Elsevier

Biochemical Engineering Journal

Volume 71, 15 February 2013, Pages 97-104
Biochemical Engineering Journal

Regular article
An o-aminobenzoic acid film-based immunoelectrode for detection of the cardiac troponin T in human serum

https://doi.org/10.1016/j.bej.2012.12.005Get rights and content

Abstract

An amperometric immunosensor for cardiac troponin T detection in human serum troponin T, a marker considered as “gold standard” for acute myocardial infarction diagnosis, is described. A stable carboxylic film to covalently bind antibodies against cTnT onto electrode surface was achieved with electropolymerization of the o-aminobenzoic acid. A fractional factorial study was performed to optimize the electropolymerization parameters. Cyclic voltammetry assays were carried out for characterize steps of the modified electrode surface. The obtained calibration curve at −0.05 V by amperometry presented a good linear response range from 0.05 to 5.0 ng mL−1 cTnT with a correlation coefficient of 0.992 (n = 6) and 0.016 ng mL−1 detection limit. The electrodes showed a good stability upon the analytical responses retaining 91.6% of its initial response after 18 days. This sensor showed outgoing results regarding sensitivity allowing reliable measurements of the cTnT at levels of clinical significance for acute myocardial infarctions diagnosis.

Highlights

► An immunosensor based on polymer film was developed for cardiac troponin T (cTnT). ► Anti-cTnT was immobilized on o-aminobenzoic acid film. ► A detection limit of 16.0 pg mL−1 troponin T was achieved. ► It was analyzed the troponin T in human serum.

Introduction

Acute myocardial infarction (AMI) is the rapid development of myocardial necrosis caused by a critical imbalance between oxygen supply and demand of the myocardium [1]. This usually results from plaque rupture with thrombus formation in a coronary vessel, resulting in an acute reduction of blood supply to a portion of the myocardium [2]. Troponins are the most specific and sensitive biochemical markers of myocardial cell injury and therefore have been highlighted as the “gold standard” testing for the AMI [3], [4]. The troponins remain abnormal for 4–10 days after the onset of AMI [5], with the concentration peak closely correlated with the infarct size [6], [7].

Analytical methods to improve the timely diagnosis of this disease have been important to appropriate disposition and treatment of AMI patients [8]. Rapid and safe tests implies on a significant reduction in mortality and also in the hospital stay. Immunoassays have become the better technique in clinical diagnostics for determining cardiac and other clinically relevant biomarkers. Currently, the conventional methods for detection of troponin T (cTnT) include enzyme immunoassay [9], fluoroimmunoassay [10] and electrochemiluminescence immunoassay (ECLIA) [11]. However, these methods require laboratory equipments with proper instrument, multi-step processings of samples and well-trained personnel, leading to considerable time consuming and expense to the overall detection. Immunosensors consisting of an antigen or antibody coupled to transducer to generate a quantitative response by immunocomplexing have been pointed as attractive alternatives. They can provide faster and more practical diagnosis due to their portability possible being indicated when a quick therapeutic procedure is required.

Recently, several attempts for determination of cTnT by immunosensors have been performed by different transducers [12], [13], [14] based on surface plasmon resonance and piezoelectric techniques. Although these sensing instruments show in particular some features as fast response and the non-labeling, they are difficult to miniaturize and to operate in the field as point-of-care device [15], [16], [17]. Electrochemical transducers based on amperometric responses generated by enzymes conjugated to the antibodies spend more time to analyze, they has been pointed out as more attractive due to easier compatibility and lower cost [12].

In an attempt to overcome difficulties regarding antibodies immobilization using a simple technique without compromising the high sensitivity required, given the fact that detection levels for the clinical diagnosis of AMI are in level of few nanograms per mL of cTnT, herein a conductive polymer was employed to anchor antibodies against cTnT [18], [19]. The use of polymer films deposited on the electrode surface has been showed as alternative to a stable and irreversible immobilization of the biomolecules, because they can provide reactive groups such as single bondCOOH or single bondNH2 [20], [21], [22] to link these biomolecules. Polymeric films can be obtained by dip- or spin-coating, electrodeposition, plasma polymerization and electropolymerization [23], [24]. Among these methods, the electropolymerization has been the most used [25]. It consists of non-manual electrochemical addressing of polymer deposition in which the main advantage hence is a higher reproducibility of the obtained film. This technique results in more stable and homogeneous electroactive areas that implies on more reliability in the developed sensors. Since the active functional groups are provided by these polymers, it is possible to generate strong linkages of between biomolecules and the coated electrode surface without lost of the film conductivity [26], [27]. Other interesting propriety of polymers to biosensor is about the possibility to enhance the electron transfer between the electroactive species and to the electrode surface [28], [29].

In this work, the poly-o-aminobenzoic acid, (poly-o-ABA), that is polyaniline derivative with carboxyl groups was used to covalent bind antibodies by their amino reactive groups of the side chains. The poly-o-ABA, a conductor polymer was electropolymerized on a glassy carbon electrode using the sweep linear voltammetry technique. The modified electrode provides abundantly carboxyl groups, acting as functional group to covalently immobilize the monoclonal antibodies against cTnT through an amide bond.

Section snippets

Reagents and materials

The cTnT, mouse monoclonal antibody against cTnT (mAb-cTnT) and peroxidase conjugated mouse monoclonal antibody against cTnT (mAb-cTnT-HRP) were purchased from Calbiochem (USA). The o-ABA, ethanolamine, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), hydroquinone, sulfuric acid (H2SO4) and potassium ferricyanide (K3Fe(CN)6) were acquired from Sigma–Aldrich (USA). Hydrogen peroxide (H2O2) (30%, v/v) and ethanol were acquired from Synth (São Paulo,

Multivariate optimization of the electropolymerization procedure

In order to optimize the analytical method to attain the best sensitivity as well as to reduce the number of assays, a fractional factorial design associated with Doehlert matrix was used. The o-ABA concentration, scans number of the electropolymerization, scan rate and electrolyte concentration (H2SO4) were established according to the results obtained from the 24−1 fractional factorial design. From the Pareto chart, more significant effects of the electropolymerization were scan rate and H2SO4

Conclusion

The poly(o-ABA) was successfully electropolymerized to the electrode surface and optimal parameters were determined by a factorial analysis. The homogenous film formed on the carbon electrode surface showed to be efficient for a covalent binding of the monoclonal antibodies against cardiac troponin T by an amide bond. This functionalized film of a polyaniline derivate acted as not only a matrix for protein immobilization but as also an excellent conductive film to promote an efficient

Acknowledgements

This work was supported by the National Council of Technological and Scientific Development (CNPq) Brazil agency. A.B. Matos, PhD student is grateful to CAPES (Brazil) by scholarship during this work. The assistance of the PROCAPE (Cardiac Emergency of Pernambuco, Recife – PE, Brazil) is also acknowledged.

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