDC solution (4mg/ml) is joined in mixed liquor, sample lucifuge in 37 ℃ of incubators is reacted 2 hours, then 4 ℃ are spent the night, 2.5mL reaction mixture (is comprised to 1.0mL quantum dot, 1.0mL PBS, 400uL antibody, 100uL EDC) the centrifugal 2min of 5000r/min, get supernatant, the ultra filtration membrane that supernatant is 100000Da with molecular cut off carries out ultrafiltration repeatedly, remove small-molecule substance and unreacted CdTe quantum dot, collect the CdTe-antibody coupling matter that ultra filtration membrane is held back, then use PBS(pH7.4) dissolve.4 ℃ keep in Dark Place for subsequent use.
being coated with of embodiment 3 optical fiber and capture antibody
By polystyrene optical fiber surface, first use cleaning fluid (the dense HCL:70-90% volumes of aqueous ethanol of 36-38% is than being 1:1) to clean 10 minutes, clean 10 minutes with 70% concentrated sulphuric acid again, next in ultrapure water, boil 10 minutes, make optical fiber surface there is hydrophilic polarization layer.Then the optical fiber of handling well is put into the anti-listeria monocytogenes monoclonal antibody of 15 μ g/ml (deposit number CGMCC No.6252, China Committee for Culture Collection of Microorganisms of depositary institution common micro-organisms center, No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, address, preservation date on June 20th, 2012, the strain of anti-listeria monocytogenes monoclonal antibody hybridoma cell) solution in soak 2h, take out with deionized water and clean, obtain being coated with capture antibody optical fiber probe.
To be coated with capture antibody optical fiber probe and put into solution to be measured, if there is the Listeria Monocytogenes reacting with capture antibody on optical fiber probe in this solution, thereby because this bacterium can form compound molecule with capture antibody specific binding; And then optical fiber probe is put into the 100 μ g/ml that contain nano-quantum point mark and detect antibody-solutions, keep 10min, the detection antibody Listeria Monocytogenes that capture antibody is combined on optical fiber in this solution is by immune response combination, thereby nano-quantum point is fixed on optical fiber, then detects by biologic sensor for fast travelling waves of optical fibre (biologic sensor for fast travelling waves of optical fibre and optical fiber provide by Chinese Academy of Sciences's Changchun optical precision optical machinery and CAS Institute of Physics).
determining of embodiment 5 detection sensitivities
To singly increase listeria spp in Half-Fraser fluid nutrient medium, cultivate 18 h～24 h for 30 ℃.Then carry out secondary and increase bacterium, get 0.1 ml and increase for the first time the culture of bacterium, join in 10 mL Fraser nutrient solutions, put 35 ℃ and cultivate 24 h.TSA-YE agar plate is counted.It is 3 × 10 that the optical fiber that Sheet is increased to listeria spp monoclonal antibody inserts respectively concentration
1cUF/mL, 3.3 × 10
2cUF/mL, 3 × 10
3cUF/mL, 3 × 10
4cUF/mL, 3 × 10
5cUF/mL and 3 × 10
6cUF/mL single increases hatches 10 min in listeria spp solution and adds negative control simultaneously, and PBS cleans after three times the how anti-10min that reacts with mark quantum dot, and measurement result as shown in Figure 1.When single increasing listeria spp cell concentration is 3 × 10
1-3 × 10
6when CUF/mL, positive signal can be detected; When cell concentration is less than 3 × 10
1when CUF/mL, result is negative.
embodiment 6 specific tests
The method that detects Listeria monocytogenes with the immune optical fiber biosensor of setting up is to known Listeria monocytogenes (ATCC19111), C.perfringens (ATCC13124), sheep listeria spp (ATCC11387), listeria innocua (ATCC19119), staphylococcus aureus (ATCC25923), Bacterium enteritidis (ATCC13076), salmonella typhi (ATCC13311), Escherichia coli (ATCC25922), Bacillus cereus (ATCC11778)), Escherichia coli O 157 (ATCC35150), the bacteriums such as campylobacter jejuni (ATCC33291) carry out specificity cross matching, establish positive control simultaneously, blank.Result only has Listeria Monocytogenes to detect as shown in Figure 2, has stronger signal.
embodiment 7the artificial detection of adding sample
Get in the Half-Fraser fluid nutrient medium that chicken 25g adds 225mL, add respectively Listeria monocytogenes suspension, make its concentration reach respectively 5CFU/mL, 10CFU/mL, 20CFU/mL, 30CFU/mL, 40CFU/mL, 50CFU/mL.After homogeneous, get 2mL supernatant, boiling sterilization, respectively get 1 mL for detection of.Every group of test repeats 3 times.Testing result is as shown in table 1, can be detected if its detection sensitivity can reach single listeria spp that increases that 30 CFU/mL contain 30CFU/mL in sample.
Claims (5)
1. detect a single method that increases listeria spp based on biologic sensor for fast travelling waves of optical fibre, it comprises:
1) CdTe quantum dot and single coupling that increases listeria spp polyclonal antibody
Getting water-soluble CdTe quantum dots mixes with PBS damping fluid, single listeria spp polyclonal antibody that increases joins in above-mentioned mixed liquor, then will add freshly prepd EDC solution, biased sample lucifuge in 37 ℃ of incubators is reacted 2 hours, then 4 ℃ are spent the night, and reaction mixture is centrifugal, and supernatant carries out ultrafiltration repeatedly with ultra filtration membrane, remove small-molecule substance and unreacted CdTe quantum dot, collect the CdTe-polyclonal antibody conjugate that ultra filtration membrane is held back;
2) preparation of capture antibody optical fiber probe
By polystyrene optical fiber surface, first clean with hydrochloric acid and alcohol mixeding liquid, then use sulfuric acid cleaned, next in ultrapure water, boil, make optical fiber surface there is hydrophilic polarization layer; Then the optical fiber of handling well is put into single solution that increases listeria spp monoclonal antibody and soaked, take out with deionized water and clean, obtain being coated with capture antibody optical fiber probe;
3) single detection that increases listeria spp
CdTe-polyclonal antibody conjugate is made to aqueous solution, capture antibody optical fiber probe is inserted in solution to be measured, and then capture antibody optical fiber probe is put into CdTe-polyclonal antibody conjugate and make aqueous solution, detect with biologic sensor for fast travelling waves of optical fibre.
2. a kind of method that detects single increasing listeria spp based on biologic sensor for fast travelling waves of optical fibre according to claim 1, is characterized in that: the biased sample described in step 1), comprises 1.0mL quantum dot, 1.0mL PBS, 400uL antibody, 100uL EDC.
3. according to claim 2ly a kind ofly detecting single method that increases listeria spp based on biologic sensor for fast travelling waves of optical fibre, it is characterized in that: step 2) described optical fiber is polystyrene optical fiber.
4. according to claim 3ly a kind ofly detecting single method that increases listeria spp based on biologic sensor for fast travelling waves of optical fibre, it is characterized in that: step 2) solution of described monoclonal antibody is 15 μ g/ml.
5. a kind of method that detects single increasing listeria spp based on biologic sensor for fast travelling waves of optical fibre according to claim 4, is characterized in that: it is that 100 μ g/ml detect polyclonal antibodies that the CdTe-polyclonal antibody conjugate described in step 3) is made aqueous solution.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104133061A (en) * | 2014-08-12 | 2014-11-05 | 吉林出入境检验检疫局检验检疫技术中心 | Method for detecting golden glucose coccus by using immune optical-fiber evanescent-wave biosensor |
| CN112083159A (en) * | 2019-06-13 | 2020-12-15 | 首都师范大学 | Evanescent wave aptamer sensor and method for detecting small molecules by applying evanescent wave aptamer sensor |
| WO2021018605A1 (en) * | 2019-07-26 | 2021-02-04 | Borealis Ag | Method for removing ink or other foreign materials from the surface of an article |
| WO2023098087A1 (en) * | 2021-12-01 | 2023-06-08 | 西湖大学 | Method for measuring plasma concentration of small molecule drug |
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| US20050111805A1 (en) * | 2003-06-09 | 2005-05-26 | Erik Hertz | Optical fiber with quantum dots |
| CN101118216A (en) * | 2007-09-07 | 2008-02-06 | 中国科学院长春光学精密机械与物理研究所 | Optical fibre biological detecting probe, manufacturing method and use thereof |
| CN101393201A (en) * | 2008-11-06 | 2009-03-25 | 中国科学院长春光学精密机械与物理研究所 | Multi biological target rapid detecting system and use thereof |
| CN102507948A (en) * | 2011-11-15 | 2012-06-20 | 吉林出入境检验检疫局检验检疫技术中心 | Method using liquid phase chip to detect mononuclear cell proliferation listeria |
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| US20050111805A1 (en) * | 2003-06-09 | 2005-05-26 | Erik Hertz | Optical fiber with quantum dots |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104133061A (en) * | 2014-08-12 | 2014-11-05 | 吉林出入境检验检疫局检验检疫技术中心 | Method for detecting golden glucose coccus by using immune optical-fiber evanescent-wave biosensor |
| CN112083159A (en) * | 2019-06-13 | 2020-12-15 | 首都师范大学 | Evanescent wave aptamer sensor and method for detecting small molecules by applying evanescent wave aptamer sensor |
| WO2021018605A1 (en) * | 2019-07-26 | 2021-02-04 | Borealis Ag | Method for removing ink or other foreign materials from the surface of an article |
| WO2023098087A1 (en) * | 2021-12-01 | 2023-06-08 | 西湖大学 | Method for measuring plasma concentration of small molecule drug |
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