CN104634847A

CN104634847A - Electrochemical immunosensing method for detecting Listeria monocytogenes

Info

Publication number: CN104634847A
Application number: CN201310581188.9A
Authority: CN
Inventors: 高志贤; 彭媛; 程超男; 宁保安; 白家磊
Original assignee: Institute of Hygiene and Environmental Medicine Academy of Military Medical Sciences of Chinese PLA
Current assignee: Institute of Hygiene and Environmental Medicine Academy of Military Medical Sciences of Chinese PLA
Priority date: 2013-11-14
Filing date: 2013-11-14
Publication date: 2015-05-20

Abstract

The invention discloses an electrochemical immunosensing method for detecting Listeria monocytogenes, belonging to the field of the biotechnological detection. The surface of a gold electrode is activated electrochemically and then is immersed in 1% of 3-mercaptopropionic acid ethanol solution to be self-assembled to form a monomolecular film, a capture antibody is combined to the surface of the electrode after activation, an enzyme amplification signal is introduced by a sandwich method, and an enzymatic electroactive substance is subjected to oxidation reduction to obtain a current response signal, therefore, a method for detecting the Listeria monocytogenes by use of the electrochemical immunosensor adopting three electrodes as a system is established; the method has good sensitivity, specificity and stability; the detection system is small, the reagent is saved, the detection cost is reduced remarkably, the method has great potential in application and practical sample detection due to no influence from the turbidity of the sample.

Description

A kind of electrochemistry immuno-sensing method for detecting Listeria monocytogenes

Technical field

The present invention relates to a kind of based on sandwich method electrochemistry immuno-sensing employing chronoamperometry detection Listeria monocytogenes, belong to biotechnology detection technique field.

Background technology

Listeria monocytogenes is the pathogenic bacteria of a kind of important zoonosis and food origin disease, the listeriosis of people, animal can be caused, after infecting, main manifestations is meningitis, septicemia, miscarriage etc., this bacterium, up to 30% ~ 70%, is classified as food four one of pathogenic bacteria greatly of the nineties by case fatality rate in the world.FDA, WHO etc. organize and have in succession set up corresponding mechanism or specified that corresponding strategy goes pollution, the propagation of this bacterium of prevention and corntrol.WHO has delivered for 1988, and " food source property listeriosis " advises book, instructs whole world various countries how to prevent LM to pollute and poisoning, advises that NSF is strengthened monitoring.In recent years, many countries reported that the food poisoning being separated to Lee Salmonella or cause because of food pollution Lee Salmonella from numerous food broke out example.In the end of the year 1999, Michigan, USA there occurs serious together Listeria and infects the food poisoning that food causes, and 21 people are dead because of " hot dog " of this fungi pollution edible and cold cuts, and other 22 state 97 people infection, 6 pregnant woman miscarry.In July, 2011, U.S.'s muskmelon, by Listeria contamination accident, causes 30 people dead, and pregnant woman's miscarriage, have 146 people and catch an illness, patient is distributed in 28 states.Since November calendar year 2001, China's quality testing department repeatedly detects the pathogenic bacteria such as Listeria monocytogenes from more than the 30 batch of nearly kiloton pig secondary product such as Pigs Kidney, pork tripe, pig ear, float of the 20 Duo Jia meat-packing plants such as the U.S., Canada, France, Ireland, Belgium, Denmark import.These break out in a large number and the epidemiology case of distributing shows, the food poisoning that Listeria monocytogenes causes has been a global public health problem.Zero tolerance attitude is held for the Listeria monocytogenes in food by the current U.S., and Canadian standard is 100CFU/g food.Therefore, detection technique is significant in field of food safety fast to set up Listeria monocytogenes scene.

Detection method mainly classic method, the Enzyme Linked Immunoadsorbent Assay (ELISA) and PCR (PCR) etc. of current Listeria monocytogenes.In these methods, conventional culture methods result accurately and reliably, but wastes time and energy, and obtaining result usually needs 5-7 days; ELISA method is simple to operate, high specificity, and reaction sensitivity is high, but needs pre-treatment or the enrichment of carrying out sample before analysis; Pcr analysis needs DNA extraction and amplification etc., complex steps, and easily occurs false positive.In a word, these methods all need the cost of well-trained professional and costliness and longer analysis time.Recent years, due to response fast, simple and sensitive, cheap and high specific, electrochemical immunosensor is subject to the people's attention.Electrochemical sensing detection method, by introducing the method for electroactive material, by electrode surface sensitive element to the identification of object and absorption, is transformed into a kind of electric signal, has rapid sensitive, advantage to sample nondestructive.Because it is for the turbidity no requirement (NR) of sample, therefore be easy to apply and the detection in actual sample.On electrode, the fixing means of antigen-antibody has very important impact for the stability of detection signal, reappearance and sensitivity.Fixing means conventional at present has the modification of LB film, polymeric membrane, albumin A, gold nano grain and self assembled monolayer etc.Self assembled monolayer preparation is simple, easy forms ultra-thin orderly unimolecular layer.By at electrochemical immunosensor electrode surface self-assembled monolayer sessile antibody, target acquisition thing, introduce enzymatic electrochemical signals method, its committed step is the determination of the preparation and determination methods method of immunosensor.

Summary of the invention

The object of this invention is to provide a kind of electrochemistry immuno-sensing method based on sandwich method for detecting Listeria monocytogenes.

Technical scheme of the present invention is summarized as follows:

Prepare a method for immunosensor at the working electrode surface of electrochemical sensor, concrete steps are as follows:

(1) by gold electrode by after sanding and polishing process, then carry out cyclic voltammetric cleaning, then at 0.05MH with sulfuric acid solution ₂sO ₄carry out in solution activating (voltage range-0.2V ~ 1.6V, sweep speed 50mV/s), until obtain a level and smooth reduction peak at about 0.94V, ultrapure washing, nitrogen dries up; This is the naked gold electrode handled well.

(2) gold electrode handled well is placed in the ethanolic solution self assembly of 1%3-mercaptopropionic acid, ambient temperature overnight, ethanol is washed, ultrapure washing, and nitrogen dries up; This is self-assembling electrode.

(3) electrode assembled is activated 30min in room temperature, ultrapure washing, nitrogen dries up; This is activated electrode.

(4) drip Listeria monocytogenes monoclonal antibody solution at the electrode surface activated, hatch 1h for 37 DEG C, PBS washes, and nitrogen dries up; This is the electrode combining monoclonal antibody.

(5) electrode 37 DEG C of closed 40min, PBS of combining monoclonal antibody are washed, nitrogen dries up; Be stored in 4 DEG C, be suspended from above PBS, for subsequent use.This is the immunosensor prepared.Cyclic voltammetry and electrochemical impedance spectroscopy is all adopted to characterize after above steps process completes.

Characterizing solution is 10mL 2.5mM Fe (CN) ₆ ^3-/ Fe (CN) ₆ ^4-(1:1) the 0.01M PBS solution containing 0.1M KCl.

Representation system is three-electrode system, and gold electrode is working electrode, and platinum plate electrode is to electrode, and Ag/AgCl (3M KCl solution) electrode is contrast electrode.

Based on a method for the sandwich method electrochemical immunosensor detection Listeria monocytogenes that enzyme amplifies, concrete steps are as follows:

(1) the Listeria monocytogenes solution of series concentration is prepared, at 37 DEG C, 1h is hatched respectively with the immunosensor of preparation, and then react with resist that HRP marks, hatch 30min for 37-DEG C, finally detect with chronoamperometry on electrochemical sensor; more Take PBS as contrast, detect at identical conditions, the current-responsive value obtained is negative value, calculates the difference of current-responsive, draws the typical curve of LM concentration and current-responsive difference, the computing method range of linearity and lowest detectable limit.

(2) with the aqueous solution regeneration 5min of the glycocoll-HCl (pH2.0) of 0.1M, the Listeria monocytogenes somatic antigen that removing combines, with the LM solution of this cycle detection same concentration, the regenerability of the immunosensor of checking preparation.

(3) result electrochemical sensor obtained compared with the result of plate count, the reliability of verificating sensor.

(4) the method is applied to the experiment of detection milk sample mark-on, the final defining method range of linearity: 10 ³-10 ⁶cFU/ml, lowest detectable limit: 10 ²cFU/ml.

Substrate solution: 10mL contains the PBS solution (0.01M, pH7.0) of the 0.1M KCl of 0.24mM thionine solution and 6mM superoxol; Nitrogen blows 5min deoxygenation.

Chronoamperometry optimum configurations: voltage constant at-0.3V, current duration 200S.

Accompanying drawing explanation

Fig. 1 electrochemical immunosensor and detection system schematic diagram

Fig. 2 gold electrode activation figure

Fig. 3 electrochemical immunosensor cyclic voltammetric and electrochemical impedance spectroscopy phenogram

The response criteria curve that Fig. 4 electrochemical response signal and Listeria monocytogenes concentration are drawn

The curve that in Fig. 5 electrochemical response signal and milk, mark-on is tested

Embodiment

Embodiment one: immunosensor is prepared on electrochemical sensor electrodes surface

(1) gold electrode is drawn a circle on 2000 orders, 3000 orders, 5000 order sand paper polishing or at Piranha solution (H ₂o ₂/ H ₂sO ₄, 1:3, v/v) and middle immersion 5min, ultrapure washing, then successively by 1.0 μm, 0.3 μm, 0.05 μm A1 ₂o ₃the polishing on chamois leather of powder furnishing suspending liquid, ultrapure washing; By the gold electrode of cleaning at 0.1M H ₂sO ₄cyclic voltammetry scan (voltage range-0.2V ~ 1.2V, sweep speed 50mV/s) cleaning is carried out, until obtain stable cyclic voltammogram in solution; Then at 0.05M H ₂sO ₄carry out in solution activating (voltage range-0.2V ~ 1.6V, sweep speed 50mV/s), until obtain a level and smooth reduction peak at about 0.94V, ultrapure washing, nitrogen dries up;

(2) gold electrode handled well is placed in the ethanolic solution self assembly of 2mL 1%3-mercaptopropionic acid, ambient temperature overnight, ethanol is washed, ultrapure washing, and nitrogen dries up;

(3) electrode assembled is placed in the aqueous solution room temperature activation 30min of 2mL0.4M EDC-0.1M NHS, ultrapure washing, nitrogen dries up;

(4) drip at the electrode surface activated the Listeria monocytogenes monoclonal antibody solution that 10 μ L dilutabilitys are 1:1000, hatch 1h for 37 DEG C, PBS washes, and nitrogen dries up;

(5) electrode combining monoclonal antibody is placed in the PBS solution of 1mL1%OVA, 37 DEG C of closed 40min, PBS wash, and nitrogen dries up; Be stored in 4 DEG C, be suspended from above PBS, for subsequent use.

Embodiment two: the sandwich method electrochemical immunosensor amplified based on enzyme detects Listeria monocytogenes

(1) immunosensor prepared and blank solution PBS are reacted at 37 DEG C, and then at 37 DEG C, react 30min with resisting of marking of HRP more, finally detecting with chronoamperometry, the value obtained is negative value;

(2) the Listeria monocytogenes solution (10 of series concentration is prepared ⁷cFU/ml, 10 ⁶cFU/ml ... 10 ¹cFU/ml), at 37 DEG C, 1h is hatched respectively with the immunosensor of preparation, and then react with resist that HRP marks more, hatch 30min for 37 DEG C, finally on electrochemical sensor, carry out detection with chronoamperometry obtains current-responsive value, with 0.01M glycocoll-HCl regeneration of waste liquor 5min, the LM that removing combines, circulates to 10 with this ¹cFU/ml-10 ⁷lM in CFU/ml concentration range detects.

(3) calculate the difference of current-responsive, the logarithm value of LM concentration and the typical curve of current-responsive difference: y=548.0x-727.2, R2=0.9883, the range of linearity is 10 ²cFU/ml-10 ⁶cFU/ml.

(4) simultaneously with the staphylococcus aureus of same concentration, salmonella, Shigella and Escherichia coli for contrast, carry out Electrochemical Detection under the same conditions, the electrochemical immunosensor of result display preparation has good specificity.

Claims

1. prepare a method for immunosensor at electrochemical sensor working electrode surface, its feature is made up of following steps:

(1) polished on 2000 orders, 3000 orders, 5000 order sand paper by gold electrode, ultrapure washing, then uses 1.0 μm, 0.3 μm, 0.05 μm Al successively ₂o ₃powder is polishing on chamois leather, ultrapure washing;

(2) by the gold electrode of cleaning at 0.1M H ₂sO ₄cyclic voltammetry scan (voltage range-0.2V ~ 1.2V, sweep speed 50mV/s) cleaning is carried out, until obtain stable cyclic voltammogram in solution; Then at 0.05M H ₂sO ₄carry out in solution activating (voltage range-0.2V ~ 1.6V, sweep speed 50mV/s), until obtain a level and smooth reduction peak at about 0.94V, ultrapure washing, nitrogen dries up;

(3) gold electrode handled well is placed in the ethanolic solution self assembly of 2mL 1%3-mercaptopropionic acid, ambient temperature overnight, ethanol is washed, ultrapure washing, and nitrogen dries up;

(4) electrode assembled is placed in the aqueous solution room temperature activation 30min of 0.4M EDC-0.1M NHS, ultrapure washing, nitrogen dries up;

(5) drip 10 μ L Listeria monocytogenes monoclonal antibody solution at the electrode surface activated, hatch 1h for 37 DEG C, PBS washes, and nitrogen dries up;

(6) electrode combining monoclonal antibody is placed in the PBS solution of 1mL1%OVA, 37 DEG C of closed 40min, PBS wash, and nitrogen dries up; Be stored in 4 DEG C for subsequent use.

2., based on a method for the electrochemical immunosensor detection Listeria monocytogenes of self-assembled monolayer, its feature is made up of following steps:

(1) immunosensor of preparation is placed in Listeria monocytogenes somatic antigen solution, hatch 1h for 37 DEG C, PBS washes; Then answer with the many anti-reflective of the Listeria monocytogenes of horseradish peroxidase-labeled, hatch 30min for 37 DEG C, PBS washes, and nitrogen dries up;

(2) substrate solution of 10mL is configured: the PBS solution (0.01M, pH7.0) of the 0.1M KCl containing 0.24mM thionine solution and 6mM superoxol; Nitrogen blows 5min deoxygenation;

(3) replace Listeria monocytogenes antigenic solution to be worth in contrast with PBS, adopt the response of chronoamperometry sensed current signal, voltage constant at-0.3V, duration 200S.With the aqueous solution regeneration 5min of the glycocoll-HCl (pH2.0) of 0.01M, the Listeria monocytogenes somatic antigen that removing combines, circulates with this and carries out the detection of variable concentrations thalline;

(4) current signal recorded and corresponding Listeria monocytogenes concentration drawing standard curve is utilized; Mark-on experiment is carried out to germ-free milk sample, the final defining method range of linearity: 10 ³-10 ⁶cFU/ml, lowest detectable limit: 10 ²cFU/ml.