AISI 304 Stainless Steel as a Transducer Substrate in Electrochemical Biosensors for Medical Applications

Ricardo Adriano Dorledo de Faria*1, Luiz Guilherme Dias Heneine2, Vanessa de Freitas Cunha Lins1 and Tulio Matencio3 1Department of Chemical Engineering, Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, Minas Gerais 30270-901, Brazil 2Department of Applied Immunology, Fundação Ezequiel Dias (FUNED), Belo Horizonte,Minas Gerais 30510-010, Brazil 3Department of Chemistry, Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, Minas Gerais 30270-901, Brazil


Introduction
For many reasons, the material sciences have focused in studies of electroanalysis, exploring the knowledge of conductive materials that can be used as sensitive elements for composing sensors in order to specifically recognize analytes of interest [1]. According to the IUPAC [2], the material responsible for providing a measurable output signal by means of a biological reaction on its surface is defined as a "transducer" and it is a crucial component of a biosensor.
In electrochemical biosensors, the transduction phenomena occurs due to oxidative and reductive processes, and the charge transferring can be correlated to the concentration of a target molecule, providing a selective and sensitive detection in a simple and easyto-use format [3,4]. Electrochemical transduction has received larger attention due to its simplicity, low cost of instrumentation and possibility of application as miniaturized systems. Thus, among innumerous applications of biosensors, medical field has particular interest on it for requiring quick, portable and simple devices for providing early and efficacious diagnosis [5]. Nowadays, point-ofcare sensors represent about 57% of the biosensors market and it requires cost-effective platforms and materials [6]. Thereby, many studies have focused on developing high-performance biosensors, whose analytical response strongly depends on the properties of their transducers [7]. In the light of this context, Stainless Steels (SSs), which are alloys of iron containing at least 11% w/w Cr, are promising candidates for biomedical applications, since they have high corrosion resistance, great biocompatibility and low cost [8,9].
Due to these interesting characteristics, some researches have been devoted to the use of SSs in the field of biosensors for various applications [8,10,11].
The present study aimed to evaluate the employment of 304 SS as an electrochemical transducer for biosensors of medical rel-Stainless Steels (SS) are promising candidates to constitute the transducer substrate of devices for electrochemical sensing mainly due to their high electrical conductivity, biocompatibility and relative low cost. In this study, the corrosion resistance of SS was examined in four solutions that represent the environment of biosensors in real applications: 0.01 M Tris buffer (pH 7.4), an electrolyte containing Tris and 0.1M KCl as supporting source of ions, Hanks' solution to simulate body fluids and, also, a solution to represent the electrolyte commonly used to performed Cyclic Voltammetry (CV). Potentiodinamic anodic polarization, Chronoamperometry and images of Optical Microscopy revealed that the Hanks' and CV solutions were the most aggressive media to the SS electrodes, which remained chemically stable in both Tris and electrolyte during 16h under the influence of critical potentials. When functionalized to recognize venom from Crotalus snakes, the SS suffered an increase of the diameter of the capacitive arc in the EIS experiment as a result of the detection of the target analyte, corroborating it is a promising electrochemical transducer for application in biosensors. evance. For this purpose, the corrosion resistance of the material was analyzed in four media where biosensors are commonly employed at their work condition: Tris buffer, which is used to prepare biological solutions and to store sensors in appropriate conditions of salt concentration and pH [12][13][14]; an electrolyte containing potassium chloride, which is an aqueous solution commonly used in the literature due to its good conductivity to perform electrochemical measurements [15,16]; Hanks' solution, which is a synthetic solution that simulates body conditions and it is one option constantly considered in corrosion studies of environmental body influence [17][18][19][20]; and a ( ) ( ) which is a medium commonly used for Cyclic Voltammetry studies, electrochemical technique that is largely employed in biosensors researches [19,21,22] or even in Faradaic Electrochemical Impedance Spectroscopy (EIS) [24].

Preparation of the Transducer Substrate
A copper wire was welded on the backside of the 304 SS (geometrical area of 1 cm²), which has chemical composition of Cr The pH and conductivity of these solutions were measured at 25°C by using a digital pHmeter pHtek model Phs 3b and a conductivity meter mCA 150 (MS TECNOPON). Open Circuit Potential (OCP) was monitored for 1 hour before each anodic polarization measurement. The polarization tests were carried out at a scanning rate of 1 mV.s -1 until the current density reached the value of 10 -3 A.cm -2 after the breakdown potential.
Chronoamperometry Test: Chronoamperometry was performed to analyze the stability of the passive layer of the 304 SS in each solution. For this purpose, the constant potentials indicated in Table 1 were previously defined by the anodic polarization test (as the intermediary potential in the passive region) and applied to the working electrode during 16 h in the different media.

Preparation of the Biosensor and Analyte Recognition
The substrate was functionalized by its immersion in an aque- To evaluate the capability of the biosensor to recognize its analyte of interest, EIS was performed before and after the incubation of the devices in a 10 µg. mL -1 crotalic venom solution, which was prepared using Hanks' solution in order to simulate the real condition of an envenomed patient. EIS was performed ranging the frequency from 10 4 to 10 -2 Hz at a 10mV amplitude potential after an

Capacitance Analysis of the SS
To assess the semiconducting properties of the SS in the various media under investigation, a Mott-Schottky test was performed in a potential range from -0.2 to +1.1 V vs Ag/AgCl with a 66 mV step height at 1000 Hz according to the methodology described by other authors who studied the electrochemical properties of SSs [27][28][29].
By means of this technique, it was possible to estimate the concentration of charge donors and acceptors in the SS.

Potentiodynamic Anodic Polarization Study
The pH and conductivity of the four media were measured and the results are shown in Table 2. All the solutions were neutral, and the conductivity values presented the more significant differences.
Because of the ions concentration in each solution, the Tris buffer was the less conductive medium and the CV solution was the one with the higher conductivity.   In a neutral aerated medium, such as employed in this work, the cathodic reaction that occur in the SS is the oxygen reduction as follows [30]: instable region where the current did not assume a progressive increasing, but it was affected probably by a sequence of formation and dissolution of the passive film. As expected, the electrolyte was more aggressive than the Tris because it contains KCl as supporting electrolyte (presenting higher conductivity), which solubilizes in the water forming Clions, anions that are responsible for the pitting corrosion in SSs [31].
In CV solution, the steel did not present a well-defined E pass , although a potential increasing occurred in density currents between 10 -5 and 10 -4 A.cm -2 , characteristic range of passivation in corrosion processes. Wolynec [32] suggests that in low current density values, around 10 -6 A.cm -2 for instance, the material does not suffer corrosion because it is protected by the passive film. The combination of the higher corrosion potential (E corr = -0.099 ± 0.002 V vs Ag/ AgCl) and pitting potential (E pit = 0.500 ± 0.061 V vs Ag/AgCl) of the steel polarized in Tris indicates that this solution was the less aggressive in comparison to the others. The CV solution was the most aggressive medium due to its higher concentration of ions (higher conductivity) that hinders the development of the passive film to protect the steel surface. Regarding the biosensing applications, the chemical stability of a material is an important parameter to be taken in account because the occurrence of oxidative/reductive reactions at the sensor's surface can compromise characteristics such as its sensitivity and reproducibility. Accordingly, the chemical stability of a material is related to its lifetime and may limit its technological application.

Chronoamperometric Behavior of the SS
In order to evaluate the stability of the 304 SS in the different media, chronoamperometry was performed in Tris, electrolyte, Hanks' solution and CV solution. The results are shown in Figure 2.
Some current fluctuations occurred in function of the time due to the exposition of the steel to the electrolyte, the Hanks' solution and the CV solution. González and Saidman [33] affirmed that this kind of behavior is characteristic of localized (pitting) corrosion.
In Hanks' solution, it is possible to observe that the steel did not

Analysis of the Semiconducting Properties of the SS
Since the Mott-Schottky relation is a function of the applied potential (E), it was possible to inspect the semiconducting features of the SS in the various media. In Eq.1 and Eq. 2, "C" refers to the space charge layer capacitance, "e" is the charge of the electron (equal to 1.6 x 10 -19 C), "ε" is the dielectric constant of the SS (ε = 12), "ε 0 " is the vaccum permittivity (8.854 x 10 -12 F.m -1 ), "A" is the surface area of the SS electrode, "E fb " is the flat band potential, "k" is the Boltzmann constant (1.38 x 10 -23 J.K -1 ), "T" is the temperature in Kelvin and "N D " and "N A " are, respectively, the donors and acceptors concentrations of the material under investigation [35,36].
In case of negative slope, it is assumed that the material is a p-type semiconductor, and in case of positive slope, the material is an n-type semiconductor. Linear relations were found between the C -2 and the applied potential in Figure 4a, which indicates that the SS possess semiconducting properties in the studied media [37].
The SSs assumed both behaviors. In potentials up to + 0.3 V vs Ag/ AgCl, the negative slope of the C -2 vs E curve associated to the p-type semiconductor arises from the inner oxides film formed on the SS surface, which are rich in chromium. On the other hand, the n-type behavior (positive slope from +0.3 to +1.1 V vs Ag/AgCl) is related to the more external layer containing iron oxides and hydroxides [38].
In SSs, the semiconducting behavior arises from the formation of the passive layer. Oxide films formed during the passivation consist of organized crystallographic structure that presents an insulating character due to its stoichiometry. However, the presence of localized defects in the film, such as the cationic vacancies ( oxygen vacancies (n Ö ) and interstitial vacancies can lead the material to behave as an extrinsic semiconductor in aqueous medium.
The cationic vacancies are electron acceptors and lead to a p-type doping, while the oxygen vacancies and interstitial cations are electron donors, which are responsible for the n-type doping [42].

Analyte
Aiming to study the recognition capability of the SS-based biosensor, EIS data was modelled to a suitable electrical equivalent circuit. A Randles model consisting of an Electrolyte Resistance (Re), a Constant Phase Element (CPE), which is used instead of a pure capacitor due to the irregularities of the substrate's surface, such as its heterogeneity and roughness [43], and leakage resistance (Rleak) was used to simulate the impedance data. The fitting quality was ensured by the low values of the statistic parameter chi-  The results revealed an increasing of the capacitive arc diameter after the incubation in the analyte solution, which is related to the difficult of charge transferring on the interface electrode-electrolyte due to the presence of pair antigen-antibody, forming a more insulating layer. Elshafey et al. [44] confirmed that the formation of the immune complex antigen-antibody hinders the capability of ions from electrolyte to penetrate its interface with the electrode, enhancing the charge transfer Resistance (Rct) in case of a faradaic sensors. In case of non-Faradaic sensors, in which there is no redox species in the electrolyte, when the antibodies present on the SS surface binds the target antigen (crotalic venom in this work), this captured analyte acts diminishing the conductivity of the transducer substrate [24]. In this sense, an expected increasing of Rleak (95.00 ± 8.71%) was observed from the biosensor before and after its incubation in the analyte solution, confirming that the SS is an adequate electrochemical transducer to be used in biosensors. In our previous publication [45], we have exploited further characteristics (limit of detection, sensitivity and selectivity) of a sensor developed by means of a SS (Crofer 22 APU) towards the detection of venom from Bothrops snakes, corroborating the usability of these material for biosensing applications.

Conclusion
The electrochemical characteristics of a 304 SS were investi- to recognize a biomolecule of interest after functionalization allow to affirm that the SS is a promising transducer substrate for application in electrochemical biosensors.