CN204203160U - The colibacillary electrochemical sensor of a kind of detection - Google Patents
The colibacillary electrochemical sensor of a kind of detection Download PDFInfo
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- CN204203160U CN204203160U CN201420621074.2U CN201420621074U CN204203160U CN 204203160 U CN204203160 U CN 204203160U CN 201420621074 U CN201420621074 U CN 201420621074U CN 204203160 U CN204203160 U CN 204203160U
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Abstract
The utility model relates to sensor technical field, and particularly one detects colibacillary electrochemical sensor, is followed successively by electrode, Prussian blue-carbon nanotube-nano Au composite layer, Escherichia coli antibody layer, bovine serum albumin(BSA) confining bed from inside to outside.Preparation method is simple, stable performance, electrode reproducible, is applicable to the practical application of colibacillary detection and biology sensor industrialization in food security; Can realize, to quick online detection colibacillary in food, detecting and being limited to 3.4 × 10
cfu mL
-1.
Description
Technical field
The present invention relates to sensor technical field, particularly one detects colibacillary electrochemical sensor.
Background technology
Escherichia coli belong to Gram-negative bacteria, and formal name used at school large intestine wishes Erichsen bacterium, and whole body flagellum, can move, without gemma, be widely distributed at occurring in nature, especially exist in a large number in the enteron aisle and ight soil of warm-blooded animal.After Eskirdge in 1885 finds Escherichia coli, within the after this quite a long time, Escherichia coli are seen as harmless non-pathogenic bacteria always.Along with progress of research, Escherichia coli, according to the difference of somatic antigen, are divided into 150 many types of, mostly belong to intestinal tract normal flora clump by people, have 16 for enteropathogenic E．Coli.Escherichia coli can survive in environment certain hour to external world, and thus the soil of faecal contamination, water source may contain enteropathogenic E．Coli.Enteropathogenic E．Coli can cause serious diarrhoea and septicemia after being taken in by baby and cub (fowl), also can cause inflammation as non-pathogenic Escherichia coli enter the organ such as human gall bladder, bladder.Coliform value reflects the degree of the faecal contaminations such as food, and it is very necessary for thus detecting Escherichia coli flora number in food, medicine and potable water.
Detecting colibacillary classic method and mainly contain three kinds, is MTF method, filter membrane method and colony counting method respectively, and these methods have instrumentation complicated, need the shortcomings such as professional operator.
Summary of the invention
This research, for existing detection method Instrumental complicated operation, needs the shortcoming of professional operator, provides the colibacillary electrochemical sensor of a kind of detection.
The present invention is obtained by following steps:
The colibacillary electrochemical sensor of a kind of detection, is followed successively by electrode, Prussian blue-carbon nanotube-nano Au composite layer, Escherichia coli antibody layer, bovine serum albumin(BSA) confining bed from inside to outside.
Described electrochemical sensor, preferably Prussian blue-carbon nanotube-nano Au composite layer thickness is 90 ± 5nm, and Escherichia coli antibody layer thickness is 120 ± 10nm, and bovine serum albumin(BSA) enclosed layer thickness is 100 ± 10nm.
Described electrochemical sensor, preferably Prussian blue-carbon nanotube-nano Au composite layer is obtained by following steps:
(1) multi-walled carbon nano-tubes carries out carboxylated;
(2) nm of gold is prepared;
(3) product using step (1) and (2) to obtain prepares Prussian blue-carbon nanotube-nano Au composite.
Described electrochemical sensor, preferably Prussian blue in Prussian blue-carbon nanotube-nano Au composite: carbon nano-tube: golden mass ratio is 2:2:1.
Described electrochemical sensor, preferably
(1) after electrochemical sensor rinses with phosphate buffer, hatch in E.coli solution, again rinse with PBS, hatch completely after drying in Escherichia coli bacteria liquid, the Escherichia coli antibody of HRP mark is dripped after dry, the antibody of HRP mark, by the strong recognition capability between object, is modified at electrode surface;
(2) three-electrode system is adopted, take Ag/AgCl as contrast electrode, be to electrode with Pt electrode, modified electrode is working electrode, with the PBS containing p-dihydroxy-benzene and hydrogen peroxide for the detection of work end liquid employing differential pulse voltammetry, current potential is set to-0.2 to 0.6 V, pulse width 0.05V, pulse width scanning is 0.06 S, detects.
The preparation method of described electrochemical sensor, comprises the following steps:
(1) prepare Prussian blue-carbon nanotube-nano Au composite, be added drop-wise to treated electrode surface, drying at room temperature, obtain Prussian blue-carbon nanotube-nano Au composite layer;
(2) on Prussian blue-carbon nanotube-nano Au composite layer, unmarked Escherichia coli antibody is dripped, dry, obtain Escherichia coli antibody layer;
(3) at Escherichia coli antibody layer outer cladding bovine serum albumin(BSA), bovine serum albumin(BSA) confining bed is obtained.
Described preparation method, preferably the preparation method of Prussian blue-carbon nanotube-nano Au composite is as follows:
1) multi-walled carbon nano-tubes is carboxylated,
2) nm of gold is prepared,
3) Prussian blue-carbon nanotube-nano Au composite is prepared.
Described preparation method, preferred multi-walled carbon nano-tubes is carboxylated to be obtained by following steps:
Take the multi-walled carbon nano-tubes of 200 mg, add in the concentrated sulphuric acid of 160.0 mL and the mixed liquor of red fuming nitric acid (RFNA), wherein the volume ratio of the concentrated sulphuric acid and red fuming nitric acid (RFNA) is 3:1, mix, centrifugal, go supernatant to add deionized water washing after centrifugal, continue centrifugal, until supernatant is in neutral, be drying to obtain.
Described preparation method, preferably Prussian blue-carbon nanotube-nano Au composite is obtained by following steps:
The carboxylated multi-walled carbon nano-tubes of (1) 4 mg is distributed to the FeCl of 40.0 mL 0.01 M
3in solution;
(2) while stirring by the K of 40.0 mL 0.01 M
3fe(CN)
6dropwise adds the FeCl that step (1) obtains
3in solution;
(3) solution step (2) obtained is constantly centrifugal under 10000 r/min conditions, removes supernatant, until supernatant is colourless;
(4) sediment is through collection, drying, and being distributed to massfraction is in the 4.0 mL chitosan aqueous solution of 0.1 %, obtains the amino group modifying PB-CNTs;
(5) centrifugal segregation residue shitosan under 10000 r/min conditions;
(6) add in gold colloid stir 2 evenly by completing amido modified PB-CNTs, centrifuging and get final product.
Described preparation method, the concentration of preferred bovine serum albumin(BSA) is 0.1%.
Principle of work of the present invention:
First gold electrode is modified synergistic matter PB-CNTs-Au compound, not only can promote electrode surface electro transfer, and the connection of specific groups between synergistic matter can ensure their layer assembly.The amino of golden nanometer particle and capture antibody, passes through Au-NH
2effect, antibody modification on electrode.Then, antibody and Escherichia coli have single-minded recognition capability, and Escherichia coli just can successfully be modified on electrode.At the colibacillary other end, the Escherichia coli antibody of HRP mark, by the specific recognition capability with object, is also successfully modified.In testing process, by the p-dihydroxy-benzene (HQ) in the HRP catalysis detection end liquid on electrode and hydrogen peroxide (H
2o
2) redox produce electric signal, connect electrochemical workstation, take Ag/AgCl as contrast electrode, be to electrode with Pt electrode, current potential is set to-0.2 to 0.6 V, pulse width 0.05V, and pulse width scanning is 0.06 S, adopt differential pulse voltammetry technology to read the change of electric signal, the size according to the electric current of electrode surface generation plays the effect detected object.The amount of HRP fixing on electrode and the colibacillary amount of detected material of modification have direct relation, and detected material is more, and the amount of fixing HRP is also more, and the electric signal that catalysis produces is also stronger.
The PB-CNTs-Au composite conductivity that the present invention adopts is strong, becomes the excellent material building sensor; Use horseradish peroxidase (HRP), by with H
2o
2with the reaction of HQ, amplifying signal; Have employed the detection model of sandwich type, introduce antibody at the two ends detecting thing E.coli respectively, detect more sensitive; The transducer sensitivity of preparation is high, and detection speed is fast; Detect the method for E.coli, simple to operate, quick, sensitive, be convenient to Site Detection.
Beneficial effect of the present invention:
1, due to use gold electrode, its electrode is easy, miniaturization, portable, can repeatedly use;
2, responding layer uses surface modification technology fixing on the working electrode (s, and optimize the consumption and concentration that use material, the requirement of obtained sandwich type electrode pair environment temperature is not obvious, uses under room temperature;
3, preparation method is simple, stable performance, electrode reproducible, is applicable to the practical application of colibacillary detection and biology sensor industrialization in food security;
4, the process costs making electrode is low, is applicable to requirement inexpensive in industrialization;
5, take gold electrode as the sandwich type electrochemical sensing system that immobilization carrier fixes based on PB-CNTs-Au compound, can realize, to quick online detection colibacillary in food, detecting and being limited to 3.4 × 10cfu mL
-1.
Accompanying drawing explanation
Fig. 1 is the Making programme figure of electrochemical sensor of the present invention, and lower right corner curve map is the current-responsive that this sensor produces respectively when Escherichia coli exist and do not exist;
Fig. 2 is embodiment 1 testing result figure;
Fig. 3 is embodiment 2 testing result figure;
Fig. 4 is embodiment 3 testing result figure.
Embodiment
Below in conjunction with specific embodiment, the present invention is further described:
First Prussian blue-carbon nanotube-nano Au composite is prepared:
1) multi-walled carbon nano-tubes is carboxylated,
2) nm of gold is prepared,
3) Prussian blue-carbon nanotube-nano Au composite is prepared.
Multi-walled carbon nano-tubes is carboxylated to be obtained by following steps:
Take the multi-walled carbon nano-tubes of 200 mg, add in the concentrated sulphuric acid of 160.0 mL and the mixed liquor of red fuming nitric acid (RFNA), wherein the volume ratio of the concentrated sulphuric acid and red fuming nitric acid (RFNA) is 3:1, mix, centrifugal, go supernatant to add deionized water washing after centrifugal, continue centrifugal, until supernatant is in neutral, product is put in 70 DEG C of dryings in baking oven, puts into Refrigerator store for subsequent use.
Nm of gold is obtained by following steps:
(1) chlorauric acid solution of 1 mL1% is joined in 100 mL deionized waters boil;
(2) joined fast in the chlorauric acid solution of backflow by the sodium citrate of 2.5 mL1%, continuation refluxes more than half an hour, until solution becomes peony from light yellow;
(3) uniform liquid represents the success of preparation without precipitation, puts into Refrigerator store after cooling.
Prussian blue-carbon nanotube-nano Au composite (PB-CNTs-Au compound) is obtained by following steps:
The carboxylated multi-walled carbon nano-tubes of (1) 4 mg is distributed to the FeCl of 40.0 mL 0.01 M
3in solution;
(2) while stirring by the K of 40.0 mL 0.01 M
3fe(CN)
6dropwise adds the FeCl that step (1) obtains
3in solution;
(3) solution step (2) obtained is constantly centrifugal under 10000 r/min conditions, removes supernatant, until supernatant is colourless;
(4) sediment is through collection, drying, and being distributed to massfraction is in the 4.0 mL chitosan aqueous solution of 0.1 %, obtains the amino group modifying PB-CNTs;
(5) centrifugal segregation residue shitosan under 10000 r/min conditions;
(6) add completing amido modified PB-CNTs in gold colloid and stir, centrifuging;
(7) nano composite material obtained is distributed in 2.0 mL deionized waters, puts into Refrigerator store.
Prussian blue in Prussian blue-carbon nanotube-nano Au composite: carbon nano-tube: golden mass ratio is 2:2:1.
embodiment 1
A preparation method for sandwich type electrochemical sensor of the present invention, comprises the following steps:
First a, 5 gold electrodes carry out polishing in the oxidation aluminium paste of 0.3 and 0.05 um, until in minute surface, rinse with intermediate water;
B, freshly prepd PB-CNTs-Au compound ultrasonic process 50 min, 10 uL are prepared PB-CNTs-Au compound and dripped at electrode surface, at room temperature dried overnight;
After c, stand-by intermediate water rinse several times, the PBS solution of 10 uL capture antibodies drips at electrode surface, and 60min is dry, rinses electrode remove unconjugated antibody with intermediate water and PBS;
D, 10 uL BSA (0.1%) are used to enclosed-electrode surface does not have combined site.
Detection method is as follows:
E, electrode are hatched respectively, and are rushed with PBS damping fluid fully stir cleaning in PBS damping fluid after in Escherichia coli bacteria liquid
F, Escherichia coli due to and capture electrode between specific binding and be attached on electrode,
G, PBS rinse, and drip the Escherichia coli antibody that 10 μ L mark with HRP after dry, and the antibody of HRP mark, by the strong recognition capability between object, is modified at electrode surface.
H, the end of at, liquid pH is respectively 6.0, and 6.5,7.0,7.5,8.0, detect in the damping fluid of 8.5, as shown in Figure 2, optimum results is that pH is respectively 7.5 best to testing result.
Damping fluid used in said method is prepared by method: take Na
2hPO
47.1 g, KCl 0.2 g and KH
2pO
46.8 g, KCl 0.2 g be dissolved in respectively in 500 mL intermediate waters, obtain two kinds of solution pH meter Mixed adjustments, obtaining pH value is 7.4, and concentration is the PBS damping fluid of 0.01 M.
embodiment 2
Detect a preparation method for colibacillary electrochemical sensor, step is as follows:
First a, 5 gold electrodes carry out polishing in the oxidation aluminium paste of 0.3 and 0.05 um, until in minute surface, rinse with intermediate water;
B, freshly prepd PB-CNTs-Au compound ultrasonic process 50 min, 10uL prepared PB-CNTs-Au compound and dripped at electrode surface, at room temperature dried overnight;
After c, stand-by intermediate water rinse several times, the PBS solution of 10 uL capture antibodies drips at electrode surface, and 60min is dry, rinses electrode remove unconjugated capture antibody with intermediate water and PBS;
D, 10uL BSA (0.1%) is used to enclosed-electrode surface does not have combined site.
Detection method:
E, electrode hatch 20,40,60,90 fully stir cleaning in PBS damping fluid after in Escherichia coli bacteria liquid, and 120 min also use PBS wash buffer;
F, Escherichia coli due to and capture electrode between specific binding and be attached on electrode.
G, PBS rinse, and drip the Escherichia coli antibody that 10 μ L mark with HRP after dry, and the antibody of HRP mark, by the strong recognition capability between object, is modified at electrode surface.
H, be contrast electrode with Ag/AgCl, be to electrode with Pt electrode, current potential is set to-0.2 to 0.6 V, pulse width 0.05V, pulse width scanning is 0.06 S, adopts differential pulse voltammetry technology to read the change of electric signal, detects object, testing result as shown in Figure 3, selects colibacillary best incubation time 60min.
embodiment 3
Detect drafting and the practical application of the typical curve of the preparation method of colibacillary electrochemical sensor, step is as follows:
A, 8 gold electrodes first 0.3 and 0.05um oxidation aluminium paste in carry out polishing, until in minute surface, rinse with intermediate water;
B, freshly prepd PB-CNTs-Au compound ultrasonic process 50 min, 10 uL are prepared PB-CNTs-Au compound and dripped at electrode surface, at room temperature dried overnight;
After c, stand-by intermediate water rinse several times, the PBS solution of 10 uL capture antibodies drips at electrode surface, and 60min is dry, rinses electrode remove unconjugated capture antibody with intermediate water and PBS;
D, 10uL BSA (0.1%) is used to enclosed-electrode surface does not have combined site.
Detection method:
E, electrode are respectively 0 in concentration, 7.8 × 10,7.8 × 10 fully stir cleaning in PBS damping fluid after
2, 7.8 × 10
3, 7.8 × 10
4, 7.8 × 10
5, 7.8 × 10
6, 7.8 × 10
7cfu mL
-1escherichia coli bacteria liquid in hatch 60min use PBS wash buffer;
F, Escherichia coli due to and capture electrode between specific binding and be attached on electrode.
G, PBS rinse, and drip the Escherichia coli antibody that 10 μ L mark with HRP after dry, and the antibody of HRP mark, by the strong recognition capability between object, is modified at electrode surface.
H, be contrast electrode with Ag/AgCl, be to electrode with Pt electrode, current potential is set to-0.2 to 0.6 V, pulse width 0.05V, and pulse width scanning is 0.06 S, differential pulse voltammetry technology is adopted to read the change of electric signal, detect object, as shown in Figure 4, a to h is respectively 3.4 × 10 to testing result, 7.8 × 10,7.8 × 10
2, 7.8 × 10
3, 7.8 × 10
4, 7.8 × 10
5, 7.8 × 10
6, 7.8 × 10
7cfu mL
-1in Fig. 4, we can see, at e. coli concentration 7.8 × 10 to 7.8 × 10
6cfu mL
-1time, logarithm and the size of current of coliform concentration are proportional, and upper left corner accompanying drawing is matched curve I=33.68 log [C
e. coli (cfu mL
-1)]+7.19, meanwhile, we are at 7.8 × 10 cfu mL
-1concentration basis on continue to lower Concentration Testing, after testing when concentration is lower than 3.4 × 10 cfu mL
-1time, the relation of electric current and concentration no longer meets matched curve rule just, and the Monitoring lower-cut that therefore the electric current minimum point namely in figure can obtain the method is 3.4 × 10 cfu mL
-1.
I, the method adopting mark-on to reclaim, detect milk and mineral water sample, replicate determination 3 times, the recovery is 91.9%-103%, and compare with traditional detection method colony counting method, error is less than 13%, and the reliability of this sensor detecting method is described, and process after milk and mineral water medial error within the scope of there is no Escherichia coli, belong to qualified samples.
Above-described embodiment is the present invention's preferably embodiment; but embodiments of the present invention are not by the restriction of embodiment; other is any do not deviate from Spirit Essence of the present invention and principle under make change, modification, combination, substitute, simplify and all should be equivalent substitute mode, be included within protection scope of the present invention.
Claims (5)
1. detect a colibacillary electrochemical sensor, it is characterized in that being followed successively by electrode, Prussian blue-carbon nanotube-nano Au composite layer, Escherichia coli antibody layer, bovine serum albumin(BSA) confining bed from inside to outside.
2. electrochemical sensor according to claim 1, it is characterized in that Prussian blue-carbon nanotube-nano Au composite layer thickness is 90 ± 5nm, Escherichia coli antibody layer thickness is 120 ± 10nm, and bovine serum albumin(BSA) enclosed layer thickness is 100 ± 10nm.
3. electrochemical sensor according to claim 1, is characterized in that Prussian blue-carbon nanotube-nano Au composite layer is obtained by following steps:
(1) multi-walled carbon nano-tubes carries out carboxylated;
(2) nm of gold is prepared;
(3) product using step (1) and (2) to obtain prepares Prussian blue-carbon nanotube-nano Au composite.
4. electrochemical sensor according to claim 3, is characterized in that in Prussian blue-carbon nanotube-nano Au composite Prussian blue: carbon nano-tube: golden mass ratio is 2:2:1.
5. the electrochemical sensor according to any one of claim 1-4, is characterized in that
(1) after electrochemical sensor rinses with phosphate buffer, hatch in E.coli solution, again rinse with PBS, hatch completely after drying in Escherichia coli bacteria liquid, the Escherichia coli antibody of HRP mark is dripped after dry, the antibody of HRP mark, by the strong recognition capability between object, is modified at electrode surface;
(2) three-electrode system is adopted, take Ag/AgCl as contrast electrode, be to electrode with Pt electrode, modified electrode is working electrode, with the PBS containing p-dihydroxy-benzene and hydrogen peroxide for the detection of work end liquid employing differential pulse voltammetry, current potential is set to-0.2 to 0.6 V, pulse width 0.05V, pulse width scanning is 0.06 S, detects.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104407132A (en) * | 2014-10-24 | 2015-03-11 | 济南大学 | Electrochemical sensor for detection of Escherichia coli and preparation method thereof |
| CN105424771A (en) * | 2015-12-16 | 2016-03-23 | 江南大学 | Application of nanogold-carbon nano tube-chitosan composite membrane cell sensor to detection of toxicity of food-borne pathogenic bacteria |
| CN105486738A (en) * | 2015-11-30 | 2016-04-13 | 中国人民解放军第三军医大学第一附属医院 | Multi-channel composite magnetic control electrochemical immunosensor and preparation method and application thereof |
| CN109239173A (en) * | 2018-09-21 | 2019-01-18 | 中南大学 | A kind of electrochemical method of detection bacterium activity and concentration |
| CN110907519A (en) * | 2019-11-29 | 2020-03-24 | 中南大学 | Preparation and detection method of electrochemical biosensor for escherichia coli |
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2014
- 2014-10-24 CN CN201420621074.2U patent/CN204203160U/en not_active Expired - Fee Related
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104407132A (en) * | 2014-10-24 | 2015-03-11 | 济南大学 | Electrochemical sensor for detection of Escherichia coli and preparation method thereof |
| CN104407132B (en) * | 2014-10-24 | 2016-05-25 | 济南大学 | A kind of colibacillary electrochemical sensor and preparation method thereof that detects |
| CN105486738A (en) * | 2015-11-30 | 2016-04-13 | 中国人民解放军第三军医大学第一附属医院 | Multi-channel composite magnetic control electrochemical immunosensor and preparation method and application thereof |
| CN105486738B (en) * | 2015-11-30 | 2019-02-26 | 中国人民解放军第三军医大学第一附属医院 | Composite magnetic controlled electrochemical immunosensor of multichannel and its preparation method and application |
| CN105424771A (en) * | 2015-12-16 | 2016-03-23 | 江南大学 | Application of nanogold-carbon nano tube-chitosan composite membrane cell sensor to detection of toxicity of food-borne pathogenic bacteria |
| CN105424771B (en) * | 2015-12-16 | 2018-12-04 | 江南大学 | A kind of method of quick detection gram negative pathogenic bacteria toxicity |
| CN109239173A (en) * | 2018-09-21 | 2019-01-18 | 中南大学 | A kind of electrochemical method of detection bacterium activity and concentration |
| CN109239173B (en) * | 2018-09-21 | 2019-12-20 | 中南大学 | Electrochemical method for detecting activity and concentration of bacteria |
| CN110907519A (en) * | 2019-11-29 | 2020-03-24 | 中南大学 | Preparation and detection method of electrochemical biosensor for escherichia coli |
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