COLLEGE OF BASIC AND APPLIED SCIENCES 

UNIVERSITY OF GHANA 

 

 

 

DEVELOPMENT OF NOVEL ELECTROCHEMICAL BIOSENSORS FOR 

CLINICAL DIAGNOSIS OF INFECTIOUS DISEASES 

 

BY 

FRANCIS DZIDEFO KRAMPA 

(10363861) 

 

 

THIS THESIS IS SUBMITTED TO THE SCHOOL OF GRADUATE STUDIES IN 

PARTIAL FULFILMENT OF THE REQUIREMENT FOR THE AWARD OF 

DOCTOR OF PHILOSOPHY DEGREE IN MOLECULAR CELL BIOLOGY OF 

INFECTIOUS DISEASES 

 

 

 

DEPARTMENT OF BIOCHEMISTRY, CELL AND MOLECULAR BIOLOGY  

 

JULY 2019 

 





ii 

ABSTRACT 

In the field of biomedical diagnostics, rapid and effective monitoring of important 

analytes is an essential goal, and for this reason, significant progress has been made 

towards developing high performing analytical tools. Despite these advancements, the 

majority of the techniques are not suitable for routine uptake in resource-limited regions 

due to technical and infrastructural challenges. Clinical diagnostics is particularly 

affected since it is not always available at the point-of-need, notably for infectious 

diseases which is the key concern to public health in these regions. It is therefore 

necessary to explore efficient and robust analytical techniques that address the 

deficiencies of traditional diagnostic methods in preparedness for outbreaks and field-

readiness. Electrochemical biosensors provide an attractive means to analyze the 

content of a biological sample due to the direct conversion of a biological event to an 

electronic signal. The thesis aimed at applying advances in electrochemical biosensor 

technologies in the development of diagnostic devices for point-of-care testing of 

infectious diseases. Given that the transducer is of uttermost importance in 

electrochemical sensors, the suitability of screen-printed electrodes was assessed for 

use as base transducers. Following this step, nanocomposites made from conductive 

polymers; poly(3,4-ethylenedioxythiophene):poly(4-styrenesulfonate) (PEDOT:PSS),  

1-ethyl-3-methylimidazolium tetrafluoroborate ([EMIM][BF4]), nafion (Naf) and

graphene nanoplatelets  (GNPs) were used to modify the electrode surfaces in order to 

enhance their electrochemical performance. Characterization of screen-printed carbon 

electrodes (SPCE) modified with IL/PEDOT:PSS and GNPs/Naf showed nano-porous 

surfaces with enhanced electrocatalytic properties of up to 40-fold compared to the bare 

unmodified electrodes. The modified surfaces were applied to detect trace levels of 

electroactive analytes in environmental and biological samples. The PEDOT:PSS/IL 



iii 

was utilised in conjunction with amperometry towards sensitive detection of catechol 

and differential pulse voltammetry applied at the GNPs/Naf for simultaneous analysis 

of dopamine (DA) and N-acetyl-p-aminophenol  (APAP) in their binary mixtures. The 

sensors showed excellent selectivity and sensitivity toward the target analytes, with 

limit of detection of 23.7 µM for catechol and 0.13 µM and 0.25 µM for DA and APAP, 

respectively. Subsequently, three strategies for immobilizing antibodies (physisorption, 

covalent binding and polydopamine assisted binding) on screen-printed micro gold 

electrode surfaces were evaluated for the development of a malaria immunosensor. A 

sensitive and efficient biosensor was achieved for Plasmodium falciparum histidine-

rich protein-II (PfHRP-II), aided by the robust covalent coupling between anti-PfHRP-

II antibodies and an amine layer glutaraldehyde crosslinking on a screen-printed gold 

microelectrode. The sensor which was built on a label-free impedimetric format was 

highly reproducible with a low detection limit of 38.0 pg/mL. Based on the efficiency 

of the covalent immobilisation, the method was repurposed to detect surface antigens 

of hepatitis B virus. The sensor possessed sufficient sensitivity and selectivity for 

hepatitis B surface antigens in buffer with a low detection limit of 0.7 ng/mL. These 

findings demonstrate that the novel nanocomposite-modified sensing platforms could 

be effective conductive supports in enhancing electroanalysis. The impedimetric 

immunosensors are a promising inexpensive alternative for label-free analyte detection. 

Together, these strategies could be integrated into high performing portable self-

contained instruments for point-of-care diagnostic application in resource-limited 

settings.  



iv 

 

ACKNOWLEDGEMENT 

Foremost, I thank Professor Gordon Awandare, Dr Prosper Kanyong, Dr Jonathan 

Adjimani and Dr Osbourne Quaye who supervised this work. I am deeply indebted to 

Dr Prosper Kanyong for making this ‘relatively new’ field an enjoyable journey, turning 

me from a novice to a more balanced and pragmatic electrochemist within a short time. 

I acknowledge the indirect inspiration from his recommendation ‘Wilful Blindness’ 

which I read alongside preliminary work on the malaria biosensor.  

I wish to thank The West Africa Centre for Cell Biology of Infectious Pathogens 

(WACCBIP) who, through funding from the World Bank, sponsored my training and 

this research work. I owe Professor Awandare a debt of gratitude for starting the 

collaboration with Dr Kanyong and for grafting the ‘Diagnostics Group’ into the Cell 

Biology and Immunology Laboratory of WACCBIP. This research could not have been 

started or completed successful without his unrelenting financial support for the 

purchase of very expensive reagents/items. 

Many thanks to Dr Yaw Aniweh for lending an intellectual insight into practically 

anything scientific and to Professor Andrew Anthony Adjei without whose mentorship 

I would not have reached this point. Special thanks to Professor Lisa Hall of the 

Cambridge Analytical Biotechnology- Hall Laboratory, Cambridge University who 

hosted my experiential learning and to Cassie Henderson whom I worked closely with. 

To my colleagues who have become my dearest friends during this adventurous time, 

it has been a pleasure. 

I am honoured to have a dedicated supportive and encouraging family without whom I 

could not have come this far. 

 



v 

 

TABLE OF CONTENT 

 

DECLARATION ........................................................................................................ i 

ABSTRACT ............................................................................................................... ii 

ACKNOWLEDGEMENT ........................................................................................ iv 

LIST OF FIGURES .................................................................................................... x 

LIST OF TABLES ................................................................................................. xiii 

LIST OF ABBREVIATIONS ................................................................................. xiv 

LIST OF SYMBOLS .............................................................................................. xvi 

 ................................................................................................................. 1 

INTRODUCTION........................................................................................................ 1 

 Infectious diseases at a glance .............................................................................. 1 

 Infectious Diseases Diagnostics: The problem of the developing world ............. 2 

 The need for improved diagnostic tests................................................................ 3 

 Point of Care Tests (PoCTs) ................................................................................ 4 

 Biosensors as PoCTs ............................................................................................ 8 

 Aim and Objectives .............................................................................................. 9 

 Scope and organisation of the thesis .................................................................... 9 

 ............................................................................................................... 11 

LITERATURE REVIEW ......................................................................................... 11 

 Introduction to biosensors .................................................................................. 11 

 Components of a biosensor ................................................................................ 12 

2.2.1 The bioreceptor ............................................................................................ 13 

2.2.2 Transducer ................................................................................................... 14 

2.2.3 Signal analysis or processing unit ............................................................... 15 

 The theoretical background of electrochemical methods in biosensing ............ 15 

2.3.1 Principles of electrochemistry and electrochemical transduction ............... 16 



vi 

2.3.1.1 The faradaic and non-faradaic processes .............................................. 16 

2.3.1.2 The Faradaic process............................................................................. 17 

2.3.1.3 Mass transport mechanisms .................................................................. 18 

2.3.1.4 Kinetics of electron transfer .................................................................. 19 

2.3.2 Methods in electrochemistry ....................................................................... 21 

2.3.2.1 Cyclic Voltammetry .............................................................................. 22 

2.3.2.2 Electrochemical Impedance Spectroscopy ........................................... 24 

 Physical properties and setup of electrochemical sensing system setup ............ 26 

2.4.1 Electrode materials ...................................................................................... 27 

2.4.2 Electrode designs and modifications ........................................................... 28 

2.4.2.1 Screen-printed electrodes ...................................................................... 28 

2.4.2.2 Conductive polymers ............................................................................ 29 

2.4.3 Strategies for immobilizing biological receptors ......................................... 32 

2.4.4 Performance criteria of biosensor responses: analytical properties............. 35 

 Electrochemical biosensors ................................................................................ 36 

2.5.1 Amperometric sensors ................................................................................. 36 

2.5.2 Potentiometric Sensors ................................................................................ 37 

2.5.3 Conductometric biosensors .......................................................................... 37 

2.5.4 Impedimetric biosensors .............................................................................. 38 

 Immunosensors................................................................................................... 38 

 Immunoassay formats of electrochemical Immunosensing ............................... 40 

 ............................................................................................................... 44 

RESULTS OF SPECIFIC OBJECTIVES 1 AND 2 ............................................... 44 

 Development and characterisation of electrochemical transducers for biosensing 

application ................................................................................................................ 44 

3.1.1 Abstract ........................................................................................................ 45 

3.1.2 Introduction ................................................................................................. 46 



vii 

 

3.1.3 Experimental ................................................................................................ 48 

3.1.3.1 Apparatus and reagents ......................................................................... 48 

3.1.3.2 Fabrication of GNPs-Naf/SPE .............................................................. 49 

3.1.3.3 Fabrication of PEDOT:PSS/20%IL/SPCE ........................................... 50 

3.1.3.4 Sessile contact angle measurement ....................................................... 51 

3.1.4 Results and discussion ................................................................................. 51 

3.1.4.1 Optimization of the percentage of IL in PEDOT:PSS/IL composite .... 51 

3.1.4.2 Characterisation of SPCE and PEDOT:PSS/20%IL/SPCE .................. 54 

 Cyclic voltammetry ........................................................................ 54 

 Electrochemical Impedance Spectroscopy ..................................... 56 

3.1.4.3 Scanning Electron Microscopy and profilometry ................................. 57 

3.1.4.4 Sessile contact angle measurements ..................................................... 58 

3.1.4.5 Electrochemical characterization of SPE and GNPs-Naf/SPE ............. 59 

3.1.4.6 Application of PEDOT:PSS/20%IL/SPCE to catechol analysis .......... 63 

 Cyclic voltammetry ........................................................................ 63 

 Chronoamperometry....................................................................... 66 

 Amperometry in stirred solution .................................................... 67 

3.1.4.7 Stability of PEDOT:PSS/20%IL/SPCE ................................................ 68 

3.1.4.8 Analysis of natural water samples ........................................................ 69 

3.1.4.9 Electrochemical behaviour of DA and APAP at SPE and GNPs-

Naf/SPE............................................................................................................. 70 

3.1.4.10 DPV analysis of DA and APAP at GNPs-Naf/SPE ............................ 73 

3.1.4.11 Analysis of DA and APAP in binary mixtures at GNPs-Naf/SPE ..... 75 

3.1.4.12 Analytical application of GNPs-Naf/SPE ........................................... 77 

 Conclusion .......................................................................................................... 78 

 ............................................................................................................... 79 

RESULTS OF SPECIFIC OBJECTIVES 3 &4 ..................................................... 79 



viii 

 

Overview .................................................................................................................. 79 

 Manuscript 3: Development of a simple sensitive impedimetric immunosensor 

for detection of Plasmodium falciparum histidine rich protein-II ........................... 80 

4.1.1 Abstract ........................................................................................................ 81 

4.1.2 Introduction ................................................................................................. 82 

4.1.2.1 Parasite development in humans, biomarkers, and diagnosis ............... 82 

4.1.2.2 Detection of PfHRP-II in Clinical Samples .......................................... 86 

4.1.3 Materials and methods ................................................................................. 90 

4.1.3.1 Reagents ................................................................................................ 90 

4.1.3.2 Solutions ............................................................................................... 90 

4.1.3.3 Apparatus and instrumentation ............................................................. 91 

4.1.3.4 Fabrication of the PfHRP2 immunosensor ........................................... 91 

 Electrodes preparation .................................................................... 91 

 Preparation of the sensing layer ..................................................... 91 

 Preparation of ePDA/SPGµE ......................................................... 92 

 Assembly of the GA-cys/SPGµE sensing layer ............................. 92 

 Capture antibody immobilization ................................................... 93 

4.1.3.5 Detection of malaria antigen PfHRP-II and evaluation of the analytical 

performance ...................................................................................................... 94 

4.1.3.6 Electrochemical studies ........................................................................ 94 

4.1.4 Results and discussion ................................................................................. 95 

4.1.4.1 Electrochemical characterisation of the SPGµE ................................... 95 

4.1.4.2 Assembly of the sensing layer .............................................................. 98 

4.1.4.3 Analytical performance of the PfHRP-II immunosensor.................... 103 

4.1.4.4 Reproducibility and stability of the malaria immunosensor ............... 107 

4.1.4.5 Conclusions ......................................................................................... 108 

 Manuscript 4: Ultrasensitive impedimetric immunosensor for the detection of 

hepatitis B using gold microelectrodes .................................................................. 109 



ix 

 

4.2.1 Abstract ...................................................................................................... 110 

4.2.2 Introduction ............................................................................................... 111 

4.2.3 Materials and methods ............................................................................... 113 

4.2.3.1 Reagents and apparatus ....................................................................... 113 

4.2.3.2 Fabrication of the immunosensor........................................................ 113 

4.2.3.3 Detection of HBsAg ............................................................................ 114 

4.2.4 Results ....................................................................................................... 115 

4.2.4.1 Characterisation of the immunosensor ................................................ 115 

4.2.4.2 Detection of HBsAg ............................................................................ 116 

4.2.4.3 Selectivity ........................................................................................... 118 

4.2.5 Conclusions ............................................................................................... 120 

 ............................................................................................................. 121 

DISCUSSIONS, CONCLUSIONS AND FUTURE PERSPECTIVE ................. 121 

 General discussions .......................................................................................... 121 

 Conclusions ...................................................................................................... 125 

 Perspective and further work............................................................................ 126 

REFERENCES ....................................................................................................... 129 

 Appendixes: Publications ................................................................................. 154 

 

 

 

 

 

 

 

 

 

 



x 

 

LIST OF FIGURES 

Figure 1. 1 Infectious disease death rates per 100,000 people in 2017 ......................... 1 

Figure 2. 1 Forecasted growth and demand for biosensors ......................................... 12 

Figure 2. 2 Schematic layout of a biosensor components applied for the detection of 

biomarkers.................................................................................................................... 13 

Figure 2. 3 An integrated circuit design of a basic 3 electrode sensing system. ......... 16 

Figure 2. 4 Illustration of the rate-determining steps of a faradaic process ................. 17 

Figure 2. 5 The methods used in electrochemical immunosensor developments. ....... 21 

Figure 2. 6 A typical cyclic voltammogram within a reversible redox species. .......... 23 

Figure 2. 7 A Nyquist plot of an ideal electrochemical cell. ....................................... 26 

Figure 2. 8 Schematic representation of an IgG. ......................................................... 39 

Figure 2. 9  Strategies of electrochemical biosensors .................................................. 42 

Figure 2. 10 An electrochemical cell and screen-printed electrode. ............................ 26 

Figure 2. 11 Structures of some conducting polymers commonly used in biosensors and 

pathway of electron transfer......................................................................................... 30 

Figure 2. 12 Covalent immobilisation via free amino groups on antibodies ............... 33 

Figure 2. 13 Physiosorbed and oriented immobilization strategies of antibodies ....... 35 

FIgure 3. 1 Fabrication of the GNPs/Naf/SPCE .......................................................... 50 

Figure 3. 2 Fabrication of the PEDOT:PSS/20%IL/SPCE sensor. .............................. 51 

Figure 3. 3 Cyclic voltammograms of different IL% in PEDOT:PSS ......................... 53 

Figure 3. 4 Voltammetric responses of 20% IL in PEDOT:PSS ................................. 55 

Figure 3. 5 Nyquist plots observed for EIS at bare and modified SPCE ..................... 57 

Figure 3. 6 Scanning electron micrographs and corresponding surface roughness for 

bare and PEDOT:PSS/20%IL modified electrodes. .................................................... 58 



xi 

 

Figure 3. 7Scanning electron micrographs and Raman spectrum of bare and GNPs-Naf 

modified electrodes ...................................................................................................... 59 

Figure 3. 8 Nyquist plots for EIS at bare and GNPs-Naf modified electrodes. ........... 60 

Figure 3. 9 Electrochemical characterization of the GNPs-Naf/SPE sensor ............... 62 

Figure 3. 10 Electrochemical detection of catechol at PEDOT:PSS/20%IL/SPCE .... 64 

Figure 3. 11 Detection of catechol by amperometry in stirred solution ...................... 67 

Figure 3. 12 Stability of PEDOT:PSS/20%IL/SPCE towards catechol ....................... 68 

Figure 3. 13 Voltammetric detection of APAP and DA. ............................................. 71 

Figure 3. 14 Analysis of DA and APAP at GNPs-Naf/SPE by differential pulse 

voltammograms (DPV) ................................................................................................ 74 

Figure 4. 1 Developmental cycle of human Plasmodium species in a mammalian host 

and the strategies used in detecting parasite specific markers. Redesigned from Scherf 

et al. 2008. .................................................................................................................... 83 

Figure 4. 2 Immobilization strategies applied for the PfHRP-II sensor. ..................... 93 

Figure 4. 3 Electrochemical characterisation of the printed gold microelectrode. ...... 97 

Figure 4. 4 Nyquist of EIS at different stages of PfHRP-II sensor development. ....... 99 

Figure 4. 5 Nyquist plot of BSA-blocked Impedimetric responses of anti-PfHRP-

II/ePDA/SPGµE ......................................................................................................... 100 

Figure 4. 6 Impedimetric responses of physiosorbed antibodies to PfHRP-II .......... 101 

Figure 4. 7 Impedimetric responses of covalently crosslinked antibodies to PfHRP-II

.................................................................................................................................... 102 

Figure 4. 8 Impedimetric responses of the anti-PfHRP-II/GA-cys/SPGµE sensor ... 104 

Figure 4. 9 Impedimetric responses of the anti-PfHRP-II/GA-cys/SPGµE sensor ... 105 

Figure 4. 10 Variations in electrodes batches used for the PfHRP-II sensor. ............ 107 

 



xii 

 

Figure 5. 1 Schematic representation of the stepwise development of the HBsAg 

immunosensor ............................................................................................................ 114 

Figure 5. 2 Nyquist plots observed for EIS at at different stages of HBsAg sensor 

development. .............................................................................................................. 116 

Figure 5. 3 Impedimetric response of the anti-HBsAb/GA-cys/SPGµE sensor ........ 117 

Figure 5. 4 Selectivity of the Hepatitis B immunosensor. ......................................... 119 

Figure 6. 1 Schematic of a label-free multiplexed immunosensor ............................ 128 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 



xiii 

 

LIST OF TABLES 

Table 1.1 Advantages and disadvantages of test-systems based on the lateral flow 

immunoassay platform………………………………………………………………..7 

Table 2. 1 Important analytical parameters of biosensors ........................................... 35 

Table 3. 1 Recovery of spiked catechol from natural water samples .......................... 69 

Table 3. 2 Comparison of graphene-based electrodes for determination of DA and 

APAP ........................................................................................................................... 76 

Table 3. 3 Recovery of DA and APAP from fortified urine samples (n=3) ................ 77 

Table 4. 1 A summary of the selected sensors developed for PfHRP-II detection is:

.................................................................................................................................... 106 

Table 5. 1 Selected sensors developed for HBsAg detection .................................... 117 

 

 

 

 

 

 

 

 

 

 

 

 

 



xiv 

 

LIST OF ABBREVIATIONS 

AC Alternating current 

Ag/AgCl Silver-silver chloride 

AuNP Gold nanoparticle 

APAP N-acetyl-p-aminophenol 

BSA Bovine serum albumin 

CE Counter Electrode 

CP Conductive polymer 

DA Dopamine 

DC Direct current 

DPV Differential pulse voltammetry 

EIS Electrochemical impedance spectroscopy 

ELISA Enzyme linked immunosorbent assay 

GCE Glassy carbon electrode 

GO Graphene oxide 

ICT Immunochromatographic test 

LAMP Loop mediated isothermal amplification 

LDH Lactate dehydrogenase 

LoD Limit of detection 



xv 

 

mAb Monoclonal antibody 

PBS Phosphate buffered saline 

PCR Polymerase chain reaction 

PDDA Poly (allylamine hydrochloride), 

PEDOT:PSS Poly(3,4-ethylenedioxythiophene):poly(4-styrenesulfonate) 

PfHRP-II Plasmodium falciparum histidine rich protein-II 

PoC Point of care 

RE Reference Electrode 

RDT Rapid diagnostic test 

RSD Relative standard deviation 

SAM Self-assembled monolayer 

SD Standard deviation 

SEM Scanning electron microscope 

SERS Surface-enhanced Raman spectroscopy 

SPE Screen Printed electrode 

SPCE Screen Printed carbon electrode 

SPGµE Screen Printed gold microelectrode  

WE Working Electrode 

WHO World Health Organization 

 



xvi 

 

LIST OF SYMBOLS 

% Percent 

~ Approximately 

< Lesser than 

= Equal to 

> Greater than 

≥ Greater than equal to 

°C Degree Celsius 

µ Micro 

A Ampere 

F Faraday constant  

fM Femtomolar 

g Gram 

Hz Hertz 

k Kilo 

kcat Turnover number 

Kd Dissociation constant 

KD Volmer constant 

L Litre 

m Meter 

M Molarity 

n Number or moles of electrons; Nano 



xvii 

 

p Pico 

R2 Regression coefficient 

Rct (Δ Rct) Charge transfer resistance (difference in Rct) 

Rs Solution resistance 

T Temperature 

V Volt 

v/v Volume/Volume 

υ Scan rate 

ω Angular frequency 

w/v Weight/Volume 

Z Impedance 

Zw Warburg impedance 

Ω Ohm 

 

 

 

 

 

 



1 

 

  

INTRODUCTION 

 

 Infectious diseases at a glance 

The demand for systems that detect pathogens or evidence of their existence spans from 

medical diagnostics, environmental monitoring, food quality to pharmaceutical and 

industrial application. Among these, medical diagnostics is of precedence as it 

implicates the health of the general population (Lee et al., 2010; Mahato et al., 2016; 

Morrison et al., 2007; Raba et al., 2013). 

Infectious diseases of various aetiologies are a leading cause of death worldwide with 

the biggest burden in low-income countries, predominantly in young children (Figure 

1.1). About 230 million cases, excluding HIV/AIDS and tuberculosis, were recorded 

globally in 2016 as Disability-Adjusted Life Years (DALY). One DALY equalling one 

lost year of a healthy life. 

 

Figure 1. 1 Infectious disease death rates per 100,000 people in 2017 

Global Burden of Diseases (https://ourworldindata.org/burden-of-disease). 

https://ourworldindata.org/burden-of-disease


2 

 

Intervention and control programs have included mainly vaccination, education and 

improvements in primary healthcare delivery. However, healthcare systems are still 

hindered by the appropriate management of patients who report to facilities. Principal 

among these challenges is the lack of rapid and efficient diagnostics (Blaschke et al., 

2015; Caliendo et al., 2013). Consequently, patients are sometimes treated based on 

clinical presentations, which tends to be ineffective in illnesses such as fevers where 

clinical features are non-specific. For example, cases of bacterial infections may not be 

guided by appropriate antibiotic therapy without accurate diagnostic tools. Treating 

such patients presumptively is susceptible to misdiagnosis, extra cost to patients with 

overtreatment, or increased potential of antimicrobial resistance. Reports have 

documented high proportions of bacterial infections in febrile children treated for 

malaria at a time where guidelines from the World Health Organisation (WHO) 

recommended presumptive malaria treatment in febrile presentations (Brent et al., 

2006; Nadjm et al., 2010).  

 

 Infectious Diseases Diagnostics: The problem of the developing world 

The challenges in pathogen detection or relevant biomarkers suggestive of disease is 

often localised to under-resourced areas (Blaschke et al., 2015). In contrast to the 

developed countries, there seems to be a lack of clear policy in the healthcare delivery 

systems of low-middle income countries despite the underlying factor of infectious 

disease burden. A key area of this neglect is access to diagnostics or diagnostic facilities 

(Peeling & Mabey, 2010). Allegedly, there is little budget allocated to diagnostics in 

the general healthcare investments (Kobusingye et al., 2005; Nkengasong et al., 2010). 

Healthcare facilities are sparsely distributed in sub-Sahara Africa with the majority in 



3 

 

urban areas but  ~80% of its population live in rural areas (Alkire et al., 2014). Poorly 

distributed laboratory facilities may inconvenience patients from rural areas who have 

to travel several hours and return later (up to weeks) for results. The distance and cost 

may become a deterrent to compliance or follow-up.  

Where laboratories do exist, the accuracy of testing tools and the general turnaround 

time could be affected by outdated methods that are lacking in robustness or samples 

requiring storage until adequate numbers are attained before analysis. Considering the 

pre-existing erratic power outages in these regions, the quality of specimen may be lost 

during storage. Routine tests lack regulatory standards and may be used without 

evidence of their effectiveness. Other challenges to laboratories include lack of 

specialists, inadequate supply-chain management for consumables and reagents, 

inconsistent water supply, poor laboratory setup or architecture and poor maintenance 

culture of equipment. It is therefore reasonable to speculate that some clinicians neglect 

laboratory services and resort to presumptive therapy due to  loss of confidence in test 

results (Peeling & Mabey, 2010; Ronald et al., 2006).  

 

 The need for improved diagnostic tests 

Testing tools are indispensable because they are directly implicated in a patient’s 

clinical outcome. Accurate, rapid and affordable diagnostic tests are required to inform 

suitable therapy, assess prognosis and assist in disease surveillance (Caliendo et al., 

2013). It is important for diagnostic tests to remain architecturally and operationally 

simple, without the need for additional equipment and sample processing. This would 

facilitate self-testing and enable uptake in underdeveloped areas by community health 

workers or even pharmacists who administer over-the-counter drugs for treatment.  



4 

 

There are about 1,400 pathogens that infect humans (Taylor et al., 2001), however, most 

diagnostic tests are specific to a single disease or pathogen. The situation is further 

complicated by the emergence of new strains and sub-populations which affect the 

sensitivity of tests. New testing tools capable of multiplexed detection or that 

discriminate between species diversity are therefore required.  

The improved sensitivity offered by molecular testing and the ability to detect multiple-

markers in parallel is useful in identifying targets from the diversified pathogen sub-

populations. Detection of multiple specific pathogens and drug-resistant markers in 

parallel would be an immediate breakthrough in guiding informative therapy in critical 

systemic infections where results from cultures may delay. Yet, most molecular 

methods in clinical diagnostics still require a level of sophistication that restricts 

widespread adoption in under-resourced laboratories. 

 

 Point of Care Tests (PoCTs) 

To increase access to testing, Point of Care Tests (PoCTs) are needed to replace 

laborious and sophisticated methods. PoCT is defined as “a medical test that is 

conducted at or near the site of patient care” (Kost, 2002; Price, 2001). 

Immunochromatographic lateral flow assays, also called rapid diagnostic tests (RDTs), 

have transformed the diagnosis of infectious diseases (Koczula & Gallotta, 2016). 

These portable paper-based devices often embedded in a plastic cassette platform can 

provide results within 5–30 minutes. Its principle is similar to enzyme-linked 

immunosorbent assay (ELISA) and involves the use of nitrocellulose paper for capillary 

suction of biological samples containing a target. Upon addition of a sample, target 

analytes interact with antibodies conjugated onto colloidal gold prefixed at the sample 



5 

 

chamber. The sample then migrates upstream via the nitrocellulose paper towards a 

zone where a second antibody specific to the analyte has been immobilised to form an 

immunocomplex; confirmation of successful formation of the immunocomplex is 

indicated by the presence of a coloured line.  Several commercial RDTs have greatly 

increased access to screening and testing in developing countries; notably for malaria 

(Mason et al., 2002), hepatitis B (Pereira et al., 2015), syphilis (Herring et al., 2006), 

HIV (Bristow et al., 2014) to mention a few. They are simple to perform, do not require 

instrumentation, disposable, inexpensive and most importantly, fast turnaround time. 

Yet, despite the obvious attractiveness of the RDTs, the substantial disadvantages of 

immunoassay constrain the expansion of practical applications of these diagnostic 

platforms in infectious disease diagnostics (Table 1). 

New PoCTs would be particularly useful for diseases such as tuberculosis, where 

culture methods can take up to six weeks before a complex treatment regime spanning 

a minimum of 6 months can begin. For diseases that require quantitation of parameters 

(mostly viral infections; viral titres), PoCTs tend to be lacking or not readily accessible 

where they are urgently needed to provide better care. The limited availability of PoCTs 

could be attributed to capital investments required in developing diagnostic tests, where 

a perceived fear of losses seems to discourage the private sector from committing 

substantial investments even into products with high commercial prospects (WHO, 

2011). All new test devices including PoCTs are required to meet the ‘A.S.S.U.R.E.D’ 

standards of the WHO (Drain et al., 2014). These set of features symbolise; A: 

affordability to persons at risk of infection, S: sensitivity with minimal false-negatives, 

S: specific with minimal few false-positives, U: User-friendliness and simplicity of 

testing with minimal training, R: rapidity of turnaround time to enable treatment at first 

assessment, R: robustness without samples requiring storage, E: equipment-free, and 



6 

 

D: expedited delivered to areas of need. In spite of recent technological reforms and 

advances in diagnostics, most developing countries where infectious diseases are 

prevalent are incapable of technology uptake. Untrained personnel, under-resourced 

facilities, intermittent power supply and limited funding are some underlying factors 

which could further exacerbate the disease burden. Also noteworthy, some diagnostic 

tests (test kits) used in developing countries have not been rigorously evaluated (D Bell 

& Peeling, 2006). Considerable progress has been made towards diagnosing diseases 

such as malaria with a high degree of reliability (Krampa et al., 2017b). However, 

several analytical parameters (turnaround time, assay complexity, low throughput) are 

still unmet, hence these promising diagnostic technologies are unsuitable for uptake 

particularly in areas where they are most needed. Research has focussed on self-

contained, high performing PoCTs using biosensing models. Biosensors have 

significant advantages of superior diagnostics; cost efficiency and rapid turnaround 

time as has been reported by growing amounts of published work (Dias et al., 2014; 

Vigneshvar et al., 2016). These relatively new class of PoCTs have the features required 

to bridge the gaps and pre-existing limitations in ensuring highly accurate diagnostics 

(Sampath & Ecker, 2004).  

 

 

 

 

 

 



7 

 

Table 1: Advantages and disadvantages of test-systems based on the lateral flow 

immunoassay platform [Adopted from (Andryukov et al., 2020)]. 

 

Advantages Disadvantages 

– Cheap, rapid, inexpensive, and 

easy to apply tests.  

– Suitable only for primary screening and 

require confirmation of positive results by 

independent methods. 

– Long shelf-life of test systems – Special equipment (scanners, 

reflectometers, CCD cameras) and 

software are required to obtain quantitative 

results. 

– Test systems do not require 

special temperature conditions 

for storage. 

– Technological improvement of the method 

increases cost and duration of the analysis. 

– No additional special 

equipment is required. 

– In a competitive format, response 

negatively correlates with concentration. 

– Possibility of multiplexed 

formats of test systems 

– Possible technical errors in application of 

specimen may affect the accuracy and 

reproducibility of result. 

– They do not need qualified 

personnel 

– Increase in sensitivity of tests is based on 

the use of gold, silver, or enzyme 

nanoparticles, which limits shelf-life, 

increases cost of analysis, and breaks the 

one-step rule of application. 

– They can be used by general 

practice physicians or patients 

at home 

– Tested specimen must be in the form of a 

solution. Preliminary dissolution of dry 

specimens is mandatory. 

– Visual result is clear and easily 

– distinguishable 

– When the analyte content in the solution is 

low, the specimen needs to be 

concentrated. 

– Tests are usually sold as kits 

with a set of all the items 

needed to perform the test 

 

 

                                        

 

      

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    



8 

 

 Biosensors as PoCTs 

Biosensors started with Clark’s ground-breaking oxygen sensor in 1956 and the 

amperometric enzyme electrode for glucose later in 1962 (Yoo & Lee, 2010). Today, 

improvements in its analytical parameters coupled with the capability of miniaturisation 

and multiplexing have exemplified the modern biosensor as a promising and powerful 

approach to early disease diagnosis. They are self-contained analytical devices in which 

a biological component that generates a recognition event is intimately associated with 

a physical element that transduces the recognition event, and responds selectively to the 

concentration or activity of a chemical species (Buerk, 1993; Chaubey & Malhotra, 

2002). They can be applied to probe analytes qualitatively and quantitatively in 

complex matrices (blood, urine, saliva, tissue, cell cultures, food and environmental 

samples) (Zhang & Ning, 2012). The components and classifications of biosensors are 

detailed in Chapter II (Section 2.2). 

Transduction of the target-receptor binding event is translated through an optical, mass 

or electrochemical readout. An example of optical transduction is the visual 

immunochromatographic RDT which is interpreted by a colour change. However, 

optical sensing is associated with poor sensitivity, the need for optically transparent 

samples/materials and expensive auxiliary equipment. Electrochemical transduction 

offers better sensitivity, low cost instrumentation and is easily miniaturised which can 

facilitate translation into PoCTs as achieved by glucometers (Clausmeyer et al., 2014; 

Lee, 2008; Patel et al., 2016; Stradiotto et al., 2003). Some of its drawbacks include 

additional instrumentation for readouts and difficult interpretation of results. 

 

 



9 

 

 Aim and Objectives 

In the pursuit of a PoCT for infectious diseases, we hypothesised that electrochemical 

biosensors allow for sensitive detection and quantification of analytes. The overall aim 

was to design a label-free, disposable electrochemical biosensor array, based on 

nanostructured surfaces, with the capacity for the simultaneous clinical diagnosis of 

malaria and hepatitis B.  

The aim was addressed through four (4) objectives; 

Objective 1: Evaluation of the suitability of in-house screen-printed carbon electrodes 

(SPCE) and screen-printed gold microelectrodes (SPGµE) as a base transducer for 

electrochemical sensors. 

Objective 2: Modification of the surface properties of the SPCE such as the deposition 

of conductive nanocomposites to enhance its electroanalytical performance 

characteristics. 

Objective 3: Immobilisation of biological components onto the surface of the 

nanostructured SPCE and SPGµE to develop electrochemical biosensors for malaria 

and hepatitis B diagnosis. 

Objective 4: Optimisation of the assay working conditions and application of the 

optimised assay for the simultaneous analysis of the biomarkers in clinical samples. 

 

 Scope and organisation of the thesis 

The thesis details the research performed towards the development of electrochemical 

biosensors which are a powerful tool in analytical chemistry.  It was based largely on 

proofs-of-concept using in-house screen-printed macro carbon electrodes and gold 

microelectrodes for sensitive detection and quantification of analytes. Chapter 2 



10 

 

highlights the basic components of biosensors, the stages of their development and 

strategies for bioreceptor immobilisation. A theoretical overview of the principles 

underlying the methods used during this work, such as voltammetry and 

electrochemical impedance spectroscopy is also presented. 

In Chapter 3, the potential applicability of the modified surfaces as electrochemical 

sensing platforms is demonstrated. Two specific sensors are presented, including the 

design, fabrication and characterisation of the base transducers and their subsequent use 

for the analysis of catechol, a priority pollutant in water, as well as the simultaneous 

analyses of dopamine and acetaminophen in urine. These organic compounds are easily 

oxidizable and reducible and electrochemical detection provides an easy procedure for 

direct and selective detection. 

Chapters 4 and 5 describe the development of a label-free impedimetric biosensor for 

malaria and hepatitis B respectively. Markers for malaria and hepatitis B; Plasmodium 

falciparum histidine-rich protein-II (PfHRP-II) and hepatitis B surface antigens 

(HBsAg) were selected as a proxy for infectious disease immunosensors and to 

demonstrate multi-parallel analyte detection. These diseases were selected because, 

despite the introduction of several intervention strategies including an effective 

hepatitis B vaccination, the estimated overall burden of both diseases remain in sub-

Saharan Africa. In order to maintain the orientation and functionality of bioreceptor 

molecules, different methods of controlled immobilisation of antibodies were 

evaluated. The capacity of the sensor for multiplexed detection of malaria and hepatitis 

B was also demonstrated.   

Finally, an overview of findings and conclusions that provide suggestions for future 

work are detailed in Chapter 6.  



11 

 

  

LITERATURE REVIEW 

 

 Introduction to biosensors 

Any device with the ability to translate measurable signals to the desired outcome falls 

within the category of a sensor. This begins with the five senses of the human body. 

Selected servants were also required to taste the food served to kings in order to detect 

poisoning and caged canaries were deployed into coal mine shafts as audible warning 

systems to alert miners of toxic gases. Prior to the breakthrough in laboratory testing 

for diabetes, ants were used as sensors for diabetic urine and even before that, 

physicians and nurses had to taste urine from their patients for traces of sugar.  

Sensors measure a variety of physical (temperature, mass, pressure or distance) and 

chemical (pH) parameters while biosensors are chemical sensors in which a biological 

element is used to generate a biochemical mechanism (Thévenot et al., 2001; Turner et 

al., 1987). These properties have been applied to biological and non-biological matrices 

with a focus on medical diagnostics. 

Owing to the demand for rapid testing and personalised healthcare, the global market 

for biosensors is forecasted to grow from the current $19.2 billion to $31.5 billion by 

2024 (Figure 2.1), the largest market share being for PoC devices.  

 



12 

 

 

Figure 2. 1 Forecasted growth and demand for biosensors 

The global biosensors market by type, product, technology, application and geography 

forecasted to 2024. 

https://www.marketsandmarkets.com/Market-Reports/biosensors-market-798.html 
 

 

 Components of a biosensor 

The core components of a biosensor are the bioreceptor or biorecognition molecule, 

transducer and a signal analysis system (Morrison et al., 2007; Perumal & Hashim, 

2014). Biosensors can be classified either according to the mechanisms of 

biorecognition or by the mode of signal transduction employed. A general layout and 

classifications are outlined in Figure 2.2. 



13 

 

  

Figure 2. 2 Schematic representation of a biosensor.  

 

 

Biosensors vary based on their transducers and bioreceptors. The interaction of analytes 

with bioreceptor components (antibody, aptamer, nucleic acid and antigen) in different 

biosensors is converted to quantifiable signals through the transducer and then analysed 

in a system.  

 

2.2.1 The bioreceptor 

Biosensors differ from chemical sensors because they incorporate biological molecules 

capable of recognising an analyte. The bioreceptors are placed in close association with 

the transducer where after interaction with an analyte, produces a desired signal. The 

biological component can be antibodies, proteins (peptides, enzymes or antigens), 



14 

 

aptamers, nucleic acids (DNA or RNA), phages, cells or cellular components 

(receptors, organelles) and/or tissues (Figure 2.2). The bioreceptor confers specificity 

and selectivity to the biosensor given that they select for specific analytes and thus, can 

prevent interference of other materials in the complex sample matrix. Biosensors can 

also be classified based on the bio-recognition process. For example, biosensors based 

on immuno-diagnostic methods are called immunosensors. They employ either an 

antibody as capture for antigens or vice versa. Where aptamers are used as the 

bioreceptor, the biosensor is termed an aptasensor and a genosensor where a nucleic 

acid probe is used to target DNA or RNA. Others include enzymatic biosensors, whole-

cell biosensors, phage biosensors, molecularly-imprinted polymer (MIP)-sensors and 

affibody sensors. 

 

2.2.2 Transducer 

The transducer transfers biological recognition events into a quantifiable signal, which 

could be an electrochemical, optical, thermal or piezoelectric response. A key 

determining parameter of sensitivity in biosensors is the accurate correlation of any 

measured signals to the actual quantity of the target. Thus, transducers are vital 

components of biosensors. A sub-classification of biosensors according to their 

transduction procedures is illustrated in Figure 2.2. Further discussions pertaining to 

transducers in this chapter are limited to electrochemical transduction.   

 



15 

 

2.2.3 Signal analysis or processing unit 

After signals in response to the biorecognition event are generated, they are transformed 

into a desirable output. For example, visual optical indicators based on colour changes 

have been used in conjunction with enzymatic biosensors where a substrate’s 

transformation is indicative of enzyme catalysis or inhibition (De Carvalho, 2011; 

Justino et al., 2015). In other setups such as whole-cell biosensors, analytical 

information can be obtained by evaluating the metabolic status of microbes and tissue-

culture cells including; growth inhibition, substrate uptake, bioluminescence, cell 

viability, cell respiration or specific gases produced (De Carvalho, 2011; Di Gennaro 

et al., 2011; Wang et al., 2014). 

Portable, digital battery-operated devices are attractive digital readouts, and the 

popularity of mobile phones has prompted the interest to develop applications and 

accessory devices for integration of biosensors into smartphones (Gawali & Wadhai, 

2018; Ionescu, 2017; Lillehoj et al., 2013; Liu et al., 2014). 

 

 The theoretical background of electrochemical methods in biosensing 

Electrochemistry encompasses the study of electron transfer reactions between an 

electrode in contact with a solution (Fisher, 1996; Manahan, 2010). This differs from 

chemical reactions where electrons are transferred between molecules within the bulk 

solution. Electrochemical reactions occur primarily at the electrode-solution interface 

and require at least two conducting electrodes (working, WE and reference, RE) in 

contact with electroactive species, making up an electrochemical cell. The desired 

electrochemical reactions occur at the WE while the indispensable RE monitors current 

and potential fluctuations of the WE under zero-current (Fisher, 1996). Methods used 



16 

 

in electrochemistry either control (potentiostatic) or measure (potentiometric) the 

potential in electrochemical systems. 

 

Figure 2. 3 An integrated circuit design of a basic 3 electrode sensing system.  

The potential of the WE and RE are sustained at the same level by balancing the current 

at CE (Kellner et al., 2015). The transfer of electrons between electrodes and solution 

is initiated by application of a potential to the system which will, in turn, produce the 

current. 

 

 

2.3.1 Principles of electrochemistry and electrochemical transduction 

2.3.1.1 The faradaic and non-faradaic processes 

During an electrochemical reaction, the faradaic and non-faradaic processes occur at 

the electrode surfaces. The faradaic, or charge transfer process involves the movement 

of electrons between electrode-electrolyte. The non-faradaic process takes into account 

the occurrences that change the electrical double layer such as adsorption, desorption 

(or voltammetric stripping), changes in solution composition, biorecognition, or 

electrode surface area alterations. Non-Faradaic effects, if uncontrolled during 

experiments can interfere with the charge transfer processes. Formation of an electrical 

double layer, for example, is characterized by condensation of ions at the electrode-

solution interface to compensate for excess charges on the electrode. 



17 

 

2.3.1.2 The Faradaic process 

Faradaic processes are monitored through the transfer of electrons during the oxidation-

reduction reaction of an analyte, denoted as in equation 1 which occurs under a 

thermodynamically or kinetically favourable potential region.  

 𝐴 + 𝑛𝑒−  ⇌ 𝐵 (1) 

Where A is the oxidized species, n the moles of electrons, and B for the reduced species.  

Redox reactions do not occur spontaneously, thus they require a potential to provide 

the energy for the Faradaic process, controlled by mass-transport and electron transfer 

(shown in Fig. 3.2). The Nernst equation describes the relationship between 

electrochemical potential and the relative activity of chemical species, where Ecell is the 

potential of the process under scrutiny, 𝐸𝑐𝑒𝑙𝑙
0  is the standard cell potential, R is the molar 

gas constant, T is the absolute temperature, n is the number of electrons transferred, F 

is the Faraday constant, and Q is the ratio of ion concentration at the anode to ion 

concentration at the cathode (In this case of  Eqn. 1, Q= [A]/[B]). 

 𝐸𝑐𝑒𝑙𝑙 =  𝐸𝑐𝑒𝑙𝑙
0 −  

𝑅𝑇

𝑛𝐹
 𝑙𝑛𝑄    =     𝐸𝑐𝑒𝑙𝑙

0 −  
0.059

𝑛𝐹
 𝑙𝑛𝑄 (2) 

 

 

Figure 2. 4 Illustration of the rate-determining steps of a faradaic process 

It involves mass transport of electroactive species (A) to the surface of the electrode 

where it is reduced (B) and transported back into the bulk solution. 

 



18 

 

2.3.1.3 Mass transport mechanisms 

Mass transport is critical to the observable current since it is responsible for conveying 

molecules to and from the electrode’s surface (Kissinger et al., 2019). Factors that 

influence mass transport of species include diffusion coefficient, concentration 

gradient, potential gradient, and analyte charges. When diffusion, convection and 

migration occur concurrently, it complicates the mass-transport process making it 

difficult to relate the current output to the analyte concentration.  

Working within a still undisturbed solution or high scan rates (up to 100 mV/s) may 

help to minimise the effects of convection on mass transport. Migration can be 

decreased with concentrated supporting electrolytes to reduce the electrical field of 

charge; however, high concentrations of supporting electrolyte (often KCl in excess of 

0.1M) which is added to annul the effects of migration and potential drop (Compton et 

al., 2013) could further compound the diffusion. 

Diffusion, therefore, becomes the major contributor to mass transport effects. If the 

reaction is limited by diffusion alone as seen in amperometric systems, the relationship 

between current and analyte concentration is governed by Cottrell behaviour (equation 

3)  

 𝐼 = 𝑛𝐹𝐴𝐷𝐶√
𝐷

𝜋𝑡
  (3) 

Given; n: number of electrons, F: Faraday’s constant, A: electrode area, D: diffusion 

layer, C; concentration and t: time. 

Since diffusion occurs spontaneously from a high to a low concentration, the Stokes-

Einstein equation is used to describe the diffusion coefficient (D), relating it to 

temperature (T), the viscosity (η), and hydrodynamic radius (R) of the diffusive species: 



19 

 

 𝐷 =
𝑘𝑇

6𝜋𝜂𝑅
 (4) 

k is the Boltzmann constant. 

 

2.3.1.4 Kinetics of electron transfer 

Electrons move across a charge gradient at the electrode-electrolyte interface, the 

mechanism requires electrons in the valence orbitals of reduced species to have higher 

energy than electrons at the electrode surface oxidation to occur, and the vice versa for 

reduction. When mass transport is high, the electrochemical process is controlled by 

the rate of electron transfer between the electrode and the electroactive species (kf and 

kb for the forward and backward reactions respectively). In a reversible reaction (Eqn. 

1), the kinetics of electron transfer is faster than the mass transport rate (ks >> km) 

hence the electron transfer reaction at the electrode surface, depending on the 

concentrations of A and B, is rapid and in equilibrium according to Nernst (under STP).  

Considering that an anodic and cathodic reaction occurs on the same electrode, the 

electron transfer process controls the relationship between electrode potential and 

current response.  

 𝐴 + 𝑛𝑒−    

𝑘𝑓

⇌
𝑘𝑏

    𝐵 (5) 

Given the rates of the forward 𝑘𝑓 and backward 𝑘𝑏reactions are; 

 𝑘𝑓 = 𝑘0 exp (
−𝛼𝐹

𝑅𝑇
[𝐸 − 𝐸𝑓

0]) ;   𝑘𝑏 = 𝑘0 exp (
−(1 − 𝛼)𝐹

𝑅𝑇
[𝐸 −  𝐸𝑓

0])    (6) 

k0 is the overall electron transfer rate constant, E is the cell potential, kf and kb are the 

rates of the forward and backward reaction, respectively, 𝐸𝑓
0 and 𝐸𝑏

0 the standard 



20 

 

potentials of the forward and backward reactions, respectively. The combined mass 

transport coefficients and electron transfer rate constants can be expressed in terms of 

the overall flux in an electrochemical system, given by the Butler-Volmer equation 

(Dreyer et al., 2016). 

 𝑗 = 𝑗𝑜 ∙ [exp (
𝛼𝑎𝑛𝐹

𝑅𝑇
𝜂) −  exp (− 

𝛼𝑐𝑛𝐹

𝑅𝑇
𝜂)] (7) 

Where; j is the electrode current density, jo is the current density, η is activation 

overpotential, ac and aa are the cathodic and anodic charge transfer coefficients 

respectively. 

The current-potential relationship within a kinetically controlled experiment differs 

from diffusion-controlled systems. This is because high mass transports of electroactive 

species to the electrode are controlled by the rate of electron transfer between the 

electrode and the electroactive species.  When an electrochemical system is reversible, 

k0 of both oxidation and reduction is high and the current responses become limited by 

mass transport (k0 > mt). Voltammetric experiments involving such systems show two 

separate waves (oxidation and a reduction wave) in a current-potential plot. In an 

irreversible reaction, mass transport is sufficiently high so that the current response is 

limited only by the rate of electron transfer (mt > k0). This is because the rate of the 

forward reaction is greater than the product of the mass transport coefficient and the 

rate of the backwards reaction which slows the kinetics of electron transfer compared 

with the mass transport rate. In such circumstances, little current flows around the 

formal potential for the redox couple, therefore large potentials are required at the anode 

and cathode to drive the electrochemical reaction. Quasi-reversible electrochemical 

reactions have mt ~ k0.  

 



21 

 

2.3.2 Methods in electrochemistry  

Electrochemical methods are classified as bulk and interfacial methods. Interfacial 

methods can occur when no current flows across the cell (static mode) or an under 

controlled current and potential (dynamic mode). Voltammetry is most frequently used. 

It is based on voltage, current and time relationships in a three-electrode 

electrochemical system (Rackus et al., 2015; Trojanowicz et al., 2003; Uslu & Ozkan, 

2011). Either under quiescent or hydrodynamic conditions. Hydrodynamic conditions 

are beneficial to mass transport and improvements in detection limits. Application of 

injection analysis systems is also beneficial in improving the sensitivity and sample 

throughput of amperometric methods (Felix & Angnes, 2010; Trojanowicz & 

Kołacińska, 2016). A classification of electrochemical methods used in sensing is 

outlined in Figure 2.5. 

 

Figure 2. 5 The methods used in electrochemical immunosensor developments. 



22 

 

2.3.2.1 Cyclic Voltammetry 

Cyclic voltammetry (CV) provides electrochemical properties of transducer materials, 

thermodynamic and kinetic information about electroactive species. It involves 

applying a potential sweep to an electrochemical system and then measuring the 

resulting current (Compton et al., 2013). An illustration of the CV process and its 

corresponding cyclic voltammogram of a reversible redox process is illustrated in 

Figure 2.6. At the start of the potential ramp (to) there is no electrode-electrolyte 

interaction. As the potential of the electrode increases towards the standard potential of 

the cell half, the faradaic process of the reaction is activated and catalyses an anodic 

reaction. This is characterised by an increased current in response to electron transfer 

between the electrode and reactants in the bulk until reactants decrease sufficiently. The 

peak oxidation current (Ipc), is indicative of a build-up of products in the diffusion layer, 

causing the current response to diminish. At a switching potential (t1/2), and the 

phenomenon is reversed if the reaction is reversible to the stop potential (t1) where the 

concentration of the reactants is assumed to be equal to the bulk solution and the 

concentration of the products is assumed to be nil. 



23 

 

 

Figure 2. 6 A typical cyclic voltammogram within a reversible redox species. 

The traces show oxidation and reduction reaction pathways of the electroactive species 

in an electrochemical cell. The extrapolations represent the peak currents (Ipa and Ipc) 

and peak potentials (Epa and Epc) for the anodic/reduction and cathodic/oxidation 

reactions. 

 

The duration of the scan must be adequate for the redox reaction to occur during CV. 

The differences between peaks (ΔEp) identifies the reversibility of a redox couple, 

independent of the scan rate. Assuming standard conditions, a redox couple produces a 

theoretical separation of ~57 mV (Eqn. 8) for a one-electron transfer process. Larger 

ΔE values for voltammograms with distinctive anodic and cathodic peaks are 

suggestive of quasi-reversible reactions. 

 ∆𝐸 =  𝐸𝑝𝑎 −  𝐸𝑝𝑐  =  
0.057

𝑛
 (8) 

Where n is the number of electrons transferred in the half-reaction. 

 



24 

 

The formal potential of the cell is found in the middle of Epa and Epc, hence; 

 𝐸0 =  
𝐸𝑝𝑎 −  𝐸𝑝𝑐

2
 (9) 

In an ideal reversible reaction, Ipa and Ipc are similar and defined in by the Randles-

Ševćik equation (Ngamchuea et al., 2014), as   

 
𝐼𝑝 = ±0.4463𝑛𝐹𝐴𝐶√(

𝑛𝐹𝑣𝐷

𝑅𝑇
) 

(10) 

Where; Ip is the voltammetric peak current, D is the diffusion coefficient of the analyte, 

υ is the scan rate, C is the concentration of the analyte. 

Convection is the major limitation where at very low scan rates, current response is 

more attributable to the Brownian movement of electroactive species than mass 

transport. Using extremely high scan rates also induce a larger Nernst diffusion layer 

due to an overcharged electrode surface. 

 

2.3.2.2 Electrochemical Impedance Spectroscopy  

Electrochemical impedance spectroscopy (EIS) is used in monitoring processes which 

change the conductivity of an electrochemical system. It can monitor both non-faradaic 

(capacitive accumulation from charge separation at the electrode interface) and faradaic 

processes of the charge transfer between the electrode and electroactive species 

(Prodromidis, 2010). Impedance (Z) is defined as the opposition force to an electrical 

current in a circuit, thus a measure of the circuit’s resistance (R) to electrical current 

flow. Impedance (Eqn. 11) differs from resistance in that, it applies a small sinusoidal 

potential (AC potential) of fixed frequency and does not obey the Ohms law 

(Brockman, 2012; Lasia, 2014).  



25 

 

 𝑍𝜔 =  
𝐸𝜔

𝐼𝜔
 (11) 

Where; 𝐸𝜔 is the frequency-dependent potential and 𝐼𝜔 the frequency-dependent 

current. The transformation of angular frequency, ω, to a linear frequency, f (in Hz), is 

expressed as 

 𝜔 = 2𝜋𝑓 (12) 

Application of EIS in biosensing takes into account resistance (a real component of 

impedance, Z', Eqn. 13) and capacitance (an imaginary component of impedance Z", 

Eqn. 14). The data is often described in Nyquist plots (Z' vs. Z")  

 𝑍′ =  𝑅𝑠 +  
𝑅𝐶𝑇

1 + 𝜔2𝑅𝑐𝑡
2𝐶𝐷𝐿

2 (13) 

 𝑍′ =  𝑅𝑠 +  
𝑅𝐶𝑇

1 + 𝜔2𝑅𝑐𝑡
2𝐶𝐷𝐿

2 (14) 

Where; RS is the solution resistance, RCT is the charge transfer resistance and CDL is the 

double-layer capacitance.  

The Rct accounts for the resistance impeding the charge transfer when the electrons 

cross the electrode-electrolyte interface (Preiss et al., 2013).  It is determined by the 

diameter of the semicircle obtained in the Nyquist plot. A Rs occurs between the WE 

and RE at high frequencies and is dependent on the electrolyte concentration, electrode 

material and geometry, temperature and geometry of the electrode. The Nyquist plot is 

fitted into a simplified Randles circuit (Randviir et al., 2012) where the individual 

components can be extracted (Figure 2.7). The Warburg component (Warburg 

impedance (ZW)) accounts for the diffusion of the ions in the electrochemical reaction 

(Prodromidis, 2010). 



26 

 

 

Figure 2. 7 A Nyquist plot of an ideal electrochemical cell. 

The plot is represented as real impedance (Z') versus imaginary impedance (–Z") and 

an equivalent Randles equivalent circuit used in modelling the data for a reversible 

redox species consisting resistance of the solution phase (Rs), the capacitance of the 

double layer (CDL), charge-transfer resistance (RCT) at the electrode surface and 

Warburg diffusion element (W).  

 
 

 Physical properties and setup of electrochemical sensing system setup  

Electrochemical sensors can employ the concept of a three-electrode system consisting 

of a working electrode (WE), auxiliary or counter electrode (CE) and a reference 

electrode (RE); depicted in Figure 2.10. 

 

Figure 2. 8 An electrochemical cell and screen-printed electrode. 

An electrochemical cell (A) consisting of working, counter and reference electrodes; 

and a screen-printed carbon electrode (B) with a carbon composite working and counter 

electrode and an Ag/AgCl reference electrode. 

Electric contacts 

A B 



27 

 

The reaction of interest occurs at the WE while the RE monitors the potential 

differences generated within the cell. The CE combines to stabilise and facilitate 

passage of current by adjusting for errors in the potentials resulting from a polarised 

WE. The RE helps to monitor the potentials generated by the other electrodes and 

electrolyte (Rani et al., 2018). Electrochemical cells are controlled by a potentiostat and 

monitored for characteristics such as current potential, impedance, capacitance, to 

mention a few (Faulkner & Bard, 2002). Not all methods require all three electrodes to 

be present within the cell. Potentiometric methods can apply only the WE and RE.  

 

2.4.1 Electrode materials   

The materials used for a WE depend upon the analyte as it could improve or impede 

the desired electroanalytical signals. It confers electrochemical characteristics to the 

sensor since it interacts directly with the reaction. A dropping mercury electrode traced 

back to Heyrovsky’s invention in detecting metallic and organic compounds is probably 

the first documentation of a WE (Adams, 1958). Mercury’s wide cathodic potential 

range and renewable surface conferred these properties; however, its use was 

discouraged due to toxicity. Electrodes made from noble, chemically stable, conductive 

metals such as platinum and gold are the most appropriate for electroanalysis, but 

expensive to research and mass production.  

The need for alternative low budget electrodes has led to the investigation of variant 

carbon allotropes of graphite and diamond as appropriate electrode materials in 

classical electrochemical analysis. Examples include glassy carbon electrode (GCE), 

carbon fibres, highly ordered pyrolytic graphite (HOPG), single and multi-walled 

carbon nanotubes (SWCNTs and MWCNTs respectively) and boron-doped diamond 



28 

 

electrode (BDDE). Among these, carbon-based electrodes have the additional benefit 

of having a large potential window, low background current and rich surface chemistry. 

RE usually consist of silver metal coated with a layer of silver chloride (Ag/AgCl) and 

suitable materials for CE are platinum or silver. 

 

2.4.2 Electrode designs and modifications 

2.4.2.1 Screen-printed electrodes 

The concurrent advances in nanotechnology and the quest for portable electrochemical 

devices have focussed on miniaturised electrochemical transducers, particularly printed 

electrodes. Screen printing has become the core of portable electrodes which could also 

be obtained. Fabrication of screen-printed electrodes (SPEs) requires depositing a 

layer/layers of conductive ink unto an inert surface such as ceramic, glass fibre or 

polyvinyl chloride (PVC) (Ha et al., 2005). Inexpensive mass production of disposable 

SPEs is made possible by automated and semi-automated systems (Randviir et al., 

2014). Miniaturised electrodes can also be achieved through inkjet printing, gravure 

and flexography (Österholm et al., 2016; Pudas et al., 2005). 

SPEs have revolutionised the integration of transducers into portable diagnostic 

devices. The commercial success of glucose sensors can be partly linked to advances 

in printing technology (Newman & Turner, 2005). The advantages of SPEs overcome 

the drawbacks of conventional electrodes as they are simpler to set-up and allow for 

independent tests thereby preventing errors related to contamination. Disposability after 

single usage prevents them from carry-over contaminations from re-usage and 

miniaturisation offers a distinct functional feature of a small sample and reagent 

volumes. Furthermore, SPEs can be efficiently printed on inexpensive substrates 



29 

 

according to any design with the possibility of multiple electrodes that allow for multi-

parallel testing. These attributes make them potential candidates for cost-efficient 

portable products. A traditional three-electrode setup and a miniature screen-printed 

electrode is shown in Figure 2.10. 

 

2.4.2.2 Conductive polymers 

In electrochemical sensors, the electrodes act as the source/captor of electrons 

transferred between the electrode and the analytes in solution. To play this role 

efficiently, electrodes need to possess excellent electrical conductivity. While many 

electroactive species produce identical electrochemical signals, electrodes may also 

respond differently depending on the potentials applied. 

Modification to obtain a heterogeneous nanostructured electrode surfaces is aimed at 

enhancing signals through increasing the active surface area and working potential 

window of electrodes (Shirakawa et al., 1977; Shrivastava et al., 2016), and to facilitate 

immobilisation of biorecognition elements (ElKaoutit, 2014; Gerard et al., 2002).  

The use of conductive polymers (CPs) has the potential in protecting electrode surfaces 

from interfering materials in a sample matrix while ensuring maximum redox activity. 

CPs consist of a group of organic and inorganic compounds with very specific 

properties. Common examples used in electrochemical research are polyaniline 

(PANI), polypyrrole (PPy), polythiophene, PEDOT:PSS (Krampa et al., 2017) and their 

derivatives (Shrivastava et al., 2016).  

 



30 

 

 

Figure 2. 9 Structures of some conducting polymers commonly used in biosensors 

and pathway of electron transfer. 

(Deng et al., 2013; Gerard et al., 2002; Ruecha et al., 2014)  

 

The unique properties that make CPs suitable for sensor fabrication include lightweight, 

flexibility, scalability, resistance to corrosion, and ease of customisation for particular 

needs (Shrivastava et al., 2016).  

Nanocomposites composed of CPs along with other nanomaterials of electrochemical 

significance can act synergistically as a highly active electroactive compound better 

than the individual components. Nanocomposites have been demonstrated to possess 

improved electroanalytical properties that address some limitations of pure CPs 

(Prakash et al., 2013) 



31 

 

Nanomaterials have been used alongside CPs in nanocomposites, examples include 

metal nanoparticles, carbon nanotubes (CNTs), graphene, MIPs, and ionic liquids (ILs). 

The interest in using polymer nanocomposites in electrochemical sensor designs has 

increased owing to their ability to promote electron transfer, large surface area, and 

high electrical conductivity. These attributes directly impact on a sensor’s sensitivity 

and detection limits. Metal and metal oxide nanoparticles possess advantageous 

features such as their small size; unique Physico-chemical and electronic properties and 

easy manipulation (Siangproh et al., 2011). 

A range of chemically modified electrodes have been applied in electrochemical 

sensors. The incorporation of nanocomposite combinations into sensor designs has led 

to enhanced electrocatalytic efficiency, giving rise to several significant applications in 

analyte detection. Some examples of the nanocomposites include; TiO2 nanoparticles 

and TiO2 nanotubes (TNTs)-PANI (Zhu et al., 2015), ZnO–PPy (Devi et al., 2011), 

PANI–ZnO (Jain et al., 2014), PANI-graphene (Fan et al., 2011), PPy–graphene (Gao 

et al., 2014), poly(ionic liquid)-functionalized polypyrrole–graphene oxide nanosheet 

(PIL–PPy–GO) (Mao et al., 2015), PEDOT–rGO (W. Wang et al., 2014), PEDOT–

graphene (Lu et al., 2013), PPy–MWCNT (Singh et al., 2012), PANI–MWCNT 

(Karolia et al., 2015), oligo(phenylene ethynylene) and chemically reduced graphene 

oxide (OPE-NH2–rGO) (Deng et al., 2013), PANI–polyvinylpyrrolidone (PVP)-

graphene (Ruecha et al., 2014), PVP–AuNP–Grp (Wang et al., 2015), PANI–AuNP–

chitosan–graphene sheet (Li Wang et al., 2014), polyaniline nanofibers (PANI-NF)–

AuNP (Spain et al., 2011), and PPy–pyrrolepropylic acid–rGO (Wang et al., 2015) and  

graphene nanoplatelets-PEDOT:PSS (Cataldi et al., 2018). 



32 

 

2.4.3 Strategies for immobilizing biological receptors 

Advances in new recognition elements for biosensors developments coupled with the 

application of nanotechnology have greatly improved the analytical performance 

(sensitivity, selectivity, the limit of detection (LoD)) and signal-to-noise ratio) of 

biosensors (Justino et al., 2013). The methods used to immobilise bioreceptors in sensor 

designs directly influences its efficiency and operational performance (Justino et al., 

2015). While biomolecules are required to remain tightly bound to the surface of the 

transducer throughout its operation without desorption, it is equally important that they 

retain their structure and orientation for maximum biological activity after being 

immobilised. Biomolecule stability after immobilisation affects accuracy of 

measurements, operational lifetimes and is responsible for inconsistencies in sensor-to-

sensor reproducibility. It is therefore imperative to incorporate successful 

immobilisation approaches into sensor designs in order to assure maximum 

performance. The classical strategies used in bioreceptor immobilisation include: 

adsorption, covalent linkage, entrapment, cross-linking and affinity–(Physical or 

chemical) (Liebana & Drago, 2016; Prieto-Simon et al., 2008). A combination of the 

strategies can be applied depending on the biomolecule, the transducer or mode of 

detection.  

Sometimes, the final application of the sensor influences the choice of immobilization 

technique. The focus turns to the characteristics desired from the sensor rather than 

producing the ideal biosensor. For instance, a trade-off in stability for improved 

sensitivity and cost; or reproducibility while overcoming the difficulty of the 

immobilisation process. 

Physical adsorption or passive adsorption is the method in which recognition molecules 

(often proteins) are directly attached to transducer surfaces via non-covalent 



33 

 

interactions. Although this form of attachment is simple and maintains the bioactivity 

after the immobilisation, non-covalent bonding like electrostatic force, ionic bond, 

hydrogen bond, and hydrophobic interaction are weak bonds and result in a weak 

attachment (Wang et al., 2008). The problem arises considering that initially adsorbed 

particles may be desorbed during assay procedures like washing (Ben Rejeb et al., 1998; 

Martín-Palma et al., 2004). Moreover, the method is still prone to random orientation 

(Sadir et al., 2014). In covalent attachment, irreversible interaction are formed between 

functional groups of biomolecules and a modified transducer (Rusmini et al., 2007; Yu 

et al., 2015). Chemical coupling agents such as carbodiimides and succinimidyl esters 

(frequently applied for carbodiimide/N-hydroxysuccinimide protocols) are used to 

generate carboxylic groups on electrode surfaces that bind with free amino groups of 

antibodies or protein, as shown in Figure 2.12 (Lai et al., 2011). 

 

Figure 2. 10 Covalent immobilisation via free amino groups on antibodies. 

(a) activation of carboxylic groups (COOH) on the WE with carbodiimides and 

succinimyl esters; (b) amine surface (NH2) activation using isothiocyanates, epoxides, 

or aldehydes; (c) alcohol surface activation periodate oxidation, isothiocyanates, 

epoxides, aldehydes, and cyanogen bromide. 

 

 

Different reactive linkers like thiol derivatives (Azrilawani Ahmad & Moore, 2012; 

Limbut et al., 2006; Nassef et al., 2008), 3-aminopropyl)triethoxysilane (APTES) 

(Wilson, 2005), 3-glycidoxypropyl)-trimethoxysilane (GPTMS) (Wei et al., 2009), 3-

mercaptopropyltrimethoxy-silane (MPTMS) (Mansur et al., 2005), diazonium cation 



34 

 

(Corgier et al., 2005) can be used to generate functional groups on electrodes that bind 

proteins through cross-linking with aldehydes or epoxides. Amine and alcohol groups 

on the proteins are activated by aldehydes (mostly glutaraldehyde) and epoxides. The 

disadvantage associated with covalent linkages and cross-linking is that harsh coupling 

chemicals products may denature biomolecules and result in the loss of activity. 

To ensure high stability and bioactivity, biomolecules can be entrapped within a gel or 

conductive polymer. Thin-film sol-gels and hydrogels, commonly, chitosan, dextran 

and carboxymethyl cellulose (Wu et al., 2006) are porous matrix with excellent 

biocompatibility and have been applied to entrap antibodies in electrochemical 

immunoassays (Dai et al., 2003; Du et al., 2003; Tan et al., 2006, 2007; Tripathi et al., 

2006). Although entrapment methods do not predispose biomolecules to chemical 

reactions that may affect their activity, the formation of gel layers may inhibit signal 

transduction. 

In remedying the partial or complete loss of biomolecule activity that results from 

random orientation and structural deformation, captured biorecognition molecules can 

be immobilised in a manner that exposes the recognition sites to a sample solution. 

Oriented immobilisations strategies that have improved immunosensor’s performance 

include interactions between a functional group and an affinity tag on a protein 

sequence such as avidin-biotin (Yang et al., 2008), protein A/G (Arora et al., 2006; 

Fowler et al., 2007), lectin-carbohydrate (Zeng et al., 2012), metal cation–chelator and 

DNA-directed immobilisation (Boozer et al., 2005).  

 



35 

 

 

Figure 2. 11 Physiosorbed and oriented immobilization strategies of antibodies  

(a) randomly immobilised antibodies; immobilisations through different bioaffinity 

systems involving (b) avidin-biotin, (c) protein A/G and (d) and DNA-directed. 

 

2.4.4 Performance criteria of biosensor responses: analytical properties 

Table 2. 1 Important analytical parameters of biosensors 

Analytical 

properties 
Description References 

Selectivity 
Specificity of the bioreceptor towards a target 

analyte amidst other contaminants 

(McNaught & 

McNaught, 1997; 

Umezawa et al., 2007) 

Sensitivity 
A function of the slope of the calibration curve 

gradient versus concentration 
(Thevenot et al., 2001) 

Linearity 
The accuracy of measurements for increasing 

analyte concentrations.  
(Stephen, 2004) 

Dynamic/linear 

range 

Defined from a series of analyte concentrations for 

which the measurement changes linearly between 

the lower and upper limits of quantification. 

(Thevenot et al., 2001) 

Reproducibility 

Drifts in a series of responses over time 

symbolising the sensor’s capacity to produce 

identical responses in repeated experiments.  

(Thevenot et al., 2001) 

Stability 

Disturbances in or around biosensing system that 

may predispose responses to errors. The 

bioreceptor and the transducer are of particular 

concern which can be affected by operational 

procedures like temperature and lengthy 

incubation. 

(Thevenot et al., 2001) 

 

 



36 

 

 Electrochemical biosensors  

Electrochemical biosensors are based on the concepts of electrochemistry, hence 

specific interactions between an electrode and the solution at its surface. It relies on the 

measurement of electrical charges from reactions of electroactive species occurring at 

an electrode-electrolyte interface using redox reactions. This provides a medium for 

electrons to flow between the two phases of different conductivity and produce signals 

which can be directly proportional to the species/analyte’s concentration. A theoretical 

background underlying electrochemistry and electrochemical transduction is detailed 

in Section 2.3.1. 

The popularity of electrochemical biosensors for clinical analysis has increased steadily 

due to key advantages in their design, assay simplicity, and superior analytical 

performance over conventional laboratory methods (Belluzo et al., 2008; Wang, 2008). 

These qualities make it suitable for POC application amidst continued efforts to 

improve and miniaturise electrochemical systems for portable devices. Electrochemical 

biosensors are categorised into their mode of signal transduction after the 

biorecognition event, namely; amperometric, potentiometric, impedimetric and 

conductometric (Gerard et al., 2002). 

 

2.5.1 Amperometric sensors 

Amperometric sensors apply a fixed potential between a sensing and a reference 

electrode to measure the current produced by an electroactive species as a function of 

time. The current which is as a result of electron transfer from oxidation/reduction 

reaction of an analyte is proportional to its concentration. This method has evolved 

through three generations. Initially, electrochemical responses from a reaction were 



37 

 

measured as the products diffused to the transducer. In order to enhance signals, specific 

mediators were employed between the reaction and transducer. The current generation 

probes the occurrence of the reaction itself without the need for mediators or product 

diffusion to the transducer (Saxena & Malhotra, 2003; Wang, 2008). The main 

drawback of amperometric methods is its dependence on a fixed potential which can 

result in interferences from other electroactive species in a sample matrix leading to 

false signals and low specificity (Lowe, 2008; Perumal & Hashim, 2014). The 

integration of mediators, selectively permeable membranes or sample dilution has been 

used to circumvent or minimise interferences (Belluzo et al., 2008; D’Orazio, 2003; 

Shinde et al., 2012). 

 

2.5.2 Potentiometric Sensors 

Potentiometric methods measure the potential difference in the form of charge build-

up that accumulates between the transducer and a reference electrode upon analyte-

receptor binding (Grieshaber et al., 2008). Ion-selective electrodes are mostly applied 

in potentiometric sensors used in clinical chemistry for physiologically relevant 

electrolytes. However, the method is less sensitive and slower than amperometry 

(Makarychev-Mikhailov et al., 2008). 

 

2.5.3 Conductometric biosensors 

Variant concentrations of electroactive species influence electrical conductivity, 

forming the basis of conductometric sensors where analytical information is obtained 

from the conductivity of electrolyte species within an electrochemical cell. 



38 

 

Conductometric methods have been employed in modelling inexpensive devices for 

food quality monitoring (Arora et al., 2011) but are faced with limited sensitivity 

attributable to interfering signals from the buffer (Jdanova et al., 1996). 

 

2.5.4 Impedimetric biosensors 

Impedimetric transduction is based on the measurement of conductivity when an 

electrical frequency sweep is applied to an electrode (McGuinness & Verdonk, 2009). 

It functions by analyte-bioreceptor interactions which can be monitored by changes in 

either capacitance and electron transfer resistance (RCT) across a transducer surface 

caused by analyte binding.  The impedance changes across the transducer surface 

changes with increasing analyte concentrations, making impedimetric systems highly 

sensitive and selective because they rely on specific receptor/ligand interactions and 

not affected by other analytes in a sample matrix. An important advantage over the 

other electrochemical methods is that, it is suitable for both label-free  detection of 

analytes with or without the need for a redox probe  (Parkinson & Pejcic, 2005; Pejcic 

et al., 2006). It is however, prone to variable reproducibility, and has the likelihood of 

nonspecific binding events occurring (Bogomolova et al., 2009; Daniels & Pourmand, 

2007). 

 

 Immunosensors 

Immunosensors are the most researched affinity biosensors. Due to its reliance on the 

theoretical basis of immunoreaction where an antibody or an antigen is used as a 

biorecognition agent, immunosensors are to an extent mistakenly associated with 



39 

 

biomedical application or disease diagnosis alone. A good accounts of their widespread 

applications involving antibodies immobilised as probes for the detection of pesticide 

residues (Jiang et al., 2008), drug compounds (Gandhi et al., 2015) toxins (Wang & 

Wang, 2008) and in food and drink industries (Thakur & Ragavan, 2013) have been 

well documented. 

The specificity of the selected antibody towards the target is its most significant feature 

and immunoreaction complex can be monitored via a labelled or label-free assay 

format. Antibodies select for antigens ranging from small molecules to macromolecules 

in a given sample. Five classes of antibodies (IgG, IgA, IgE, IgM, and IgD) are defined 

based on structures and biological roles. IgG is abundant in blood and extracellular fluid 

and binds many kinds of pathogens, therefore, immunoassays have often employed its 

use. IgGs have a “Y-shaped” consisting of four polypeptide units (2 heavy chains and 

2 light chains) (Figure 2.8); The upper segments of the Y-shape, denoted as Fab 

fragments, are the sites that bind with antigen. 

 

Figure 2. 12 Schematic representation of an IgG. 

 

 

 



40 

 

Among immunoglobulins, IgG, the most widely used in the developed immunosensors. 

Abs are bivalent and can bind with two specific antigens according to size, shape, and 

chemical compatibility. The Ag-binding site, the paratope binds the complementary 

region on the antigen called the epitope. Antibodies are classified either as monoclonal 

antibodies (mAbs) or polyclonal antibodies (pAbs). mAbs are produced from identical 

immune B cells and are used as a primary antibody in immunosensors to target a single 

epitope of an antigen. Owing to their monovalent affinity they are highly specific 

towards an antigen. On the other pAbs hand are a heterogeneous mixture of 

immunoglobulins produced by different B cell clones. Each pAbs them can recognize 

and bind to different epitopes of a specific antigen. (Lipman et al., 2005). 

 

 

 Immunoassay formats of electrochemical Immunosensing 

The distinction between immunosensors and immunoassays is based on the site of 

immunocomplex between the antibody and the antigen.  In immunoassay systems such 

as the conventional Enzyme-Linked-Immunosorbent Assay (ELISA), the 

immunocomplex processes takes place elsewhere whereas in immunosensors, the 

formation of the immunocomplex and diagnosis occur on the same platform (Wang et 

al., 2008). In ELISA the detectable signal is a colour change readable by an optical 

transducer (Mistry et al., 2014). The optical immunoassay approach is liable to 

drawbacks depending to the type of measurements applied (direct ELISA, indirect 

ELISA, competitive ELISA, and sandwich ELISA). These limitations can be associated 

with the potential false signals arising from coloured samples, a relatively long analysis 

time, a requirement of power-intensive light sources and detectors, as well as sample 

size and usage problem outside the classical diagnostic laboratory (Arduini et al., 2016). 



41 

 

In this regard, the use of immunosensors is a promising alternative to optical 

immunoassay approach for diagnosis of clinically important analytes due to high 

sensitivity and selectivity (Diaconu et al., 2013; Karunakaran et al., 2015). Furthermore, 

they provide the possibility of progression of immunoreactions at detector surfaces in 

real time. The use of electrochemical immunosensors simplifies the analysis with rapid 

and reliable signals. 

Immunosensors are categorised as labelled and label-free formats (H Ju et al., 2011), 

illustrated in Figure 2.9. Labelling strategies require tagging immunoreagents with 

molecules such as fluorophores, magnetic beads and enzymes that produce detectable 

analytical signals upon immunoreaction. Label-free formats, on the other hand, do not 

require any labels during the immunoreaction. Labelled assays could be homogeneous 

or heterogeneous. A homogeneous immunoassay does not require unbound reagents to 

be separated from the immunocomplex reactions during operational procedures; similar 

to agglutination (Englebienne et al., 2000), capillary electrophoresis (Moser & Hage, 

2008), fluorescence polarisation (Nielsen et al., 2000), and fluorescence resonance 

energy transfer-based immunoassays (Pulli et al., 2005). 



42 

 

 

Figure 2. 13 Assay strategies applied in electrochemical biosensors. 

(A) involves a one-step label-free immunosensor with signals induced by antigen-

antibody binding, and (B) is a labelled sandwich-type immunoassay involving multiple 

steps. In the labelled assay signals are obtained from the reaction between an enzymatic 

label and a substrate.  

 

Heterogeneous immunoassay formats involve separation of unbound immune reagents 

from immunocomplexes so that only the analytes of interest remain on the transducer 

surface while unbound ones are washed away. Heterogeneous immunoassay formats, 

commonly the sandwich-type are more popular because they augment sensitivity, 

specificity and throughput. However, the numerous steps involved prolong the 

turnaround time and makes them labour intensive and can be relatively expensive due 

to the use of different chemicals.  Moreover, the analyte concentration measured is 

based on signals generated through the product of a secondary event. Labels can be 

fluorophores, magnetic beads, active enzymes with an easily detectable product, or 

anything that allows facile target conjugation and convenient detection. The labels can 



43 

 

change the binding properties of the biomolecules, which could, in turn, affect their 

binding affinity for targets (Haab, 2003). Label-free detection has been vastly applied 

in impedance sensors. Interaction between a biomolecule and a probe-functionalised 

electrode results in alterations in the electrical properties of the electrode surface. 

Therefore, no labelling is required, and real-time detection can be achieved in 

impedance sensing of proteins through immunocomplexes. 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 



44 

 

  

RESULTS OF SPECIFIC OBJECTIVES 1 AND 2 

 

 

 Development and characterisation of electrochemical transducers for 

biosensing application 

 

Overview 

To achieve objectives 1 and 2, electrochemical studies were conducted on SPCEs to 

evaluate the suitability as a base transducer. This was followed by enhancement of the 

electrode surfaces with conducting nanocomposites to improve their electroanalytical 

properties.  The modified electrodes were then applied to the detection of important 

analytes to demonstrate their suitability as electrochemical sensing platforms.   

The chapter is based on two related publications. 

 

Related publications 

1. Krampa, F., Aniweh, Y., Awandare, G., & Kanyong, P. (2017). A Disposable 

amperometric sensor based on high-performance PEDOT: PSS/ionic liquid 

nanocomposite thin film-modified screen-printed electrode for the analysis of 

catechol in natural water samples. Sensors, 17(8), 1716. doi: 10.3390/s17081716 

 

2. Krampa, F. D., Aniweh, Y., Kanyong, P., & Awandare, G. A. (2018). Graphene 

nanoplatelet-based sensor for the detection of dopamine and N-acetyl-p-

aminophenol in urine. Arabian Journal of Chemistry. 

doi:10.1016/j.arabjc.2018.10.006 

 

 

 

 

 

https://doi.org/10.1016/j.arabjc.2018.10.006


45 

 

3.1.1 Abstract 

The development and application of disposable sensors for the detection of catechol 

and simultaneous voltammetric determination of dopamine (DA) and N-acetyl-p-

aminophenol (APAP) is reported. The sensors were fabricated by drop-coating 

conducting polymer-based composite material of poly(3,4-ethylenedioxythiophene) 

(PEDOT): poly(4-styrenesulfonate) (PSS) doped with a room temperature ionic liquid 

(IL), 1-ethyl-3-methylimidazolium tetrafluoroborate ([EMIM][BF4]) and graphene 

nanoplatelets (GNPs)-Nafion (Naf) nanocomposite onto the working area of a screen-

printed electrode (SPE). The sensors were characterized by scanning electron 

microscopy (SEM), Raman spectroscopy, electrochemical impedance spectroscopy 

(EIS) and cyclic voltammetry (CV). The composites exhibited a nano-porous 

microstructure and was found to be highly stable and conductive with enhanced 

electrocatalytic properties. PEDOT:PSS/IL was utilised in conjunction with 

amperometry towards catechol, a priority environmental pollutant, and differential 

pulse voltammetry applied at the GNPs/Naf for simultaneous analysis DA and APAP 

in their binary mixtures. The sensors showed excellent selectivity and sensitivity toward 

the target analytes, with limit of detection of 23.7 µM for catechol and 0.13 µM and 

0.25 µM for DA and APAP, respectively. The catechol sensor produced recoveries 

exceeding 99.0% when applied to natural water samples and the performance of the 

APAP/DA sensor in human urine samples were found to be well over 97.0%. 

 

 

 

 

 

 



46 

 

3.1.2 Introduction 

Electrochemical transduction has received the most credit among several mechanisms 

of transduction in biosensor designs owing to its simplicity, minimal instrumentation 

cost, capability of miniaturization, and automation. The use of scree