Development of membrane based electrodes for electroanalytical applications 4

4 CHAPTER 4 Membrane-based electrochemical nanobiosensor for detection of virus 4.1 INTRODUCTION 4.1.1 West Nile Virus Domain III West Nile Virus WNV was first identified from a febr

4 CHAPTER 4

Membrane-based electrochemical nanobiosensor for detection of virus

4.1 INTRODUCTION

4.1.1 West Nile Virus Domain III

West Nile Virus (WNV) was first identified from a febrile female adult

in the West Nile region of Uganda in 1937 [1] WNV, a single-stranded positive sense RNA envelope virus, belongs to the Flaviviridae virus family WNV has wide range of animal hosts and is transmitted mainly by a mosquito vector WNV causes human illness that can progress to paralysis, encephalitis and death

WNV contains 3 structural proteins They are large envelope (E) protein,

a single nucleocapsid protein (C) and a lipid membrane protein (M) The E protein monomer folds into 3 domains: domain I, II and III Domain I has an

eight-stranded β-barrel which takes part in the conformational changes associated with the acidification in the endosome Domain II is 12β-strands and plays roles in dimerization, trimerization and fusion Domain III (DIII) adopts an immunoglobulin-like fold, and contains surface exposed loops in the

Fig 4.1 (A) Homology model of a WNV domain III protein (B)

Transmission electron micrograph of WNV particles obtained using Philips 208s transmission electron microscope Sample was stained with phosphotungstic acid for 2 min, rinsed with water, and dried before viewing mature virion [2] The sequence of WNV-DIII was well-studied [3]; it has short length of ca 100 amino acids [4] Some previous studies suggest that domain III could mediate flavivirus attachment to the host cell [5] The envelope domain III protein is chosen in this work because of its important role in the implications in virulence Immunoglobulin M antibody raised against domain III protein is used as the specific biorecognition probe for both

4.8 nm

A B

the WNV-DIII protein and WNV particle Fig 4.1 shows the structure of WNV-DIII protein and WNV particle

4.1.2 Current analysis method for West Nile Virus detection

In general, two types of tests have been developed for the diagnosis and screening of WNV They are based on either antibodies (immunoglobulin M (IgM) or immunoglobulin G (IgG)) or nucleic acid tests in body fluid or tissue Some methods for detections of WNV antibodies are complement fixation test (CF), hemagglutination- inhibition test (HI), plaque-reduction neutralization (PRNT), immune-fluorescence assay (IFA), enzyme-linked immunosorbent assay (ELISA), and the microsphere immunoassay (MIA) ELISA and MIA give high sensitivity and selectivity for WNV detection test, but it takes two or three days for the whole assay procedure while the IFA assays requires additional serum test and faces the cross-reactivity of WNV antibodies [6]

The other test for WNV diagnosis is the detection of WNV nucleic acid Because virus presents in blood or plasma samples of infected patients at very low concentration, an in vitro amplification procedure is needed to increase

WNV genetic material Several amplification tests were found to enhance the

WNV detection such as the real-time polymerase chain reaction (PCR) test, which takes few hours and has extremely low detection limit for WNV in blood and plasma samples such as 0.1 plaque-forming units (pfu) of virus [7]

4.1.3 Immunoglobulin M antibody

During acute infection, serological WNV IgM antibody detection was found to be of higher diagnostic sensitivity than viral RNA determination by reverse transcriptase PCR Moreover, immunoglobulin M (IgM) antibodies are less cross-reactive, whereas immunoglobulin G (IgG) antibodies show more cross-reactions with other flaviviruses [8] Since in our biosensor, the sensing signal is based on the specific antigen-antibody binding, the IgM was chosen

as the biorecognition element and was immobilized within the nanochannels

Immunoglobulin (Ig) is a globutin-type protein found in serum or other body fluids which possesses antibody activity Each Ig unit is built up from 2 light (L) and one heavy (H) polypeptide chains linked together by disulfide bonds (Fig 4.2(A)) Based on antigenic and structural differences in the H chains, Immunoglobulin are divided into five classes of A, G, D, M and E In the five immunoglobulin classes, immunoglobulin M is the largest antibody

and is the first antibody that appears when the body is challenged by the antigen IgM normally exists as pentamer (Fig 4.2(B)) but it can also exist as

a monomer In the pentamer form, IgM has a molecular mass of approximately

900 kD

Antigen binding sites

Light chain

Heavy chain

Variable region on light chain

Variable region

on heavy chain

Constant region on heavy chain

Constant region on light chain

Join chain

S S S S

Antigen binding sites

Light chain

Heavy chain

Variable region on light chain

Variable region

on heavy chain

Constant region on heavy chain

Constant region on light chain

Join chain

S S S S

Fig 4.2 (A) Structural region of antibody molecule (B) Structure of IgM in

the pentamer form

In this work, the monoclonal IgM antibody (H5.46) which specifically binds to E protein of WNV was employed [9] to form the interaction with the WNV proteins and viral particles during the sensing process

4.1.4 Immunosensor

Immunosensor is the most specific biosensors in which the biorecognition elements are antibodies (Ab) Immunosensors provide low detection limits and can be applied in wide range of substances In immunosensors, antigen-antibody interactions are transduced directly into

physical signals to sense antigen (Ag)

Ab + Ag ' Ab-Ag

In a typical immunosensor, antibodies which are the globular protein produced by organisms to bind foreign molecules (antigens) and mark them for elimination from the organism, are linked on a stable solid support and coupled to a transducer element Prepared antibody can be monoclonal or polyclonal; the latter is cheaper but possesses varied binding affinities due to many different types of biorecognition elements present, with poorly reproducible proportions across different preparations

The design and preparation of an optimum interface between the biorecognition elements and the transducer material are the key part of biosensor development Electrochemical immunosensors that combine specific immunoreactions with an appropriate electrochemical transduction have increased interests due to low cost, rapid response and simple-to-use procedure There are two categories of electrochemical immunosensors, one detects antigen directly, while the other senses indirectly In the indirect electrochemical immunosensors, the signal transduction is produced by the

second compound or reaction (such as mediators) Our immunosensor in this study is based on the indirect sensing mechanism

4.1.5 Nanoporous membrane based biosensors

Nanoporous membranes, comprising uniform and regularly spaced channels of nanometer dimension, have attracted great interest as a template material for the incorporation of various materials including biological molecules, metals, semiconductors, and polymers within the nanosized channels [10-12] Nanoporous membranes possess uniform pore sizes, high aspect ratio, and high surface areas, are relatively easy to prepare, and are inexpensive by comparison to conventional lithographic techniques In general, incorporation of interesting and useful materials within the membrane channels impart new physicochemical or biological properties which results in new or improved membrane applications In particular, electrochemical nanobiosensors using nanoporous membranes, such as a nanoporous semiconductor [13], porous conducting polymer [14], track-etched polymer [15, 16], and porous alumina[17, 18], have been reported

Electrochemical biosensors generally involve redox enzymes for the

conversion of substrate into product [19] The response signal at the sensing electrode is derived from the redox enzyme directly by electrically conducting linkers [20] or indirectly via redox mediator or redox reaction of the substrate

or product [21] More recently, non redox antibodies and single-stranded nucleic acids are promising biorecognition molecules because of high recognition specificity for the analyte of interest and are applicable to large numbers of nonredox active biological analytes, as reported by Wang et al and other researchers [22-26] Electrochemical sensing can be derived from the changes in physical proximity of the redox labeled biorecognition molecules to the sensing electrode [24, 25] or variation in the concentration of the electrochemical tags close to the sensing electrode, before and after binding to the analytes of interest [26] or via the gold nanoparticle amplification method [22, 23] Until now, several methods using multiarray nanopores (or nanochannels) embedded within membranes have demonstrated that rapid analysis is potentially suitable for extreme analysis of a small sample quantity For example, using an array of conical gold nanopores functionalized with thiolated-biotin [16], specific detection of streptavidin modified analytes can be achieved Silicon dioxide based nanopores are

modified using silane chemistry to influence transport of charged analytes via electrostatic interactions between the charged analytes and the charged functional groups at the modified surface [27] A recent interesting report uses the change in protein structure upon binding to target analyte to influence the electrolyte conductivity within Au-coated nanopores [15] Unlike the impedance or conductance measurement methods, we use a highly sensitive differential pulse voltammetry method to monitor the Faradaic current of redox species at the membrane-electrode interface which generates the biosensor signal in response to antigenic analytes bound within the multiarray membrane nanochannels In addition, the sensing electrode is directly coated onto the nanoporous membrane, comprising multiarray nanochannels in order

to couple the mass transport rate of the redox species within the membrane to the electrochemical reaction at the membrane-electrode interface Previous work using a nanoporous alumina membrane [18] suggested that it is ideally suited for this work because of high pore density which offers high current flux, tunable pore sizes, rigid support structure, chemical and thermal stability, and ease of preparation using electrochemical anodization of aluminum

In this work, we report the nanoporous membrane-based biosensor that

can be used to detect extremely low concentration of a virus protein and particle using a rapid sensing time of 30 min The nanobiosensor response is first optimized using the West Nile virus-domain III protein to which the immunoglobulin M (IgM) biorecognition probe binds to, with subsequent application for the direct detection of the West Nile virus (WNV) particle AC voltammetry reveals that contribution of diffusion is significant in the observed reduced biosensor response signal toward ferrocenemethanol, in the presence of the virus protein or the particle Equilibrium constants for the antigen-antibody binding are derived from the electrochemical response signal using simple Langmuir isotherm Limits of detection for the viral protein and particles are 4 pg mL-1 and 2 viral particles per 100 mL, respectively, which are similar to detection limits of viruses using PCR techniques [28, 29] Finally, we demonstrate the highly specific sensing of the WNV particle is readily achieved in a complex medium, blood serum containing other proteins

4.2 EXPERIMENT

4.2.1 Reagents and Materials

Bovine serum albumin (BSA, >98%), ferrocenemethanol (FeMeOH,

>99%), sodium dihydrogenphosphate dehydrate, phosphoric acid and

ethyleneimine were purchased from Sigma Aldrich Sodium chloride was purchased from QRëC Virus protein and particle solutions were prepared in 0.1 M phosphate buffer (pH 6.2-8.2) All chemicals and solvents used were of analytical grade and used as received Ultrapure water (Barnstead Nanopure Ultrapure Water System) was used for all preparations Anti-West Nile virus mAb H546 (IgM) was purchased from Microbix Biosystems Inc A laboratory attenuated strain of West Nile virus, Sarafend was kindly provided by E G Westaway, Sir Albert Sakzewski Virus Research Laboratory, Queensland, Australia The recombinant West Nile virus domain III protein was cloned, expressed, purified and validated according to standard procedure described elsewhere [30] West Nile virus proteins and particles were received from Prof

Ng in collaboration

4.2.2 Inactivation of virus

Inactivation of virus was performed by the addition of binary ethyleneimine to the virus suspension to give a final concentration of 0.1 M The suspension was subsequently incubated at 37 °C for eight hours with constant shaking In order to validate for the complete inactivation of all infectious viral particles in the suspension, small aliquots of the suspension

were used to perform three consecutive blind passes on Syrian golden hamster kidney adherent fibroblasts (BHK-21) by using the spent culture medium of the previous pass as inoculum The lack of cytopathic effect confirmed the complete inactivation of all infectious viral particles

4.2.3 Nanoporous membrane electrode construction and sensing

procedure

Home-made electrodes were fabricated using epoxy glue, micropipette tips and platinum wire The electrode tip was polished with 1.0 µm and 0.3 µm diameter alumina powder before sputter-coated with aluminium metal film Sub-micrometer thick aluminum films in the thickness range of 300 to 500 nm were sputter-coated onto the platinum electrodes (0.076 mm diameter) using 99.999% purity aluminum target, Denton discovery® 18 Sputtering System and sputtering power of 100 W in an atmosphere of research-grade Ar at 5×10-3 Torr Anodization of aluminum coated electrodes were conducted by using previously described method of surface contact anodization method [18] Electrochemical behaviors of the alumina modified electrodes were characterized by using cyclic voltammetry (CV) and differential pulsed voltammetry (DPV) techniques (CHI440 potentiostat/galvanostat, data

acquisition software) in the presence of 1.0 mM ferrocenemethanol in 0.1 M phosphate pH 6.8 buffer solution (refer reference[18] for details) All potentials were measured with respect to the saturated Ag/AgCl reference electrode

4.2.4 A.C Voltammetry

AC Voltammetry studies were conducted using CH Instrument electrochemical station 440 Frequency applied for Alternating Current Voltammetry (ACV) ranged from 0.7 to 5000 Hz A three-electrode system was employed which comprised 2 mm diameter platinum auxiliary electrode, saturated Ag/AgCl reference electrode and alumina membrane electrode as the working electrode Solution of 1.0 mM ferrocenemethanol in 0.1 M phosphate

pH 6.8 buffer was used as the electrolyte

4.2.5 Biosensor construction and sensing procedure for WNV domain

III protein and inactivated WNV particle

-1

5 µL of a 0.2 µg mL IgM solution was applied onto the nanoporous membrane electrode tip to adsorb IgM molecules into the membrane nanochannels After 1 hr, the electrode was rinsed and further 5 µL of a 200

µg mL-1 BSA solution was applied to the membrane electrode tip for 30 min

This is to adsorb BSA molecules at remaining empty sites of the nanochannel walls to block non-specific binding sites After this step, the electrode was rinsed again A three-electrode system was used for the measurement of electrochemical response towards redox active ferrocenemethanol, using a saturated Ag/AgCl reference electrode and a 1 cm diameter platinum disk auxiliary electrode Detection of WNV-DIII protein was carried out by immersing the biosensor in a buffer solution (0.1 M phosphate, pH 6.2-8.2) containing the virus protein and 1 mM ferrocenemethanol Concentration of WNV-DIII in the solution was varied by addition of aliquots from a 0.25 µg

mL-1 WNV domain III stock solution containing 1 mM ferrocenemethanol Differential pulse voltammograms were obtained at 30 min intervals after each addition of the antigen stock solution Biosensor detection limit was derived from the minimum antigen concentration which gives decrease of response signal in an amount equal to three times the background noise at zero antigen concentration The same detection procedures were conducted for WNV particle, using aliquots of stock solutions containning 103-105 particles per 100

mL

4.2.6 Standard Addition Method for the Detection of the WNV Particle

in Spiked Blood Serum

and extrapolated to IWNV/IWNV=0 = 1 to derive the concentration of the unknown sample

4.3 RESULT AND DISCUSSION

4.3.1 West Nile Virus Domain III protein detection using membrane-

based electrochemical biosensor

Fig 4.3 demonstrates the working principle of membrane-based electrochemical biosensor for WNV-DIII protein and particle accordingly the

specific binding of immunoglobulin M antibody with the WNV-DIII protein and particle was employed to operate the biosensor The structure of the WNV-DIII protein and the WNV particle was used as models in this study, with described dimensions in relation to the nanochannel sizes within the membrane electrode

Fig 4.4(A) shows the typical differential pulse voltammograms (DPV) of

ferrocenemethanol obtained at a membrane electrode immersed in a sensing solution containing redox active ferrocenemethanol, after each step of the biosensor preparation procedure Fig 4.4B shows that the DPV response signal of the assembled membrane biosensor is sensitive toward increasing concentrations of WNV-DIII protein Normalization of these response signals

in the presence of antigen (δiWNV-III) against those derived from the same

biosensor in the absence of antigen (δiWNV-III=0) gives good linear logarithmic relation (inset of Fig 4.4(B)) Control studies using membrane electrodes loaded with BSA which prevent protein adsorption, but without IgM, give an insignificant response toward the protein antigen Dimensions of IgM (19 nm[31]) and WNV-DIII protein (ca 4.8 nm estimated from the homology model) are comparable to the nanochannel dimensions (ca 20-100 nm) and can enter the nanochannels [18] Thus, the observed decline in response signal after each immobilization step suggests increased loading of biomolecules within the membrane electrode which likely impedes the movement of ferrocenemethanol to the underlying sensing electrode Other control studies

to detect nontarget proteins show no decrease in biosensor response signals in the presence of other proteins (lysozyme, myoglobin, and hemoglobin)

prepared in phosphate pH 7.0 buffer solutions

A

8.0 10.0 12.0 14.0 16.0 18.0 20.0 22.0 24.0

After adsorption of BSA

Response toward WNV-DIII protein

B

8.0 10.0

Fig 4.4 (A) DPV response signal obtained at a 60 min etched alumina

membrane electrode immersed in 1mM ferrocenemethanol, after each step of the sensor preparation procedure (B) DPV response signal of the biosensor in

increasing concentrations of WNV-DIII protein 6.7; 13.3; 20; 26.7 and 53.5 pg

mL-1, using the sensor preparation procedure in (A) and in the presence of 1

mM ferrocenemethanol Inset: Plot of normalized current vs log ([WNV-DIII]), [WNV-DIII] in unit of pg mL-1

5 μA

5 μA

Fig 4.5 AC Voltammetry response of the membrane electrodes for the sensing

of WNV-DIII protein 20 pg mL-1 at the frequency range from 0.7-5000 Hz, with 1.0 mM ferrocenemethanol in 0.1 M phosphate pH 6.8 buffer solution as the electrolyte

0.0 2.0 4.0 6.0 8.0 10.0 12.0

WNV-DIII protein 7 pg/mL WNV-DIII protein 20 pg/mL WNV-DIII protein 33 pg/mL

U24.9 ± 0.9

‘24.5 ± 0.6 c21.5 ± 0.6 …17.3 ± 0.4

¼14.4 ± 0.3

U24.9 ± 0.9

‘24.5 ± 0.6 c21.5 ± 0.6 …17.3 ± 0.4

¼14.4 ± 0.3

A

0.0 2.0 4.0 6.0 8.0 10.0 12.0

WNV-DIII protein 7 pg/mL WNV-DIII protein 20 pg/mL WNV-DIII protein 33 pg/mL

U24.9 ± 0.9

‘24.5 ± 0.6 c21.5 ± 0.6 …17.3 ± 0.4

¼14.4 ± 0.3

U24.9 ± 0.9

‘24.5 ± 0.6 c21.5 ± 0.6 …17.3 ± 0.4

¼14.4 ± 0.3

B

0.0 0.5 1.0 1.5 2.0 2.5

WNV particle 0.03 PFU/mL WNV particle 0.05 PFU/mL

U4.61 ± 0.60

‘6.63 ± 0.50 c4.13 ± 0.18 …1.60 ± 0.16

U4.61 ± 0.60

‘6.63 ± 0.50 c4.13 ± 0.18 …1.60 ± 0.16

B

0.0 0.5 1.0 1.5 2.0 2.5

WNV particle 0.03 PFU/mL WNV particle 0.05 PFU/mL

U4.61 ± 0.60

‘6.63 ± 0.50 c4.13 ± 0.18 …1.60 ± 0.16

U4.61 ± 0.60

‘6.63 ± 0.50 c4.13 ± 0.18 …1.60 ± 0.16

Fig 4.6 Plot of AC voltammetry responses (Ip) of the membrane electrodes as

a function of square root angular frequency (w1/2) after each step of the sensor preparation procedure for sensing of (A) WNV-DIII protein (B) WNV particle Frequency from 0.7-5000 Hz, with 1.0 mM ferrocenemethanol in 0.1 M phosphate pH 6.8 buffer solution as the electrolyte Lines are guides to the eye Inset: Cross-sectional area A calculated using Eqn 1.8

Fig 4.6 shows the effect of frequency on the AC voltammetry peak current

obtained at E from 0.7-5000 Hz At low frequencies, I1/2 p is clearly limited by frequency for all membrane electrodes From the initial slopes (at zero

frequency) of the plots, we obtain AD1/2 values using Eqn 1.8 in chapter 1

Since D is assumed to remain constant, the change in initial slopes in Fig 4.6 reflects variation in the biosensor cross-sectional area A, given as inset in Fig 4.6 Area A decreases with addition of WNV-DIII or WNV particles It is interesting to note that the values of A are significantly larger than geometric

area of the underlying sensing Pt electrode (76 µm diameter) This is likely because of the long diffusion lengths of ferrocenemethanol at low frequencies which spread the ferrocenemethanol molecules radially away from the sensing electrode region through the extensively etched and inter-connected nanochannels It is clear from Fig 4.6 that as more immunocomplex are formed within the membrane in the presence of WNV-DIII or WNV particle,

A decreases which would ‘block’ the movement of diffusing ferrocene

molecules This change in the mass transport rate of ferrocenemethanol through the nanochannels is readily monitored using the mass transport sensitive DPV technique