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One of the key issues to develop biosensing platforms concerns the processes involved in enzyme immobilization on surfaces. The understanding of their fundamentals is crucial to obtain stable and catalytically active protein layers for developing successful biosensing devices. In this respect, the advent and development of new characterization techniques, in particular at the submicron level, has allowed the study of these processes with high resolution, which has opened new routes to improve, and eventually control, enzyme immobilization on electrode surfaces. This review focuses on the application of Atomic Force Microscopy (AFM), Scanning Electrochemical Microscopy (SECM) and Quartz Crystal Microbalance (QCM) techniques in the characterization of the successive immobilization steps involved in the development of bioanalytical platforms. A common advantage of these techniques is their ability to provide important information without damaging the immobilized biological sample due to the possibility of performing measurements under physiological conditions close to the native environment of the specimens. A particular emphasis is placed on the application of these techniques to the characterization of the immobilization of enzymes on different modified and unmodified surfaces as well as on the study of protein interactions, which is a more recent and less current application.

Different enzyme immobilization approaches of Trametes versicolor laccase (TvL)onto gold surfaces and their influence on the performance of the final bioanalytical platforms are described. The laccase immobilization methods include: i) direct adsorption onto gold electrodes (TvL/Au), ii) covalent attachment to a gold surface modified with a bifunctional reagent, 3,3'-Dithiodipropionic acid di (N-succinimidyl ester) (DTSP), and iii) integration of the enzyme into a sol-gel 3D polymeric network derived from (3-mercaptopropyl)-trimethoxysilane (MPTS) previously formed onto a gold surface (TvL/MPTS/Au). The characterization and applicability of these biosensors are described. Characterization is performed in aqueous acetate buffer solutions using atomic force microscopy (AFM), providing valuable information concerning morphological data at the nanoscale level.The response of the three biosensing platforms developed, TvL/Au, TvL/DTSP/Au and TvL/MPTS/Au, is evaluated in the presence of hydroquinone (HQ), used as a phenolic enzymatic substrate. All systems exhibit a clear electrocatalytic activity and HQ can be amperometrically determined at -0.10 V versus Ag/AgCl. However, the performance of biosensors -evaluated in terms of sensitivity, detection limit, linear 2 response range, reproducibility and stability-depends clearly on the enzyme immobilization strategy, which allows establishing its influence on the enzyme catalytic activity.

In this work, we report the synthesis and characterization of different kinds of graphene nanomaterials and their applicability to the development of biosensing platforms. We have synthesized graphene oxide (GO) following a modified Hummer’s method, which has been subsequently reduced by electrochemical procedures. This reduction strategy precludes the employment of toxic solvents, leading to a product, electrochemically reduced graphene (ERG), free of contaminants. The characterization of the synthesized nanomaterials has been performed by different techniques such as X‐ray diffraction spectroscopy (XRD), Raman spectroscopy, X‐ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM) and atomic force microscopy (AFM). The information gathered by this combination of techniques confirms that i) the synthesis methodology affords the production of GO nanosheets, which present a typical lateral dimension of several hundreds of nanometers and a thickness value of 1.3±0.1 nm, ii) the reduction step has been successfully achieved leading to graphene nanosheets free of oxygen functionalities with an average lateral dimension of at least 1 micrometer and a thickness value of 2.8±0.2 nm. Once we have confirmed that both materials have been successfully synthesized, we have studied the effect of the effect of their inclusion in biosensing platforms on the analytical response, selecting a lactate oxidase based biosensor as a model system. We have demonstrated that although the incorporation of GO or ERG to the device results in an enhancement of the analytical response of the resulting biosensing platform, the former system offers slightly better analytical properties and a more reproducible response than the ERG one.

The design and characterization of a new nanostructured organic-inorganic hybrid material and its application to L-lactic acid determination are described. This material is based on the integration of the enzyme lactate oxidase (LOx) and gold nanoparticles (AuNPs) into a sol-gel 3D polymeric network derived from (3-mercaptopropyl)-trimethoxysilane (MPTS) previously formed onto a gold surface. MPTS presents the advantage of forming a 3D polymeric network containing a large number of thiol tail groups distributed throughout its structure that enable both its anchoring onto gold surfaces and the AuNPs incorporation. Moreover, this matrix provides a biocompatible environment that preserves the catalytic activity of LOx after its immobilization and allows the incorporation of a high amount of enzyme, which is expected to improve the sensitivity of the final biosensing device. Characterization of the designed biosensing platform was performed using quartz crystal microbalance (QCM), scanning electron microscopy (SEM) and atomic force microscopy (AFM) techniques. From the conjunction of these techniques, information about (i) the kinetic of LOx adsorption process in real time, (ii) the amount of LOx incorporated into the network, and (iii) the morphological characteristics at the nanometre level of the designed biosensing material was obtained. This information is very useful on the development of successful biosensing devices. Finally, the response of the biosensor to L-lactic acid was evaluated. The biosensor responds linearly to L-lactic acid in the range of 50 µM to 0.25 mM, with a sensitivity of 3.4 µA mM(-1) and a detection limit of 4.0 µM.

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The authors describe an electrochemical sensor based on the use of diamond nanoparticles (DNPs) and molybdenum disulfide (MoS) platelets. The sensor was applied to the voltammetric determination of the anticonvulsant valproic acid which was previously derivatized with ferrocene. The MoS platelets were obtained by an exfoliation method, and the DNPs were directly dispersed in water and subsequently deposited on a glassy carbon electrode (GCE). The sensor response was optimized in terms of the solvent employed for dispersing the MoS nanomaterial and the method for modifying the GCE. Sensors consisting of a first layer of MoS dispersed in ethanol/water and a second layer of DNPs give better response. The single steps of sensor construction were characterized by atomic force microscopy and electrochemical impedance spectroscopy. The differential pulse voltammetric response of the GCE (measured at +0.18 V vs. Ag/AgCl) was compared to that of sensors incorporating only one of the nanomateriales (DNPs or MoS). The formation of a hybrid MoS-DNP structure clearly improves performance. The GCE containing both nanomaterials exhibits high sensitivity (740 µA ⋅ mM ⋅ cm), a 0.27 μM detection limit, and an 8% reproducibility (RSD). The sensor retained 99% of its initial response after 45 days of storage. Graphical abstract Electrochemical sensor by co-immobilization of MoS and diamond nanoparticles (DNP). The formation of a hybrid MoS-DNP structure enhances the performance of the sensor towards valproic acid derivatized with a ferrocene group, when compared with sensors incorporating only DNP or MoS.