Nanomaterials-based electrochemical immunosensors for cardiac troponin recognition: An illustrated review

Accepted Manuscript Title: Nanomaterials-based electrochemical immunosensors for cardiac troponin recognition: An illustrated review Author: Mojgan Abdorahim, Mohammad Rabiee, Sanaz Naghavi Alhosseini, Mohammadreza Tahriri, Sara Yazdanpanah, S. Habib Alavi, Lobat Tayebi PII: DOI: Reference:

S0165-9936(16)30044-9 http://dx.doi.org/doi: 10.1016/j.trac.2016.06.015 TRAC 14787

To appear in:

Trends in Analytical Chemistry

Please cite this article as: Mojgan Abdorahim, Mohammad Rabiee, Sanaz Naghavi Alhosseini, Mohammadreza Tahriri, Sara Yazdanpanah, S. Habib Alavi, Lobat Tayebi, Nanomaterials-based electrochemical immunosensors for cardiac troponin recognition: An illustrated review, Trends in Analytical Chemistry (2016), http://dx.doi.org/doi: 10.1016/j.trac.2016.06.015. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Nanomaterials-based electrochemical immunosensors for cardiac troponin recognition: An illustrated review Mojgan Abdorahim 1, Mohammad Rabiee 1*, Sanaz Naghavi Alhosseini 1, Mohammadreza Tahriri 1,2,3*, Sara Yazdanpanah 1, S. Habib Alavi 4, Lobat Tayebi 2,5 1

Biomaterials Group, Faculty of Biomedical Engineering, Amirkabir University of Technology, Tehran, Iran 2

3

Marquette University School of Dentistry, Milwaukee, WI, 53201, USA

Dental Biomaterials Department, School of Dentistry, Tehran University of Medical Sciences, Tehran, Iran

4

School of Mechanical and Aerospace Engineering, Oklahoma State University, Stillwater, OK, USA 5

Department of Engineering Science, University of Oxford, Oxford OX1 3PJ, UK *

Corresponding author: [email protected]

*

Co-corresponding author:

[email protected]

Highlights • Novel electrochemical biosensors for cardiac troponin recognition are introduced. • Recent advances in electrochemical sensing of cardiac troponin are discussed. • Serious problems in electrochemical sensing cardiac troponin are discussed.

1

Page 1 of 41

ABSTRACT Cardiac troponins (I and T) have been recommended as the biomarkers of choice for the serological diagnosis and prognosis of Acute Myocardial Infarction (AMI) because of their high sensitivity and specificity. Sensor designing has been developed by nanotechnology revolution presenting faster detection and better reproducibility. This review highlights the nanotechnology impact on electrochemical immunosensor that have been developed for the determination of cardiac troponin and provides an overview of the various types of nano based diagnostic methods, along with significant advances over the last several years in related technologies. It is critically important to diagnose Cardio Vascular Disease (CVD) at early stages of its progression, which allows successful treatment and recovery of patients. Therefore, it is essential to develop simple and sensitive CVD diagnostic methods that can detect cardiac troponin as biomarker based on different types of nanomaterials and their developmental and implicational aspects at very low concentrations in biological fluids. Keywords: Electrochemical; Biosensor; Cardiac troponin; Acute Myocardial Infarction; Biomarker

CONTENT 1. Introduction 2. Electrochemical immunosensors 3. Conclusions and perspectives

2

Page 2 of 41

1. Introduction Nanotechnology has bestowed a great impact in the improvement and development of medicine and biotechnology، featuring nano biosensors, nano biomaterials and nano pharmaceutics [1-22]. The sensing phenomenon has made great strides by the manifestation of nanotechnology and biosensors based on different types of nanomaterials that have been

3

Page 3 of 41

developed. Nanomaterials are used as important devices for early AMI diagnosis by measuring cardiac troponin. Early diagnosis of acute myocardial infarction (AMI) is crucial for successful medical treatment of the condition and the prevention of fatality. Cardiac troponin (cTn) has a great cardio specificity compared to other biochemical markers of myocardial injury, such as creatin kinaseMB isoenzyme and myoglobin (cTn) [23, 24]. Troponin, as a complex of three regulatory troponin C, troponin I, and troponin T proteins, is integral in the functionality of skeletal and cardiac muscle contractions. Troponin I (cTnI) and troponin T (cTnT) are biomarkers for AMI diagnosis and understanding their significance is essential in interpreting and treating cardiovascular disease. Consequently, the high sensitivity and specificity of troponin makes it beneficial in AMI diagnosis. Cardiac troponin I (cTnI) is a specific marker for coronary events [25-27]. Very low or undetectable serum troponin levels have been verified in patients who are free of heart disease, and after presentation of AMI symptoms, the level of troponin increases in a 2-4 hour span and could be extended to 14 days after acute myocardial infarction [25, 28]. The troponin complex serves as a receptor for calcium ions to induce structural changes via actin and myosin, propagating contraction [29]. The three subunits of troponin complex are: troponin C, which binds calcium; troponin I, which inhibits actin-myosin interactions; and troponin T, which binds the troponin complex through tropomysin and facilitates contraction [30, 31]. The expression of Troponin C occurs in cells in both cardiac and skeletal muscle while the specific sequences of amino acids in Troponin I and T are unique to cardiac muscle. The troponin complex is broken down and protein components are released into the bloodstream following myocardial damage. Cardiomyocyte damage results in the detection of cTnI and cTnT

4

Page 4 of 41

in peripheral blood. The intracellular cTns are known as the fundamental part in AMI diagnosis as AMI is the most important reason for cardiovascular cell damage, and this demonstrates the critical value of cTns concentration, detection, release kinetics, and the supreme advantage of cTns among other biomarkers. Therefore, in AMI diagnosis, cTnI and cTnT are the preferred markers, and the differential diagnoses of elevated cTns manifest an estimate of cardiomyocyte damage irrespective of its cause. In general, electrocardiography (ECG) and cardiac troponin (cTn) detection provide the diagnostic fundament and complement the clinical assessment for patients with angina pectoris, acute chest pain and other symptoms suggestive of acute myocardial infarction. Highly sensitive detection methods for troponin concentrations are necessary due to extremely low ranges in clinical sensing [25]. Troponins are generally undetectable in healthy patients. The abnormal or elevated troponin concentration is based on a determination of the 99th percentile URL (upper reference limit) for a given assay [32]. Using this definition, the risk that a healthy person, selected at random from the general population, has an abnormal troponin level is 0.01 (1%). The coefficient of variability (CV) is a measure of the imprecision of a laboratory test if the same sample were repeatedly analyzed. With respect to serum troponin assays, a CV of <10% is considered ideal, and in general, an assay with a CV of >20% should not be utilized. An ideal assay would then be characterized by a CV of <10% at the 99th percentile URL. Unfortunately, contemporary troponin assays are at the limits of their detection at low troponin levels and are often unable to deliver precise measurements at or below the 99th percentile URL (CV >20%). In such situations, it has been recommended that a cut-off be set at the lowest point that can be measured with a CV of <10% [33, 34] .

5

Page 5 of 41

The detection of cardiac troponin (cTn) has been investigated using several methods. Electrochemiluminescence immunoassay (ECLIA)[35, 36] and enzyme –linked immunosorbent assay (ELISA) [37] have been widely used for AMI diagnosis [38]. However, the detection requires laboratory equipment with proper instruments that are not easily miniaturized, multi-step processing of samples and well-trained personnel, leading to a considerable time commitment, which would hinder a rapid diagnosis in an acute onset of myocardial infarction. Many types of biosensors based on optical detection, such as fluorescence [39, 40], surface plasmon resonance [41-44] and colorimetry [45, 46] have shown effective performance, but these methods have some limitations in sensitivity, miniaturization and cost efficiency. Thus, immunosensors based on electrochemical detection have been considered as an effective and practical method in analytical approaches especially because of simplicity, inexpensive cost, accuracy and high sensitivity for patient diagnosis [47-50]. The electrochemical approach seems to be the most appealing alternative to optical methods. A very small volume of samples (microliters to nanoliters) can be used in electrochemical biosensors without changing the sensitivity due to modern microelectronics with the possibility of miniaturization to build micro electrodes, and finally, they have the advantage of multiplexing. Both competitive and sandwich formats can be used in electrochemical immunosensors. In the competitive format, immobilized antibodies react with free antigens in competition with labeled antigens, while in sandwich format, after an interaction between immobilized

6

Page 6 of 41

antibodies and free antigens, labeled antibodies are injected and the antigen is sandwiched between two antibodies [51-53]. In general, the recognition elements (i.e. antigen or antibody) in the electrochemical immunosensor concept are immobilized on the electrode surface, and then, a secondary enzyme-labeled antibody is injected onto the electrode surface. The enzymatic reaction takes place, and the electroactive production molecules are created. Consequently, the signal is recorded by the use of bench or portable instruments that are usually capable of applying different electrochemical techniques [54, 55] . For an electrochemical immunosensor, choosing the electrode type is very important, and the sensitivity of the method, the cost of the assay and the possibility to adopt different immobilization procedures are affected by electrode type. Inert metals such as platinum [56], gold [57-59], and several forms of carbon including carbon fiber [60], epoxy graphite [61, 62], graphene [63-65] or glassy carbon [66, 67]have been used as different kinds of electrodes. For immobilizing the recognition elements on the electrode surface or the other solid supports, different methods can be used, and these procedures are the crucial part in the development of an electrochemical immunosensor [68]. In electrochemical immunosensors, when binding between antibody and antigen occurs, the current or voltage changes. Electrochemical transducers are classified as amperometric, potentiometric, conductometric and capacitative. In potentiomtric measurement, when the oxidation or reduction of the species in a sample solution occurs, the potential between a working and a reference electrode is measured via voltammeter. In amperometric

7

Page 7 of 41

measurement, by maintaining a constant amplitude voltage at a working electrode, a current is generated because of the oxidation or reduction of an electro active species. Impedimetric biosensors have been used to monitor an affinity reaction. The antibody is immobilized on the electrode surface, and when the antigen binds to the surface, the change in impedance is detected. In conductometric measurement style, the relationship between a biorecognition event and conductance is observed during the reaction, any alteration in ionic concentration affects the electrical conductivity of the solution or leads to flow a current.

2. Electrochemical immunosensors 2.1. Amperometric sensors for troponin detection Amperometric devices are categorized in electrochemical sensor section. The oxidation or reduction of electroactive species in a biochemical reaction result the current change. An amperometric system performs by continuously measuring the resulting current at a constant potential. In voltammetry, the current is measured during controlled variations of the potential [69]. In 2007, Sungho Ko et al. have developed an electrochemical immunosensor comprising an interdigitated array (IDA) microelectrode chip and a PDMS (poly dimethyl siloxan) channel for the detection of human cardiac troponin I (cTnI) [70] . To immobilize the biomolecules on a gold surface, self–assembled monolayers (SAMs) have been used. The process of direct electron transfer between electroactive species may involve a disorder by blocking the electrode surface with the SAMs and biomolecule-formed layer, and therefore, it reduces the analytical

8

Page 8 of 41

signal and sensitivity of the electrochemical sensor [71, 72]. For this reason, researchers tried to functionalize the inside of poly (dimethyl siloxane)(PDMS) channels assembled with the gold electrode chip [70, 72] . The internal surfaces of the PDMS channel, excluding the gold electrodes, were functionalized via silanization process with a surface-exposed carboxyl group. Protein G was immobilized on the silane film and was followed by anti- cTnI. Protein G binds specifically with the Fc protein of the IgG antibodies. The functionalized surface of the PDMS channel was assembled with an interdigitated array (IDA) chip. This process enhances the electron transfer mechanism and results in an increase of sensitivity due to reduction of the fouling on the surface of electrode as well as the effective immobilization of antibodies onto the surface of the PDMS channel[70] . The analyte cTnI is loaded and the detection antibody alkaline phosphatase (AP)- labeled anti cTnI and then p-amino phenylphosphate were injected and incubated. Then, a cyclic voltammogram was obtained by the oxidation peak. In this research, the effect of immobilized antibody density on the surface and electrode fouling on the electrochemical signal were examined. Results showed the limit of cTnI detection for this microchip obtained on 148 pg/ml within approximately 8 min from the injection of the analyte until the final electrochemical signal. Three separate reasons may cause this low detection limit: the antibody’s proper orientation by protein G, the best packing density reducing steric hindrance and no fouling on the gold electrodes via the surface-functionalized PDMS channels [70]. Since the 1990s, by developing the microelectronics industry, screen-printing technology has offered high-volume, inexpensive, reproducible and reliable single-use sensors. A plethora of

9

Page 9 of 41

substances can be used for electrochemical determination in screen-printing technology [73, 74]. Screen-printed electrodes (SPE) have been employed in the design of disposable sensors for use in electrochemical immunosensors [75, 76]. SPE were composed of a composite by mixing graphite powder with epoxy resin via a film technology. SPE play as a transducer for electrochemical signal generation, and in addition, they permit the attachment of different substances such as mediators, enzymes, antibody and antigen [77]. In 2010, Barbara V.M. Silva et al. have employed a modified SPE followed by streptavidinmicrosphere integration and its binding to the anti-cTnT biotinylated monoclonal antibody for cTnT detection [78]. Mouse monoclonal anti-cTnT biotin-conjugate was immobilized on the streptavidin microspheres, and then, streptavidin microspheres were immobilized onto the electrode surface, the SPE electrode via glutaraldehyde. An electrochemical immunoassay was achieved by a sandwich scheme. The electrode was incubated with cTnT solution and HRP-conjugate. Anti-cTnT was applied on the sensor surface, and amperometric signals were recorded after the peroxidase reaction from anti-cTnT –HRP. Fig. 1 shows the schematic diagram of the HRP mechanism on the electrode response [78]. (Figure 1)

The amperometric responses of SPE were achieved from a cathodic current peak at – 0.7 V potential.

10

Page 10 of 41

The results exhibited a linear increase proportional to the concentration of cTnT (0.1-10 ng/ml) [78] The clinical analysis range of the proposed immunosensor showed the desired detectable concentration of cTnT. To introduce this method as an alternative approach to cTnT assay in the clinical route with real samples, the direct determination of cTnT concentration in human serum is necessary [78]. Carbon nanotubes (CNTs) have been used as electrodes to transmit electrical signals or as sensors to detect the concentration of chemicals or biological materials. CNTs are rolled – up tubular shells of graphite sheet with cylindrical nanostructures[79] . Carbon nanotubes (CNTs) are employed in biosensors due to their fascinating mechanical and chemical properties, such as high mechanical stiffness and the possibilities to their functionalization. Also, CNTs have a great electrical, thermal and optical properties, such as electrochemical actuation, transistor behavior, piezoresistance, high thermal conductivity and high luminescence characteristics [79]. They improve the reaction rate of many electroactive species, promote a rapid electron transfer and reduce the electrode response time [80, 81]. The electroactive area in CNTs has been increased by forming a nanostructured surface and, a greater amount of biomolecules has been immobilized on the surface as a result. Conductive polymers have been used for CNT bonding on the electrode surface. The conducting polymers or electroactive, conjugated polymers, have exerted on the electrochemical sensors. They are deposited on the electrode surface area and act as an electron promoter between the polymer film and electrolyte solution [82].

11

Page 11 of 41

In 2013, S.L.R. Gomes-Filho et al. employed a nanostructured immunosensor based on carbon nanotubes supported by a conductive polymer film for detection of cardiac troponin T (cTnT) [83]. In this work, a gold surface was coated with poly ethyleneimine (PEI) as a highly cationic polymer with high density of amine groups [84, 85]. Carboxylated carbon nanotubes bound to the amine groups in a polymer film via amide linkage [86] and covalent binding between the antibodies and COOH-CNT. Fig. 2 shows the stepwise construction process of the immunosensor [83]. (Figure 2)

Stepwise modification of the electrode was characterized by cyclic voltammetry studies. In Fig. 3, the calibration curve showed a linear correlation in different concentrations of cTnT between 0.1 to 10 ng/ml. A low limit of detection was 0.33 ng/ml and a linear range between 0.1 and 10ng/ml cTnT was observed demonstrating that the detectable concentration of cTnT by use of the proposed immunosensor meets the requirement for AMI diagnosis [83].

(Figure 3) Liquid crystals (LCs) are a class of material between crystalline and liquid phases that have been used in several scientific and technological fields [87-90]. In recent years, the applications of LCs have increased substantially. Au nanoparticles have been widely used in several fields. Their unique properties, such as high surface area to volume ratio and quantum confinement effect and good biocompatibility, considerably draw attention

12

Page 12 of 41

for their ability to take part in the immobilization of biomolecules, such as enzymes and proteins in electrochemical systems[91-93]. In order to avoid agglomeration of nanoparticles, the synthesis of Au NPs requires the use of stabilizing agents[91]. In some studies, it has been reported that soluble and charged silsesquioxane poly, an attractive alternative, has been used to support and stabilize metallic nanoparticles[94-96]. These materials adsorb anionic metal complexes, such as AuCl4 – ions, make small AuNPs, and this nanoparticle can be dispersed in an aqueous medium. In 2014, Eduardo Zapp et al. developed an immunosensor based on the liquid crystal (E)-1decyl-4-[ (4-decyloxyphenyl) diazenyl] pyridinium bromide (Br-Py) and gold nanoparticles supported by the water-soluble hybrid material 3-n-propyl-4-picolinium silsesquioxane chloride (AuNP-Si4Pic(+)Cl(-)) for the detection of troponin T (cTnT), a cardiac marker for acute myocardial infarction (AMI)[97]. To detect cTnT, cyclic voltammetry and electrochemical impedance spectroscopy have been tested. When cTnT molecules bind to the electrode surface, the signal changed, and this may be related to the azo group conjugated to the heterocycles of the Br-Py structure. The result indicates the limit of detection of 0.076 ng/ml and a linear range between 0.1 and 0.9 ng/ml, and it revealed that

the proposed

immunosensor is able to detect clinical levels of cTnT [97].

In 2015, Josef Horak et al. reported enhanced electrochemical detection of a cardiac biomarker, troponin I, based on microfluidic immunochip fabricated in Vacrel® 8100 photoresist film and a highly effective surface functionalization employing polyethylenimine (PEI) [98].

13

Page 13 of 41

The flexible dry film photoresist Vacrel® 8100, has wetting properties for passive fluid control and is easily biofunctionalized. A thin film of polyethylenimine coats the surface of photoresist and acts as a matrix for immobilization of biomolecules. The surface carboxylates of the photoresist were used to biofunctionalize the microchannel on the chip by direct amine-specific coupling and modification via adsorbed and immobilized PEI in both linear (LPEI) and branched (BPEI) formation. PEI, either LPEI or BPEI, has a high density of amino groups. BPEI has primary, secondary and tertiary amino groups with a ratio of 1:2:1, whereas LPEI mostly contains only secondary amines [99]. The effect of the immobilization strategy has been investigated. The passivating layer method using immobilized PEI in both the linear (LPEI) and branched (BPEI) form, and direct immobilization methods based on EDC/SNHS (1-ethyl-3-(3-dimethylaminopropyl/carbodiimide /Nhydroxysulfosuccinimide), have been assessed in chip performance. In comparison between methods, the EDC/SNHS coupling has poor assay characteristics, and the PEI based procedures have a powerful capacity for immobilizing the large amount of biomolecules that show higher biological activity. The adsorbed LPEI has some advantages, such as simplicity and compatibility with various materials. In comparison with immobilized BPEI, failure to provide sufficient stability against a high number of washing steps has been reported. In contrast, the BPEI modified chip reached an LOD of 25 pg/ ml with 10% CV, with other assay parameters comparable to the benchtop ELISA [98]. In 2015, Satish K. Tuteja et al. employed the ultrasensitive immunosensing of cardiac troponin I (cTnI) based on 2-Aminobenzyl amine (2-ABA) functionalized graphene [100].

14

Page 14 of 41

In the recent years, graphene has gained much interest due to its unique properties, such as high specific surface ratio, high electrical conductivity, biocompatibility, high elastic behavior, excellent electrochemical properties, tunable band gap and high carrier mobility [101-105]. Tuteja et al. developed the electrochemical functionalization of graphene with 2-Aminobenzyl amine (2-ABA) as a derivative of aniline possessing an extra aliphatic amine group [100]. The interdigitated electrode surface was generated by the lithography (e-beam evaporation) of gold on a silicon chip. Graphene dispersion was dropcasted onto the working area of the interdigitated gold chip then, 2-ABA was deposited electrochemically onto the graphene chip for the desired functionalization. The cTnI antibody attached the 2-ABA functionalized graphene (f-GN) sensor via Schiff reaction-based chemistry. The proposed method showed the linear range of antigen detection between (0.01–1 ng/mL) and the limit of detection was achieved in 0.01 ng/mL[100] . In 2015, Daniela Brondani et al. developed a label-free electrochemical immunosensor based on an ionic organic molecule ((E)-4-[(4- decyloxyphenyl)diazenyl]-1-methylpyridinium iodide) and chitosan-stabilized gold nanoparticles (CTSAuNPs) for the detection of cardiac troponin T (cTnT)[106] . Chitosan – stabilized gold nanoparticles (CTS-AuNPs) were employed as a green platform for the immobilization of the monoclonal anti-cTnT antibody and the new ionic organic molecule was used as a redox probe. For construction of the immunosensor, the solution of an ionic organic molecule ((E)-4-[(4- decyloxyphenyl)diazenyl]-1-methylpyridinium iodide) or I-Py, which was synthesized by a simple and high yielding protocol, was dropped onto the clean GCE surface, and then, the CTS-AuNP–anti-cTnT dispersion was dropped onto the I-Py film formed.

15

Page 15 of 41

In order to block non-specific sites on the surface of this sensor, Glysin was treated. The electrochemical characterization for the proposed immunosensor was achieved by using cyclic voltammetry (CV) and square-wave voltammetry (SWV) techniques. In each step of the immunosensor construction, the square wave voltammograms were obtained in a PBS solution (0.01 mol /L, pH 7.4). When specific binding between antigens and the anti-cTnT immobilization on the electrode surface occurs, the electron transfers and the peak current of I-py decreases. The limit of detection for the proposed immunosensor is 0.10 ng/ml, and the immunosensor performance demonstrates a linear graph in the range of 0.2 to 1.00 ng/ml cTnT [106].

2.2. Capacitative sensor for troponin detection In electrochemical impedance biosensors, the interaction between the biological element or sensing molecules and the analyte at the interface of an electronic transducer affects the electrical properties of the system [69]. The value of the analyte can be examined by changing the capacitance or conductance and the overall impedance [107]. Different analytical methods have been proposed for various types of analytes, and today, impedance biosensors are employed due to their potential for label–free , real time, in situ detection of various analytes and the affordability of impedance analyzers and data evaluation softwares [108]. Citrate-capped gold nano particles (GNPs) are produced by an easy methodology in controlled monodispersed suspensions with merits, such as high surface-to-volume ratio, and provide a field enhancement at the surface interface [109, 110].

16

Page 16 of 41

In 2012, Vijayender Bhalla et al. used GNPs for creating a nanosized colloidal Au base matrix onto screen-printed electrodes (SPE) in order to propose a low cost, one-step, electrochemically controlled immobilization platform for diagnosis purposes (see Fig. 4) [111]. The dual roles of the colloidal GNPs method as a matrix for direct antibody immobilization and transduction properties were established. In this method, cardiac troponin I was utilizing a change in capacitance. The electrical capacitance increases at the electrode surface due to binding of highly charged antigens on the biointerface. After antibody immobilization, the GNPs antibody surface was blocked by BSA solution to avoid any nonspecific adsorption of protein [112]. Observations of the reaction between monoclonal antibody and the antigen showed that the antibody-immobilized GNP/SPE were exposed to various concentrations of cTnI. The limit of detection is 0.2 ng/ml for capacitance-based detection. The values larger than 0.4 ng/ml indicate cardiac muscle tissue injury or the clear onset of myocardial infarction [113, 114].

(Figure 4)

Non-labeled affinity biosensors have been developed in both academia and industry[115] . The main technology of label-free affinity biosensors is surface plasmon resonance(SPR)[116] devices, mass-sensitive piezoelectric devices [117], field effect transistor (FET) sensors[118] and electrochemical impedance biosensors[119, 120] that are currently in use or have already been developed.

17

Page 17 of 41

In 2009, elder A.de Vasconcelos et al. investigated a simple device structure and simplified measurement scheme for developing a low cost, label-free, point of care capacitive biosensor [121]. The device has been tested successfully with both IgG and TnT analytes. In this work, an Al film, 80 nm thick, was thermally evaporated from a tungsten boat onto the SiO 2 layer and two Al strips (electrode) on to the SiO2 layer were generated by photolithograph. For immobilizing anti-TnT, a self-assembled monolayer (SAM) of thiols is formed, and anti-TnTs were immobilized by using glutaraldehyde via the anti –TnT amino groups in crosslink with aldehydes [121]. For monitoring antibody-antigen interaction, an increase of low capacitance between two planar Al electrodes was used. The device has potential for the fabrication of low cost, labelfree capacitive biosensors. After device fabrication, the capacitance actually becomes constant in all frequencies. But after antibody-antigen interaction, the device capacitance at low frequencies increases. Fig. 5 showed the steps of antibody immobilization on Al electrodes [121].

(Figure 5)

A schematic of measurement setup and device structure and location of antibody-antigen are shown in Fig. 6 [121]. Fig. 6I shows the position of a device on a vacuum chuck, inside a shielded dark box and contacted by two micromanipulator probes. The structure of the device and antibody-antigen interactions are shown in Fig. 6II and Fig. 6III shows the curve of capacitance

18

Page 18 of 41

versus frequency after device fabrication (a), after anti-IgG immobilization(b), and after pipette dropping of IgG solution onto the electrode (c).

(Figure 6)

TnT concentration level was detected in the range of 0.07ng/ml to 6.38ng/ml in human blood serum and in the range 0.01ng/ml to 5 ng/ml in phosphate buffer saline [121].

2.3. Potentiometric sensor for troponin detection In an electrochemical cell, the potentiometric measurement style is measured by the accumulation of a charge potential at working electrode compared to the reference electrode while no significant current or zero flows between them[69]. Molecular imprinting (MI) techniques are 3-D or 2-D imprints of a certain molecule that ordinarily are made from synthetic materials built with vinyl functional derivatives, which generate a rigid polymer matrix [122, 123] . The template molecules are removed without disrupting matrix geometry. Plastic antibodies may be prepared by using molecular imprinting (MI) techniques via inexpensive reagents and have some advantages, such as stable and reproducible materials of quick response (<30s) [124]. Multiwalled carbon nanotubes (MWCNTs) offer excellent thermal conductivity and electrical conductivity [125]. In 2011, Felismina T.C. Moreira et al. designed new MI materials interacting selectively with TnT on the surface of MWCNT and participating in subsequent potentiometric transduction

19

Page 19 of 41

[126]. TnT bind to the surface of carbixylated MWCNTs, and for the purpose of creating a rigid structure

around

the

template,

acrylamide

(AAM,

functional

monomer),

N,N´

-

methylenebisacrylamide (NNMBA ,cross-linker) and ammonium persulphate (ApS, initiator) react under mild conditions. Fig. 7 shows the step process for synthesizing the plastic antibody, which consists of linking the protein to the surface of the CNT, filling the vacant places around it with a suitable rigid structure and removing the protein after this[126] .

(Figure 7) In 2011, A. J. Saleh Ahammad et al. reported electrochemical biosensors for cardiac biomarker troponin I detection [127]. For this reason, gold nanoparticles [128] have been used for immobilizing anti-troponin mAb, and the native environment of the enzyme has been created by using gold nanoparticles(GNPs) [127] . GNPs have been deposited on indium tin oxide (ITO), and, by self-assembly, a specific monoclonal antibody against human cardiac troponin I (cTnI) has been modified on the surface of GNPs coated ITO electrode via linker molecules including cystamine and glutaraldehyde. The target, human cTnI, has been captured by the immobilized antibody on the surface of GNPs through immune binding reaction. The secondary antibody conjugated HRP (horseradish peroxidase) binds to another part of human cTnI and interacts with the antigen in a sandwich way. The interaction between the antigen and antibody has been detected by measuring open circuit potential (OCP) [127] .

20

Page 20 of 41

This electrode system cannot determine the low concentration of cTnI with a very high sensitivity, but it shows meaningful signals at the clinical concentration ranges of cTnI. Fig. 8 shows the modification process of gold nanoparticle-modified ITO electrode to immobilize HRPconjugated antibody[127] . (Figure 8)

2.4. Conductometric sensor for troponin detection The conductometric method measures the ability of an analyte or medium to conduct an electrical current between electrodes or reference nodes. In Conductometric method, the changes in conductance of an electrode as a result of immobilization of enzymes, complementary antibody-antigen pairs and any target molecules onto the electrode surface directly monitored [69]. Organic materials in contrast with inorganic materials are more easily modified with biomolecules, and for functionalization with bio-recognition elements, such as antibodies, inorganic materials require complicated processing conditions. Organic nanomaterials and conducting polymers such as poly aniline (PANI) and poly pyrrole (PPy) are appealing for electrical, mechanical or biomedical applications, and they have some advantages, such as controllable conductivity, mechanical flexibility and exceptional bioaffinity [129]. In 2012, Innam Lee et al. developed a single PANI nanowire-based biosensor for the following cardiac biomarkers: myoglobin (Myo), cardiac troponin I (cTnI), creatine kinase-MB (CK-MB), and b-type natriuretic peptide (BNP) [130] .

21

Page 21 of 41

By using the electrochemical deposition growth method between pre-patterned Au electrodes, the single PANI nanowire was directly fabricated [131, 132]. The mAbs of the cardiac biomarkers were immobilized on the PANI nanowires via covalent binding by a surface immobilization method. The cardiac biomarkers were detected by measuring the conductance alteration between the nanowires [130]. The applicable methods for cardiac troponin measurement have been summarized in Table 1. (Table 1) The early stage of AMI, because of their low cost, ease of miniaturization and the possibility to be fabricated from different conducting substrates and offer sensitivity, selectivity and reliability. However, these biosensors suffer from perturbations on the sensor surface that are influenced by different pH, ionic strength and co-existing molecules in biological fluids. The biological recognition elements used for cardiac biomarkers detection in electrochemical biosensors are antibodies. In diagnostic immunoassays similar structure endogenous molecules with the analyte exist or common cross-reactive epitopes in metabolites of the analyte occur. In general, administration of structurally similar medications take place and cause Cross-reaction and Human anti-mouse antibodies (HAMA) interference demerit. cTnI is present at ultra-low levels after the onset of AMI symptoms, highly sensitive detection methods are urgently needed for early diagnosis and intervention. Highly sensitive assays can measure cTn in the single digit range of nanograms per liter and some research assays even allow detection of concentrations

lower than 1 ng/L. Thus, they obtain a more precise calculation of the 99th

percentile of cTn concentration in reference subjects (the recommended upper reference limit [URL]). These assays measure the URL with a coefficient of variation (CV) lower than 10%. For

22

Page 22 of 41

the development of immunosensors with high sensitive for troponin detection, novel nanomaterials such as metal nanoparticles, graphene, carbon nanotubes, conductive polymers, surfactants and liquid crystals have been employed. Label free electrochemical immunosensor is attractive to use for troponin detection because of the speed of analysis and possibility of miniaturizing the analytical devices to allow portability.

3. Conclusions and perspectives In this review, we summarized recent nano-based advances in the electrochemical immunosensing of human cardiac troponin as a marker of myocardial damage in the early diagnosis of acute myocardial infarction (AMI). The detection concepts and device developments based on electrochemical detection principles for cardiac troponin have been presented. Electrochemical immunosensing development in cardiac troponin detection is a recently developed and promising technology. Although Cross-reaction is a problem in diagnostic immunoassays where endogenous molecules with a similar structure to the measured analyte exist or where metabolites of the analyte have common cross-reactive epitopes, through nanotechnology, the device design with precision for troponin detection applicable in early AMI diagnosis has a profound impact in point-of-care diagnostics and is one of many bioengineering devices that are transitioning from bench to bedside. New approaches based on electrochemical measurement can be applied for diagnostic systems as a simple and smart detection method in cardiac troponin detection. The nanotechnology

23

Page 23 of 41

coupled with a portable electrochemical analyzer exhibit major significance for point of care quantitative testing of cardiac troponin. For the development of electrochemical immunosensing of cardiac troponin, conducting new research based on new types of nanomaterials and miniaturization technology in electronic industry is demanded in the future of biomedical engineering and medicine.

References

24

Page 24 of 41

1.

2.

3. 4. 5.

6.

7.

8. 9. 10.

11.

12.

13.

14.

15. 16.

17.

Ashuri, M., et al., Development of a composite based on hydroxyapatite and magnesium and zinc‐containing sol–gel-derived bioactive glass for bone substitute applications. Materials Science and Engineering: C, 2012. 32(8): p. 2330-2339. Bodaghi, M., et al., Mechanochemical assisted synthesis and powder characteristics of nanostructure ceramic of α-Al 2 O 3 at room temperature. Materials Science and Engineering: B, 2009. 162(3): p. 155-161. Bodaghi, M., et al., Investigation of phase transition of γ-alumina to α-alumina via mechanical milling method. Phase Transitions, 2008. 81(6): p. 571-580. Bodaghi, M., et al., Synthesis and characterization of nanocrystalline α-Al 2 O 3 using Al and Fe 2 O 3 (hematite) through mechanical alloying. Solid State Sciences, 2009. 11(2): p. 496-500. Eslami, H., et al., Hydrothermal synthesis and characterization of TiO2-derived nanotubes for biomedical applications. Synthesis and Reactivity in Inorganic, Metal-Organic, and Nano-Metal Chemistry, 2015(just-accepted): p. 00-00. Eslami, H., F. Moztarzadeh, and M. Tahriri, Synthesis, characterisation and thermal properties of Ca5 (PO4) 3 (OH) 1− xFx (0⩽ x⩽ 1) nanopowders via pH cycling method. Materials Research Innovations, 2011. 15(3): p. 190-195. Eslami, H., M. Tahriri, and F. Bakhshi, Synthesis and characterization of nanocrystalline hydroxyapatite obtained by the wet chemical technique. Materials Science-Poland, 2010. 28(1): p. 5-13. Karimi, M., et al., Ammonia-free method for synthesis of CdS nanocrystalline thin films through chemical bath deposition technique. Solid State Communications, 2009. 149(41): p. 1765-1768. Kazemi, F., et al., NOVEL METHOD FOR SYNTHESIS OF METASTABLE TETRAGONAL ZIRCONIA NANOPOWDERS AT LOW TEMPERATURES. Ceramics-Silikáty, 2011. 55(1): p. 26-30. Khoshroo, K., et al., The influence of calcination temperature on the structural and biological characteristics of hydrothermally synthesized TiO2 nanotube: in vitro study. Synthesis and Reactivity in Inorganic, Metal-Organic, and Nano-Metal Chemistry, 2015(just-accepted): p. 0000. Khosroshahi, M.E., L. Ghazanfari, and M. Tahriri, Characterisation of binary (Fe3O4/SiO2) biocompatible nanocomposites as magnetic fluid. Journal of Experimental Nanoscience, 2011. 6(6): p. 580-595. Mohagheghpour, E., et al., Micro-Emulsion Synthesis, Surface Modification, and Photophysical Properties of Nanocrystals for Biomolecular Recognition. NanoBioscience, IEEE Transactions on, 2012. 11(4): p. 317-323. Mozafari, M., et al., DEVELOPMENT OF 3 D BIOACTIVE NANOCOMPOSITE SCAFFOLDS MADE FROM GELATIN AND NANO BIOACTIVE GLASS FOR BIOMEDICAL APPLICATIONS. Advanced Composites Letters, 2010. 19(2): p. 91-96. Mozafari, M., F. Moztarzadeh, and M. Tahriri, Green synthesis and characterisation of spherical PbS luminescent micro-and nanoparticles via wet chemical technique. Advances in Applied Ceramics, 2011. 110(1): p. 30-34. Poursamar, S., et al., Influence of the value of the pH on the preparation of nano hydroxyapatite polyvinyl alcohol composites. J Ceram Process Res, 2009. 10(5): p. 679-682. Shafia, E., M. Bodaghi, and M. Tahriri, The influence of some processing conditions on host crystal structure and phosphorescence properties of SrAl 2 O 4: Eu 2+, Dy 3+ nanoparticle pigments synthesized by combustion technique. Current Applied Physics, 2010. 10(2): p. 596600. Shafiei, F., et al., Nanocrystalline fluorine-substituted hydroxyapatite [Ca 5 (PO 4) 3 (OH) 1-x F x (0⩽ x⩽ 1)] for biomedical applications: preparation and characterisation. Micro & Nano Letters, IET, 2012. 7(2): p. 109-114.

25

Page 25 of 41

18. 19.

20.

21. 22. 23.

24. 25. 26.

27. 28. 29. 30.

31. 32. 33. 34.

35.

36.

Solgi, S., et al., Synthesis, Characterization and In Vitro Biological Evaluation of Sol-gel Derived Sr-containing Nano Bioactive Glass. Silicon, 2015: p. 1-8. Tahriri, M. and F. Moztarzadeh, Preparation, characterization, and in vitro biological evaluation of PLGA/nano-fluorohydroxyapatite (FHA) microsphere-sintered scaffolds for biomedical applications. Applied biochemistry and biotechnology, 2014. 172(5): p. 2465-2479. Tahriri, M., M. Solati-Hashjin, and H. Eslami, Synthesis and characterization of hydroxyapatite nanocrystals via chemical precipitation technique. Iranian Journal of Pharmaceutical Sciences, 2008. 4(2): p. 127-134. Touri, R., et al., The use of carbon nanotubes to reinforce 45S5 bioglass-based scaffolds for tissue engineering applications. BioMed research international, 2013. 2013. Zamanian, A., et al., Novel calcium hydroxide/nanohydroxyapatite composites for dental applications: in vitro study. Advances in Applied Ceramics, 2010. 109(7): p. 440-444. Etievent, J.-P.C., Sidney Toubin, Gérard Taberlet, Christian Alwan, Kifah Clement, , Use of cardiac troponin I as a marker of perioperative myocardial ischemia. The Annals of Thoracic Surgery, 1995. 59(5): p. 1192-1194. Pedrero María, C.S., Pingarrón José M., Electrochemical Biosensors for the Determination of Cardiovascular Markers: a Review. Electroanalysis, 2014. 26(6): p. 1132-1153. Babuin Luciano, J.A.S., Troponin: the biomarker of choice for the detection of cardiac injury. CMAJ : Canadian Medical Association Journal, 2005. 173(10): p. 1191-1202. Spyracopoulos Leo, L.M.X., Sia Samuel K., Gagné Stéphane M., Chandra Murali, Solaro R. John and S.B. D., Calcium-Induced Structural Transition in the Regulatory Domain of Human Cardiac Troponin C. Biochemistry, 1997. 36(40): p. 12138-12146. Takeda Soichi, Y.A., Maeda Kayo,Maeda Yuichiro, Structure of the core domain of human cardiac troponin in the Ca2+-saturated form. Nature, 2003. 424(6944): p. 35-41. Daubert Melissa A., J.A., The utility of troponin measurement to detect myocardial infarction: review of the current findings. Vascular Health and Risk Management, 2010. 6: p. 691-699. Filatov V. L., K., A. G., Bulargina T. V., Gusev N. B., Troponin: structure, properties, and mechanism of functioning. Biochemistry (Mosc), 1999. 64(9): p. 969-85. Apple Fred S., L.R., Chung Adrine Y., Berger Michael J., Murakami MaryAnn M., Point-of-Care iSTAT Cardiac Troponin I for Assessment of Patients with Symptoms Suggestive of Acute Coronary Syndrome. Clinical Chemistry, 2006. 52(2): p. 322-325. Mohammed Asim A., J.J.L.J., Clinical Applications of Highly Sensitive Troponin Assays. Cardiology in Review, 2010. 18(1): p. 12-19. Qureshi Anjum, G.Y., Niazi Javed H., Biosensors for cardiac biomarkers detection: A review. Sensors and Actuators B: Chemical, 2012. 171–172: p. 62-76. Thygesen K., A.J.S., Jaffe A. S., Simoons M. L., Chaitman B. R., White H. D., et al., Third universal definition of myocardial infarction. Circulation, 2012. 126(16): p. 2020-35. Apple F. S., P.C.A., Buechler K. F., Christenson R. H., Wu A. H., Jaffe A. S., Validation of the 99th percentile cutoff independent of assay imprecision (CV) for cardiac troponin monitoring for ruling out myocardial infarction. Clin Chem, 2005. 51(11): p. 2198-200. Shen Wen, T.D., Cui Hua, Yang Di, Bian Zhiping, Nanoparticle-based electrochemiluminescence immunosensor with enhanced sensitivity for cardiac troponin I using N-(aminobutyl)-N(ethylisoluminol)-functionalized gold nanoparticles as labels. Biosensors and Bioelectronics, 2011. 27(1): p. 18-24. Giannitsis Evangelos, K.K., Hallermayer Klaus, Jarausch Jochen, Jaffe Allan S., Katus Hugo A., Analytical Validation of a High-Sensitivity Cardiac Troponin T Assay. Clinical Chemistry, 2010. 56(2): p. 254-261.

26

Page 26 of 41

37. 38.

39.

40. 41.

42.

43.

44.

45.

46.

47. 48. 49.

50. 51. 52. 53.

54. 55.

Lequin, R.M., Enzyme Immunoassay (EIA)/Enzyme-Linked Immunosorbent Assay (ELISA). Clinical Chemistry, 2005. 51(12): p. 2415-2418. Kallempudi Sreenivasa Saravan, G.Y., A nanostructured-nickel based interdigitated capacitive transducer for biosensor applications. Sensors and Actuators B: Chemical, 2011. 160(1): p. 891898. Song Seung Yeon, H.Y.D., Kim Kangil, Yang Sang Sik, Yoon Hyun C., A fluoro-microbead guiding chip for simple and quantifiable immunoassay of cardiac troponin I (cTnI). Biosensors and Bioelectronics, 2011. 26(9): p. 3818-3824. Abdolrahim, M., et al., Development of optical biosensor technologies for cardiac troponin recognition. Analytical biochemistry, 2015. 485: p. 1-10. Kwon Young-Chul, K.M.-G., Kim Eon-Mi, Shin Yong-Beom, Lee Seok-Ki, Lee SungDong, C. MyungJe, and R. Hyeon-Su, Development of a surface plasmon resonance-based immunosensor for the rapid detection of cardiac troponin I. Biotechnology Letters, 2011. 33(5): p. 921-927. Dutra Rosa Fireman, M.R.K., Lins da Silva Valdinete, Kubota Lauro Tatsuo, Surface plasmon resonance immunosensor for human cardiac troponin T based on self-assembled monolayer. Journal of Pharmaceutical and Biomedical Analysis, 2007. 43(5): p. 1744-1750. Dutra Rosa Fireman, K.L.T., An SPR immunosensor for human cardiac troponin T using specific binding avidin to biotin at carboxymethyldextran-modified gold chip. Clinica Chimica Acta, 2007. 376(1–2): p. 114-120. Liu Jen Tsai, C.C.J., Ikoma Toshiyuki, Yoshioka Tomohiko, Cross Jeffrey S., Chang Shwu-Jen, T. Jang-Zern, and T. Junzo, Surface plasmon resonance biosensor with high anti-fouling ability for the detection of cardiac marker troponin T. Analytica Chimica Acta, 2011. 703(1): p. 80-86. Abdolrahim Mojgan, R.M., Alhosseini Sanaz Naghavi, Tahriri Mohammadreza, Yazdanpanah Sara, Tayebi Lobat, Development of optical biosensor technologies for cardiac troponin recognition. Analytical Biochemistry, 2015. 485: p. 1-10. Wu Wen-Ya, B.Z.-P., Wang Wei, Wang Wei, Zhu Jun-Jie, PDMS gold nanoparticle composite filmbased silver enhanced colorimetric detection of cardiac troponin I. Sensors and Actuators B: Chemical, 2010. 147(1): p. 298-303. Wang, J., Electrochemical biosensors: Towards point-of-care cancer diagnostics. Biosensors and Bioelectronics, 2006. 21(10): p. 1887-1892. Yazdanpanah, S., et al., Glycated hemoglobin-detection methods based on electrochemical biosensors. TrAC Trends in Analytical Chemistry, 2015. 72: p. 53-67. Bahmani, B., et al., A sulfite biosensor fabricated by immobilization of sulfite oxidase on aluminum electrode modified with electropolymerized conducting film (polyaniline). Asian Journal of Chemistry, 2009. 21(2): p. 923. Bahmani, B., et al., Development of an electrochemical sulfite biosensor by immobilization of sulfite oxidase on conducting polyaniline film. Synthetic Metals, 2010. 160(23): p. 2653-2657. Marquette Christophe A., B.L.J., State of the art and recent advances in immunoanalytical systems. Biosensors and Bioelectronics, 2006. 21(8): p. 1424-1433. Warsinke A., B.A., Scheller F. W., Electrochemical immunoassays. Fresenius' Journal of Analytical Chemistry, 2000. 366(6-7): p. 622-634. Ghindilis Andrey L., A.P., Wilkins Michael, Wilkins Ebtisam, Immunosensors: electrochemical sensing and other engineering approaches. Biosensors and Bioelectronics, 1998. 13(1): p. 113131. Ramanavičius A., R.A., Malinauskas A., Electrochemical sensors based on conducting polymer— polypyrrole. Electrochimica Acta, 2006. 51(27): p. 6025-6037. Ronkainen Niina J., H.H.B., Heineman William R., Electrochemical biosensors. Chemical Society Reviews, 2010. 39(5): p. 1747-1763.

27

Page 27 of 41

56.

57.

58.

59. 60.

61.

62.

63.

64. 65.

66.

67. 68. 69. 70.

71.

72.

Deng An-Ping, Y.H., A multichannel electrochemical detector coupled with an ELISA microtiter plate for the immunoassay of 2,4-dichlorophenoxyacetic acid. Sensors and Actuators B: Chemical, 2007. 124(1): p. 202-208. Yu Hye-Weon, L.J., Kim Sungyoun, Nguyen GiangHuong, Kim InS, Electrochemical immunoassay using quantum dot/antibody probe for identification of cyanobacterial hepatotoxin microcystinLR. Analytical and Bioanalytical Chemistry, 2009. 394(8): p. 2173-2181. Hu Kongcheng, L.D., Li Xuemei, Zhang Shusheng, Electrochemical DNA Biosensor Based on Nanoporous Gold Electrode and Multifunctional Encoded DNA−Au Bio Bar Codes. Analytical Chemistry, 2008. 80(23): p. 9124-9130. Dong Shaojun, L.J., Self-assembled monolayers of thiols on gold electrodes for bioelectrochemistry and biosensors. Bioelectrochemistry and Bioenergetics, 1997. 42(1): p. 7-13. Tian Yang, M.L., Okajima Takeyoshi, Ohsaka Takeo, A carbon fiber microelectrode-based thirdgeneration biosensor for superoxide anion. Biosensors and Bioelectronics, 2005. 21(4): p. 557564. Wring Stephen A., H., John P., Birch Brian J., Voltammetric behaviour of ascorbic acid at a graphite—epoxy composite electrode chemically modified with cobalt phthalocyanine and its amperometric determination in multivitamin preparations. Analytica Chimica Acta, 1990. 229(0): p. 63-70. Pumera Martin, A.M., Mills Chris, Merkoçi Arben, Alegret Salvador, Direct voltammetric determination of gold nanoparticles using graphite-epoxy composite electrode. Electrochimica Acta, 2005. 50(18): p. 3702-3707. Luo Jing, J.S., Zhang Hongyan, Jiang Jinqiang, Liu Xiaoya, A novel non-enzymatic glucose sensor based on Cu nanoparticle modified graphene sheets electrode. Analytica Chimica Acta, 2012. 709(0): p. 47-53. Shao Yuyan, W.J., Wu Hong, Liu Jun ,Aksay Ilhan A ,Lin Yuehe, Graphene Based Electrochemical Sensors and Biosensors: A Review. Electroanalysis, 2010. 22(10): p. 1027-1036. Kuila Tapas, B.S., Khanra Partha, Mishra Ananta Kumar, Kim Nam Hoon, Lee Joong Hee, Recent advances in graphene-based biosensors. Biosensors and Bioelectronics, 2011. 26(12): p. 46374648. Cheng Guifang, Z.J., Tu Yonghua, He Pingang, Fang Yuzhi, A sensitive DNA electrochemical biosensor based on magnetite with a glassy carbon electrode modified by muti-walled carbon nanotubes in polypyrrole. Analytica Chimica Acta, 2005. 533(1): p. 11-16. Engstrom Royce C., S.V.A., Characterization of electrochemically pretreated glassy carbon electrodes. Analytical Chemistry, 1984. 56(2): p. 136-141. Xie Yunying, C.A., Du Dan, Lin Yuehe, Graphene-based immunosensor for electrochemical quantification of phosphorylated p53 (S15). Analytica Chimica Acta, 2011. 699(1): p. 44-48. Grieshaber Dorothee, M.R., Vörös Janos, Reimhult Erik, Electrochemical Biosensors - Sensor Principles and Architectures. Sensors, 2008. 8(3): p. 1400. Ko Sungho, K.B., Jo Seong-Sik, Oh Se Young, Park Je-Kyun, Electrochemical detection of cardiac troponin I using a microchip with the surface-functionalized poly(dimethylsiloxane) channel. Biosensors and Bioelectronics, 2007. 23(1): p. 51-59. Arya Sunil K., S., Pratima R., Datta Monika, Malhotra Bansi D., Recent advances in self-assembled monolayers based biomolecular electronic devices. Biosensors and Bioelectronics, 2009. 24(9): p. 2810-2817. Noh Jaegeun, P.H., Jeong Youngdo, Kwon Seungwook, Structure and electrochemical behavior of aromatic thiol self-assembled monolayers on Au (111). BULLETIN-KOREAN CHEMICAL SOCIETY, 2006. 27(3): p. 403.

28

Page 28 of 41

73.

74. 75.

76.

77. 78.

79. 80. 81.

82. 83.

84.

85.

86.

87.

88. 89.

Hart John P, W.S.A., Recent developments in the design and application of screen-printed electrochemical sensors for biomedical, environmental and industrial analyses. TrAC Trends in Analytical Chemistry, 1997. 16(2): p. 89-103. Renedo O. D., A.-L.M.A., Martinez M. J., Recent developments in the field of screen-printed electrodes and their related applications. Talanta, 2007. 73(2): p. 202-19. Micheli L., G.R., Badea M., Moscone D., Palleschi G., An electrochemical immunosensor for aflatoxin M1 determination in milk using screen-printed electrodes. Biosensors and Bioelectronics, 2005. 21(4): p. 588-596. Viswanathan Subramanian, R.C., Vijay Anand Annadurai, Ho Ja-an Annie, Disposable electrochemical immunosensor for carcinoembryonic antigen using ferrocene liposomes and MWCNT screen-printed electrode. Biosensors and Bioelectronics, 2009. 24(7): p. 1984-1989. Taleat Zahra, K.A., Mazloum-Ardakani Mohammad, Screen-printed electrodes for biosensing: a review (2008–2013). Microchimica Acta, 2014. 181(9-10): p. 865-891. Silva Bárbara VM, C.I.T., Mattos Alessandra B, Moura Patrícia, Maria Del Pilar T Sotomayor, Dutra Rosa F, Disposable immunosensor for human cardiac troponin T based on streptavidinmicrosphere modified screen-printed electrode. Biosensors and Bioelectronics, 2010. 26(3): p. 1062-1067. Yun YeoHeung, D.Z., Shanov Vesselin, Heineman William R., Halsall H. Brian, Bhattacharya Amit, et al., Nanotube electrodes and biosensors. Nano Today, 2007. 2(6): p. 30-37. Laschi S, B.E., Palchetti I, Cristea C, Mascini M, Disposable electrodes modified with multi-wall carbon nanotubes for biosensor applications. Irbm, 2008. 29(2): p. 202-207. Janegitz Bruno C, P.R., Ghica Mariana E, Brett Christopher MA, Fatibello-Filho Orlando, Direct electron transfer of glucose oxidase at glassy carbon electrode modified with functionalized carbon nanotubes within a dihexadecylphosphate film. Sensors and Actuators B: Chemical, 2011. 158(1): p. 411-417. Belluzo María Soledad, R.M.É., Lagier Claudia Marina, Assembling amperometric biosensors for clinical diagnostics. Sensors, 2008. 8(3): p. 1366-1399. Gomes-Filho SLR, D.A., Silv, MMS, Silva BVM, Dutra RF, A carbon nanotube-based electrochemical immunosensor for cardiac troponin T. Microchemical Journal, 2013. 109: p. 1015. Liu Xianggang, Q.X., Fan Hai, Ai Shiyun, Han Ruixia, Electrochemical detection of DNA hybridization using a water-soluble branched polyethyleneimine–cobalt (III)–phenanthroline indicator and PNA probe on Au electrodes. Electrochimica Acta, 2010. 55(22): p. 6491-6495. Hong Hyobong, L.S., Kim Taewan, Chung Myungae, Choi Cheljong, Surface modification of the polyethyleneimine layer on silicone oxide film via UV radiation. Applied Surface Science, 2009. 255(12): p. 6103-6106. Shen Mingwu, W.S.H., Shi Xiangyang, Chen Xisui, Huang Qingguo, Petersen Elijah J, et al., Polyethyleneimine-mediated functionalization of multiwalled carbon nanotubes: synthesis, characterization, and in vitro toxicity assay. The Journal of Physical Chemistry C, 2009. 113(8): p. 3150-3156. Han Yuqi, C.Z., Cao Dong, Yu Jianhui, Li Haozhi, He Xiaoli, and L.Y. Zhang Jun, Lu Huihui, Tang Jieyuan, Side-polished fiber as a sensor for the determination of nematic liquid crystal orientation. Sensors and Actuators B: Chemical, 2014. 196: p. 663-669. Lagerwall Jan PF, S.G., A new era for liquid crystal research: Applications of liquid crystals in soft matter nano-, bio-and microtechnology. Current Applied Physics, 2012. 12(6): p. 1387-1412. Bushby Richard J, K.K., Liquid crystals that affected the world: discotic liquid crystals. Liquid Crystals, 2011. 38(11-12): p. 1415-1426.

29

Page 29 of 41

90. 91. 92. 93. 94.

95.

96.

97.

98.

99. 100.

101. 102.

103. 104.

105. 106.

107.

Woltman Scott J, J.G.D., Crawford Gregory P, Liquid-crystal materials find a new order in biomedical applications. Nature materials, 2007. 6(12): p. 929-938. Campbell Fallyn W, C.R.G., The use of nanoparticles in electroanalysis: an updated review. Analytical and bioanalytical chemistry, 2010. 396(1): p. 241-259. Pingarrón José M, Y.-S.P., González-Cortés Araceli, Gold nanoparticle-based electrochemical biosensors. Electrochimica Acta, 2008. 53(19): p. 5848-5866. Iost Rodrigo M, C.F.N., Layer-by-layer self-assembly and electrochemistry: applications in biosensing and bioelectronics. Biosensors and Bioelectronics, 2012. 31(1): p. 1-10. de Menezes Eliana W, N.M.R., Arenas Leliz T, Dias Silvio LP, Garcia Irene TS, Gushikem Yoshitaka, Gold nanoparticle/charged silsesquioxane films immobilized onto Al/SiO2 surface applied on the electrooxidation of nitrite. Journal of Solid State Electrochemistry, 2012. 16(12): p. 3703-3713. Fattori Natalia, M.C.M., Da Costa Luiz P, Strauss Mathias, Mazali Italo O, Gushikem Yoshitaka, Chemical and photochemical formation of gold nanoparticles supported on viologenfunctionalized SBA-15. Colloids and Surfaces A: Physicochemical and Engineering Aspects, 2013. 437: p. 120-126. da Silva Paulo Sérgio, G.B.C., Magosso Hérica A, Spinelli Almir, Gold nanoparticles hosted in a water-soluble silsesquioxane polymer applied as a catalytic material onto an electrochemical sensor for detection of nitrophenol isomers. Journal of hazardous materials, 2014. 273: p. 70-77. Zapp Eduardo, d.S.P.S., Westphal Eduard, Gallardo Hugo, Spinelli Almir ,Vieira Iolanda Cruz, Troponin T Immunosensor Based on Liquid Crystal and Silsesquioxane-Supported Gold Nanoparticles. Bioconjugate chemistry, 2014. 25(9): p. 1638-1643. Horak Josef, D.C., Qelibari Edvina, Bakirci Hüseyin, Urban Gerald, Polymer-modified microfluidic immunochip for enhanced electrochemical detection of troponin I. Sensors and Actuators B: Chemical, 2015. 209: p. 478-485. Vicennati Paola, G.A., Ortaggi Giancarlo, Masotti Andrea, Polyethylenimine in medicinal chemistry. Current medicinal chemistry, 2008. 15(27): p. 2826-2839. Tuteja Satish K. , K.M., Suri C. R., Paul A. K., Deep Akash, One step in-situ synthesis of amine functionalized graphene for immunosensing of cardiac marker cTnI. Biosensors and Bioelectronics, 2015. 66(0): p. 129-135. Han Melinda Y, Ö.B., Zhang Yuanbo, Kim Philip, Energy band-gap engineering of graphene nanoribbons. Physical review letters, 2007. 98(20): p. 206805. Novoselov KSA, G.A.K., Morozo SVb, Jiang Da, Katsnelson MIc, Grigorieva IVa, D. SVb, and F. AAb, Two-dimensional gas of massless Dirac fermions in graphene. nature, 2005. 438(7065): p. 197-200. Zhang Yuanbo, T.Y.-W., Stormer Horst L, Kim Philip, Experimental observation of the quantum Hall effect and Berry's phase in graphene. Nature, 2005. 438(7065): p. 201-204. Berger Claire, S.Z., Li Xuebin, Wu Xiaosong, Brown Nate, Naud Cécile, Mayou Didier, Li Tianbo, Hass Joanna, Marchenkov Alexei N, Electronic confinement and coherence in patterned epitaxial graphene. Science, 2006. 312(5777): p. 1191-1196. Pumera, M., Electrochemistry of graphene: new horizons for sensing and energy storage. The Chemical Record, 2009. 9(4): p. 211-223. Brondani Daniela, P.J.V., Westphal Eduard, Gallardo Hugo, Dutra Rosa Amalia Fireman, Spinelli Almir, Vieira Iolanda Cruz, A label-free electrochemical immunosensor based on an ionic organic molecule and chitosan-stabilized gold nanoparticles for the detection of cardiac troponin T. Analyst, 2014. 139(20): p. 5200-5208. Khan Raju, D.M., Chitosan/polyaniline hybrid conducting biopolymer base impedimetric immunosensor to detect Ochratoxin-A. Biosensors and Bioelectronics, 2009. 24(6): p. 1700-1705.

30

Page 30 of 41

108. 109.

110. 111.

112.

113.

114. 115. 116.

117. 118.

119.

120. 121.

122. 123.

124. 125.

Yang Liju, B.R., Electrical/electrochemical impedance for rapid detection of foodborne pathogenic bacteria. Biotechnology Advances, 2008. 26(2): p. 135-150. Hutter Eliza, P.M.-P., Detection of DNA hybridization by gold nanoparticle enhanced transmission surface plasmon resonance spectroscopy. The Journal of Physical Chemistry B, 2003. 107(27): p. 6497-6499. De Vlaminck Iwijn, V.D.P., Lagae Liesbet, Borghs Gustaaf, Local electrical detection of single nanoparticle plasmon resonance. Nano letters, 2007. 7(3): p. 703-706. Bhalla Vijayender, C.S., Sharma Priyanka, Nangia Yogesh, Suri C Raman, Gold nanoparticles mediated label-free capacitance detection of cardiac troponin I. Sensors and Actuators B: Chemical, 2012. 161(1): p. 761-768. Singh Rajdeep, S.I.I., Minimizing nonspecific adsorption in protein biosensors that utilize electrochemical impedance spectroscopy. Journal of The Electrochemical Society, 2010. 157(10): p. J334-J337. Relos Rene P, H.I.K., Beilman Greg J, Moderately elevated serum troponin concentrations are associated with increased morbidity and mortality rates in surgical intensive care unit patients. Critical care medicine, 2003. 31(11): p. 2598-2603. Kay, S.E., Clozapine associated pericarditis and elevated troponin I. Australian and New Zealand Journal of Psychiatry, 2002. 36(1): p. 143-144. Rogers, K., Principles of affinity-based biosensors. Molecular Biotechnology, 2000. 14(2): p. 109129. Boozer Christina, K.G., Cong Shuxin, Guan HannWen, Londergan Timothy, Looking towards label-free biomolecular interaction analysis in a high-throughput format: a review of new surface plasmon resonance technologies. Current Opinion in Biotechnology, 2006. 17(4): p. 400-405. Janshoff Andreas, S.C., Label-free detection of protein-ligand interactions by the quartz crystal microbalance, in Protein-Ligand Interactions. 2005, Springer. p. 47-63. Maehashi Kenzo, K.T., Kerma Kagan, Takamura Yuzuru, Matsumoto Kazuhiko, Tamiym Eiichi, Label-free protein biosensor based on aptamer-modified carbon nanotube field-effect transistors. Analytical Chemistry, 2007. 79(2): p. 782-787. Lee Jung A, H.S., Kwak Juhyoun, Park Se Il, Lee Seung S, Lee Kwang-Cheol, An electrochemical impedance biosensor with aptamer-modified pyrolyzed carbon electrode for label-free protein detection. Sensors and Actuators B: Chemical, 2008. 129(1): p. 372-379. Daniels Jonathan S, P.N., Label-Free Impedance Biosensors: Opportunities and Challenges. Electroanalysis, 2007. 19(12): p. 1239-1257. de Vasconcelos Elder A., P.N.G., Pereira Cintya O., da Silv, Valdinete L., da Silva Jr Eronides F., Dutra Rosa F., Potential of a simplified measurement scheme and device structure for a low cost label-free point-of-care capacitive biosensor. Biosensors and Bioelectronics, 2009. 25(4): p. 870876. Mosbach Klaus, R.O., The Emerging Technique of Molecular Imprinting and Its Future Impact on Biotechnology. Nat Biotech, 1996. 14(2): p. 163-170. Chen Lingxin, X.S., Li Jinhua, Recent advances in molecular imprinting technology: current status, challenges and highlighted applications. Chemical Society Reviews, 2011. 40(5): p. 29222942. Hoshino Yu, S.K.J., The evolution of plastic antibodies. Journal of Materials Chemistry, 2011. 21(11): p. 3517-3521. Klingeler Rüdiger, H.S., Büchner Bernd, Carbon nanotube based biomedical agents for heating, temperature sensoring and drug delivery. International journal of hyperthermia, 2008. 24(6): p. 496-505.

31

Page 31 of 41

126.

127.

128.

129. 130.

131.

132. 133.

134.

135.

Moreira Felismina T. C., D.R.A.F., Noronha João P. C., Cunha Alexandre L., Sales M. Goreti F., Artificial antibodies for troponin T by its imprinting on the surface of multiwalled carbon nanotubes: Its use as sensory surfaces. Biosensors and Bioelectronics, 2011. 28(1): p. 243-250. Ahammad AJ Saleh, C.Y.-H., Koh Kwangnak, Kim Jae-Ho, Lee Jae-Joon, Lee Minsu, Electrochemical detection of cardiac biomarker troponin I at gold nanoparticle-modified ITO electrode by using open circuit potential. International Journal of Electrochemical Science, 2011. 6(6): p. 1906-1916. Norouzi Parviz, L.B., Faridbod Farnoush, Ganjali Mohammad Reza, Hydrogen Peroxide Biosensor Based on Hemoglobin Immobilization on Gold Nanoparticle in FFT Continuous Cyclic Voltammetry Flow Injection System. Int J Electrochem Sci, 2010. 5(11): p. 1550-1562. Ahuja Tarushee, M.I.A., Kumar Devendra, Biomolecular immobilization on conducting polymers for biosensing applications. Biomaterials, 2007. 28(5): p. 791-805. Lee Innam, L.X., Huang Jiyong, Cui Xinyan Tracy, Yun Minhee, Detection of cardiac biomarkers using single polyaniline nanowire-based conductometric biosensors. Biosensors, 2012. 2(2): p. 205-220. Yun Minhee, M.N.V., Vasquez Richard P, Lee Choonsup, Menke Erik, Penner Reginald M, Electrochemically grown wires for individually addressable sensor arrays. Nano Letters, 2004. 4(3): p. 419-422. Lee Innam, P.H.I., Park Seongyong, Kim Moon J, Yun Minhee, Highly reproducible single polyaniline nanowire using electrophoresis method. Nano, 2008. 3(02): p. 75-82. Silva Bárbara V. M., C.I.T., Silva Mízia M. S., Dutra Rosa F., A carbon nanotube screen-printed electrode for label-free detection of the human cardiac troponin T. Talanta, 2013. 117(0): p. 431437. Mattos Alessandra B., F.T.A., Kubota Lauro T., Dutra Rosa F., An o-aminobenzoic acid film-based immunoelectrode for detection of the cardiac troponin T in human serum. Biochemical Engineering Journal, 2013. 71(0): p. 97-104. Zhou Fang, L.M., Wang Wei, Bian Zhi-Ping, Zhang Jian-Rong, Zhu Jun-Jie, Electrochemical Immunosensor for Simultaneous Detection of Dual Cardiac Markers Based on a Poly(Dimethylsiloxane)-Gold Nanoparticles Composite Microfluidic Chip: A Proof of Principle. Clinical Chemistry, 2010. 56(11): p. 1701-1707.

32

Page 32 of 41

Figure captions Figure 1. The HRP mechanism on the electrode response [78] Figure 2. Immunosensor schematic diagram. (a) Bare Au , (b) PEI film formation, (c) COOH–CNT assembly, (d) anti-cTnT immobilization, (e) Glycine blocking step [83] Figure 3. Calibration plot of immunosensor in different concentrations of cTnT [83] Figure 4. For antibody immobilization, the enhanced surface was produced using GNP-modified SPE surface by electrodeposition to form a nano-sized colloidal Au base matrix. For passivating the immobilized monoclonal antibody layer, the BSA solution was used as a blocking agent. As cTnI (highly charged molecules) are specifically recognized by monoclonal antibodies, the capacitance changes [111] Figure 5. Antibody immobilization of Al electrode: For anti-IgG: Glutaraldehyde + anti-IgG mixture treated on the surface (I, a); Glycine free aldehyde groups blocking step (I, b). Selfassembled monolayers of thiol treated on the Al surface for anti-TnT step (II, a); aldehyde reacting with amino groups (II, b) and glycine blocking procedure for free aldehyde groups (II, c) [121] Figure 6. A schematic of measurement setup and device structure and location of antibodyantigen [121] Figure 7. Troponin T molecular imprinting. The protein link to carbon nanotubes via a two step process of diimide-actived amidation (A) and protein removal imprinting step (B) [126] Figure 8. The modification process scheme of gold nanoparticle to modify ITO electrode for the immobilization of HRP-conjugated antibody [127]

33

Page 33 of 41

Figures Figure 1

Figure 2

34

Page 34 of 41

Figure 3

35

Page 35 of 41

Figure 4

36

Page 36 of 41

Figure 5

Figure 6

37

Page 37 of 41

Figure 7

38

Page 38 of 41

Figure 8

39

Page 39 of 41

Tables Table 1. Summarized Cardiac Troponin measurement methods Biomarker

Immobilization strategies

Human cardiac troponin (cTnT)

NH2-CNTSPEs/polyethyleneterephtalate(P ET)/NHS-EDC-anti-cTnT/glycine/ cTnT Au/polyethyleneimine (PEI)/carboxylated CNTs (COOHCNT)/anti-cTnT/glycine/ cTnT GCE/o-aminobenzoic acid(polyo-ABA)/EDC/NHS/anticTnT/ethanolamine/cTnT SPE/polyethylene terephtalate (PTE)/anticTnT/biotin/glutaraldehyde (glu)/ streptavidin microsphere/glycine/cTnT/HRPconjugated anti-cTnT İnterdigitatedarray(IDA)chip/poly (dimethylsiloxane)(PDMS)/NHS/ BSA/anticTnI/proteinG/cTnI/alkalinephos phatase(AP)-labeledantiCTnI/enzyme substrate(PaPP) PDMS-GNP composite/anti-cTnI and anti-CRP (Ab1)/BSA/CdTe and ZnSe quantum dots-anti-cTnI and anti-CRP (Ab2) SPE/AuNPs/anti-cTnI/BSA/cTnI indium tin oxide (ITO)/ Gold nanoparticles (GNPs)/ anticTnI/cTnI/HRP –conjugated anticTnI Al electrode/self –assembled

cTnT

cTnT

cTnT

Cardiac troponin I (cTnI)

cTnI

cTnI

cTnI

cTnT

Assay principle

Linear range

Limit of Detection

Ref.

Amperometric

0.0025– 0.5 ng/mL

0.0035 ng/ mL

[133]

Amperometric

0.1– 10ng/mL

0.033 ng/ mL

[83]

Amperometric

0.05– 5 ng/mL

0.016 ng/ mL

[134]

Amperometric

0.1– 10ng/mL

0.2 ng/mL

[78]

Amperometric

0.2ng/ mL– 10 μg/mL

148 pg/mL

[70]

Amperometric

0.01– 50 μg/l

5 amol

[135]

Capacitance

0.2– 12.5ng/ mL

0.2 ng/mL

[111]

Open Circuit Potential

1-100 ng/ml

[127]

capacitance

0.07-6.38 ng/ml

[121]

40

Page 40 of 41

cTnT

cTnI

cTnT

cTnI

cTnT

cTnI

monolayer/glutaraldehyde/ anticTnT/glysin/cTnT TnT/ carboxylated MWCNT/ acrylamide (AAM), N,N_-methylenebisacrylamide potentiometric (NNMBA, cross-linker) and ammonium persulphate (APS, initiator)/ cTnT Au electrode/ PANI nanowire integrated with microfluidic conductance channels/ anti-cTnI/cTnI GCE/ (E)-1-decyl-4-[(4decyloxyphenyl)diazenyl]pyridini um bromide (Br-Py)film/gold cyclic nanoparticles (AuNP) stabilized voltammetry in a water-soluble 3-n-propyl-4and picolinium silsesquioxane impedance chloride (Si4Pic+Cl−)/anticTnT/glycine/ cTnT microfluidic channel/EDC and SNHS/ Branched polyethylenimine (BPEI)/BPEI activation with GA/anticTnI/BSA/cTnI/biotinylated detection antibody/GOx-avidin GCE/I-Py/ CTS-AuNP/anticTnT/glycine/cTnT interdigitated electrode surface/graphene-ABA nano composite/anti-cTnI/cTnI

1.4120.68µg/ml

0.16µg/ml

[126]

250fg/ml

[130]

0.076ng/m l

[97]

25pg/ml

[98]

0.2-1ng/ml

0.1ng/ml

[106]

0.1-1ng/ml

0.01ng/ml

[100]

0.1-0.9ng/ml

Amperometric

Cyclic voltammetry cyclic voltammetry and impedance

41

Page 41 of 41