Master’s Programme in Master’s Thesis

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Master’s Programme in Chemical, Biochemical and Materials Engineering

Optimization and characterization of a flexMEA multi-target electrochem- ical aptasensor for the detection of malaria biomarkers

Reetta Penttinen

Master’s Thesis 2022

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i Author Reetta Penttinen

Title of thesis Optimization and characterization of a flexMEA multi-target electro- chemical aptasensor for the detection of malaria biomarkers

Programme Chemical, Biochemical and Materials Engineering Major Biotechnology

Thesis supervisor Prof. Silvan Scheller

Thesis advisor(s) Dr. Gabriela Figueroa Miranda Collaborative partner Forschungszentrum Jülich

Date 26.10.2022 Number of pages 103 + 10 Language English Abstract

This thesis aims to optimize and characterize a multi-target testing method for malaria detection. Malaria is a mosquito-borne infectious disease caused by Plasmodium parasites. It is a leading cause of death and disease in many developing countries. Electro- chemical aptamer-based biosensors (aptasensors) offer lower cost, more mobile and easier to manufacture and operate detection tests compared to other testing methods such as microscopy and polymerase chain reaction. Aptamers are artificially prepared short single-stranded oligonucleotides which are used as bioreceptors for aptasensors.

Chronocoulometry, cyclic voltammetry and differential pulse voltammetry (DPV) were used for fabrication optimization and parameter analysis and DPV was used for characterization and detection. Diffusion based spontaneous self-assembled monolayer (SAM) formation was used as biofunctionalization method.

By using four different aptamers synthetized with multiple thiol groups, 2008s, pL1, LDHp11 and 2106s, a multi-target aptasensor was fabricated by SAM deposition forming an aptamer/PEG mixed monolayer offering high sensitivity, selectivity, and specificity. The resulting multi-target aptasensors have sensitivities > 80% at 5 parasites/μL, clearly surpassing the WHO’s clinical minimum of > 75% at 200 parasites/μL. Furthermore, the multi-target aptasensors have also demonstrated specifici- ties of 100% for all the aptamers, therefore sufficing the WHO’s clinical minimum of 90% for malaria detection. Long-term stability of the aptasensors was enhanced with commonly used stabilizer pullulan. The experiments were pullulan was introduced to the aptasensors showed great promise of enhanced stability.

Fabrication of the flexMEA multi-target electrochemical aptasensor for malaria biomarker detection was further optimized with aptamers synthetized with multiple thiol groups and the resulting aptasensor was characterized based on its sensitivity, selectivity, specificity, and stability. Combination test that could detect and distinguish between P. falciparum and P. vivax is in high demand since distinguishing between the parasites is a key factor for correct treatment and preventing the parasites from developing further resistance towards antimalaria medication, a phenomenon recently observed in certain malaria-endemic areas. With further research, this detection method that detects not only HRP-2 but also PfLDH and PvLDH biomarkers for P. falciparum and P. vivax detection has potential to offer a low-cost, high affinity and sensitive approach for highly specific malaria detection.

Keywords malaria detection, electrochemical aptasensor, multi-target detection, multielectrode arrays, lactate dehydrogenase, multi-thiol, histidine rich protein, pullulan, self-assembled monolayer, polyethylene glycol, aptamer

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ii Tekijä Reetta Penttinen

Työn nimi Taipuisan, montaa elektrodia käyttävän sähkökemiallisen aptasensorin optimointi ja luonnehtiminen malaria biomarkkereiden havaitsemiseen

Koulutusohjelma Chemical, Biochemical and Materials Engineering Pääaine Biotechnology

Vastuuopettaja/valvoja Professori Silvan Scheller Työn ohjaaja(t) Tohtori Gabriela Figueroa Miranda Yhteistyötaho Forschungszentrum Jülich

Päivämäärä26.10.2022 Sivumäärä103 + 10 Kieli englanti

Tiivistelmä

Tämän diplomityön tavoite on malarialoisten aiheuttaman malariainfektion havaitsemiseen käytetyn testausmenetelmän valmistuksen parantaminen ja luonnehtiminen. Tämä testausmenetelmä hyödyntää useita malarialoisten proteiineja tartunta- lähteen tunnistamiseksi. Malaria on yksi johtavista kuolin- ja sairastumissyistä kehi- tysmaissa. Sähkökemialliset aptasensorit tarjoavat edullisemman, liikkuvamman ja helpommin valmistettavan ja käytettävän testausmenetelmän kuin mikroskopia ja polymeraasiketjureaktio. Valmistuksen parantamiseen ja analysointiin käytettiin biosensoreille tyypillisiä sähkökemiallisia luonnehtimis- ja testausmenetelmiä. Dif- fuusioon perustavaa itsestään kokoontuvan monomolekulaarisen kerroksen muo- dostumista käytettiin biotoiminnallisuuden saavuttamiseksi.

Neljä aptameeria, 2008s, pL1, LDHp11 ja 2106s, toimivat työssä reseptorimole- kyyleinä. Nämä aptameerit syntetisoitiin käyttäen useita tioliryhmiä aptameeri/PEG sekalaisen kerroksen muodostamiseksi, mikä tarjosi korkean signaalin, havaintoky- vyn, selektiivisyyden ja havaitsemistarkkuuden. Tuotetun aptasensorin havaintokyky oli yli 80% kun parasiittejä oli 5/ μL ylittäen selkeästi WHO:n kliinisen minimin, yli 75% kun parasiittejä on 200/ μL. Sen lisäksi, aptasensorin havaitsemistarkkuus oli 100% kaikkien aptameerien kohdalla täyttäen WHO:n kliinisen vaatimuksen, 90%.

Pitkäaikaisen vakauden parantaminen pullulaanilla näytti myös lupaavia tuloksia.

Taipuisan useita elektroneja sisältävän kokoonpanon käyttöä useita malaria bio- markkereita havaitsevana sähkökemiallisena aptasensorina paranneltiin käyttämällä useita tioliryhmiä ja lukuisia piirteitä havaitsevia menetelmiä tutkittiin tämän dip- lomityön aikana. Yhdistelmätesteille, jotka pystyvät erottamaan P. falciparumin ja P. vivaxin välillä, on tällä hetkellä tarvetta ja kysyntää. Parasiittien erottaminen on tärkeää oikean hoidon tarjoamisen kannalta. Parasiittien erottelu ja oikea diagnoosi estää parasiittejä kehittämästä vastustuskykyä malarialääkkeitä vastaan, sillä eri parasiittien aiheuttama infektio hoidetaan oikealla tavalla. Tutkimalla lisää tätä me- netelmää, joka käyttää HRP-2:n lisäksi PfLDH:ta ja PvLDH:ta biomarkkereina P.

falciparumin ja P. vivaxin havaitsemiseen, on mahdollista tarjota edullinen, korkea affiniteettinen ja tarkka menettelytapa malarian havaitsemiselle.

Avainsanat malaria, malariatestaus, sähkökemiallinen aptasensori, monikohtei- nen havaitseminen, monen elektrodin joukko, taipuisa polymeerisubstraatti, laktaatti dehydrogenaasi, usea tioliryhmä, pullulaani

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Preface

This thesis would not have been possible without the great people in my life, and I would like to thank them all.

Thank you, Professor Silvan Scheller, for supervising this thesis and helping me to stay on the time frame with your supervising methods. Thank Dr. Gabriela Figueroa Miranda for teaching me everything there is so far to know about malaria and aptasensors and inspiring me to become better scientist. I would also like to thank Dr. Dirk Mayer, who offered insightful ideas how to overcome challenges and acting as the donor for the (nonin- fected) blood. Furthermore, I would like to thank all my colleagues at the IBI-3 for ensuring a splendid work atmosphere and great companionship.

We built some great friendships, and I am forever grateful that you made my life in Germany meaningful. A special thanks goes in this regard to Fer- dinando Catania as my office colleague and peer support.

To my family I extend my utmost gratitude for everything. I would like to thank my sister Heta for being my biggest inspiration, supporter, and critic.

I would like to thank my parents for making it possible for me to live my dream during this period. Thank you to my father Hannu for always believ- ing in me and my abilities and to my mother Marjut for always being there for me regarding if I need you or not. Thank you to all my friends in Finland for supporting me during this journey and staying in my life. A special thanks to Jyri Jokinen, who inspired me to pursue a degree in engineering.

This journey was filled with moments of joy, inspiration and success which felt even better when shared with. It was also filled with moments of challenges and despair. In both highs and lows, the support of my colleagues, friends and family mattered the world to me. So, thank you all for being part of this journey and as Jesse Pinkman once said, “Yeah science!”

Aachen, 26 October 2022 Reetta Penttinen

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Symbols and abbreviations

Symbols

𝑩̅ σn

Q Qmax

keq

cⁿ S Au λD

n ΔG R T [R]

[O]

F E E⁰ C m Nana

ip

A D v CA

Ti Ag Cl I Q NA

z

mean for the blank signal value

standard effort of the blank measurement signal of the aptasensor

maximal saturation value of the curve

= 𝐾₀^𝑛,

concentration power homogeneity factor n ∈ [0, 1]

sulphur gold

Debye length number of electrons Gibbs free energy change

gas constant ≈ 8.314 [J K^-1 mol^-1] cell temperature

concentration of reduced species concentration of oxidized species Faraday constant ≈ 96485 [C/mol]

open circuit potential standard electrode potential

concentration of the electrochemical active species in electrolyte solution mas or mass transfer coefficient, m = D/δ or number of nucleotides moles of the analyte

peak current area

diffusion coefficient for the electroactive species scan rate

concentration of the electroactive species in the electrode titanium

silver chlorine current charge

Avogadro’s number ≈ 6.02214076 ∙ 10²³ charge of redox molecules

Abbreviations

CE CC CV DPV

Counter Electrode Cyclic Voltammetry Chronocoulometry

Differential Pulse Voltammetry

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6 DTPA

EDL flexMEA HRP-2 LDH LF LoD MCH PCR PEG PfLDH POC PvLDH RDT RE SAM SELEX ssDNA TCEP WE WHO

1,2-Diathiane-4-O-dimethoxytrityl 5- [(2-cyano- ethyl)-N, N- diisopropyl)]-phosphoramidite

Electric Double Layer

Flexible Multielectrode Array Histidine Rich Protein 2 Lactate Dehydrogenase Langmuir-Freundlich Limit of detection 6-mercapto-1-hexanol Polymerase Chain Reaction

Poly(ethylene glycol) methyl ether thiol

Plasmodium falciparum Lactate Dehydrogenase Protein Point-of-Care

Plasmodium vivax Lactate Dehydrogenase Protein Rapid Diagnostic Test

Reference Electrode Self-assembled monolayer

Systematic Evolution of Ligands by Exponential Enrichment Single-Stranded DNA

Tris (2-carboxyethyl) phosphine hydrochloride Working Electrode

World Health Organization

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1 Introduction

Malaria is one of the oldest infectious diseases in human history. Malaria has a high statistical incidence rate, and it is therefore considered a major public health challenge. In 2020, there were 241 million recorded malaria cases and 626 909 deaths caused by malaria [1]. Malaria is the leading cause of morbidity and mortality in many tropical and developing countries.

Developing countries with tropical environment are most affected by malaria due to their poor infrastructure, especially inadequate healthcare and lack of control and prevention of diseases. [1] Malaria is transmitted via Anopheles mosquitos, which can only reproduce in moisture-rich environ- ment tropical climate provides [2]. In 2020, 82% of the cases and 95% of the deaths were recorded in the World Health Organization (WHO) African Region, followed by WHO South-Asian Region with 10% of the cases and 2% of the deaths [3]. WHO strongly advocates its policy of "test, treat and track". This policy aims to improve the quality of care and surveillance of malaria prevalence. In addition, WHO recommends that all suspected malaria cases should be confirmed by diagnostic testing before treatment is administered. [4] The early detection of malaria is critical to provide fast and sufficient antimalaria treatment.

Malaria is a vector-borne disease caused by Plasmodium parasites. There are five known species of protozoan Plasmodium parasites, of which Plas- modium falciparum and Plasmodium vivax are the most common cause of malaria morbidity and mortality. Vectors are living organisms that can transmit infectious pathogens between humans, or from animals to humans. In the case of malaria, infected female Anopheles mosquitos function as vectors and transmit the Plasmodium parasite to humans by biting. Ma- laria can be completely cured with an early diagnosis and treatment. [5-8]

Malaria infection caused by P. falciparum is potentially deadly if not treat- ed rapidly and effectively [6] and it is associated with a higher case fatality rate than P. Vivax infection [3]. More than 90% of all malaria cases are caused by P. falciparum infected mosquitos in Africa. The highest burden of

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malaria is concentrated in sub-Saharan Africa where P. falciparum-based malaria is the main cause of disease and death. It affects particularly young children since a considerable proportion of older children and adults have acquired functional immunity against the parasite. P. falciparum and P.

vivax are nearly equally prevalent in South-East Asia, as well as in North and South America. P. vivax-based malaria infection is less virulent and only caused 2% of the total malaria cases in 2020. [3] However, P. vivax parasites form dormant liver stages, hypnozoites, which create a reservoir of non-replicating, persistent parasites. These hypnozoites re-activate peri- odically and lead to new symptomatic blood stage infections without new exposure to parasite-infected mosquitoes. These infections require special treatment. [9]

There has been accelerated decrease in malaria cases between 2000 and 2019. In 2020, the malaria case incidence increased after almost two dec- ades of declining case trend. However, this increase was found to be associated with disruption to services during the COVID-19 pandemic. The decrease of malaria cases, particularly P. falciparum based, is critical factor to consider regarding the increased drug resistance of the parasites. In the Greater Mekong subregion, P. falciparum parasites have developed partial resistance to artemisinin, which is the core compound of the currently best available antimalarial drugs. There are concerns that this could become common characteristic for P. Falciparum parasites worldwide. [3] If malaria diagnosis is based on clinical symptoms, it can be misdiagnosed as other diseases such as enlarged spleen [10] or typhoid fever [11]. This can further lead to increased drug resistance of parasites and poor treatment of other febrile illnesses. The parasites have been proven to develop resistance to antimalaria drugs and therefore is it essential to confirm malaria infection and further differentiate between the causing parasite species. By identify- ing the correct parasite species, treatment options can be correctly guided and the use of antimalaria medicines rationalised. [6, 7] The increased resistance can complicate malaria diagnosis due to gene mutations in the parasites and therefore making the detection of malaria biomarkers more diffi-

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cult. Without correct diagnosis, the treatment will be compromised in the future due to the increased drug resistance of the parasites.

The method to diagnose malaria must be accurate and easily available at the point-of-care (POC) since it is typically needed in remote areas where malaria is most common. Microscopy test is a fast and most simple method for diagnosis since the parasites can be detected in blood via microscopy.

However, microscopy-based diagnosis or sufficient training in microscope handling is not extensive available in malaria-endemic countries due to their poor infrastructure. [12] There are multiple commercially available rapid diagnostic tests (RDTs) that are used for the detection of malaria parasites. The detection is based on monoclonal antibodies acting as receptor molecules binding specifically to Plasmodium antigens. [3]

The majority of WHO recommended RDTs detect only P. falciparum ex- cept for few tests also targeting P. vivax [12-15]. Lately, deletions in the pfhrp2 and pfhrp3 genes of the parasites have been found. Therefore, RDTs based on histidine-rich protein 2 (HRP-2) are unable to detect the malaria infection anymore. HRP-2 is an abundant protein exclusively expressed by P. falciparum parasites. It is the target antigen for the most widely used malaria RDTs. If P. falciparum parasites no longer express these genes, the current state of malaria diagnostics will suffer major setback. Alternative tests independent of HRP-2 are mostly based on the detection of lactate dehydrogenase (LDH) of the parasites. LDH is an enzyme of the anaerobic metabolic pathway which catalyzes the reversible conversion of lactate to pyruvate by reducing NAD+ to NADH and contrariwise [16]. Anerobic metabolic pathway produces energy-carrying molecule adenosine triphosphate (ATP) without oxygen. ATP detains chemical energy from molecules obtained from nutrition and therefore provides as energy source for cellular processes. [17] Furthermore, there is a limited amount of WHO- prequalified non-HRP-2 combination tests that could detect and distinguish between P. falciparum and P. vivax. Therefore, the development of non-HRP-2-reliant and parasite distinguishing test is in high demand. A combination of receptors targeting the two main malaria biomarkers, HRP-

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2 and LDH, could be of great advantage in confirming the infection by ex- cluding false-positive tests and discriminating between the two main parasites. [3]

Antibodies are the most used molecules for biomarker recognition in current commercially available RDTs. However, antibodies as biomarker detection molecules have many drawbacks. [14] Their production cost is considerably high, and they have low thermostability. They have limited potential for chemical modifications to directly adapt towards other diagnostic platform technologies. Aptamers have proven to be promising alternative for antibodies in diagnostics. [18] Aptamers are mostly short, single- stranded oligonucleotide sequences of artificial RNA or single-stranded DNA (ssDNA). [19] Compared to antibodies, their synthesis process is sim- pler and more cost-efficient, and they have higher thermostability. Ap- tamers are smaller, less prone to denaturation and already chemically modified, and they recognize and bind to a target analyte with high specificity and strong affinity. [18, 19] Therefore, aptamers are promising solution to solve the drawbacks associated with antibodies used for RDTs. Various detection technologies applying different transducers have been proposed by implementing aptamers as receptor molecules. [18-28] One of these is electrochemical ssDNA aptamer-based sensor. These sensors have already been developed for different pathogen biomarker detection, such as malaria parasites [26] and SARS-CoV-2 virus [29]. Electrochemical sensors based on aptamers are relatively novel detection methods; the fabrication process of aptasensors by self-assembled monolayers (SAMs) often requires optimization of the receptor layer according to the aptamer-target binding charas- teristics.

The objective of this thesis was to characterize and optimize a previously developed flexMEA multi-target aptasensor for the detection of malaria biomarkers [26]. To this end, the fabrication of flexible multielectrode arrays (flexMEAs) functionalized with aptamers that target both HRP-2 protein expressed by P. falciparum and LDH proteins, expressed by both P. falci- parum (PfLDH) and P. vivax (PvLDH) was optimized and the resulting ap-

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tasensor was characterized by electrochemical measurements. The long- term thermal stability of the aptasensor was attempted to be enhanced with pullulan. Since RDTs for malaria are mostly required mostly in countries with tropical environment, it is a necessity for the RDT to be stable in high temperatures. For this thesis, the aptamers were synthetized with multiple thiol groups in attempt to establish more stable and high-density mixed SAM and in such a way improve the sensitivity, selectivity, and specificity of the aptasensor. Since multiple electrodes of the aptasensor have the same receptor multiple redundant signals are obtained, which reduces device specific variations and grants the average of the signals, thus improving the reliability of the sensor output. The utilization of flexMEAs allows the functionalization of multiple aptamers on one aptasensor by diving the electrode array into different sets of electrodes which can be utilized for biomarker detection. This expands the application of the highly sensitive and selective electrochemical multi-target biosensor for malaria detection based on PfLDH, PvLDH, and HRP-2 discriminatory recognition by specific aptamer/target binding events. These characteristics combined with POC capabilities offer a new outlook for sensing device, which allows an early detection and therefore, treatment towards the specific pathogens. Efficient and specific POC testing can also prevent further spread of malaria and limit the development of the parasites’ immunity. The characterization was performed with electrochemical measurements.

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2 Literature review

2.1 Aptamer

Aptamers are artificially obtained short single-stranded oligonucleotides either in the form of peptides, RNA or DNA. The name aptamer derives from the Latin word aptus, meaning to fit because these oligonucleotides bind to specific molecules through its secondary structures and the Greek word merus, meaning part [30]. Aptamers are specific oligonucleic acid sequence with a range of 30 to 100 nucleotides and they recognize specific ligands [22]. Aptamers can form a variety of shapes due to their tendency to form helices and loops. They can bind to various target analytes, such as carbohydrates, cytokines, enzymes, growth factors, immunoglobulins, peptides, diverse types of proteins, small molecules and toxins, metal ions. Ap- tamers can also bind to more complex structures, such as drugs, material surfaces or even whole cells. Target recognition and binding of aptamers is based on three-dimensional, shape-dependent interactions that include base-stacking, electrostatic interactions, hydrophobic molecules, and insertion into a host lattice (intercalation). [31-34] Aptamers are hydrophilic molecules and therefore binding to hydrophilic molecules such as water or hydrogen would disturb the formation between aptamers and their target molecules [35]. Aptamers are relatively novel interest in the field of thera- peutics and diagnostics, being only reported first time in 1990 [36].

Antibodies have been widely used as biosensor receptor molecules. How- ever, aptamers have multiple favourable qualities compared to antibodies regarding the choice as biosensor receptor molecule in POC testing. They are more thermally stable and have lower molecular weight. Furthermore, their chemical production is more accurate and reproducible. The stability of aptamers has been proven to be solid under various harsh conditions.

Aptamers do not lose their bioactivity at elevated temperatures or extreme pH values nor do their functionality degrade during long-time storage. Un- like antibodies, the thermal denaturation is reversible for aptamers. Ap- tamers have a wide range of molecular targets as previously stated in this

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chapter. [36] The production of aptamers does not involve animal suffering since the reproduction of aptamers requires only biochemistry laboratory.

[19] Figure 1 presents the schematic diagram of aptamer functions.

Figure 1. The formation of aptamer's 3D structure followed with target binding to the specific biomarker. The biomarker can be of various kinds and sizes, e.g., polypeptide, protein, or cell. Figure adapted from [37].

Systematic Evolution of Ligands by Exponential Enrichment (SELEX) is used to isolate the most specific high affinity aptamer to the desired target analyte in vitro [30]. The SELEX approach can be used for RNA and DNA strands [30, 38]. A random library with up to 10¹⁵ individual oligonucleotide strands is exposed to the target analyte and a target reaction time is left for the target-DNA-analyte complex formation. Afterwards, the non-bound DNA is either washed off the solution or separated from the solution to isolate the bound complex. The separated target-DNA complex is denatured via chemical treatment or with high temperature. After denaturation, the DNA strands are extracted and amplified using polymerase chain reaction (PCR). The PCR amplified DNA strands are exposed to the target again and the cycle restarts. Depending on the specific SELEX-method, the cycle will be repeated over various loops. The variation of base pairs of the oligonucleotides increases under the approach, thus the final resulting oligonucleotide sequence can be used as an aptamer which is highly specific to its target molecule. [30, 38] Figure 2 presents the SELEX process.

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Figure 2. Schematic of the SELEX process which involves 1. repeated cy- cles of incubation the library with the targets, 2. removal of unbound sequences and recovery of the bound oligonucleotides, 3. amplification of the bound sequenced via PCR. [39]

Through the described SELEX process, the 2008s aptamer was obtained in the group of Dr. J. Tanner. The 2008s aptamer has been reported to recognize specifically the malaria biomarker PfLDH. The crystal structure of the formed PfLDH–2008s aptamer complex has two specific binding loops in PfLDH which are recognized by the 2008s hair-pin structure. These loops are absent in normal human lactate dehydrogenase. [13] 2008s was found to have some cross-selectivity versus PvLDH. [26] The group also found another aptamer called 2106s which selectively detects HRP-2. HRP- 2 protein is exclusively produced by P. falciparum parasites. The 2106s aptamer forms a stem-loop secondary structure binging to HRP-2. [40] The group of C. Ban discovered that the pL1 aptamer binds to both PfLDH and PvLDH with high affinity [41]. pL1 folds into a hairpin-bulge and recognizes PvLDH via shape complementary [42]. LDHp11 aptamer was generated against a unique species-specific epitope of PfLDH. The LHDp11 aptamer is

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highly and exclusively selective towards PfLDH. The use of different aptamers with different target analytes offers a possibility to not only detect but also discriminate between PfLDH and PvLDH proteins and therefore between P. falciparum and P. vivax infections. [43] The malaria infection can be confirmed via protein detection. The discrimination between the proteins informs which parasite has caused the malaria infection and therefore provides specific diagnosis which can be utilized for correct treatment methods.

2.2 Aptasensor

2.2.1 Electrochemical aptasensor

Aptasensor is a class of biosensors. Biosensor is a device that selectively transforms chemical information into a signal which can be analyzed. Like all biosensors, aptasensor is composed of two main components; a biological recognition system, bioreceptor, and a physiochemical transducer. [44]

The bioreceptor detects the target analyte. The transducer recognizes the interactions of the receptor-analyte complex and translates the information into a measurable physical signal. [45] There are numerous approaches for both main biosensor components. For aptasensors, the receptor molecule for the analyte recognition element is an aptamer. The binding event can be based on folding, fusing, splitting, and structural binding of the aptamers.

[20] The aptamers need to be fined to function as a receptor to specific target analytes. The binding of aptamers and their ligands often relies on secondary and tertiary structures of the nucleic acid [46, 47]. The aptamer in- teracts with the target analyte specifically or selectively. The transducer converts the biorecognition event into a measurable signal which is later analyzed. The signal correlates with the transducer type, and it is typically proportional to the analyte-bioreceptor binding interaction on the surface of the transducer. The aptamer is usually modified according to the transducer for immobilization. The transducer can be acoustic wave devices, electrochemical sensors, fluoresce and luminescence sensors, or electrical

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sensors. [48] Figure 3 presents the schematic diagram of biosensor detection system.

Figure 3. Target analyte binds to its specific bioreceptor, such as cell, tis- sue, organelle, enzyme, antibody, or aptamer. The binding effect can be detected with physicochemical variables, and each of these variables activate a signal in their specific transducer. Each of these signals leads to corre- sponding signal e.g., electrical signal, which can be later analyzed for the purpose of biosensing. Figure adapted from [44].

Electrochemical transducers have multiple benefits, including their ability to be miniaturized and integrated in microsystems and their manufac- turing is cost efficient with only few required materials compared to other complex setups such as optical ones. They are highly sensitive and easy to read with fast response time and their use does not require expensive laboratory equipment, high-voltage power supplies nor light sources. The advantages that electrochemical transduction mechanism offers combined with the favorable characteristics of aptamers described in Section 2.1, provide extremely practical, portable, and affordable systems. These are desired qualities for POC tests, especially in malaria-endemic countries, making it a noteworthy application for POC testing. [49-52] POC testing is defined as form of medical diagnostic at the time and place of patient care [53]. The challenges with aptasensors include possible conformational changes through the immobilization of aptamers and the necessary backfill molecules. This will be discussed further in Subsection 2.3.

There are various electrochemical methods available, and the signal obtained with the different methods will depend on the type of aptamer-

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analyte complex formed on the sensor transducer. The first developed type of electrochemical aptasensor was sandwich-type, where a pair of receptors bind to the different sites of the same target. [54] Displacement-type electrochemical aptasensors are also common and they are based on the affinity to the target analytes of aptamers through their structural bonding. [55]

Folding-based electrochemical aptasensors were used in the scope of this thesis. Folding-based electrochemical aptasensor is based on the structural binding of aptamers to the target analyte. Once the aptamer is immobilized on the electrode, the structure of aptamer layer changes after the aptamer- analyte complex is formed. This binding event can be detected with different electrochemical methods. The conformational change conducts to a change in electron transfer resistance, and therefore to a changed impedance at the electrode surface. This can be measured either directly as the impedance change [56], or as the measurable change of the electrode current or potential [57, 58]. The redox marker can be functionalized in the aptamer, or it can be present in the solution the aptasensor is measured.

The redox marker is attracted closer or forced further away from the electrode and therefore increases or decreases the current exchange. [59-61]

FlexMEAs were used as electrochemical aptasensor platform in this thesis. The flexible and disposable platform can integrate multiple individually addressable sensor electrodes with fast electrode kinetics and low noise re- cording capabilities. [62] Since the flexMEAs are fabricated on soft polymer substrates, the fabrication costs decreases and the mechanical tension caused by the biological matter can be adaptable [63, 64]. The multi-target detection is possible since the electrode arrays are divided into different sets which can be incubated in different bioreceptor solutions.

2.2.2 Characterization of the Aptasensor

A biosensor is characterized based on how it reacts to its target analyte. To fabricate a fully functional aptasensor, certain criteria need to be fulfilled. If the specific performance requirements are fulfilled, the aptasensor can be further enabled for successful clinal implementation. The specific charac-

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teristic values are known as figures of merit, and they can be used to assess the performance and applicability of biosensors [65].

Limit of detection (LoD) is a value characterizing the lowest analyte concentration likely to be reliably distinguished from the blank. LoD is defined as in Equation 2.1.

𝐿𝑜𝐷 = 𝐵̅ + 3 ⋅ 𝜎_𝑛 (2.1)

, where 𝐵̅ stands for the mean for the blank signal value and σn for the standard error of the blank measurement. If blank is not available, the signal value of the lowest measured concentration is used as a blank. This is the case if the blank measurement is used as reference value and therefore a value is measured as blank instead. [66]

Dynamic range of detection defines the range of analyte concentration in which the biosensor has a linear response. Dynamic range of detection demonstrates if the aptasensor can cover the detection of the analyte in a relevant clinical concentration range. This ranges from the lowest concentration (LoD) to a saturation concentration, where the signal stops changing linearly to the analyte [56]. A biosensor based on the absorption of the target analyte on a heterogeneous surface of the absorbent can be described by the Langmuir-Freundlich (LF) absorption isotherm as in Equation 2.2 [67].

𝑄(𝑐) =^𝑄^𝑚𝑎𝑥^⋅𝑘^𝑒𝑞^⋅𝑐^𝑛

1+𝑘_𝑒𝑞⋅𝑐^𝑛 (2.2)

, where Q stands for the signal, Qmax for the maximal saturation value of the curve, n the homogeneity factor with a value from [0, 1], c for concentration and keq = K₀ⁿ, where K0 stands for the medium binding affinity between receptor and analyte [68].

The sensitivity defines the change in the sensor response for a given change in analyte concentration. It is calculated from the slope of the linear dynamic range of the calibration curve. The sensitivity is mainly limited by the intrinsic resolution of the used transducer platform to convert the binding event into a detectible signal [69]. The sensitivity of the biosensor used with clinical samples can also be defined as the number of positive signal responses among the total number of positive samples [70].

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Selectivity defines the ability of the biosensor to identify or discriminate the target analyte in a mixture of other interference molecules, especially in more complex systems. Selectivity can be defined qualitatively, as an extent on which other substances interfere with the determination of the target analyte or quantitatively as a value, which can weigh the interference in the measurement procedure. [56] Selectivity is an important criterion for biosensor since usually biological media to be measured are complex and con- tain different substances. A key factor for the functionality of the biosensor is its ability to generate a signal to solely the analyte-receptor reaction event. The two main factors determining the selectivity are the specificity of the aptamer-analyte reaction and the noise signal through non-specific adsorption of molecules on the surface of the electrode. The latter can be prevented in electrochemical biosensors functionalizing the electrode surface with blocking molecule thus ensuring that the only reactions occurring during the measurement are the ones between the receptor and its analyte. [71]

Selectivity of the biosensor for clinal samples can also be defined as the number of negative signals among the total number of negative control samples. [70].

Stability defines the window of time on which an unattended well- preserved biosensor can deliver an expected response. After this window of time, a strong drift or no reliable signal will be measured. The stability can also be applied as a correction factor of drift as a function of the storage time. [26]

2.3 Functionalization of the aptasensor

The functionalization of the aptasensor is conducted by immobilizing the aptamers onto the electrically active surface to maximize the aptamer-target protein binding event and implementing backfill molecules to minimize non-specific binding and adsorption events. There are various methods to conduct these steps. One of the methods is based on physical adsorption via electrostatic interaction, hydrophobic exclusion or London and van der Waals forces. This is not an optimal method since aptamers desorb from the

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surface thus causing low stability of the biomolecules. Another method is the attachment of avidin to biotin conjugation. With this method, the formed layer has been found to be flexible, stable, and repeatable. [72] The functionalization of the aptasensors with biomolecules can also be achieved by chemisorption. Chemisorption is based on the covalent bonding between a functional group of the biomolecule and the surface of the supporting structure [73].

In the scope of this thesis, the aptasensor functionalization was achieved by chemisorption, more specifically with self-assembled monolayers (SAMs). The SAMs are formed firstly by self-assembling the thiolated aptamer onto gold substrate by using a thiol-alkane linked to the aptamer sequence and subsequently, by introducing backfill molecules. This results in a mixed monolayer which adapts the optimal receptor density on the surface. The immobilization via gold-sulfur bond is highly implemented in electrochemical aptasensor fabrication. The immobilization is achieved by mixed SAM, where thiol-labeled aptamers and back-fill molecules form a stable bond onto a gold surface. [74-78]

2.3.1 Formation of the Self-assembled Monolayer

The immobilization of aptamers is critical for the aptamers to retain their biophysical characteristic and thus binding abilities. The immobilization ensures accessibility, controlling the surface density and the stability of the surface-bound aptamers. Implementing the necessary backfill molecules for the electrically active surface is crucial for minimizing nonspecific binding and adsorption events. Possible complex interactions on the immobilized layer and conformational changes through the immobilization may influ- ence the performance of the produced aptasensor. Furthermore, the interactions of the electrochemically active species, the aptamers, and the backfill molecules represent a complex system and their interactions between each other must be understood for proper sensor preparation. [26, 79]

To achieve the desired balance between high load, minimal nonspecific interactions, and preferred orientation, mixed two-component ap-

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tamer/PEG SAMs are used as recognition and shielding components. The thiol-labeled aptamers form a stable bond onto the gold surface, thus forming SAM on the aptasensor. The formation of SAM is based on the spontaneous absorption of the biomolecules into gold surface from a solution. [60, 80] SAMs present dense monolayers formed via rigid intermolecular pack- ing [76, 77, 81]. The aptamer receptors are often immobilized together with shorter backfill molecules into mixed monolayers to achieve the optimal receptor density on the surface. The optimal receptor density ensures that there is enough space for the three-dimensional conformation of the aptamers with their target, thus ensuring high binding efficiency. The blocking molecule is implemented to avoid non-specific adsorption, thus effectively reducing false-positive signals during the diagnosis. [75-78, 82] The approach of SAMs formed with gold-sulfur bonds is widely used in the fabrication of electrochemical aptasensors as both aptamers [83] and blocking molecules [84] can be immobilized onto the gold surface with the assistance of the thiol groups of the molecules.

2.3.2 Multi-Thiol Anchoring

In this thesis, the aptamers were synthetized with multiple thiol groups to assemble thiol-gold SAM via anchoring for the aptasensor. By using multiple thiol groups, the aim was to improve the stability of the binding and avoid aggregation and phase separation within the mixed SAM which can degrade the sensitivity of the aptasensor [26].The aptamers have four 1,2- Diathiane-4-O-dimethoxytrityl 5- [(2-cyano- ethyl)-N, N-diisopropyl)]- phosphor amidite (2xDTPA) units. Structural formula of DTPA linked to oligonucleotide can be seen in Figure 4.

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Figure 4. The chemical structure of 1,2-Diathiane-4-O-dimethoxytrityl 5- [(2-cyano- ethyl)-N, N-diisopropyl)]-phosphoramidite, DTPA linked to oligonucleotide.

DTPA was developed for the use in automated solid phase synthesis of DNA and RNA oligonucleotides to improve the attachment of these oligonucleotides to thiol-reactive surfaces. The attachment is possible also to proteins or other molecules like halogens, iodoacetamides or pyridyl disul- fides. DTPA is inserted to the aptamer at the 5’ position internally and it can be inserted in series with two or more dithiol-groups to increase the efficiency of the ligand-surface interaction. DTPA modified oligonucleotides have already been applied to nanostructures and silver and gold electrodes.

They have proven to be thermally stable and endure high chemical stress, especially with multiple DTPA molecules. The gold surfaces of the electrodes that are functionalized via thiol anchoring are subjected to stress by other thiol-containing molecules in the environment. The other molecules may possibly displace the original thiol anchored loading of the surface.

This displacement is common for mono-thiol anchored functionalization but the use of multiple DTPAs reduces this displacement event. The use of two or more DTPA units has been proven to increase the thermal and chemical stability compared to monothiol and single DTPA. [85]

Tris (2-carboxyethyl) phosphine hydrochloride (TCEP) is used to reduce the disulfide-protecting bond (See Figure 5) and each insertion results in two thiol groups coupling with the gold surface. [85] The thiol groups allow

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the formation of a strong bond between S-Au group and thus the deposition of the biomolecules onto the gold substrate. The thiol group loses one hydrogen atom and binds covalently to three gold atoms. This enables the di- rect binding of the thiol groups of the aptamer to the gold surface of the electrode, resulting to the immobilization of the aptamer-gold SAM. [85-95]

Figure 5. TCEP functions as reducing agent in the reduction of organic disulfide bonds, forming covalent bonds between the sulfide group and other molecule. [96]

2.3.3 Implementing the Backfill Molecules and Forming the Ap- tamer/PEG Mixed Monolayer

The minimization of non-specific binding and adsorption events is another key factor for a functional aptasensor, especially when it is applied to complex matrices, like human serum or blood in situ. In situ measurements are conducted within the complex medium, in which the interference of the other components may affect the selectivity and sensitivity of the aptasensor. Therefore, the other components may impair the reliability of quantita- tive signals by non-specific binding or screening real target detection. To block the undesired components from the surface of the aptasensor, the transducer is blocked with materials or molecular films. Hence, the spaces in between the receptors can be filled without affecting their target analyte binging capabilities. Suggested compounds for backfill molecules include alkanethiols, zwitterionic molecules, peptides, polyethylene glycols and oth- ers. [97] 6-mercapto-1-hexanol (MCH) has been used as a blocking molecule in various aptasensors. [79, 87, 93, 97-100] However, the capability to test in biological fluids has been challenging with MCH as backfill molecule [97] .

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Polyethylene glycol is a hydrophilic material widely used in biomedical devices and aptasensors [79, 97, 101-107]. It has a low interfacial energy in aqueous medium, thus leading proteins close to polyethylene glycol face thermodynamically unfavourable interactions causing repulsion. [71] In this thesis, poly(ethylene glycol) methyl ether thiol (PEG) of 2000 kDa was used as a blocking molecule to avoid biofouling from other molecules present in the human blood. Figure 6 presents the chemical structure of polyethylene glycol and PEG.

a) b)

Figure 6. The chemical structure of a) polyethylene glycol and b) poly(ethylene glycol) methyl ether thiol 2000 (PEG). The key difference lies in the sulfide, which in PEG’s case helps to form a mixed monolayer of aptamer/PEG on the gold surface of the electrode.

After the aptamer-gold SAM has been formed, the remaining uncovered areas of the electrode are blocked with PEG to form a mixed aptamer/PEG monolayer which further prevents the non-specific reactions between the electrode and all the substances of a complex medium. The aptamer/PEG mixed monolayer has been found to have higher resistance to biofouling than alkanethiol films and to enhance the tolerance to matrix complex [26].

Each PEG molecule contains one sulfur atom, which helps the thiol- terminal group anchor the PEG to gold surface. PEG has been proved to enhance the dynamic range, limit of detection and tolerance against complex matrix for aptasensors without affecting their sensitivity [26]. In previ- ous studies, a phase separation between aptamer and PEG was observed, meaning the well-mixed monolayer separated into two phases [26]. This increased the local aptamer density and thus degraded the binding capability to target proteins by restricting the conformational degree of freedom of the aptamers [26] which is a key factor for aptamer-binding. [97] By implementing aptamers with multiple thiol groups, the stability of the mixed

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monolayers is expected to improve and thus the phase separation of aptamers and PEG to reduce or even be completely prevented.

2.4 Malaria Biomarkers

The electrochemical aptasensor can detect malaria protein biomarkers since they are upregulated for the demanding metabolic rate of the infecting Plasmodium parasites. One of the main proteins for malaria diagnosis is the essential energy-converting enzyme PLDH, which is expressed by all malaria parasite species [12, 62, 108]. Most common biomarker for malaria detection is HRP-2, which is an abundant protein exclusively expressed by P.

falciparum parasites. It can be found in red blood cells, serum, cerebrospi- nal fluid, and urine of P. falciparum infected patients. There are many dis- advantages of using only HRP-2-based detection methods to detect malaria.

HRP-2 is persevered in host circulation for several weeks after parasite clearance, potentially resulting in persistent positive signal and therefore unnecessary treatment. This has been one of the reasons for the parasites to develop resistance towards antimalaria treatment. Deletions in pfhrp2 and pfhrp3 genes of the P. falciparum parasites have been found in certain WHO malaria-endemic areas and therefore, some P. falciparum infections have become undetectable by testing methods based on HRP-2 protein expression. [109] A diagnostic device with a combination of receptors simul- taneously targeting not only HRP-2 but another malaria biomarker PfLDH and a third biomarker, PvLDH, expressed exclusively by P. vivax, offers a promising approach in discriminating between P. falciparum and P. vivax parasites. The combination of multiple bioreceptors targeting the biomarkers of P. falciparum offers a detection method which is not merely restricted to HRP-2 protein expression. The concentration levels of these three analyte proteins correlate with parasitemia and can thus reflect parasite levels [12, 62, 108, 110, 111].

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2.5 Long-term Stabilizers

Long-term maintenance of both viability and activity of biosensor is required for the development of biomolecule-based POC testing. Various techniques to conserve both the analytes and the sensors have been re- searched. Long-term stability enhancing methods include cultivating the biomolecules in cultures where the culture conditions remain in steady state over time [112], foam, freeze, spray and vacuum drying [113] and immobilization in biocompatible polymers of organic or inorganic origin [114].

Pullulan (Figure 7) is an organic biocompatible polymer used for long- term stability test of the biosensor. Pullulan is a linear, nonionic, water- soluble, powder-like polysaccharide. It consists of three glucose units connected by α-1,4 glycosidic bonds, maltotriose units, which are connected to each other via α-1,6 glycosidic bond [115]. Bernier discovered pullulan in 1958. It is produced from the fungus Aureobasidium pullulans by a simple fermentation procedure from various feedstocks consisting of starch. [116]

Figure 7. The chemical structure of pullulan. [115]

Because of its unique connection pattern with glycosidic linking, pullulan has distinctive physical properties beneficial for the long-term stability of the aptasensor [113, 117, 118]. It can be derivatized easily to regulate its solubility or introduce reactive groups [116]. It can be chemically altered to be

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either partially soluble or fully insoluble in water [115, 116, 119-122], or to produce pH sensitivity [116]. Furthermore, it is adhesive [116, 122] and bio- degradable [119, 122]. Pullulan can develop fibers [119, 122] and films [116, 119-122]. The films have low thickness, and they are lucid and isolated against oxygen [116, 119-122]. Pullulan is resistant against enzymes such as amylases [116, 122], fructosyltransferase [122], glucose oxidase [116, 122], glucosidase [122] and invertase [116, 122], which are all present in blood, which is the medium when the aptasensor is used to detect malaria. This compound has also proven to be hemocompatible [119, 120, 123], noncar- cinogenic [119, 120, 124], nonimmunogenic [116, 119, 120, 124] and nontox- ic [119, 120, 124]. These aspects make pullulan a prospective source for medical and diagnostic use.

Due to its higher water solubility and low viscosity, pullulan has count- less industrial applications. These include the use as an adhesive [116, 119- 125], a blood plasma substitute [116, 122], a film [116, 120, 122], a flocculant [116], a food additive [116, 122], and an assistant biomolecule in many biomedical applications [116, 119, 120, 122-125]. Pullulan exhibits oxygen bar- rier property [116, 122], excellent moisture retention [116], stability over a wide range of pH values [116] and resistance to heat [120, 122, 123].

Pullulan has shown to be effective at protecting labile biomolecules from oxidation and thermal degradation. [113, 117, 118] Reagents enhanced with pullulan have remained active even at 90°C [113]. The temperature stability of the aptasensor is of relevance since high temperature is common in malaria-endemic countries. Due to this and numerous of its other favorable properties, pullulan was chosen for the long-term stabilizing of the aptasensor. In this thesis, pullulan was used to enhance the shelf life of the aptasensor, particularly in dry conditions.

2.6 Electrochemistry

Electrochemistry is defined as “the study of electricity and how it relates to chemical reactions” [126]. Electrochemical reactions involve electric charges moving between electrodes and an electrolyte. An electrochemical ap-

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tasensor usually requires a general setup consisting of a set of electrodes, which are electrically or chemically stimulated, and the resulting electrical response is measured. This general setup consists of working electrode (WE), a reference electrode (RE), and, in many cases, a counter electrode (CE) [127]. The fundamental reactions and environment of the electrochemical active parts, the electrodes, and the functionalized layer of the aptasensor will be introduced before describing the electrochemical analysis techniques.

2.6.1 Electrode-Electrolyte Interface

The transport of charge between an electronic conductor (electrode) and an ionic conductor (electrolyte) can be measured through various types of electrochemical techniques in the electrochemical system [69]. The charge is transported through the electrode by the movement of electrons. Typical electrode materials are carbon, liquid and solid metals, and semiconduc- tors. In electrochemical aptasensors the electrolyte is a solution with charged molecules and ions. The charge is carried by the movement of con- tained ions in the electrolyte. Typical electrolytes are liquid solutions containing anionic and cationic species, most commonly in water. The electrolyte system must be sufficiently conductive for the electrochemical experi- ment to be useful in an electrochemical cell. The application of electric fields causes that electrodes and electrolytes interact with each other. The electrode can be charged to a specific electric potential, generating surface charges and at the electrode-electrolyte interface. This applied potential forces charge transfer across the interface changing the interface until the chemical potential compensates the applied electrical potential. The charge transfer causes an electric field, which attracts counter-ions of the electrolyte to decrease the potential energy of the system. The layer of ions set up at the interface is called electric double layer. The electric double layer (EDL) is established via the diffusion transport of the ions, which are the energy carriers in the electrolyte. [128]

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The electrode-electrolyte interface can be modeled by the characteristics of a double-plate capacitor. At specific potential, a charge exists on the electrode and in the electrolyte. These charges can be determined as potentials.

EDL is determined as the assembly of charged species and oriented dipoles on the electrode-solution interface. The most basic approach to EDL was proposed by Helmholtz in 1853 [129]. EDL was demonstrated as a simple plate capacitor between the charged surface of the electrode and charged ions covering the electrode. This capacitor mediates the potential difference between the metal electrode and the electrolyte, and the sandwiched ions functioned as dielectric material. In 1913, Chapman and Gouy followed Helmholtz’ basic principle of capacitor and expanded the concept of the Helmholtz layer by substituting the rigid double layer by a diffuse phase.

The diffusion phase accounts for the movement of the ions through the electrolyte solution. Both approaches assumed that the charge distribution throughout the electrolyte and on their respective layers is in continuity independent of the molecular nature of the ions. In 1924, Stern continued the development of the EDL model by defining the ions as point charges.

The molecular behavior of the ions was modeled more closely which pro- vided a to some degree unrealistic yet calculable model of a static phase.

More throughout model of diffuse phase was proposed, describing ions through molecular statistics, called Gouy-Chapman layer.

However, these models did not consider the fact that ions or charges on the electrode may be interchanged, or other charged molecules could reach the electrode surface. In 1947 Grahame developed the EDL model to a multi-layer approach to take these possible interactions into account. [130]

Closest layer to the electrode, Inner Helmholtz Plane (IHP), is formed when charged molecules and partially dissolved ions are closely adsorbed to the metal surface. Then, a second Outer Helmholtz Plane (OHP) layer is formed of non-specifically bound ions with a hydration shell and solvent molecules with specific dipole direction. Solvated ions can approach the electrode surface only to a certain distance, in which the OHP layer is formed. Subse- quently, the diffuse double layer is formed, in which charged molecules

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function as point charges with molecular behavior. The diffuse double layer begins from OHP and continues into the solution until the surface effect is no longer present.

Out of the different distribution of charges, a specific potential gradient results for each layer. This potential is applied to electrode, and it can be applied as a characteristic value to determine how far the potential reaches into the electrolyte. This value is called the Debye length (λD) and it is a measure of the thickness of the diffuse double layer. At the Debye length, the potential decays to e⁻¹ of its value on the electrode. The electric double layer accounts for both the interface between the electrodes and an electrolyte as part a of circuit. [131] The Debye length is highly dependent on the concentration of ions in the electrolyte solution.

The transition in electric potential from one conducting phase to another occurs almost entirely at the interface. The high gradient of the transition indicates the existence of high electric field at the interface. High electric field at the interface affects the behavior of charge carriers in the interfacial region. The magnitude of the potential difference at the interface affects the relative energies of the carriers in the two phases, thus controlling the direction and the rate of charge transfer. The potential of the electrode is defined by its Fermi level, which represents the energy level that has a 50% proba- bility of being occupied. The sign of the potential is determined in relation to a reference potential, which is referred to the standard hydrogen electrode since potentials cannot be directly measured. The energy of electrons can be increased by imposing more negative potentials. The electrons can reach a potential high enough to transfer into vacant electronic states on species in the electrolyte. The transfer of electrons from electrode to electrolyte solution is called a reduction current. The energy of electrons can also be decreased by imposing more positive potential. If the Fermi levels of electrons in the electrode are lower than that of the species in the electrolyte, the electrodes will find more favorable energy on the electrode, causing them to transfer to the electrode. The transfer of electrons from electrolyte to the electrode is called an oxidation current. [132]

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2.6.2 Electrochemical Cell

An array of electrode-electrolyte interfaces is called electrochemical cells.

Electrochemical cells are defined as two electrodes separated by at least one electrolyte phase. The overall chemical reaction in chemical cell consists of two independent half reactions which are reduction and oxidation. These independent half reactions describe in detail the chemical changes at the two electrodes.

Figure 8. A schematic presentation of an electrochemical biosensor with three electrode system including reference (RE), working (WE) and counter electrode (CE) in electrolyte solution. The potential difference is obtained from potentiostat, and the obtained current is measured with it. [133]

An electrochemical biosensor is a system of different electrodes, where on each surface exists an electrode-electrolyte interface (Figure 8). A three- electrode system is most common for electrochemical biosensors. In the three-electrode system, the working electrode (WE) functions the as the transduction element of the biological interaction. It is the stimulator or receiver of the signal to be measured. The reference electrode (RE) has a constant composition which delivers constant potential, to be able to apply a certain potential or current change in WE. RE is typically made of silver/silver chloride (Ag/AgCl) and stands at a close distance from where the reaction occurs to provide a potential that is proportional to the known and stable solution. The use of RE allows normalizing the measurements. Using