AU2021104855A4

AU2021104855A4 - Molecularly imprinted polymer for detecting bacteria, and its preparation and use in bacterial detection

Info

Publication number: AU2021104855A4
Application number: AU2021104855A
Authority: AU
Inventors: Jikui WU; Junling Zhang; Min Zhu
Original assignee: Shanghai Ocean University
Current assignee: Shanghai Ocean University
Priority date: 2021-08-03
Filing date: 2021-08-03
Publication date: 2021-09-30
Anticipated expiration: 2029-08-03

Abstract

(FIG. 1) The present disclosure is related to the field of biochemistry, and in particular, to a molecularly imprinted polymer for detecting bacteria and its preparation and use in bacterial detection. A method for preparation of a molecularly imprinted polymer for detecting bacteria comprises: (a) adding bacteria serving as a template molecular that is used for imprinting, a functional monomer, and a dopant to a buffer solution at a predetermined ratio to form a mixed liquid, into which electrodes are immersed to conduct an electrochemical polymerization reaction so as to form a conducting polymer on surfaces of the electrodes; and (b) placing the electrodes having the conducting polymer formed thereon in an elution solution to remove the template bacteria, followed by drying of the electrodes with a protective gas, so as to obtain a molecularly imprinted polymer for detecting the bacteria on the surfaces of the electrodes. By combining the mechanism of effective improvement of PPy conductivity by CuPcTs with the mechanism of specific recognition of the template bacteria by a molecularly imprinted polymer, a molecularly imprinted polymer is provided which has good capability for recognizing a target bacteria species with high sensitivity and specificity. -1/4 8 CsPyC~~-I+.cl 6- GCEs/PPy-CuPcTs-MIP+ci * CsP4-uc--I ME6OEO MMM N 2- ~m MuU 00 60 A' 5 50B 90.0 60- 60 20 90 60 30 0.05 0.10 01')5 0.20 025 5 to Is 20 Cu~cTsIC (mmol/nL) Electropolymerization C 500 D01o0 400- 80 300- 60 C: 200- 40 100 20 0 04 0.5 1.0 1.5 2.0 1.0 1.5 2.0 2.5 Template Removal Time h Recognition Time/h FIG.2

Description

-1/4

8 CsPyC~~-I+.cl

6- GCEs/PPy-CuPcTs-MIP+ci

* CsP4-uc--I MMM ME6OEO

N 2- ~m MuU

00

60

A' 5 50B 90.0

60- 60

20 90

60 30

0.05 0.10 01')5 0.20 025 5 to Is 20

Cu~cTsIC (mmol/nL) Electropolymerization C500 D01o0

400- 80

300- 60 C:

200- 40

20

100

0 04 0.5 1.0 1.5 2.0 1.0 1.5 2.0 2.5 Template Removal Time h Recognition Time/h

FIG.2

MOLECULARLY IMPRINTED POLYMER FOR DETECTING BACTERIA, AND ITS PREPARATION AND USE IN BACTERIAL DETECTION TECHNICAL FIELD

[01] The present disclosure is related to the field of biochemistry, and in particular, to a molecularly imprinted polymer for detecting bacteria, and its preparation and use in bacterial

detection.

BACKGROUNDART

[02] Standards for pathogen limits for food are integral to food safety standards, and

National Food Safety Standard - Pathogen Limits for Food is the first standard that specifies the

limits of pathogens in food. In particular, this standard specifies the limits of the most common

kinds of pathological bacteria, including Salmonella, Staphylococcus aureus (S. aureus), Vibrio

Parahemolyticus (VP), Listeria monocytogenes (L. monocytogenes), and Escherichia coli (E.

coli), in foods including meat products, aquatic products, grain products, ready-to-eat fruit and

vegetable products, beverages, and frozen drinks. According to this standard, the limits of

csalmonella and Staphylococcus aureus in the foods are zero and 100 CFU/g, respectively.

[03] Conventional techniques for bacterial detection, such as enzyme-linked immunosorbent

assay (ELISA), radioimmunoassay (RIA), and real-time quantitative polymerase chain reaction

(qPCR), suffer from a number of disadvantages, such as very slow response speed, complex

sample preparation, bulky and expensive devices, and limited sensitivity and selectivity.

Therefore, it is of great importance for public health and food safety to provide a faster, less

costly, and more sensitive and specific method for detecting pathogens.

SUMMARY

[04] In view of the above problems, embodiments of the present disclosure provide a

molecularly imprinted polymer for detecting bacteria and a method for preparation thereof, as

well as a method for detecting bacteria by using the molecularly imprinted polymer with high

sensitivity and specificity.

[05] Accordingly, one embodiment provides a method for preparation of a molecularly imprinted polymer for detecting bacteria, comprising steps of:

[06] (a) adding bacteria serving as a target molecular or a template that is used for imprinting (hereinafter also referred to as the template bacteria), a functional monomer, and a

dopant to a buffer solution at a predetermined ratio to form a mixed liquid, into which

electrodes are immersed to conduct an electrochemical polymerization reaction so as to form a

conducting polymer on surfaces of the electrodes; and

[07] (b) placing the electrodes having the conducting polymer formed thereon in an elution

solution to remove the template bacteria, followed by drying of the electrodes with a protective

gas, so as to obtain a molecularly imprinted polymer for detecting the bacteria on the surfaces

of the electrodes.

[08] The template bacteria may be Listeria monocytogenes (L. monocytogenes),

Staphylococcus aureus (S. aureus), Salmonella, or Escherichia coli 0157:H7 (E. coli 0157:H7)

species.

[09] The dopant may be copper tetrasulfonatophthalocyanine (CuPcTs).

[10] The functional monomer may be pyrrole.

[11] The predetermined ratio of the template bacteria, the functional monomer, and the

dopant may be 500:17:(0.05-0.25) by volume, preferably 500:17:0.1 by volume.

[12] The buffer solution may be a 0.1 mol/L potassium chloride solution containing 1 mmol/L potassium ferricyanide (K3[Fe(CN) 6]) and 1 mmol/L potassium hexacyanoferrate(II)

(K4Fe(CN) 6).

[13] The electrochemical polymerization reaction in the step (a) may be conducted via cyclic

voltammetry. In particular, when the electrodes are immersed in the mixed liquid containing the

template bacteria, the functional monomer, and the dopant, scan parameters and the number of

electropolymerization cycles are set to predetermined values so as to carry out the

electrochemical polymerization reaction in the buffer solution.

[14] Further, the scan parameters may include a scan rate and a circulation voltage, which may be set to 0.05 V/s and - 0.4 to +0.7 V, respectively. The number of electropolymerization cycles may be set to 5 to 20, preferably 15. The polymerization reaction may be conducted at room temperature.

[15] The elution solution used in the step (b) may be an acetic acid solution containing 5

% by weight of sodium dodecyl sulfate (SDS).

[16] The electrodes used in the step (a) may be glassy carbon electrodes, which may be pretreated by grinding and polishing surfaces of the electrodes using a suspension of aluminium oxide particles having a particle size of 0.05 to 0.3 m and subjecting the electrodes to ultrasonic cleaning using deionized water for 30 seconds to 1 minute and then using an organic solvent, such as ethanol or acetone, for a further 30 seconds to 1 minute, followed by drying with a protective gas.

[17] Another embodiment provides a molecularly imprinted polymer for detecting bacteria prepared according to the method as described above.

[18] A further embodiment provides a method for detecting bacteria, comprising:

[19] detecting bacteria in a sample by using the molecularly imprinted polymer as described above, and measuring impedance via electrochemical impedance spectroscopy (EIS) measurement to determine recovery of the bacteria.

[20] The EIS measurement may be performed at room temperature with a frequency of 0.1 to 100,000 Hz and an amplitude of 5 mV.

[21] The bacteria in the sample may be L. monocytogenes, S. aureus, Salmonella, or E. coli 0157:H7 species.

[22] The electrochemical polymerization can enable the thickness of the conducting polymer film to be controlled and thus a uniform surface imprinting layer to be formed on the electrodes, and also enable the surface imprinting layer to be securely bound to the electrodes. Polypyrrole (PPy) is known to have low nonspecific adsorption ability, good conductivity, and excellent stability, and can be readily formed by polymerization of pyrrole monomers at mild conditions in an efficient manner. Furthermore, PPy has sensitivity to active sites on the outside of the bacterial cells, and thus has high selectivity and versatility for various bacteria.

[23] We have experimentally found that the use of CuPcTs (which is a conductive supramolecular material) as a dopant in the preparation of the molecularly imprinted polymer

can further improve the sensitivity and the specificity of the molecularly imprinted polymer to

the template bacteria. CuPcTs is known as a good organic semiconductor material, and we have

also found that the conductivity of PPy doped with CuPcTs is two orders of magnitude higher

than pure (undoped) PPy, that is, the conductivity of PPy doped with CuPcTs is substantially

increased.

[24] We therefore use pyrrole as the functional monomer. As such, the mechanism of

effective improvement of PPy conductivity by CuPcTs can be combined with the mechanism of

specific recognition of the template bacteria by the molecularly imprinted polymer. In this way,

the molecularly imprinted polymer prepared may have a higher selectivity for a target

molecular, and the sensitivity and accuracy of bacteria detection by using such molecularly

imprinted polymer can be improved.

[25] The present disclosure provides several advantages over prior art. By combining the mechanism of effective improvement of PPy conductivity by CuPcTs with the mechanism of

specific recognition of the template bacteria by a molecularly imprinted polymer, a molecularly

imprinted polymer is provided which has good capability for recognizing a target molecular

with high sensitivity and specificity. The method for preparation of the molecularly imprinted

polymer according to the present disclosure is commercially feasible, rapid, simple, and

efficient. Further, the molecularly imprinted polymer of the present disclosure can be used to

detect bacteria with high sensitivity and specificity.

[26] Definitions of specific embodiments of the present disclosure as claimed herein follow.

[27] According to a first specific embodiment, there is provided a method for preparation of

a molecularly imprinted polymer for detecting bacteria, comprising steps of:

(a) adding bacteria serving as a template molecular that is used for imprinting, a

functional monomer, and a dopant to a buffer solution at a predetermined ratio to form a mixed

liquid, into which electrodes are immersed to conduct an electrochemical polymerization

reaction so as to form a conducting polymer on surfaces of the electrodes; and

(b) placing the electrodes having the conducting polymer formed thereon in an elution

of the electrodes;

wherein the template bacteria is Listeria monocytogenes, Staphylococcus aureus,

Salmonella, or Escherichia coli 0157:H7 species;

the dopant is copper tetrasulfonatophthalocyanine; and

the functional monomer is pyrrole.

[28] According to a second specific embodiment, there is provided a molecularly imprinted

polymer prepared according to the method of the first aspect.

[29] The term "comprise" and variants of the term such as "comprises" or "comprising" are used herein to denote the inclusion of a stated integer or stated integers but not to exclude any

other integer or any other integers, unless in the context of usage an exclusive interpretation of

the term is required.

BRIEF DESCRIPTION OF THE DRAWINGS

[30] FIG. 1 is a graph showing electrochemical impedance spectroscopy (EIS) data for

GCEs/PPy-CuPcTs-MIP immersed in a 0.1 mol/L KCl solution containing 1 mmol/L

K3[Fe(CN) 6] and 1 mmol/L K 4 Fe(CN) 6 and EIS data for GCEs/PPy-CuPcTs-MIP immersed in a

0.1 mol/L KCl solution containing 1 mmol/L K3[Fe(CN) 6] and 1 mmol/L K 4 Fe(CN) 6 after

recognitionof anE. coli0157:H7 species;

[31] FIG. 2 shows optimization of parameters including (A) concentration of CuPcTs, (B)

polymerization cycles, (C) template removal time, and (D) recognition time;

[32] FIG. 3A shows the impedance spectra of GCEs/PPy-CuPcTs-MIP for detection of E.

coli 0157:H7 at series of gradient concentrations from 10 to 108 CFU/mL;

[33] FIG. 3B shows the standard curve of the EIS response versus log concentration of E. coli 0157:H7;

[34] FIG. 4 shows the EIS response of different biosensors of GCEs/PPy-CuPcTs-MIP to the respective template bacteria and the other three interfering bacteria species; and

[35] FIG. 5 is a graph showing EIS data for GCEs/bare, GCEs/PPy, and GCEs/PPy-CuPcTs immersed in a 0.1 mol/L KCl solution containing 1 mmol/L K3[Fe(CN) 6] and 1 mmol/L K 4Fe(CN) 6 .

DETAILED DESCRIPTION OF THE EMBODIMENTS

[36] The molecularly imprinted polymer (which was actually formed on the surfaces of the electrodes) used in the following examples 1 to 10 was prepared as follows.

[37] Surfaces of glassy carbon electrodes were ground and polished with a suspension of aluminium oxide particles having a particle size of 0.05 to 0.3 m. Thereafter, the electrodes were ultrasonically cleaned in deionized water and ethanol each for 30 s, and then dried with nitrogen. 17 L of pyrrole (functional monomer), 0.1 L of CuPcTs (dopant), 0.5 mL of a solution containing an E. coli 0157:H7 concentration of 106 CFU/mL (template bacteria), and 4 mL of a buffer solution (that is a 0.1 mol/L KCl solution containing1 mmol/L K3[Fe(CN) 6] and 1 mmol/L K4 Fe(CN) 6) were well mixed and introduced into a 5 mL-beaker. The glassy carbon electrodes were then immersed in the mixed liquid in the beaker to conduct electrochemical polymerization at room temperature via cyclic voltammetry under the conditions of a scan rate of 0.05 V/s, a cyclic voltage of from -0.4 V to +0.7 V, and 15 electropolymerization cycles. After completion of the polymerization, the electrodes were removed from the beaker and rinsed several times with deionized water and then dried with nitrogen. Then, the electrodes were subjected to elution with SDS/HAc (5 % w/v) for 1.5 h to remove the template bacteria, and were rinsed several times with deionized water and then dried with nitrogen. A molecularly imprinted polymer was thus obtained on the surfaces of the electrodes (such electrodes are also referred to herein as the modified electrodes or the biosensor GCEs/PPy-CuPcTs-MIP).

[38] Example 1

[39] 9 mL of drinking water was well mixed with 1 mL of phosphate buffered saline (PBS, pH=7.4) containing E. coli 0157:H7 to prepare artificially contaminated samples having

concentrations of 10', 104, and 105 CFU/mL, respectively. A volume of 250 L of each sample

was transferred into a 1-mL centrifuge tube. The modified electrodes were immersed in each

sample solution for 2 h, and EIS measurement was then performed thereon. The specificity of

the molecularly imprinted polymer formed on the surfaces of the electrodes to E. coli 0157:H7

and recovery of E. coli 0157:H7 were investigated. The results are shown in Table 1.

[40] Example 2

[41] 9 mL of orange juice was well mixed with 1 mL of PBS (pH=7.4) containing E. coli 0157:H7 to prepare artificially contaminated samples having concentrations of 103, 104,

and 105 CFU/mL, respectively. A volume of 250 L of each sample was transferred into a 1-mL

centrifuge tube. The modified electrodes were immersed in each sample solution for 2 h, and

EIS measurement was then performed thereon. The specificity of the molecularly imprinted

polymer formed on the surfaces of the electrodes to E. coli 0157:H7 and recovery of E.

coli 0157:H7 were investigated. The results are shown in Table 1.

[42] Example 3

[43] Some constituents of milk, such as lipids and proteins, may interfere with detection of

bacteria, so the milk to be used was centrifuged and filtered before use. In particular, the milk

was centrifuged to give a supernatant, which was filtered through a sterile microporous

membrane. 9 mL of the filtered supernatant was well mixed with 1 mL of PBS (pH=7.4)

containing E. coli 0157:H7 to prepare artificially contaminated samples having concentrations

of 103, 104, and 105 CFU/mL, respectively. A volume of 250 L of each sample was transferred into a 1-mL centrifuge tube. The modified electrodes were immersed in each sample solution

for 2 h, and EIS measurement was then performed thereon. The specificity of the molecularly

imprinted polymer formed on the surfaces of the electrodes to E. coli 0157:H7 and recovery of

E. 0157:H7 were investigated. The results are shown in Table 1.

[44] Table1 Detection ofE. coli 0157:H7 in the Samples Added Measured Recovery RSD /(CFU/mL) /(CFU/mL) /% /(%, n=3)

103 9.81x102 98.1 2.49

Example 1 104 9.79x103 97.9 1.10

105 1.008x105 100.8 3.37

103 1.026x103 102.6 1.21

Example 2 104 9.07x 103 90.7 3.53

105 1.01x105 101.0 1.43

103 9.61x102 96.1 2.35

Example 3 104 9.53x103 95.3 1.52 105 1.022x105 102.0 2.30

[45] The contents of E. coli 0157:H7 in the samples were determined by using the biosensor GCEs/PPy-CuPcTs-MIP. The recovery of E. coli 0157:H7 in drinking water was 97.9 to 100.8

% with a relative standard deviation (RSD) of from 1.10 to 3.37 %; the recovery of E. coli 0157:H7 in orange juice was 90.7 to 102.6 % with RSD of from 1.21 to 3.53 %; and the

recovery of E. coli 0157:H7 in milk was 95.3 to 102.2 % with RSD of from 1.52 to 2.35 %, as

shown in Table 1. These indicate that the biosensor GCEs/PPy-CuPcTs-MIP is suitable for detecting E. coli 0157:H7 in real samples.

[46] Example 4

[47] The modified electrodes were immersed in a buffer solution (that is a 0.1 mol/L potassium chloride solution containing 1 mmol/L K3[Fe(CN) 6 ] and 1 mmol/L K4Fe(CN) 6), and EIS measurement was then performed thereon. The results are shown in FIG. 1.

[48] Surfaces of glassy carbon electrodes were ground and polished with a suspension of aluminium oxide particles having a particle size of 0.05 to 0.3 m. Thereafter, the electrodes were ultrasonically cleaned in deionized water and ethanol each for 30 s, and then dried with nitrogen. 17 L of pyrrole (functional monomer), 0.1 L of CuPcTs (dopant), 0.5 mL of a solution containing an E. coli 0157:H7 concentration of 106 CFU/mL (template bacteria), and 4 mL of a buffer solution (that is a 0.1 mol/L KCl solution containing1 mmol/L K3[Fe(CN) 6] and 1 mmol/L K4 Fe(CN) 6) were well mixed and introduced into a 5 mL-beaker. The glassy carbon electrodes were then immersed in the mixed liquid in the beaker to conduct electrochemical polymerization at room temperature via cyclic voltammetry under the conditions of a scan rate of 0.05 V/s, a cyclic voltage of from -0.4 V to +0.7 V, and 15 electropolymerization cycles. After completion of the polymerization, the electrodes were removed from the beaker and rinsed several times with deionized water and then dried with nitrogen. Then, the electrodes were subjected to elution with SDS/HAc (5 % w/v) for 1.5 h to remove the template bacteria, and were rinsed several times with deionized water and then dried with nitrogen. A molecularly imprinted polymer was thus obtained on the surfaces of the electrodes.

[49] A volume of 250 L of a solution containing an E. coli 0157:H7 concentration of 106 CFU/mL was introduced into a 1-mL centrifuge tube. The modified electrodes were immersed in the solution in the tube for 2 h for recognition of E. coli 0157:H7, and EIS measurement was then performed thereon. The measurement results are shown in FIG. 1.

[50] FIG. 1 is a graph showing EIS data for the biosensor GCEs/PPy-CuPcTs-MIP immersed in the buffer solution and EIS data for the biosensor GCEs/PPy-CuPcTs-MIP immersed in the buffer solution after recognition of E. coli 0157:H7 (106 CFU/mL). It can be seen from this figure that, after recognition of E. coli0157:H7 (106 CFU/mL), the biosensor GCEs/PPy-CuPcTs-MIP had an impedance of 9,000 Q, which was considerably higher than that of the biosensor GCEs/PPy-CuPcTs-MIP before the recognition, which was 50 Q.

[51] Example 5

[52] A volume of 250 L of a solution containing an E. coli 0157:H7 concentration of 106 CFU/mL was introduced into a 1-mL centrifuge tube. The modified electrodes were immersed in the solution in the tube for 2 h for recognition of E. coli 0157:H7, and EIS measurement was then performed thereon. The measurement results are shown in FIG. 2. It can be seen from FIG. 2 that, the sensing performance of the biosensor GCEs/PPy-CuPcTs-MIP was gradually improved as the concentration of CuPcTs was increased from 0.05 to 0.10 mol/L; while, when the concentration of CuPcTs was further increased to 0.15 to 0.25 mol/L, its reactivity to the template bacteria was reduced. The optimal concentration of CuPcTs was thus determined to be

0.10 mol/L.

[53] Example 6

[54] A volume of 250 L of a solution containing an E. coli 0157:H7 concentration of 106

CFU/mL was introduced into a 1-mL centrifuge tube. The modified electrodes were immersed

in the solution in the tube for 2 h for recognition of E. coli 0157:H7, and EIS measurement was

then performed thereon. The measurement results are shown in FIG. 2. It can be seen from FIG.

2 that the sensing performance of the biosensor GCEs/PPy-CuPcTs-MIP was gradually

improved as the number of the polymerization cycles were increased from 5 to 15; while, when

the number of the polymerization cycles was further increased to 20, its reactivity to the

template bacteria was reduced. The optimal number of polymerization cycles was thus

determined to be 15.

[55] Example 7

[56] A volume of 250 L of a solution containing an E. coli 0157:H7 concentration of 106

2 that the elution time could influence the removal of the template bacteria. In particular, the Ret

value was decreased as the elution time was increased from 0.5 to 1.5 h; while, when the elution

time was further increased to 2 h, the Rt value did not vary any longer. The optimal elution

time was thus determined to be 1.5 h.

[57] Example 8

[58] A volume of 250 L of a solution containing an E. coli 0157:H7 concentration of 106

in the solution in the tube for 1 to 2.5 h for recognition of E. coli0157:H7, and EIS

measurement was then performed thereon. The measurement results are shown in FIG. 2. It can be seen from FIG. 2 that the recognition time is a further parameter that may influence the sensing performance of the sensor. In particular, the intensity of the response signal was gradually increased as the recognition time was increased from 1 to 2 h; while, when the recognition time was further increased to 2.5 h, the intensity of the response signal did not vary significantly any longer. The optimum recognition time was thus determined to be 2 h.

[59] Example 9 - Establishment of Standard Curve

[60] A solution containing E. coli 0157:H7 was diluted with PBS (pH=7.4) by 10-fold serial dilution to prepare standard samples having concentrations of 10, 102, 10 , 104, 105, 106, 107,

and 108 CFU/mL, respectively. A volume of 250 L of each standard sample was introduced

into a 1-mL centrifuge tube. The modified electrodes were immersed in the solution in the tube

for 2 h for recognition of E. coli 0157:H7, and EIS measurement was then performed thereon.

[61] A standard curve was established based on the EIS response of the biosensor

GCEs/PPy-CuPcTs-MIP at different concentrations of E. coli0157:H7. FIG. 3A shows the

impedance spectra of GCEs/PPy-CuPcTs-MIP for detection of E. coli 0157:H7 at series of

gradient concentrations from 10 to 108 CFU/mL; and FIG. 3B shows the standard curve of the

EIS response versus log concentration of E. coli 0157:H7. The standard curve was linear in the

range of 10 to 108 CFU/mL, and the equation was: AR/R(Q)=10.83logioC - 9.40, with a

correlation coefficient R 2 =0.9938. The minimum detection limit was 10 CFU/mL. In order to

accurately determine the concentration of E. coli 0157:H7 in a sample, the concentration of E.

coli0157:H7 in the sample should not exceed 108 CFU/mL. If the concentration of E.

coli 0157:H7 in a sample is not within such range, the sample may be diluted or concentrated.

[62] Example 10

[63] 250 L of each of solutions containing E. coli 0157:H7, L. monocytogenes, S. aureus,

and Salmonella, respectively, at a concentration of 106 CFU/mL, were introduced into a 1-mL

centrifuge tube. The modified electrodes were immersed in the solution in each tube for 2 h for

recognition of the respective bacteria species, and EIS measurement was then performed

thereon. The measurement results are shown in FIG. 4.

[64] Example 11

[65] Surfaces of glassy carbon electrodes were ground and polished with a suspension of aluminium oxide particles having a particle size of 0.05 to 0.3 m. Thereafter, the electrodes

were ultrasonically cleaned in deionized water and ethanol each for 30 s, and then dried with

nitrogen. 17 L of pyrrole (functional monomer), 0.1 L of CuPcTs (dopant), 0.5 mL of a

solution containing a L. monocytogenes concentration of 106 CFU/mL (template bacteria), and

4 mL of a buffer solution (that is a 0.1 mol/L potassium chloride solution containing 1 mmol/L

K3[Fe(CN) 6] and 1 mmol/L K 4 Fe(CN) 6) were well mixed and introduced into a 5 mL-beaker.

The glassy carbon electrodes were then immersed in the mixed liquid in the beaker to conduct

electrochemical polymerization at room temperature via cyclic voltammetry under the

conditions of a scan rate of 0.05 V/s, a cyclic voltage of from -0.4 V to +0.7 V, and 15

electropolymerization cycles. After completion of the polymerization, the electrodes were

removed from the beaker and rinsed several times with deionized water and then dried with

nitrogen. Then, the electrodes were subjected to elution with SDS/HAc (5 % w/v) for 1.5 h to

remove the template bacteria, and rinsed several times with deionized water and then dried with

nitrogen. A molecularly imprinted polymer was thus obtained on the surfaces of the electrodes.

[66] 250 L of each of solutions containing E. coli 0157:H7, L. monocytogenes, S. aureus, and Salmonella, respectively, at a concentration of 106 CFU/mL, were introduced into a 1-mL

thereon. The measurement results are shown in FIG. 4.

[67] Example 12

[68] Surfaces of glassy carbon electrodes were ground and polished with a suspension of

aluminium oxide particles having a particle size of 0.05 to 0.3 m. Thereafter, the electrodes

solution containing a S. aureus concentration of 106 CFU/mL (template bacteria), and 4 mL of a

buffer solution (that is a 0.1 mol/L potassium chloride solution containing 1 mmol/L

The glassy carbon electrodes were then immersed in the mixed liquid in the beaker to conduct electrochemical polymerization at room temperature via cyclic voltammetry under the conditions of a scan rate of 0.05 V/s, a cyclic voltage of from -0.4 V to +0.7 V, and 15 electropolymerization cycles. After completion of the polymerization, the electrodes were removed from the beaker and rinsed several times with deionized water and then dried with nitrogen. Then, the electrodes were subjected to elution with SDS/HAc (5 % w/v) for 1.5 h to remove the template bacteria, and rinsed several times with deionized water and then dried with nitrogen. A molecularly imprinted polymer was thus obtained on the surfaces of the electrodes.

[69] 250 L of each of solutions containing E. coli 0157:H7, L. monocytogenes, S. aureus, and Salmonella, respectively, at a concentration of 106 CFU/mL, were introduced into a 1-mL centrifuge tube. The modified electrodes were immersed in the solution in each tube for 2 h for recognition of the respective bacteria species, and EIS measurement was then performed thereon. The measurement results are shown in FIG. 4.

[70] Example 13

[71] Surfaces of glassy carbon electrodes were ground and polished with a suspension of aluminium oxide particles having a particle size of 0.05 to 0.3 m. Thereafter, the electrodes were ultrasonically cleaned in deionized water and ethanol each for 30 s, and then dried with nitrogen. 17 L of pyrrole (functional monomer), 0.1 L of CuPcTs (dopant), 0.5 mL of a solution containing a Salmonella concentration of 106 CFU/mL (template bacteria), and 4 mL of a buffer solution (that is a 0.1 mol/L potassium chloride solution containing 1 mmol/L K3[Fe(CN) 6] and 1 mmol/L K 4 Fe(CN) 6) were well mixed and introduced into a 5 mL-beaker.

[72] 250 L of each of solutions containing E. coli 0157:H7, L. monocytogenes, S. aureus, and Salmonella, respectively, at a concentration of 106 CFU/mL, were introduced into a 1-mL

thereon. The measurement results are shown in FIG. 4.

[73] FIG. 4 shows EIS response of the different biosensors of GCEs/PPy-CuPcTs-MIP to the

respective template bacteria and the other three interfering bacteria species. It can be seen from

this figure that in the case of the template bacteria of E. coli 0157:H7, the EIS response of the

biosensors to E. coli 0157:H7 was higher than the response to the other interfering bacteria

species. Similarly, in the cases that the template bacteria was L. monocytogenes, S. aureus, or

Salmonella, the EIS response of the biosensors to the template bacteria was also higher than the

response to the other interfering bacteria species. Thus, the biosensor GCEs/PPy-CuPcTs-MIP

had high selectivity and versatility for the various bacteria speciess.

[74] Comparative Example 1

[75] Surfaces of glassy carbon electrodes (GCEs) were ground and polished with a

suspension of aluminium oxide particles having a particle size of 0.05 to 0.3 [m. Thereafter, the

electrodes were ultrasonically cleaned in deionized water and ethanol each for 30 s. The

electrodes so obtained was referred to as GCEs/bare herein. The GCEs/bare were then

immersed in a buffer solution, that is a 0.1 mol/L potassium chloride solution containing 1

mmol/L K3[Fe(CN) 6 ] and 1 mmol/L K 4 Fe(CN) 6, and EIS measurement was then performed

thereon.

[76] Comparative Example 2

[77] Surfaces of glassy carbon electrodes were ground and polished with a suspension of

were ultrasonically cleaned in deionized water and ethanol each for 30 s, and then dried with nitrogen. 17 L of pyrrole (functional monomer) and 4 mL of a buffer solution (that is a 0.1 mol/L potassium chloride solution containing 1 mmol/L K3[Fe(CN) 6] and 1 mmol/L K4 Fe(CN) 6

) were well mixed and introduced into a 5 mL-beaker. The glassy carbon electrodes were then

immsered in the mixed liquid in the beaker to conduct electrochemical polymerization at room

temperature via cyclic voltammetry under the conditions of a scan rate of 0.05 V/s, a cyclic

voltage of from -0.4 V to +0.7 V, and 15 electropolymerization cycles. After completion of the

polymerization, the electrodes were removed from the beaker and rinsed several times with

deionized water and then dried with nitrogen. The electrodes so modified were referred to as

GCEs/PPy herein. Then, the GCEs/PPy were immersed in a buffer solution (that is a 0.1 mol/L

potassium chloride solution containing 1 mmol/L K3[Fe(CN) 6 ] and 1 mmol/L K4 Fe(CN) 6), and

EIS measurement was then performed thereon.

[78] Comparative Example 3

[79] Surfaces of glassy carbon electrodes were ground and polished with a suspension of

nitrogen. 17 L of pyrrole (functional monomer), 0.1 L of CuPcTs (dopant) and 4 mL of a

removed from the beaker and were rinsed several times with deionized water and then dried

with nitrogen. The electrodes so modified were referred to as GCEs/PPy-CuPcTs herein. Then,

the GCEs/PPy-CuPcTs were immersed in a buffer solution (that is a 0.1 mol/L potassium

chloride solution containing 1 mmol/L K3[Fe(CN) 6] and 1 mmol/L K 4 Fe(CN) 6), and EIS

measurement was then performed thereon.

[80] FIG. 5 shows EIS data for GCEs/bare, GCEs/PPy, and GCEs/PPy-CuPcTs in the buffer solution. It can be seen from this figure that GCEs/bare, GCEs/PPy, and GCEs/PPy-CuPcTs had an impedance of 150, 200, and 40 Q, respectively. Thus, the impedance of GCEs/PPy-CuPcTs was 3.75 times lower than GCEs/bare, and was 5 times lower than GCEs/PPy.

Claims

1. A method for preparation of a molecularly imprinted polymer for detecting bacteria,

comprising steps of:

reaction so as to form a conducting polymer on surfaces of the electrodes; and

of the electrodes;

wherein the template bacteria is Listeria monocytogenes, Staphylococcus aureus,

Salmonella, or Escherichia coli 0157:H7 species;

the dopant is copper tetrasulfonatophthalocyanine; and

the functional monomer is pyrrole.

2. The method according to claim 1, wherein the predetermined ratio of the template

bacteria, the functional monomer, and the dopant is 500:17:(0.05-0.25) by volume.

3. The method according to claim 1, wherein the electrochemical polymerization reaction

in the step (a) is conducted via cyclic voltammetry, that is, when the electrodes are immersed in

the mixed liquid, scan parameters and the number of electropolymerization cycles are set to

predetermined values so as to carry out the electrochemical polymerization reaction.

4. The method according to claim 3, wherein the scan parameters include a scan rate and a

cyclic voltage, which are set to 0.05 V/s and -0.4 to +0.7 V, respectively; and wherein, the

number of electropolymerization cycles is set to 5 to 20.

5. A molecularly imprinted polymer prepared according to the method any one of claims 1

to 4.