An ultrasensitive Cystatin C renal failure immunosensor based on a PPy/CNT electrochemical capacitor grafted on interdigitated electrode

https://doi.org/10.1016/j.colsurfb.2020.110834Get rights and content

Highlights

  • An interdigitated immunosensor for Cystatin C was successful developed.

  • Polypyrrole/carbon nanotube electrochemical capacitor was In situ synthetized.

  • IDE immunosensor responses were obtained by changes on phase angle.

  • Cutoff was established at 28 ng/mL CysC in real serum samples.

Abstract

An interdigitated immunosensor for Cystatin C detection based on polypyrrole/carbon nanotube electrochemical capacitor is described. Cystatin C (CysC) is powerful biomarker for early acute renal failure and one predictive for cardiovascular risk, sepsis, cancer and death. Recently, electrochemical immunosensors based on interdigitated electrodes (IDE) have been successfully focused on development of point-of-care testing, due to their miniaturization facilities and higher sensitivity as compared with the screen-printed electrochemical sensing. Herein, a polypyrrole/carbon nanotube nanoyhibrid film was grafted on two gold fingers by electropolymerization obtaining a supercapacitor. Anti-CysC antibodies were immobilized on the IDE by covalent entrapment via ethylenediamine bifunctional agent, followed by glycine blocking in acid and alkaline medium. Under low frequency, capacitive effect of antigen-antibody interaction were observed by double layer capacitance, and analytical responses of this IDE immunosensor to CysC serum were obtained by changes on phase angle a linear range up to 300 ng/mL. The cutoff was calculated for serum samples showing a total reducing of non-specific binding at approximately 28 ng/mL CysC. This immunosensor based on interdigitated electrode (IDE) is a potential tools as portable device,with possibility to use as a practical and rapid test for CysC diagnostic in samples of serum.

Introduction

Kidney diseases affects more than 12 % of the world population and are considered a serious public health problem that can lead to renal substitutive therapy or kidney failure, resulting in a strong impact on the patient's quality of life and death [1,2]. Recently, Cystatin C (CysC) has gained attention being considered as a powerful early biomarker of acute kidney injury characterized by rapid decline in glomerular filtration rate [3,4]. The serum levels of CysC can be correlated to the presence and degree of renal injury, with the advantage to be less affected by gender, age, muscle mass and also does not suffer delayed in acute failure, and others [5,6]. Recent studies have reported the CysC also as biomarker for risk of cardiac attack [7,8], neurological disorders [9], cancer [10,11], risk of death [6,12] and sepsis [13]. Thus, monitoring of CysC helps in making important therapeutic decisions, being mandatory the development of practical and rapid tests. Currently, the dosage of cystatin C is performed by enzyme immunoassays [4], nephelometry, turbidimetry [14] and other more sophisticated methods, such as electrospray chromatography [15]. However, these techniques require skilled personals, sophisticated equipment and are commonly processed in a central laboratory, which can hamper patient access or may delay the patient management [16]. Point-of-care testings represent a good alternative for CysC, and some lateral flow testings were developed in this sense; however, low sensitivity and limitations regarding the quantifying are main drawbacks. In this sense, immunosensors can be a good strategy due to attending all these demands and, can be offered at a lower cost.

Recently, impedance immunosensor based on interdigitated electrode (IDE) has shown as one of attractive analytical possibilities for point-of-care testings with good sensitivity and reproducibility [17,18], with great advantages, especially as compared with screen-printed electrode technologies [19]. Interdigitated interdigitated electrode (IDE) consists of two interconnected, but non-contacting combs, obtained by evaporating the metal in a piezoelectric substrate [20]. When the IDE is excited by an electrical frequency, specific interactions between antigen-antibodies are detected by oscillations in the electromagnetic field generated by perturbations on the electrode surface (Fig. 1(a)). The impedance of biomolecule interactions (ZBiom) measured on the IDE immunosensor depends on the Faradaic and non-Faradaic processes occurred at the electrode surfaces. ZBiom depends on the double layer processes, the electrode-electrolyte interface capacitance and resistance between the electrodes on the medium conductivity [21]. An illustrative model of electrical circuit derived is showed in Fig. 1(b). In Faradaic process, the perturbations are resulting in charges (e.g., electrons) transferred across the metal-solution interface that are measured by Resistance of charge transfer (RCT). Under some conditions, no charge transfer reactions occur, however processes such as adsorptions and desorptions can occur modifying the electric surface potential, that result in a differential capacitance with relation to the surface charge. This differential capacitance, denoted as CDL is the capacitance of double layer. In the IDE immunosensor, ZBiom is dependent on the CDL and the Capacitance of dielectric (CDE). When the frequency is high enough, the current passes through CDE instead of RCT [22]. However, it well-established that biomolecular interactions are better observed at low frequencies [18], and in this case, the ZBiom does not depend on the CDE [17].

One of first works using capacitive immunosensor based on non-faradaic impedance was performed in a planar device with electrodes formed by two strips evaporated on a silicon oxide substrate, measuring the direct capacitance [23]. Recently, 3D capacitor electrodes with a high charge storage capacity by containing more material loading in the third dimension have gained great attention for non-Faradaic sensor applications [24,25]. Electrochemical capacitors (EC) can be produced by combining different materials and grafted on the IDE surface electrodes, resulting in the 3D electrochemical capacitor to feasible more sensitive immunosensors. EC is one of the most promising energy storage technologies offered by green chemistry and it has challenged the crescent demand required by portable/mobile electronics, electrical vehicles, biosensors, etc. [26]. The fundamental mechanism for higher energy storage of EC is first, derivated from the electrostatic accumulation of charge in the electrical double layer, and due to the enlargement of surface area; therefore charge storage takes place at the electrode/electrolyte interface. The second mechanism is based on reversible redox reactions occurring on electrode materials [27]. Interest in the EC based on the conducting polymer-carbon hybrid nanomaterials have been intensified due to they possess new or improved physicochemical properties, resulting from the synergism of organic and inorganic components that interact at the molecular level level [28]. They can form a three-dimensional conducting grid, increasing the surface area and allow an abundance of reaction sites with the possibility of large charge storage and mechanical stability [[29], [30], [31]]. Different conductive organic polymers such as polyanyline (PANI), polythiophene (PTh), polypyrrole (PPy) and their derivatives have been employed for electrochemical applications [27,32]. Although these polymers have very similar characteristics in terms of conductivity, arrangments with different materials can produce peculiar behaviors. Studies comparing PPy and PANI in the formation of the nanowires have demonstrated that PPy has advantages, due to has promoted nanocomposites with more supercapacitive effects [33], besides due to also have a high conductivity, storage ability and doping/dedoping [[34], [35], [36]]. The microstructural uniformity achieved by the CNT integrations to the PPy is derived from strong π- π bonds between PPy conjugated structure and the CNT sidewall graphitic [37]. Without CNTs, intercalation–deintercalation of anions leads to a change in the polymer volume, reducing the long term stability of PPy and diminished charge propagation and capacitance. Consequently, the cycle-life of PPy is poor compared with PPy carbon-based supercapacitors [38]. In synthesis, EC of PPy/CNT nanohybrid compound has advantage of ensuring better reproducibility, simpler synthesis and controlled thickness [39]. PPy/CNT can be in situ grafted to electrode surfaces, forming EC by means of the oxidation of aromatic compounds,with advantage to be readily electrosynthetized from a range of aqueous and non-aqueous solvents [40,41]. Herein, a PPy/CNT electrochemical capacitor was assembled on a IDE immunosensor for cystatin C detection through monitoring CDL by phase angle changes,achieving a high sensitivity, label-free and rapid responses.

Section snippets

Reagents

CysC, monoclonal antibodies anti-CysC, pyrrole (98 %), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS) and ethylenediamine (EDA) 98 % were obtained from Sigma-Aldrich®(St. Louis, Missouri, EUA). Amine multi-walled carbon nanotube (CNT) was acquired from Dropsens®. Glycine was purchased at Fluka Analytical®(Charlotte, Carolina do Norte, EUA) H2SO4 and, potassium ferrocyanide (K4Fe(CN)6) were obtained from Vetec® (São Paulo-Brasil). The potassium

Synthesis of the PPy/CNT electrochemical capacitor

Due to several factors affecting the performance of EC, two electrochemical techniques by grafting the PPy/CNT were used, chronoamperometry (0.8 V) and the cyclic voltammetry (30 cycles, with the potential window between −0.2 and + 1.2 V at 50 mV/s scan rate. For both techniques, the polymerization was successful performed with increase in electroactive area and charge storage. Nevertheless, it was observed a more defined redox peaks (Fe(CN)63−/Fe(CN)64-) by Cyclic voltammetry than using the

Conclusions

A immunosensor based on a PPy/CNT supercapacitor grafted on the IDE electrode, with large charge storage capacity and high surface area reached was developed to CysC detection. Linear range and LOD achieved allows this use in clinical range and cutoff is relevant for use in renal failures. Moreover, this immunosensor has potential for applying to the point-of-care immunoassays, helping detection of biomolecule with the high diagnostic sensitivity e specificity. The efficient blocking agent

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This research was supported by CNPqgrants number 471065/2014-5 and 440605/2016-4 and CAPES brazilian science funding agencies. We also acknowledge Dr. Angelo Luis Gobbi from Brazilian Nanotechnology National Laboratory /CNPEM, Campinas, Brazil for his notable technical support in this work. P.A.B. Ferreira thanks the research agency of Pernambuco, Brazil, (FACEPE) by scholarship.

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