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Biosensors and Bioelectronics 24 (2009) 3461–3466

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Biosensors and Bioelectronics
journal homepage: www.elsevier.com/locate/bios

Highly sensitive amperometric immunosensor for the detection of
Escherichia coli
K. Abu-Rabeah a,1 , A. Ashkenazi a,1 , D. Atias b , L. Amir c , R.S. Marks a,d,e,∗
a

Department of Biotechnology Engineering, Ben-Gurion University of the Negev, P.O. Box 653, Beer-Sheva 84105, Israel
Department of Virology, Ben-Gurion University of the Negev, P.O. Box 653, Beer-Sheva 84105, Israel
Department of Chemistry, Ben-Gurion University of the Negev, P.O. Box 653, Beer-Sheva 84105, Israel
d
National Institute for Biotechnology in the Negev, Ben-Gurion University of the Negev, P.O. Box 653, Beer-Sheva 84105, Israel
e
ILSE Katz Center for Meso and Nanoscale Science and Technology, P.O. Box 653, Beer Sheva 84105, Israel
b
c

a r t i c l e

i n f o

Article history:
Received 28 February 2009
Received in revised form 25 April 2009
Accepted 27 April 2009
Available online 3 May 2009
Keywords:
Escherichia coli (E. coli)
Electrochemistry
Immunosensor
␤-d-Galactosidase
Alginate-polypyrrole (Alg-Ppy)

a b s t r a c t
With the aim of detecting rapidly the presence of Escherichia coli (E. coli), a disposable amperometric
immunosensor was developed based on a double layered configuration at the transducer surface, consisting first of a polypyrrole-NH2 -anti-E. coli antibody (PAE) inner layer followed by an alginate-polypyrrole
(Alg-Ppy) outer packing layer. In the presence of the substrate p-aminophenyl ␤-d-galactopyranoside
(PAPG), the bacterial enzyme, ␤-d-galactosidase produces the p-aminophenol (PAP) product, also generating an amperometric signal due to PAP electrooxidation by potentiostating the glassy carbon (GC)
electrode at 0.22 V. The operational procedure consists in first adding the test sample containing the
bacteria, then coating it with Alg-Ppy to ensure the confinement of the released enzyme and the analyte
(being generated by the enzymatic catalysis) to the electrode active surface. This procedure facilitates
the diffusion of the substrate within the complex and thus creates a higher oxidation level of the PAP
enabling a detection limit of 10 colony forming units (CFU)/ml. The immunosensor setup demonstrates
an improved detection limit of more than 10 times less bacteria detected than other immunosensing
techniques without the need for multi step pretreatments of the test sample and/or incubation as found
in some of the existing methods.
© 2009 Elsevier B.V. All rights reserved.

1. Introduction
Detection of microorganisms is important in food and water
safety, while the presence of Escherichia coli (E. coli) is used as a
potential marker for the presence of pathogens originating from
humans and warm-blooded animals (Tryland and Fiksdal, 1998).
Estimating the number of coliforms is essential due to enteric disease, such as enterohemorrhagic strains of E. coli (Buchanan, 1997;
Tokarskyy and Marshall, 2008) contracted from food or polluted
coliform water supplies which still constitute public health concerns (Lin et al., 2008; Tokarskyy and Marshall, 2008).
Conventional procedures for E. coli detection and monitoring
are mainly based on cultures grown on differential agar media followed by counting the number of target organisms in the sample,
a procedure that can take 1–3 days (George et al., 2000; Lin et al.,
2008). The time-consuming drawback increases the motivation for

∗ Corresponding author at: Department of Biotechnology Engineering, BenGurion University of the Negev, P.O. Box 653, Beer-Sheva 84105, Israel.
Tel.: +972 8 6477182; fax: +972 8 6472857.
E-mail address: rsmarks@bgu.ac.il (R.S. Marks).
1
Both authors contributed equally to this work.
0956-5663/$ – see front matter © 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.bios.2009.04.042

the development of rapid and sensitive methods aiming at estimating E. coli (nucleic acid assays and immunological techniques
with chromogenic, fluorogenic, or chemiluminogenic substrates)
(Datsenko and Wanner, 2000; Feng and Hartman, 1982; Gehring et
al., 2004; Loge et al., 2002), usually showing a good linear correlation with those obtained from traditional methods (George et al.,
2000; Tryland and Fiksdal, 1998; Venkateswaran et al., 1996).
Biosensors are also being developed for the detection of
pathogenic bacteria including E. coli (Ivnitski et al., 1999; Leonard
et al., 2003; Tokarskyy and Marshall, 2008). Hence biosensors
using electrochemical (Abdel-Hamid et al., 1999a,b; Lin et al., 2008;
Mittelmann et al., 2002; Palenzuela et al., 2004), piezoelectric (Su
and Li, 2004), acoustic (Howe and Harding, 2000) and optical (Pyle
et al., 1995) transducers were applied for E. coli detection. The
number of electrochemical biosensing platforms (Bakker, 2004;
Mehrvar and Abdi, 2004) for the detection of coliform contamination have grown rapidly. For example, endogenous enzymatic
activity such as ␤-d-galactosidase which participates in lactose
metabolism was used as a general marker for monitoring coliforms
(Mittelmann et al., 2002; Tryland and Fiksdal, 1998), while some
studies have used indirect sandwich enzyme-linked immunoassay
with amperometric immunosensing (Abdel-Hamid et al., 1999a,b;
Lin et al., 2008). In all these methods, E. coli bacteria could be

the kinematic viscosity of the solution. ω. E. ı. pH 7. sodium p-toluene sulfonate (NaPTS) (152536) and 1. pyrrole (py) (13170-9). Eq. The cultures were then diluted with phosphate-buffered saline (PBS). Electropolymerized Alg-Ppy fills the porous alginate with polypyrrole filaments reducing possible leaching of bacterial contents that was made available by lysis. This system was tested with a model E. viscosity of 1% (w/v) solution is 39 cP) was provided by FMC Biopolymer (Norway). coli.8 mg/ml was introduced and the current resulting from ␤-dgalactosidase activity was collected and measured by PGSTAT 30 equipped with GPES4 software. the substrate concentration. coated with Ppy-NH2 .5 mM.62nFAC 0 DS 2. rapid.3. the substrate PAPG in final concentration of 0. simple to fabricate and cost effective was thus developed. whose value depends on the permeability Pm of the membranes (Eqs. 2. The enzyme catalyzed PAPG to form PAP which is then oxidized at the electrode surface. n. To start detection.2. coli polyclonal antibody (BO357) was purchased from Dako. (3) and (4)). a saturated Ag–AgCl–KCl electrode (Ag/AgCl) as reference electrode and a platinum wire as counter electrode. The system is entrapped in an Alg-Ppy matrix (Ionescu et al. This step is followed by induced bacterial lysis and the subsequent release of ␤-d-galactosidase which in the presence of PAPG produces PAP that is then oxidized on the electrode thereby creating a current (Mittelmann et al. Rabbit anti-E. 2) presents a linear behavior with the same slope as for a bare electrode with a positive intercept.. with the mass transport for a RDE coated with different electro-inactive membranes.. The amperometric detection was carried out in 15 ml of an aqueous solution of 0.1 ferrocenedicarboxylic acid (FeCN) (1293-87-4) were purchased from Aldrich. 2005). coli antibody is carried out through carbodiimide chemistry (Abu-Rabeah et al. Experimental PGSTST30 electrochemical workstation: a conventional electrochemical cell (Metrohm) consisting of GC disk electrodes as working electrode (diameter 3 mm—polished with 1 ␮m diamond paste MECAPREX Press PM). specific. K. where the first represents the current flow under the same conditions. The permeability (Pm ) of the coatings was estimated using RDE experiments at different rotation rates. 2. The results were analyzed and examined by applying Eqs. coli K-12 MG1655. 2005) which will confine the enzymatic catalysis to the electrode surface facilitating the diffusion and oxidation of the PAP. Abu-Rabeah et al. coli BW25113 with no ␤-d-galactosidase activity and Pseudomonas aeruginosa were grown over night in Luria Bertani (LB) medium at 37 ◦ C with shaking. Amperometric detection of ˇ-d-galactosidase activity ␤-d-Galactosidase activity was detected using PAPG as a substrate. a plot of 1/Ilim versus 1/ω1/2 (Fig. / Biosensors and Bioelectronics 24 (2009) 3461–3466 detected at ranges of concentrations between 102 and 108 CFU/ml without preincubation of the original sample. the partition equilibrium constant of the substrate between solution and membrane. lithium perchlorate (431567). the thickness of the membrane. 1979). The immobilization of the antiE. 2. Chemicals and reagents PAPG (A9545). The method is based on electropolymerization and coating of glassy carbon (GC) electrodes with polypyrrole-amine (Ppy-NH2 ). the diffusion constant of the substrate in the membrane and the film thickness. (1)–(4) (Gough and Leypoldt.1 M Tris–HCl (pH 7) as a redox probe. coli strain K-12 MG1655. . Only the first term of the equation is dependent upon the rotation rate of the RDE. The relative deviation of the values was <5%. the number of exchanged electrons and C0 . supplemented with IPTG to a final concentration of 0. Eq. Use of a secondary antibody was not necessary because detection was based on the activity of the intrinsic ␤-d-galactosidase enzyme.2. average molecular weight of 128 kDa. N-ethyl-N -(3-dimethylaminopropyl)carbodiimide hydrochloride (EDAC) (E1769). 2. the electrode surface. AFM micrographs were taken with AFM systems manufactured by Nanonics Imaging Ltd. coli.1 M Tris–HCl buffer electrolyte (pH 7). perchloric acid (17931) and hydroxy-2. There is still room to improve on the detection limits of environmental samples.. (1) is composed of two terms. An immunosensor that is very sensitive. lysozyme (Amresco 0663).22 V versus the reference electrode until reaching equilibrium.5-dioxopyrrolidine-3-sulfonicacid sodium salt (sulfo-NHS) (5648) were purchased from Fluka..4.5 h at 37 ◦ C while shaking until reaching logarithmic phase. 1 1 1 = + Ilim Is Im 2/3 Is = 0. acetonitrile (7505-8) was purchased from Bio Lab. in the absence of a membrane. 2005) later enabling the specific attachment of E. Jerusalem. The data acquisitions were achieved using GPES and FRA software. The experiment was begun by cultures inoculated into Erlenmeyer flasks containing 10 ml of LB medium. A description of the variation of the steady state limiting current Ilim is shown. A.. .3462 K. Therefore. The modified electrode was potentiostated at 0. and Tris–HCl (77-861) were purchased from Sigma. however.5. The experiments were conducted at different rotation speeds using FeCN (2 mM) in 0. The second term accounts for the diffusion of the substrate in the membrane and depends on the product of the partition equilibrium constant K. (2)). and Alg-py and py-NH2 were synthesized according to the protocols described in literature (Abu-Rabeah et al. and is therefore characteristic of the diffusion of the substrate in the bulk solution (Levich current. coli including O157:H7. These cultures were grown during 2–2. Permeability studies The permeability of a GC bare electrode. (1) Terms Ds and Dm are the diffusion coefficients of the substrate in the bulk solution and in the membrane respectively. introducing the appropriate capture antibodies would allow for the detection of any wild type strains of E. Sodium alginate (Alg) Protanal LF 10/60 (Laminaria hyperborea. 2002). Electrochemical instrumentation The voltametric and the amperometric measurements as well as the electropolymerization process were carried out using a −1/6 ω1/2 (2) Im = nFAKC 0 Dm = NFC 0 Pm ı (3) Pm = KDm (cm s−1 ) d (4) 2. 102 and 106 CFU/ml coatings were examined by a rotating-disk electrode (RDE) equipped with a rotation controller.1. isopropyl ␤-d-thiogalactopyranoside (IPTG) (16758). Bacterial culture Bacterial strains of wild type E. to concentrations ranging from 1 CFU/ml up to 107 CFU/ml and analyzed later by the electrochemical amperometric technique. the rotation rate of the RDE. We report an alternative immunosensing approach for the amperometric detection of E.

2(1a). 1. The impedance values increased significantly when the electrode was treated with the entrapping layer of the Alg-Ppy (Fig. 102 and 106 CFU/ml. 1. Permeability and AFM study The permeability of the immunosensor with different concentrations of E. The permeability of the electrode Fig. 2008). 3. Abu-Rabeah et al.93 V to retain the polymer conductivity. Different electrode configurations were investigated in the presence of Fe(CN)6 3− /Fe(CN)6 4− as a redox probe. thereafter the values were fitted by randles circuit based on Nyquist plots over the frequency range 1–105 Hz.1 M pyrrole in double distilled water and acetonitrile (volume ratio 7:3 respectively). 2 × 10−2 and 16 × 10−3 cm s−1 for GC coated with Ppy-NH2 . The operational procedure consists in first adding the sample. coli was performed with 50 ␮l of lysozyme solution (100 ␮g/ml) during 1 h at 37 ◦ C enabling the release of ␤-d-galactosidase from the bacteria.7 × 103 ) resulting from the immobilization of macromolecules on the transducer surface which inhibit the redox probe diffusion and increase the charge transfer resistance. coli antibody to Ppy-NH2 by incubation of the polymerized electrode in 1 ml PBS solution containing EDAC. (6) the Alg-Py matrix was electropolymerized on the surface of the working electrode by applying a constant voltage of 0.3 mg/ml and sulfo-NHS. Both lysis and antibody attachment procedures were performed in optimized conditions. inset) (R(p) = 0. The permeability of the electrode coated with 102 CFU/ml (Fig. 0. coli immunosensor The construction of the amperometric immunosensor consisted in: (1) electropolymerizaion of the Ppy-NH2 on the surface of the working electrode by applying a constant voltage of 0. 3. The amount of linked antibody was calculated while considering the charge density and the surface area of the electrode.93 V in 15 ml of 0.1. 2(3a)). This diffusion barrier will confine the resulting products of the enzymatic catalysis for oxidization at the transducer surface.1 M NaPTS and 1 M perchloric acid. 3. The impedance of the setup with higher bacteria concentration (106 CFU/ml) and entrapping Alg-py layer showed the highest charge transfer resistance (R(p) = 9 × 103  (Fig. (3) the antibody-linked electrode was incubated in 1 ml solution of E. Results and discussion The new disposable immunosensor configuration exhibits a double layer configuration in the transducer surface. (2) linking of anti-E. Polymerization was monitored by charge accumulation on the electrode surface. It is noteworthy that the increase in the charge transfer resistance could be correlated directly to diffusion limitations through the polymeric coating matrix. The high frequency (low impedance) semicircular regions in Ppy-NH2 modification have a very small radius of curvature. 0.6.1 M lithium perchlorate aqueous solution.1 M py-NH2 .93 V in a 15 ml solution composed of 0. and thereafter will be coated with Alg-Ppy to confine the released enzyme and the ana- 3463 lyte (being generated by the enzymatic catalysis) to the electrode active surface. Schematic diagram of the fabrication and chemical processes of the electrochemical immunosensor. coli in different concentrations for 1 h at 37 ◦ C with gentle shaking and then washed with Tris–HCl buffer. namely. These immunosensor working electrode probes were then used in the amperometric detection of ␤-d-galactosidase activity to estimate the bacterial concentrations of the test samples. Polymerization time was around 10 min and reaches a steady charge of Q = ∼90 mC. 2(3c)) was 13 times lower than the value calculated for the electrode coated with Ppy-NH2 only (Fig. 2(1b).5 × 103 ). (4) lysis of the attached E. and presented in Fig.5 × 10−1 .2. 0. 2.1 M Tris–HCl buffer) and then cross-linking for 30 min with a further drop of 10 ␮l of 0. / Biosensors and Bioelectronics 24 (2009) 3461–3466 2. respectively. Preparation of the amperometric E. (5) creation of an entrapment layer on the surface was achieved by dropping 20 ␮l of Alg-py (2% (w/v) in 0. this could be explained by the existence of a higher concentration of bacteria reducing the diffusion of the redox probe to the oxidizing sites on the transducer surface. Adding a volume greater than 20 ␮l of Alg-Py will lead to an unstable entrapment layer.3 mg/ml and 30 ␮l of polyclonal antibody for 2 h at 37 ◦ C with mild shaking before a PBS wash. 2(1d)). where the voltage was set at 0. But that of the electrode coated with PAE (Fig. and if the target bacteria are present then they are specifically attached by the PAE layer. indicating low resistance of the charge transfer (Fig.K. 2(1c)). 0. Electrochemical characterization of poly(Alg-py-PAE) Electrochemical impedance spectroscopy (EIS) was used as a sensitive method to probe the interface properties of surfacemodified electrodes (Geng et al. . set around Q = ∼3 C and also by the formation of a thick and stable polymer layer..1 M CaCl2 . 2(1b)). This setup showed higher charge transfer resistance (R(p) = 4 × 103 ) than the setup without Alg-Ppy layer (Fig. coli in the presence of FeCN as a redox probe was measured showing the following results: 3. inset) showed an expected higher value (R(p) = 1. a PAE inner capture layer and an Alg-Ppy outer confinement layer described in Fig.

2002). antibody and 106 CFU/ml of E.b. Calibration curve of E. the Ppy chains within the matrix improve the charge transfer and present superior platform when compared with unmodified alginate. The linear correlation coefficient (0. (c) 102 CFU/ml.. 3. Abu-Rabeah et al. Background levels of the current were also measured (filled triangle).1) alginate coated in GC electrode and (4.1 M Tris–HCl (pH 7) for GC bare electrode.3464 K. 3. coli (no lysozyme and Alg-Ppy matrix).2) Alg-Ppy coated and polymerized upon GC electrode. Mittelmann et al. The detection limit of the immunosensor was 10 CFU/ml. (d) 106 CFU/ml. The electrode coated with alginate and Alg-Ppy was probed by AFM techniques (Fig. 1999a. The experiments were run at three independent times with immunosensors that were made in different days and still showed good reproducibility. The current values were taken after 3 min from the addition time of PAPG.3. coli concentrations (1–107 CFU/ml) were tested and investigated by the immunosensor probe to set a correlation between the current level and the bacterial concentrations in order to explore the sensitivity of the applied approach. however the permeability value still enabled a good diffusion compared to the values presented for an electrode coated with only Ppy-NH2 .95) shows high matching between both current and concentration when it was drawn on a logarithmic scale. coated with 106 CFU/ml was further reduced (Fig... 2008. The permeability values were further supported by the impedance charge resistance values described before. GC electrode coated with Ppy-NH2 . However. (1) Impedance spectra of the immunosensor with different concentrations of E. (2) Equivalent electrical circuit of EIS data. 3. (b) Electrode coated with Ppy-NH2 . coli in the presence of Fe(CN)6 3− /Fe(CN)6 4− as a redox probe. coli ranging from 1 CFU/ml until 107 CFU/ml (filled squares). 2(3d)) by the increase of the biological material. GC electrode coated with 102 CFU/ml and 106 CFU/ml and entrapped in Alg-Ppy layer. These results are depicted in Fig. / Biosensors and Bioelectronics 24 (2009) 3461–3466 Fig. Reducing the number of bacteria to 1 CFU/ml gave a 35% overlap between the developed signal and the background noise of the instrument. Hamid et al. The dense morphology of the Alg-Ppy tightens the polymeric matrix on the electrode surface and decreased the permeability as shown before. Immunosensor calibration and sensitivity Various E. (4) AFM micrographs of (4. (3) Permeability measurements in the presence of 2 mM FeCN in 0. 2. Lin et al. Inset plot: Impedance spectra of: (a) electrode coated just with Ppy-NH2 . The layer of Alg-Ppy was shown to be more compact with smooth morphology and less thickness than the layer of natural unmodified alginate. . which is sensitive to detect the concentration of more than 10 times less bacteria than other immuno-sensing techniques that were published recently (Abdel- Fig. 2(4)).

4. coli antibody Sulfo-NHS and EDAC E. aeruginosa that is phylogenetically remote from E. Inset plot: Control experiments were preformed by the amperometric detection of 107 CFU/ml E. coli BW25113 (with no ␤-d-galactosidase activity) (䊉) were analyzed by the immunosensor with PAPG as a substrate. E. coli. presented a similar current response shape to that of E. Then the attached cells are lysed and entrapped by a conductive Alg-Ppy composite to confine the catalytic reaction on the transducer surface. food safety and environmental control. and the PAPG response decreased to 45% after 30 days. experiments were performed with a mutant strain of E. The longtime stability of the immunosensor activity was examined in this study too.. coli.7 Sensor + + + + − + − 283 + + + − + + + 11. coli (107 CFU/ml) lacking ␤-d-galactosidase activity and it showed no current (Fig.K. specific and sensitive assay has been described for the detection of intact cells of E. contributing to the sensitivity of the immunosensor. the bacterial separation and measurement are performed in several steps with a manual transfer step in between resulting in sample loss and errors due to contamination. coli immunosensor for ␤-dgalactosidase activity. coli K-12 MG1655 () and 107 CFU/ml E. In addition. coli with lysozyme.5 times in the current when compared to the complete modified sensor. 3. The experiment for long-time stability was conducted daily for this time. When it was not in use. both polypyrrole networks formed by electropolmerization and coating (Alg-py) at the electrode surface. when we used P. coli. The resulting current (1. Similarly. the immunosensor was stored in a PBS buffer solution at 4–8 ◦ C. Conclusions A rapid. In many analytical systems. 2005). providing a relative standard deviation (RSD) value of 10%. coli cultures PAPG was used as a substrate for ␤-d-galactosidase. Three different enzyme electrodes were tested independently for the PAPG amperometric response. 4. for his valuable assistance and . and the current response recorded as depicted in the amperometric curves in Fig. Abu-Rabeah et al. in particular. an efficient and reproducible immobilization process of the proposed biosensing matrix. coli that were analyzed by the immunosensor without lysozyme (Fig. Finally the reproducibility of the immunosensor fabrication was evaluated via the comparison of the response current to 0. The resulting curves with a concentration of bacteria of 107 CFU/ml showed that the fast current developed by the catalytic reaction of the enzyme clearly indicates a high current level measured after 3 min (1 ␮A) that could be directly related to the special construction of the immunosensor. Acknowledgments The authors gratefully acknowledge Dr. The polarization time of the sensor was 3 h/day. strengthen the structure mechanically from moderate stirring. 4. current levels dramatically decreased.. The chemical attachment of the antibody to the Ppy-NH2 film polymerized onto the electrode surface and attachment of an anti-E.5 nA) implied that no bacteria were attached as the activating linkers and therefore also the capture antibodies were Table 1 Effect of the different modifications on the response current in the electrochemical immunosensor. To check the high specificity of the E.4. chromatography or immunofiltration) which dramatically improves speed. It is expected that this extremely sensitive immunosensor will be helpful for the easy monitoring of water quality (Nicholas et al. inset). 2001). The importance of the different modifications to the immunosensors sensitivity was further supported by running several controls to examine the applied procedure (Table 1).8 mg/ml PAPG of different electrodes. In order to increase assay sensitivity prior to the assay. Omitting the entrapping layer of Alg-py caused a decrease of 3. coli antibody was examined by running a control experiment without the coupling reagents. This immunosensor set up has shown a high sensitivity (10 CFU/ml) as well as specificity and was shown to be stable up to one month. it would be desirable to concentrate the bacteria into a smaller volume which requires a long period of time for such a multistep procedure.5 + + + + − − + 1. Our approach does not require separation (electrophoresis. the potential was set at 0. but approximately 600 times lower. Immunosensor performance in identifying E. This coating layer is aimed at increasing the sensitivity of the obtained current by preventing the diffusion of the catalytic products (PAP) out into the phase solution being agitated around and to allow them to be oxidized with greater probability at the GC surface. while still being selective and sensitive with minimal sample manipulation and a short measurement time (3 h) from the moment the modified electrode has been exposed to the bacterial sample. USA.5 + + + + − + + 1000 Current values were taken after 3 min from the addition of the substrate PAPG to the monitoring system. sulfo-NHS and EDAC. The technique is based on the immobilization of the bacterial sample via specific capture antibodies to a GC electrode surface. The detection is done within a short 3 h period from the dipping of the modified electrode into the bacterial sample without the need for a preincubation period.22 V. K-12 MG1655 without lysozyme (). coli K-12 MG1655 (107 CFU/ml) Pseudomonas aeruginosa (107 CFU/ml) Lysozyme Alg-Ppy matrix Current (nA) + + − + − + + 1. Boris Polyak from Drexel University. The activity of ␤-d-galactosidase is dramatically increased from its basal levels by the lysis of the cells followed by a large amount of secreted enzymes. / Biosensors and Bioelectronics 24 (2009) 3461–3466 Fig. This indicates. Time is thus saved by avoiding concentrating the bacteria. 4). Electropolymerized Alg-Ppy fills the porous alginate with polypyrrole filaments reducing possible leaching of bacterial contents that was made available by lysis. We sought to increase sensitivity by coating the electrode with an Alg-Ppy layer assigned to better entrap the enzyme onto the electrode surface and confine the enzymatic reaction in close proximity to the electrode surface (Ionescu et al. 3465 missing and all the surface was washed away. College of Medicine. 4. Controls Ppy-NH2 Anti-E. Amperometric detection of 107 CFU/ml E.

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