An ultrasensitive molecularly-imprinted human cardiac troponin sensor

Highlights

• The human cardiac troponin T (TnT) molecularly-imprinted human cardiac troponin sensor is reported.

• Cardiac biomarker TnT was used as template with electrochemical polymerisation of functional monomer, o-phenylenediamine.

• The analytical performance of the sensor was evaluated in both buffer and serum.

• The detection limit of sensor was found to be 9 pg/mL.

Abstract

Cardiac troponin T (TnT) is a highly sensitive cardiac biomarker for myocardial infarction. In this study, the fabrication and characterisation of a novel sensor for human TnT based on a molecularly-imprinted electrosynthesised polymer is reported. A TnT sensitive layer was prepared by electropolymerisation of o-phenylenediamine (o-PD) on a gold electrode in the presence of TnT as a template. To develop the molecularly imprinted polymer (MIP), the template molecules were removed from the modified electrode surface by washing with alkaline ethanol. Electrochemical methods were used to monitor the processes of electropolymerisation, template removal and binding. The imprinted layer was characterised by cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and atomic force microscopy (AFM). The incubation of the MIP-modified electrode with respect to TnT concentration resulted in a suppression of the ferro/ferricyanide redox process. Experimental conditions were optimised and a linear relationship was observed between the peak current of [Fe(CN)₆]³⁻/[Fe(CN)₆]⁴⁻ and the concentration of TnT in buffer over the range 0.009–0.8 ng/mL, with a detection limit of 9 pg/mL. The TnT MIP sensor was shown to have a high affinity to TnT in comparison with non-imprinted polymer (NIP) electrodes in both buffer and blood serum.

Introduction

Acute myocardial infarction is one of the leading cause of death in the world (Friess and Stark, 2009, Iwanaga and Miyazaki, 2010). Rapid identification and diagnosis of acute myocardial infarction is vital for effective medical treatment and disease management. The most commonly employed diagnostic methods currently used in the recognition of myocardial infarction is electrocardiography (ECG) but the diagnostic sensitivity of the initial ECG is about 50% for detecting myocardial damage (Gibler et al., 1990). The assessment of cardiac marker elevation is therefore needed for the precise diagnosis. A range of cardiac biomarkers have been evaluated with respect to their incremental cardiac diagnostic and prognostic value (Wang et al., 2006). Among them cardiac troponins have higher sensitivity and specificity with respect to heart function.

Cardiac troponins consist of a complex of troponin C (TnC), troponin I (TnI) and troponin T (TnT). Both TnI and TnT are identified as specific biomarkers for myocardial tissue (Kemp et al., 2004). They are released into the bloodstream from injured muscle cells during cardiac ischaemia with no overlap with skeletal muscle troponins under normal conditions (Sarko and Pollack, 2002). Several methods have been shown to be able to detect the troponin T including enzyme linked immunosorbent assay (ELISA) (Katus et al., 1989), immunochromatographic (Penttila et al. 1999), electrochemiluminescence immunoassay (Asano et al., 2012), surface plasmon resonance (SPR) (Dutra and Kubota, 2007, Dutra et al., 2007), carboxylic polyvinyl chloride-coated quartz crystal microbalance (QCM) immunosensor (Wong-ek et al., 2010) and potentiometric molecularly imprinted polymer (MIP) sensor (Moreira et al., 2011). The majority of these methods relies on the use of natural antibodies with the limitations of storage stability, sensitivity and detection limits (Table 1). These constraints may be overcome by replacing the natural antibodies with stable synthetic analogues. One promising way of making artificial receptors is the molecular imprinting of polymers. Molecularly imprinted materials have attracted significant attention due to their low cost, high affinity and mechanical and chemical stability (Holthoff and Bright, 2007, Xu et al., 2009). The sensitivity and detection limit of TnT MIP sensor are still challenges to be overcome.

Molecular imprinting is a process by which appropriate functional monomers and cross-linking agents are polymerised in the presence of a template molecule or its derivative to form a cast-like shell. Initially, the monomers form a complex with the template through covalent or non-covalent interactions. The template can be removed after polymerisation. Thus, the MIP contains binding sites, which are complementary to the target molecule in size, shape, and position of functional groups, and which are held in place by the cross linked polymer matrix (Li et al., 2010, Madhuri et al., 2011, Tiwari et al., 2011, Kumar et al., 2011, Tiwari et al., 2012a, Tiwari et al., 2012b). In essence, a molecular memory is imprinted on the polymer, which is capable of selectively binding of target (Haupt 2010). Different methods are available for the imprinting of polymers (Hedborg et al., 1993, Poma et al., 2010, Sergeyeva et al., 1999). One of the simplest approaches is one-step electro-polymerisation of MIP film attached to electrode surfaces (Cheng et al., 2001, Madaras and Buck, 1996, Sallacan et al., 2002, Kumar et al., 2011). The thickness of film can be well-controlled by the amount of charge passed. This approach facilitates miniaturisation, one of the major goals of chemical sensor technology (Malitesta et al., 1990), and addresses ready formation of recognition elements as single elements or arrays. o-Phenylenediamine (o-PD) is easily electropolymerised on various substrate materials and forms films with good chemical and mechanical stability (Malitesta et al., 1999). The presence of neutral or protonated –NH₂ groups may be responsible for interactions with either single-stranded oligodeoxyribonucleotide, enzymes or molecules, when used as molecular templates (Losito et al., 2003, Tiwari et al., 2012a, Tiwari et al., 2012b). Hence, o-PD is well suited to molecular imprinting, offering hydrophilic, hydrophobic and basic recognition sites via electrostatic interactions (Malitesta et al., 1990).

In the present manuscript, we describe the first human cardiac TnT MIP sensor based on molecularly imprinted electrosynthesised polymer. Cardiac biomarker TnT was used as template molecule for electrochemical polymerisation of functional monomer, o-PD. The analytical performance of the sensor was evaluated in both buffer and blood serum. This work is intended to lead to a robust biomimetic human TnT sensor to quickly diagnose myocardial infarction.