Chemical and Biological Sensors Based on Electrochemical Detection Using Conducting Electroactive Polymers

Microchim. Acta 143, 123–137 (2003)

DOI 10.1007/s00604-003-0065-6

Original Paper

Chemical and Biological Sensors Based on ElectrochemicalDetection Using Conducting Electroactive Polymers

Sean Brahim1, Ann M. Wilson2;3, Dyer Narinesingh3, Emmanuel Iwuoha4,

and Anthony Guiseppi-Elie1;2;�

1 Departments of Chemical Engineering and The Center for Bioelectronics, Biosensors and Biochips (C3B),

Virginia Commonwealth University, School of Engineering, P.O. Box 843038, 601 West Main Street, Richmond,

Virginia 23284-3038, USA2 ABTECH Scientific, Inc., 911 East Leigh Street, Richmond, Virginia 23219, USA3 Department of Chemistry, University of the West Indies, St. Augustine, Republic of Trinidad and Tobago4 Department of Chemistry, University of the Western Cape, Bellville, CapeTown, South Africa

Received May 18, 2003; accepted June 12, 2003; published online November 17, 2003

# Springer-Verlag 2003

Abstract. The electrochemical behavior of compo-

sites of conducting electroactive polyaniline (PAn)

and polypyrrole (PPy) formulated within cross-linked

hydrogel networks was investigated by cyclic voltam-

metry and electrochemical impedance spectroscopy

(EIS). Composite PAn gels displayed similar anodic

charge density compared to the pristine conducting

polymer (80 mC=cm2 and 84 mC=cm2, respectively),

suggesting a similar degree of electroactivity between

the two systems. Composite gels of PAn displayed

fast cation transport with Kþ diffusivity (Dappt¼5.31�10�7 cm2s�1) that were three orders of magni-

tude larger than that of pristine PAn (Dappt¼3.12�10� 10 cm2s�1), while PPy composite gels

showed similar ferrocene anion diffusivity (Dappt¼7.05�10�5 cm2s�1) compared to electropolymerized

PPy (Dappt¼ 6.54�10�5 cm2s�1). The electrochemi-

cal interactions between CYP2D6, a cytochrome

P450 isoenzyme, and fluoxetine mediated by electro-

active polyaniline films on glassy carbon electrodes

(GCEs) were investigated. Cyclic voltammograms

indicate that PAn is an effective mediator of CYP2D6

activity under anaerobic conditions. An analytical

interrogation methodology based on small-amplitude,

pulsed DC was developed and incorporated into the

Electroconductive Polymer Sensor Interrogation Sys-

tem (EPSIS). Polypyrrole membranes were rendered

biospecific by either copolymerization of pyrrole (Py)

with 4-(1-pyrrolyl) butyric acid (4PyBA), followed by

direct conjugation with 5-(biotinamido)pentyl amine

(5BPA), or by reacting 4PyBA with 5BPA to form

pyrrolyl-biotin conjugates. The biotinylated PPy was

made responsive to glucose or urea by exploiting

strong biotin-streptavidin binding to either strepta-

vidin-glucose oxidase or biotin-urease conjugates.

These bioactive conducting polymer membranes were

demonstrated as conductimetric glucose and urea bio-

sensing layers using the EPSIS. The rate of conductiv-

ity of the bioactive PPy membranes was observed

to double upon increasing glucose concentration

from 100mM (4�10�6 S cm�1 s�1) to 600mM (9�10�6 S cm�1 s�1).

Key words: Electroconductive; hydrogel; polyaniline; polypyr-

role; electroactive polymers; polymer composites; cyclic voltam-

metry; electrochemical impedance spectroscopy; EPSIS.

The recent emphasis on materials science and chem-

istry has opened new approaches to analytical sensing,� Author for correspondence. E-mail: [email protected]

influencing the design of sensors, particularly those

using electrochemical or spectroscopic transduction

methods. Two groups of materials that have received

widespread applications research have been conduct-

ing electroactive polymers (CEPs) and hydrogels.

Conducting electroactive polymers (CEPs) are

advanced materials with tremendous prospects for

use in numerous applications such as sensors, actuator

components in microsurgical tools, controlled drug

delivery systems, corrective implantable aids, life-like

prosthetic limbs, actuators and artificial muscles [1].

Since the early 1990s, CEPs have been extensively

reported to function as thin films for batteries, sensors,

ion-selective electrodes and solid-state devices [2–6].

Of all the presently available CEPs, polypyrrole (PPy)

is generally considered to be the most promising for

the development of advanced sensor devices. This is

because of its good electrical conductivity, good

environmental stability, relative ease of synthesis [2,

7] and the potential for co-polymerization without

compromise of electroactivity. Polyaniline (PAn) has

also received considerable attention as a CEP because

of its unique conduction mechanism and environmen-

tal stability. It has been used primarily as a sensing

material for vapors such as methanol, ethanol, ace-

tone, benzene, etc. [8, 9].

Another interesting class of polymer that has gen-

erated much research and investigation is hydrogels.

One of the attractive features of these materials is that

they can be readily tailored to respond to specific

environmental stimuli such as pH, temperature and

ionic strength [10]. They may also possess high levels

of hydration, a very desirable property if the polymer

is to function as a matrix for biomolecule immobiliza-

tion. Several research groups have examined the

utility of hydrogels as sensors, by themselves or

chemically modified. Hydrogels have been employed

as the sensing layer in amperometric [11], potentio-

metric [12], conductimetric [13], and fibre-optic sen-

sors [14].

Over the last few years, special attention has been

given to the use of conducting polymeric composites

as a fabricating material for sensor devices [15]. Con-

ducting polymer composites or blends have been fab-

ricated by combining polypyrrole and=or polyaniline

with a host of polymers such as poly(vinyl chloride)

[16], polycarbonate [17], poly(vinyl alcohol) [18],

polyamides and imides [19], poly(ether ketone) [20],

nafion [21], and rubber [22]. Each of these efforts has

significantly modified the physical and mechanical

properties of the conducting polymer component and

has rendered them suitable for application in different

devices. Several research groups have investigated the

composites formed from CEPs and hydrogels for sen-

sing and controlled delivery applications. Wallace

et al. [23, 24] synthesized conducting polymer com-

posites of PPy and PAn with polyacrylamide and PPy

with poly(methylmethacrylate), demonstrating these

materials as controlled delivery devices. Dharwan

et al. [25] reported on the synthesis of a polyaniline-

(acrylonitrile-butadiene-styrene) composite membrane

as a sensor material for aqueous ammonia. Park and

Park [26] investigated the electrical properties of the

conducting composite poly(methylmethacrylate-co-

pyrrolylmethylstyrene)-g-polypyrrole. Guiseppi-Elie

et al. [27–30] synthesized bioactive composites of

polypyrrole- and polyaniline-containing poly(2-

hydroxyethyl methacrylate) hydrogels. These materi-

als were demonstrated as the sensing layer of stable

biosensors for clinically important analytes, and for

the electrostimulated release of bioactive peptides,

respectively.

In the present work, we report on the electrochemi-

cal characterization of electroconductive polypyrrole

and polyaniline co-polymers and hydrogel compo-

sites, and compare their electrochemical behavior to

the pristine CEPs. These copolymers and composites

allow the formation of polymer–biopolymer assem-

blies that serve as bio-smart materials, capable of

electrochemical response to biological stimuli. We

also describe a novel sensor technology platform,

the Electroconductive Polymer Sensor Interrogation

System (EPSIS). This instrument incorporates a

patented analytical methodology for sensor interroga-

tion of conducting electroactive polymer membranes

grown on microfabricated interdigitated microsensor

electrodes. The capability of the EPSIS sensor tech-

nology is demonstrated with PPy-based conducti-

metric glucose and urea biosensors.

Experimental

Materials

The monomer, 2-hydroxyethyl methacrylate 2-(HEMA), and

the prepolymers poly(ethylene glycol)(200) monomethacrylate

(PEG 200MMA) and poly-(2-hydroxyethyl methacrylate) (pHEMA)

were obtained from Polysciences, Inc., Warrington, PA. The cross-

linker, tetraethyleneglycol diacrylate (TEGDA), inhibitor remover

columns, aniline monomer (An) and dimer dianiline (DAn), pyrrole

(Py), photoinitiator dimethoxyphenyl acetophenone (DMPA), 3-

aminopropyltrimethoxysilane (�-APS), 3-methacryloxypropyltri-

124 S. Brahim et al.

methoxysilane (�-MPS), and N-[tris(hydroxymethyl)methyl] acryl-

amide (HMMA) were all obtained from Aldrich Co. (Milwaukee,

WI). The prepolymer methoxy-PEG(5000)-epoxide was obtained

from Shearwater Polymers, Inc. (Huntsville, AL). The potassium

salt of 1-(3-methacryloxy)propylsulfonate, the dopant for the com-

posite gels, was obtained from Sigma Chemical Co. (St. Louis,

MO). The N-hydroxy succinimide ester of biotin (sulfo-NHS-LC-

Biotin), 1,3-diisopropylcarbodiimide (DIPC), N-hydroxysulfosuccin-

imide (sulfo-NHS) and 5-(biotinamindo) pentyl amine (5BPA)

were obtained from Pierce and were used as received. Platinizing

solution of chloroplatinic acid (<5.0% w=v) and lead acetate

(<0.1% w=v) (YSI 3140) was purchased from YSI, Inc. (Yellow

Springs, OH). All other reagents used, including poly(styrenesul-

fonic acid) (PSS) (MW¼ 200,000), the dopant for pristine conduct-

ing electroactive polymers, and sodium dodecylbenzenesulfonate

(DBS), were of analytical reagent grade (Aldrich Co.) and used

without further purification. Prior to formulation, HEMA, HMMA,

and TEGDA were each passed over inhibitor remover columns to

remove the polymerization inhibitor, hydroquinone monomethyl

ether (MEHQ). The working electrodes used for electrochemical

characterization studies were either planar gold electrodes (PMEs)

(part number PME Au 118; 1.0 cm�1.8 cm) or microlithographi-

cally fabricated interdigitated microsensor electrodes (IMEs) (part

number IME 1050.5-M-Pt-U) that were purchased from ABTECH

Scientific Inc. (Richmond, VA). Each IME borosilicate glass chip

consisted of a pair of opposing electrodes comprising 10mm wide

platinum digits that were ca. 5 mm long and separated by 10mm

wide free spaces. There were 50 digits on each electrode bus that

established a tortuous path of 49.60 cm of exposed glass surface

between the digits.

Surface Preparation and Functionalization of Electrodes

The PMEs and IMEs were washed in boiling trichloroethylene,

followed by boiling acetone, 3 minutes in each solvent, then ultra-

sonically washed in isopropanol followed by distilled water. This

was followed by treatment at 60 �C for 10 seconds with a solution

comprising a 1:1:5 volume ratio of aqueous ammonia (0.1 M),

hydrogen peroxide (20% volume) and distilled water (RCA-clean).

The electrodes were then ultrasonically rinsed in deionised (DI)

water. An electroactive window that was 0.425 cm in diameter was

defined on each PME and IME using adhesive backed polyimide

tape. The windowed Au PME electrodes were then modified with

4-aminothiophenol and the primary amine subsequently derivatized

with methoxy-PEG(5000)-epoxide by reaction at 110 �C for

10 min. The exposed glass of the interdigit spaces of the windowed

IMEs were surface-modified with 3-aminmopropyltrimethoxy

silane followed by surface derivatization with methoxy-PEG

(5000)-epoxide. These surface modifications were done to prevent=minimize subsequent hydrogel disbondment from the electro-

des upon immersion and repeated redox cycling in aqueous

solution.

Formulation of Electroconductive Hydrogels

A typical electroconductive gel formulation contained the mono-

mers and prepolymers in the ratio shown in Table 1. To each 5 g

batch of this formulation was added 20% by weight of DI water

(1 g) and ethylene glycol (1 g) as mixed solvent. The acrylate

monomers HEMA and TEGDA, along with the respective electro-

active monomer (Py or An and DAn), were first mixed together

with the other components and used as the receiving mixture to

dissolve the photoinitiator. An appropriate quantity (typically 3mL)

of the formulated gel-monomer mixture was then applied to the

window, and a thin film was cast over the working electrode area

by spin coating at ca. 3000 r.p.m for 5 seconds. The mixture was

immediately irradiated with U.V. light (366 nm, 2.3 watts=cm2,

Spectroline Model 330844) for 30 minutes under an inert argon

atmosphere to effect polymerization of the hydrogel component.

For hydrogel composites of polyaniline, the conducting electroac-

tive component of the composite membrane was grown within the

interstitial spaces of the pre-formed hydrogel network by two

techniques: oxidative chemical polymerization and oxidative elec-

tropolymerization. Chemical oxidation of aniline was effected by

exposure of the aniline-impregnated hydrogels to 0.10 M FeCl3(ca. 25mL, pH 1.4) at 20 �C for 1 h. Electropolymerized polyani-

line hydrogels were prepared by potentiostatic electropolymeriza-

tion (þ0.70 V vs. Ag=AgCl, 3 M Cl�) of the entrapped monomer

from deaerated 1.0 M An, 0.01 M DAn, and 2.0 M HCl held at

20 �C. The film was allowed to grow on the electrodes such that a

fully contiguous membrane was formed. Finally, a mixed mode

technique combining the above two approaches was also used to

prepare composite gels. In this scheme, electroactive aniline mono-

mer within the pre-formed hydrogel membrane was exposed to

FeCl3 prior to electropolymerization. With each technique, un-

polymerized monomer was removed by sequential washing for

30 min in each of 100% ethanol, 75%, 50%, 25% ethanol-water

mixtures, and finally in DI water.

The composition of the electroconductive PPy–hydrogel that

was selected (Table 1) corresponded to the formulation used in

the construction of amperometric biosensors [28]. This formulation

consisted of the monomers HEMA:TEGDA:Py in a ratio of

85:10:05 mol%. Immediately following UV-initiated polymeriza-

tion of the hydrophilic hydrogel network, the electrode-supported

membrane was immersed into 3 mL of a deaerated phosphate

buffered KCl solution (0.1 M NaH2PO4 containing 0.1 M KCl,

pH 7.2) that was saturated with pyrrole monomer (ca. 0.4 M).

The polypyrrole component of the composite membrane was

deposited within the interstitial spaces of the pre-formed hydrogel

network by potentiostatic electropolymerization (þ0.85 V vs.

Ag=AgCl, 3 M Cl�) for 100 seconds. This resulted in a typical

Table 1. Typical formulations of electroconductive gel dopes based

on polyaniline (PAn) and polypyrrole (PPy)

Compounds in formulae Mole% g%

PAn PPy PAn PPy

2-Hydroxyethyl methacrylate 57.85 84 50.44 74.99

N-[Tris(hydroxymethyl)methyl]

acrylamide

10 – 11.74 –

Poly(ethyleneglycol)(200)mono-

methacrylate

5 – 8.78 –

1-(3-Methacryloxy)propyl-

sulfonate

5 – 8.25 –

Tetraethylene glycol diacrylate 3 10 6.08 20.97

Poly-(2-hydroxyethyl

methacrylate)

2 – 1.74 –

2,2-Dimethoxy-2-phenylaceto-

phenone

2 1 3.43 1.72

Aniline 15 – 9.36 –

Dianiline 0.15 – 0.19 –

Pyrrole – 5 – 2.32

Total of Reagents 100 100 100 100

Water 20 –

Ethylene glycol 20 –

Chemical and Biological Sensors Based on Electrochemical Detection Using Conducting Electroactive Polymers 125

polymerization charge of ca. 90�10�3 C. The electrodes were

then rinsed with phosphate buffer (0.1 M, pH 7.0) to remove any

residual monomer.

Synthesis of 4-(1-Pyrrolyl) Butyric Acid (4PyBA)

4-(1-pyrrolyl) butyric acid (4PyBA) was synthesized using proce-

dures analogous to the methods of Kakushima [31] and Ryder [32].

Briefly, succinic anhydride (9.75 g, 97%) was added at room tem-

perature to a suspension of AlCl3 (28.0 g) in 400 mL dichloroethane

(dry). The mixture was stirred at room temperature for 20 minutes.

A solution of phenylsulfonylpyrrole (20 g, 98%) in 50 mL of

dichloroethane was added, and the mixture was stirred for 4 h at

room temperature. The reaction was quenched with about 500 g ice

and the product extracted 3 times with dichloromethane. The

dichloromethane extract was washed once with water, and the sol-

vent was removed using a rotary evaporator. Recrystallization from

dichloromethane gave the product of 4-(1-(phenylsulfonyl)-3-pyrro-

lyl)-oxobutyric acid (27.12 g). A mixture of zinc granules (300 g)

and mercuric chloride (5.0 g) in 100 mL water with 4.0 mL HCl

added was shaken at room temperature for 10 minutes. The super-

natant was decanted. To this solid was added 40 mL water, 100 mL

12 N HCl, 250 mL toluene and 20.0 g 4-(1-(phenylsulfonyl)-3-pyr-

rolyl)-oxobutyric acid. The mixture was refluxed for 36 hours during

which time 4�20 mL 12 N HCl was added. After cooling, the

organic layer was separated, and the aqueous layer was washed 3

times with toluene. The combined organic extract was evaporated to

dryness to give a solid of 4-(1-(phenylsulfonyl)-3-pyrrolyl)-butyric

acid. Under N2, the solid was stirred in a solution of 100 mL metha-

nol and 100 mL 5 N NaOH at 21 �C for 24 h and at 75 �C (bathing

temperature) for 4 h. After cooling, it was acidified with dilute HCl

to pH 5–6 and extracted with dichloromethane four times. The

combined organic extract was washed once with water. Removal

of solvent and recrystallization in dichloromethane gave a final

product of 2.9 g. mp 92–94 �C; FT-IR (KBr) 2964 (br), 1717 (br),

3400, 3075, 2915, 1466, 1353, 1293, 1246, 1210, 1177, 1082, 1058,

916, 784, 602 cm�1; lH NMR (300 MHz, CDCl3) � 8.1 (1 H, br,

NH), 7.3 (1 H, s, CO2H), 6.76 (1 H, d, H 5, pyrrole), 6.6 (1 H, d, H 4,

pyrrole), 6.13 (1 H, s, H 2, pyrrole), 2.62 (2 H, t, C4 H) 2.4 (2 H, m,

C2 H), 1.93 (2 H, quintet, C3 H).

Bioimmobilization

In one instance, biospecific membranes were prepared by copoly-

merization of pyrrole (Py) with 4-(1-pyrrolyl) butyric acid (4PyBA)

to form the conducting polymer with pendant carboxylic acid func-

tionalities on the backbone. This was followed by direct conjugation

of available surface carboxylic acid groups with 5-(biotinamido)

pentyl amine (5BPA). In another instance, a pyrrolyl-biotin conju-

gate was first prepared by reacting 0.1 M 4PyBA with 0.1 M 5BPA

in the presence of 0.1 M sulfo-NHS and 0.1 M DIPC. To prepare

polymer membranes, an electropolymerization bath was prepared

that contained 0.1 M Py, ca. 0.1 M 4PyBA or 0.1 M 4PyBA-5BPA

conjugate, 2.5 mM poly(styrenesulfonic acid) (PSS), and 2.5 mM

sodium dodecylbenzenesulfonate (DBS) at a pH¼ 3.0 and T¼20 �C. Electropolymerization of co-polymer films was accom-

plished by applying þ0.65 V vs. Ag=AgCl, 3 M Cl� to the shorted

electrodes of the IME device. The film was allowed to grow on each

electrode and also between the digits of the pair of electrodes such

that it formed an adherent and fully contiguous membrane as shown

in Scheme 1. At the end of electropolymerization, the device was

removed and rinsed thoroughly in 0.1 M KCl. EPSISTM conductivity

testing of the derivatized and functionalized polypyrrole was per-

formed using an enzyme-streptavidin conjugate that binds specifi-

cally to biotin. The biotinylated PPy-IME was made responsive to

glucose by immobilizing the enzyme glucose oxidase (GOx) to the

polymeric membrane via a GOx-streptavidin conjugate. Exploiting

the very strong biotin-streptavidin binding, a streptavidin-glucose

Scheme 1. Immobilization of biotin

to IME=PPy-hydrogel device. Reac-

tion of surface-available, N-acid

moieties of the pyrrole=4-(1-pyrro-

lyl)butaric acid polymer conjugate

with 5-(biotinamido)pentyl amine

using carbodiimide linking chemis-

try produces a biotinylated copoly-

mer at the surface


oxidase conjugate (1 mg mL�1) was allowed to couple to the bio-

tinylated surface of the polymer membrane in phosphate buffer-

ed saline, pH 7.2, at 15� for 1 h. Alternatively, streptavidin

(1 mg mL�1) was allowed to couple to the biotinylated surface of

the PPy-membrane under identical conditions, followed by subse-

quent coupling of a biotin-urease conjugate that was prepared as

described previously [33]. The response of the resulting PPy-based

conductimetric biosensors to glucose and urea, respectively, was

determined using EPSIS.

Characterization of Polymer, Copolymer and Polymer

Composite Membranes

For all electrochemical characterization studies (cyclic voltammetry,

EIS and EPSIS) composite membranes of the electroconductive

hydrogels were prepared as outlined above on cleaned microlitho-

graphically fabricated interdigitated microsensor electrodes (IMEs)

and=or gold planar metal electrodes (PMEs). Prior to polymer mem-

brane deposition, the digits of the IME (1050.5-M-Pt-U) were plat-

inized via controlled potential coulometry using a current density of

21 mA=cm2 (49 mC). The set-up for three-electrode electrochemis-

try consisted of a Perkin-Elmer Princeton Applied Research Model

273 Potentiostat=Galvanostat linked to a Gateway PC and controlled

by Perkin-Elmer PAR M272 software. Cyclic voltammetry, ampero-

metry, electrochemical polymerization and coulometry were per-

formed in a standard three-electrode cell with a miniature Ag=AgCl, 3 M Cl� reference electrode (RE 803; ABTECH Scientific,

Inc., Richmond, VA) and a platinum mesh counter electrode. Three-

electrode electrochemical impedance spectra (10 mVamplitude; sine

wave; 1 mHz–60 kHz; 20� 1 �C) were obtained using a Perkin-

Elmer PAR Model 273 Potentiostat=Galvanostat coupled with

a Solartron Schlumberger 1250 Frequency Response Analyzer

(FRA). The FRA was used in single-sine mode to cover the range

1 Hz–60 kHz, and the Model 273 was used in multi-sine mode to

cover the range 1 mHz–1 Hz. The Perkin-Elmer PAR M398 soft-

ware was used for data capture, data merging and analysis. Two-

point electrical resistance measurements of the polymer membranes

were made using a Keithley Model 2000 Multimeter. The EPSIS

(Electroconductive Polymer Sensor Interrogation System) was used

to characterize the biologically modulated changes in conductivity

of the conducting polymer films. EPSIS employs a unique and

patented [34] interrogation method specifically developed for

obtaining the conductimetric signal amplification derived from

electroconductive polymer transducers [35]. This method is most

effectively performed on electroconductive polymer transducers

formed from interdigitated microsensor electrodes (IME) onto

which are coated the chemically sensitive electroconductive poly-

mer membranes.

Results and Discussion

Cyclic Voltammetry

Cyclic voltammograms (CVs) were obtained for both

electropolymerized pristine conducting polymers and

the electroconductive composite membrane compris-

ing polyaniline and=or polypyrrole electropolymer-

ized within the UV polymerized p(HEMA)-based

hydrogel. The CV for electropolymerized polyaniline

(poly(styrene sulphonate) counter anion) on platinum

IMEs (Fig. 1a) was measured at 20 �C in dilute HCl,

pH 4.0, with no other added electrolyte, at a scan rate

of 100 mV=s. The resulting voltammogram displayed

the expected two distinct redox peaks over the poten-

tial range of �0.2 V–0.8 V vs. SCE. Using the IME

format, the conductivity of the PAn film was measured

at discrete potentials over the range of �0.2 V to 0.8 V

vs. SCE. This is shown as an overlay on Fig. 1. The

first oxidation peak at 0.2 V resulted in a concomitant

increase in the inherent conductivity of the electro-

polymerized polymer over three orders of magnitude

(<1e-4 S=cm to ca. 1e-1 S=cm), corresponding to

the formation of the conducting radical cation of

Fig. 1. Cyclic voltammograms of (A) polyaniline and (B) polypyrrole on IME devices measured in dilute HCl, pH 4.0, 20 �C


imino-1,4-phenylene (–NH�þA�C6H4–). The second

oxidation peak occurred at around 0.7 V, and this

redox phenomenon was associated with a decrease

in polymer conductivity back to initial low values

approaching 1e-5 S=cm, corresponding to the forma-

tion of the non-conducting pernigraniline (–N¼C6H4¼NC6H4–). On the cathodic half wave, the

corresponding reduction potential peaks occurred at

0.65 V and 0 V, respectively. The formal potential

E�0, associated with each pair of redox peaks, was

determined to be 0.12 V (corresponding to the first

redox pair) and 0.68 V (corresponding to the second

redox pair). The peak separation between anodic and

cathodic peak potentials (�Ep) was smaller for the

second redox couple, ca. 0.05 V, compared to 0.2 V

for the first redox couple. It is noteworthy that using

the interdigitated microsensor electrode (IME) chip

format, the conductivity displayed sharp transitions

coincident with the E�0 of these redox reactions. This

has been previously shown as a maximum between

the redox transitions [36].

The CV for electropolymerized polypyrrole, doped

with poly(styrene sulphonate), on a platinum IME was

also measured at 20 �C in dilute HCl, pH 4.0, at a scan

rate of 100 mV=s. The resulting voltammogram (Fig.

1b) displayed the characteristic two distinct redox

peaks over the potential range of �1.0 V to 1.0 V vs.

SCE. The absence of electroactivity over positive

potentials, typical of voltammograms of polypyrrole

doped with Cl�, was due to the nature of the dopant

anion incorporated into the film, as well as the choice

of solvent. The oxidation peak occurred at around

�0.45 V, which resulted in a concomitant sharp

increase in the inherent conductivity of the electropo-

lymerized polymer over three orders of magnitude

(near 1e-6 S=cm to >1e-3 S=cm). On the cathodic

half wave, the corresponding reduction peak occurred

at ca. �0.75 V, with a subsequent return to the elec-

trically insulating form of polypyrrole as shown by the

sharp reduction in conductivity. Like the polyaniline

conductivity profile, the conductivity transitions

observed for polypyrrole measured using the IME

chip format were similarly very sharp and coincident

with the E�0 of the characteristic redox reaction. The

formal potential, E�0, was determined to be �0.60 V.

The peak separation between anodic and cathodic

peak potentials (�Ep) was 0.3 V. The current ratio,

Ipc=Ipa, was ca. 1.5 at the scan rate investigated, sug-

gesting that the cathodic reaction is more facile than

the anodic reaction under these experimental condi-

tions. That is, the reduction of the PPyjPSS film,

which involves ingress of cations from solution into

the film to establish electroneutrality, is more electro-

chemically feasible and occurs more readily than the

alternate egress of cations into solution, which char-

acterizes the oxidation of the PPyjPSS film.

Cyclic voltammograms of the composite polyani-

line gels were measured in 0.1 M KCl solution, pH

5.4, at 20 �C. Well-resolved voltammograms (Fig. 2a

and b) containing one pair of redox peaks were

obtained at the different scan rates investigated (5–

500 mV=s). For the composite membranes formed

by the combined chemical and electrochemical poly-

merization of An, PAn gel–FeCl3 and EP (Fig. 2a),

the separations between anodic and cathodic peak

potentials (�Ep) increased from ca. 50 mV to

350 mV upon increasing the scan rate from 5 mV=s

to 500 mV=s. At the highest scan rate of 500 mV=s,

well-defined oxidation and reduction peaks were

observed at 500 and 150 mV, respectively. The current

ratios of cathodic and anodic peak currents, Ipc=Ipa,

did not deviate significantly from 1, suggesting redox

reversibility. The formal potential at this scan rate,

E�0, was calculated to be 0.28 V. In contrast, oxida-

tively prepared PAn films (PAn gel–FeCl3) exhibited a

formal potential of 0.27 V at 500 mV=s.

Of interest is the increase in magnitude of both

anodic and cathodic peak currents for PAn gel–FeCl3and EP compared to pristine electropolymerized poly-

aniline (PAn–EP, Fig. 1a) and PAn gel–FeCl3 compo-

site membranes (Fig. 2b). At 100 mV=s, these

increase from ca. 2 mA for polyaniline and 5 mA for

PAn gel–FeCl3 to around 25 mA for the composite

gel, indicating increased capacitative charging. A

comparison of the anodic charge density for these

three conducting polymer systems (PAn–EP, PAn

gel–FeCl3 and PAn gel–FeCl3 and EP) at 5 mV=s

reveals approximate values of 84 mC=cm2, 3 mC=cm2

and 80 mC=cm2, respectively. Anodic charge density

can be regarded as a measure of the electroactivity of

the gel membrane, thus suggesting a similar degree of

electroactivity between pristine PAn–EP and PAn

gel–FeCl3 and EP.

Multiple scan rate cyclic voltammetry was per-

formed on each of the following systems in 0.1 M

KCl solution, pH 5.4, at 20 �C using gold PMEs: (i)

electropolymerized polyaniline (PAn–EP); (ii) com-

posite hydrogel containing PAn grown using FeCl3(PAn gel–FeCl3); (iii) composite hydrogel containing

PAn grown using electropolymerization combined


with FeCl3 (PAn gel–FeCl3–EP). From these scans,

approximate apparent diffusion coefficients for the Kþ

cation were determined for each polymer system

using the Randles-Sevcik equation:

ip ¼ 2:686�105 n3=2CoxD1=2v1=2A

Developed for solution species under semi-infinite lin-

ear diffusion conditions, this equation provides only

an indication of the differences in diffusion coeffi-

cients when applied to surface-confined polymer

films. Analysis of the peak anodic and cathodic cur-

rents (Ip) as a function of scan rate (v1=2) revealed the

diffusivity (D) for Kþ that was 5.31�10�7 cm2s�1 in

PAn gel–FeCl3–EP, 3.12�10�8 cm2s�1 in PAn gel–

FeCl3, and 3.12�10�10 cm2s�1 in PAn–EP. Table 2

summarizes the apparent diffusion coefficients, Dappt,

obtained for these different materials and compared to

that of ferrocene monocarboxylic acid obtained under

similar conditions at a bare or unmodified planar gold

electrode. Thus, the electroconductive gels containing

PAn prepared by the mixed-mode technique showed

the fastest rate of cation transport of all the mem-

branes and was three orders of magnitude larger than

that of electropolymerized PAn. However, this apparent

diffusion coefficient was one order of magnitude less

Fig. 2. Cyclic voltammograms of electroconductive polyaniline hydrogels (A) synthesized by the mixed mode technique: PAn gel–FeCl3–

EP and (B) by chemical oxidation: PAn gel–FeCl3 and (C) electroconductive polypyrrole hydrogels: PPy gel–EP on IME devices measured

in 0.1 M KCl solution, pH 5.4, 20 �C (PAn gel–FeCl3–EP and PAn gel–FeCl3) and 0.1 M phosphate buffered KCl, pH 7.4, 25 �C (PPy

gel–EP)

Table 2. Apparent diffusion coefficients (Dappt) for ferrocene

monocarboxylate at different electrode surfaces

‘‘Material’’ Apparent diffusion

coefficient (Dappt)=cm2s�1

Au 3.70�10�6

Pt 3.46�10�5

AujPAn–EP 3.12�10�10

AujPAn gel–FeCl3 3.12�10�8

AujPAn gel–FeCl3–EP 5.31�10�7

PtjPPy–EP 6.54�10�5

PtjPPy gel–EP 7.05�10�5


than solution phase ferrocene monocarboxylic acid.

This confirms that electroconductive hydrogel compo-

sites offer faster redox switching speeds compared to

the pure electroactive polymers because of the signif-

icant hydration levels contributed by the hydrogel

component.

Cyclic voltammograms of the composite electro-

conductive hydrogel after electropolymerization of

incorporated pyrrole monomer at þ0.85 V vs.

Ag=AgCl for 100 s showed retained electroactivity

(Fig. 2c). Anodic and cathodic peak potential separa-

tions were observed to increase with scan rate, from

0.18 V at 10 mV=s up to 0.34 V at 100 mV=s, similar

to the �Ep for pristine electropolymerized polypyr-

role at 100 mV=s. The current ratios, Ipc=Ipa, averaged

around 1.4 over the four scan rates investigated sug-

gesting that, like the electropolymerized polypyrrole,

the cathodic reaction is more facile than the anodic

reaction for the electroconductive gel. Likewise, the

formal potential (E�0) of the composite shifted to more

negative values, �0.40 to �0.47 V, upon increasing

the scan rate to 100 mV=s. The overall impact of com-

posite formation is to force the cathodic reaction to

more negative potentials suggesting less facile egress

of anions or ingress of cations under the swollen con-

ditions of the hydrogel composite.

Multiple scan rate cyclic voltammetry was per-

formed on platinum PMEs coated with the following

membranes: (i) uncoated platinum (Pt); (ii) electropo-

lymerized polypyrrole (PPy–EP); (iii) electroconduc-

tive PPy gel (PPy gel–EP) in 0.1 M PBKCl solution,

pH 7.0 at 25 �C containing 15 mM ferrocene mono-

carboxylic acid. From these scans the apparent

diffusion coefficients for the ferrocene anion were

determined for each system as before and summarized

in Table 2. Using the peak cathodic currents, the diffu-

sivity (D) for ferrocene anion was calculated to

be 3.46�10�5 cm2s�1 at the bare Pt electrode,

6.54�10�5 cm2s�1 in pristine PPy (PPy–EP), and

7.05�10�5 cm2s�1 in PPy gel–EP. Thus the rate of

diffusion of the ferrocene carboxylate anion is ob-

served to double at platinum electrodes coated with

the electroconductive PPy gel membrane compared to

bare, unmodified electrodes.

Electrochemical Impedance Spectroscopy

Both the ‘blank’ hydrogel without any PAn and elec-

troconductive PAn–hydrogel were grown on platinum

IMEs, immersed in deaerated 0.10 M KCl at 20 �C

and subjected to frequency-dependent impedimetric

analyses at three different inquiring or offset poten-

tials; �300 mV, 215 mV and 800 mV. These potentials

were selected to interrogate the composite hydrogel

under fully reducing, intermediate redox, and fully

oxidizing electrochemical states, respectively. EIS

interrogation was performed over the frequency range

of 0.10 mHz to 100 kHz using a sine wave voltage

pattern of 10 mV peak voltage. The two electrodes

of the interdigitated array served as working and aux-

iliary electrodes, respectively, while the reference

electrode (Ag=AgCl, 3 M Cl�) was placed in the elec-

trolyte and in close proximity to the hydrogel surface.

Figure 3a shows a Bode plot of the impedance (mag-

nitude and phase) of polyaniline-free hydrogel coated

IMEs. There was almost complete superpositioning of

the impedance magnitude, jZj, at all potentials and

across the entire frequency range interrogated. Sweep-

ing over the frequency range from low (0.1 mHz) to

high (100 kHz) was accompanied by an inherent

decrease in network impedance by ca. 4 orders of

magnitude. The impedance upon approaching DC

was in all cases ca. 1 MOhm. Interrogating the hydro-

gel at the three potentials did not cause the phase

profiles to deviate significantly from each other, with

a minimum phase angle occurring near 100 Hz for all.

With the electroconductive PAn gel coated IME,

the resulting Bode plot of network impedance (mag-

nitude and phase, Fig. 3b) showed distinct profiles

depending on the inquiring potential. At �300 mV,

the profile of impedance magnitude, jZj, resembled

that for the blank hydrogel at the same potential, with

a slight reduction in impedance magnitude at frequen-

cies approaching DC. The corresponding phase plot

also resembled that for the blank hydrogel with a

phase angle minimum near 100 Hz, but there was also

the appearance of a second inflexion point around

10 kHz. At this potential, the polyaniline component

of the composite gel is fully reduced and hence elec-

trically insulating, contributing to the observed high

network impedance magnitude. Upon increasing the

offset potential to 215 mV, there was a dramatic

reduction in network impedance magnitude (>2

orders of magnitude) over the low to medium fre-

quency interrogation range compared to the profile

obtained at �300 mV. At this oxidizing potential,

there was frequency independence of network impe-

dance magnitude over the entire range investigated

and frequency independence of network phase angle

up to 1 kHz. At 800 mV, there was still further


reduction in network impedance magnitude and fre-

quency independence. The effect of applying oxidiz-

ing potentials to the composite gel is to force the

transition of the conducting polymer component from

its electrically insulating state to a conducting state,

and this is clearly reflected in the Bode plots of Fig. 3b.

For the system containing the electroconductive

PPy–hydrogel and measured across the gel (Fig. 4),

the frequency-dependent impedance profiles (jZj and

phase) were determined at three different potentials.

The three potentials that were selected were: (i) the

formal potential, E�0, (�0.47 V) (ii) E�0 ��Ep,

(�0.80 V) and (iii) E�0 þ�Ep (�0.13 V). In the fre-

quency range approaching DC, it was observed that

the network impedance with the highest value was

established with the reduced form of the electrocon-

ductive hydrogel composite (�0.80 V), corresponding

to the polymer in the electrically insulating state. The

phase angle profile at �0.80 V showed a peak phase of

ca. 68� occurring at just under 1 Hz. The other two

impedance magnitude profiles corresponding to oxi-

dized gel (�132 mV) and gel in an intermediate redox

Fig. 3. Bode plot showing impedance magnitude and phase for (A) the blank hydrogel and (B) the electroconductive polyaniline hydrogel

Fig. 4. Bode plot showing impedance magnitude and phase for the electroconductive polypyrrole hydrogel measured with the pair of

interdigitated microsensor electrodes shorted to serve as a single working electrode and using an external counter electrode (across the

polymer membrane)


state (�468 mV) displayed similar network impedance

values in this frequency range. Between the frequency

range 0.1–1000 Hz, however, the oxidized form of the

electroconductive PPy–hydrogel exhibited the highest

network impedance, with the intermediate redox state

having network impedance magnitudes between the

oxidized and reduced forms of the gel. The phase angle

plots obtained for oxidized and intermediate redox

states of electroconductive PPy–gel both showed peak

phase angles of 35� and 42� respectively at ca. 1 Hz.

Thus it can be concluded that the network impe-

dance varies with the nature of the electroconductive

gel and its redox state. Network impedance near the

DC region and low-to-mid frequency range is

observed to be lowest with the electroconductive

polyaniline in the oxidized state, where frequency

independence is observed.

Polyaniline-Based CYP2D6 Biosensors

Fluoxetine hydrochloride (Prozac+), a selective sero-

tonin re-uptake inhibitor (SSRI), is currently an effec-

tive and widely prescribed drug routinely dispensed to

adults and children for the treatment of major depres-

sive disorders [36]. Multiple cytochrome P450 isoen-

zymes, including CYP2D6, are responsible for the

conversion of fluoxetine to norfluoxetine; norfluo-

xetine’s potency and selectivity as a serotonin uptake

blocker are essentially equivalent to fluoxetine’s. The

electrochemical interactions between CYP2D6 and

fluoxetine mediated by electroactive polyaniline films

on glassy carbon electrodes (GCEs) were investigated.

Polyaniline films were prepared on previously

cleaned GCEs by potentiostatic electropolymeriza-

tion (þ0.70 V vs. Ag=AgCl, 3 M Cl�, for 300 s) from

aniline (1.0 M) dissolved in aqueous HCl (2.0 M, pH

0.3, at 20 �C). The cyclic voltammograms of poten-

tiostatically prepared polyaniline films on glassy car-

bon electrodes, GCEjPAn, in buffer with and without

solution-borne CYP2D6 under aerobic and anaerobic

conditions are shown in Fig. 5. Comparison of these

voltammograms shows a number of differences. In

the presence of the enzyme, the multiple redox peaks

observed in pure buffer become single anodic and

cathodic peaks and were observed under both anae-

robic and aerobic conditions. Also, there is a shift of

the cathodic peaks to more negative potentials under

both conditions in the presence of CYP2D6. Under

aerobic conditions the anodic wave no longer

showed the shoulder peak, a, but the peak b

appears, slightly broadened and flattened at 310 mV

(þ285 mA), at an almost identical position as in the

pure buffer. However, extensive broadening and flat-

tening was seen under anaerobic conditions giving

an anodic peak estimated at 419 mV (75 mA) and a

�Ep¼ 920 mV. Under aerobic conditions there was a

large, clearly visible cathodic peak at �557 mV

(�284 mA). Under anaerobic conditions however a

cathodic peak is only just discernible at �625 mV

(�52 mA). The cathodic peak currents, ipc, differed

by 232 mA. The data suggest that oxygen plays a

key role in the reactions of CYP2D6 at the PAn

surface, giving rise to the larger cathodic current of

284 mA. CYP2D6, like all members of the cyto-

Fig. 5. Cyclic voltammograms of GCEjPAn in pure PB KCl buffer (pH 7.5, 25 �C) and buffer solution containing CYP2D6 (14.4 mM)

vs. Ag=AgCl [3 M Cl�] collected under (A) aerobic and (B) anaerobic conditions


chrome P450 superfamily of enzymes, is primarily a

monooxygenase [37]. It takes up first one electron

and then another in the presence of oxygen and

sequentially binds the substrate to the iron protopor-

phyrin IX group [38]. The complex then undergoes a

series of cyclic oxidation-reduction reactions even-

tually resulting in the functionalization of the sub-

strate, making it more polar, and the regeneration of

the unbound CYP enzyme.

Characterization of the Biosensor

in Solution-Borne Pure CYP2D6

in the Presence of Fluoxetine

The cyclic voltammograms collected for a GCEjPAn

in anerobic CYP2D6 [14.4 mM] solution in the pre-

sence of increasing fluoxetine hydrochloride concen-

tration is shown in Fig. 6a. As the concentration of

fluoxetine was increased, the voltammograms show

increasing anodic and cathodic current along with

some shifting of the peak potential values. Two anodic

peaks were seen, labeled A and B in Fig. 6a, with

peak A appearing as a shoulder to peak B for concen-

trations in the range of 3.5 to 350mM fluoxetine hydro-

chloride. The voltammograms for 0.0mM and 700mM

fluoxetine concentrations showed no shoulder. Peak B

had an Epa value of 275 mV� 25 mV for concentra-

tions higher that 0.0mM fluoxetine, which was 419 mV.

The anodic peak current ipa of B increased with an

increase in concentration of fluoxetine, but within

the limits of experimental error the peak position

Epa did not vary with fluoxetine concentration. Two

cathodic peaks, labeled C and D in Fig. 6a, were also

noted. These peaks showed shifting, both of the posi-

tion, Epc, and height, ipc, with change in the fluoxetine

concentration.

A calibration plot of the cathodic peak current, ipc,

vs. fluoxetine concentration for peak D is shown in

Fig. 6b. An increase in the current response is noted as

the concentration of fluoxetine is increased up to

350 mM. The curve increases to a maximum at this

concentration. This confirms a catalytic reduction

process. However, as the concentration is further

increased beyond 350 mM to 700mM, double the

previous concentration, the ipc appears to begin to

decrease. Similarly, as the concentration of fluoxetine

was increased to 700 mM, there was a back shift of the

Epc to more positive potentials. These observations

suggest saturation of the enzyme or substrate or pos-

sibly product inhibition of the enzyme at fluoxetine

concentrations above 350mM.

EPSIS Analytical Methodology

EPSIS represents a unique analytical tool designed for

research and development of chemical and biological

sensor devices, instruments, and systems based on

electroconductive polymer sensor technology. Electro-

conductive polymers form effective conductimetric

transducers for chemical and biological sensors. EPSIS

combines potentiometric, potentiostatic, and superior

pulsed DC chronocoulometric capabilities into a

Fig. 6. (A) Cyclic voltammograms of CYP2D6 at a potentiostatically prepared GCEjPAn in the presence of varying fluoxetine concentra-

tions under anaerobic conditions. (B) Plot of ipc vs. [Fluoxetine] for potentiostatically prepared GCEjPAn in the presence of CYP2D6. Data

taken from Fig. 6A, cathodic peak D


powerful and versatile analytical detection and mea-

surement method that is unique to the determination

of conductimetric chemical and biological sensor

responses of electroconductive polymers. The EPSIS

analytical method involves three distinct phases:

(i) Pre-Initialization – An Undisturbed Open Circuit

Potential Measurement. This step measures, stores

and presents to the screen the interfacial potential

of the electroconductive transducer in its electro-

lyte or test environment. This measurement is

made of the complete polymer film relative to a

reversible Ag�=AgCl, Cl� electrode that also con-

tacts the electrolyte. The unperturbed open circuit

potential is diagnostic of the redox state of the

device and conveys information about the integrity

of the transducer.

(ii) Initialization – A Conditioning Electrolysis.

EPSIS applies a user-specified potential to the

electroconductive polymer transducer for a user-

specified duration or a user-specified limiting

current. Electroconductive polymer transducers

possess redox active sites that are present in vary-

ing amounts of oxidized and reduced forms. By

applying an initialization potential, EPSIS fixes

the initial redox composition of the transducer,

thereby also fixing its initial electrical conductiv-

ity and sensitivity toward biologically induced

redox changes.

(iii) Interrogation – A Nonpertubating Measurement

of the Time Dependence of the Electrical Con-

ductivity of the Transducer. Interrogation is itself

broken into a sequence of events designed to

reveal the time dependence of the electrical con-

ductivity of the transducer as it responds to

biologically induced redox changes. These four

events include the application of a non-pertubat-

ing but interrogating voltage pulse of 10–50 mV

applied between the fingers of the IME device;

measurement of subsequent transducer conduc-

tivity by integration of its current such that the

measured charge is directly proportional to the

transducer’s conductivity during the pulse peri-

od; a float period whereby the potential is com-

pletely withdrawn from the fingers of the device

and the device is allowed to spontaneously

respond to biologically induced redox changes;

and re-measurement of the open circuit potential,

which then becomes the basis for the application

of the subsequent voltage pulse. The three phases

of pre-initialization, initialization, and interroga-

tion are illustrated schematically in Fig. 7.

The four sensor interrogation steps of Pulse Appli-

cation, Conductivity Measurement, Float Period, and

Open Circuit Potential Measurement are repeated for

a user-defined number of cycles to produce a sensor

response curve. Each set of four such steps produces

a single datum point of conductivity and open circuit

potential data. Several such points obtained over a

period of time produce a response curve. The re-

sponse curve captures the change in conductivity of

the transducer as a function of time following initi-

alization.

Conductimetric Glucose and Urea

Biosensors Using the EPSIS

A general purpose H2O2-sensitive, conductimetric

transducer makes it possible to develop a wide range

of oxidoreductase enzyme biosensors such as those

based on glucose oxidase (GOx). A PPy-based, con-

ductimetric bio-transducer that is sensitive to H2O2,

one of the enzymatic products of the glucose-GOx

oxidation reaction, can be readily configured into an

immunosensor by conferring the transducer with the

specificity of biotin and exploiting strong biotin-strep-

tavidin binding in various bioassays. The EPSIS

method provides a convenient approach and the

EPSIS instrument a convenient platform for the devel-

opment of biospecific oxidoreductase enzyme biosen-

sors and for the fabrication of oxidoreductase labeled

immunosensors. We demonstrate the design, fabrica-

tion, and operation of a polypyrrole-based conduc-

timetric biosensor for glucose using the EPSIS

methodology. The principle of operation of the bio-

sensor is based on two phenomena: (i) the very strong

binding (affinity) between biotin and streptavidin,

which covalently immobilizes the GOx enzyme to

the PPy-IME substrate and confers glucose-sensitiv-

ity, and (ii) the oxidation of polypyrrole by enzy-

matically-generated H2O2, shown coupled in the

following equations:

�-D-Glucoseþ O2 �!GOx

gluconolactoneþ H2O2

H2O2 þ 2HþCl� þ PPy�! 2H2Oþ ðPPyþþ � 2Cl�Þ

The H2O2 causes oxidation of PPy resulting in a con-

comitant and stoichiometric change in inherent con-

ductivity which is recorded by the EPSIS instrument.

The above mechanisms are illustrated in Scheme 2.


Evidence for biotin availability at the surface of the

electropolymerized polypyrrole films prepared as out-

lined in the Bioimmobilization section was found

by using a streptavidin-fluorescein conjugate and

streptavidin-glucose oxidase conjugate. Streptavidin

is an extracellular protein of Streptomyces avidinii,

the functional portion of which has a molecular

weight of ca. 47 kDa, consists of four identical sub-

units, and is tetravalent in its binding with biotin.

Biotin is a common vitamin associated with the labile

binding of carbon dioxide in various carboxylation

cycles. Exposure of the biotinylated surface to the

streptavidin-GOx conjugate resulted in strong binding

of the protein to the vitamin.

The response of the conductimetric biosensor to

different glucose concentrations (100–600mM) was

investigated in acetate buffer (1 mM, pH 5.2) at

25 �C. Figure 8a shows the corresponding time-based

conductimetric response profiles as measured using

the EPSIS analytical methodology of applying

10 mV amplitude discontinuous (50 ms pulse and

500 ms off, i.e. a 100 duty cycle) square wave dc

pulses. As the concentration of glucose was increased,

there was a corresponding increase in conductivity of

the PPy membrane over all glucose concentrations

investigated. The change in initial conductivity of

the PPy membrane is evident and quantifiable after

Fig. 7. Schematic illustration of the EPSIS interrogation phases

indicating resistance, current and potential responses

Scheme 2. Principle of operation of the polypyrrole-based conductimetric biosensor for glucose based on strong biotin-streptavidin

affinity. Enzyme¼ glucose oxidase


the first few seconds of the enzymatic reaction as

shown in Fig. 8b. From this plot it can be seen that

the rate of conductivity change, as determined by the

slopes, increases with glucose concentration from

approximately 4�10�6 S cm�1 s�1 at 100mM glucose

to nearly 9�10�6 S cm�1 s�1 in response to 600mM

glucose.

The EPSIS was also used to demonstrate a conducti-

metric urea biosensor employing the biotinylated PPy

membrane grown over the interdigitated array of IMEs

and incorporating the enzyme urease through a biotin-

urease conjugate involving streptavidin coupling. The

change in conductivity of PPy, measured in DI water, is

effected through the following enzymatic reaction:

Ureaþ 3H2O��!UreaseHCO3

� þ 2NH4þ þ OH�

The enzymatic products, ammonia and OH� ions,

serve to compensate the formation of charge carriers

within the polymer, resulting in an ability to decrease

the apparent polymer conductivity. The conducti-

metric response pattern shown in Fig. 9 is, however,

complicated by a second opposing phenomenon; the

increasing concentration of anions at the polymer

electrolyte interface that serves to increase apparent

conductivity via the availability of doping anions.

With this configuration therefore, the polypyrrole

device displays a ‘‘step’’ in its response that corre-

sponds to an ON from an OFF condition at a particu-

lar urea concentration [33]. We anticipate that such a

threshold response may be engineered by changing

the activity of the enzyme within the polymer mem-

brane. Such a detector may be used to monitor the

urea content of the dialysate from patients under urea

dialysis.

Conclusion

Electroconductive composites of polyaniline and

polypyrrole within highly hydrophilic poly(hydroxy-

ethyl methacrylate)-based hydrogels have been

synthesized that possess unique electrochemical behav-

ior pertinent to the redox state of the inherently con-

ducting polymer component. The composite structure

provides for: (i) enhanced switching speeds that arise

Fig. 9. Response profiles of the urea-responsive PPy-based con-

ductimetric biosensor toward different urea concentrations as mea-

sured using the EPSIS analytical methodology. (Reprinted with

permission from [33])

Fig. 8. (A) Response profiles of the glucose-responsive PPy-based conductimetric biosensor toward different glucose levels as measured

using the EPSIS analytical methodology; (B) the change in initial conductivity of the PPy membrane (slopes) after the first few seconds of

the enzymatic reaction


from the larger apparent diffusion coefficients of ions

into and out of the membrane, and (ii) stabilization of

the bioactive recognition molecule. A novel electro-

conductive polymer sensor technology, EPSIS, was

demonstrated using bioactive membranes of functiona-

lized PPy incorporated into a conductimetric transduc-

tion mode. The analytical system was demonstrated for

glucose and urea detection.

Acknowledgements. The authors acknowledge the support of the

Virginia Center for Innovative Technology (CIT BIO-99-010) to

the VCU Center for Bioelectronics, Biosensors and Biochips (C3B).

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