Jana Šefčovičová

Immobilization in biotechnology and biorecognition: from macro- to nanoscale systems

Biological molecules such as enzymes, cells, antibodies, lectins, peptide aptamers, and cellular components in an immobilized form are extensively used in biotechnology, in biorecognition and in many medicinal applications. This review provides a comprehensive summary of the developments in new immobilization materials, techniques, and their practical applications previously developed by the authors. A detailed overview of several immobilization materials and technologies is given here, including bead cellulose, encapsulation in ionotropic gels and polyelectrolyte complexes, and various immobilization protocols applied onto surfaces. In addition, the review summarises the screening and design of an immobilization protocol, practical applications of immobilized biocatalysts in the industrial production of metabolites, monitoring, and control of fermentation processes, preparation of electrochemical/optical biosensors and biofuel cells.

A hyaluronic acid dispersed carbon nanotube electrode used for a mediatorless NADH sensing and biosensing

A biocompatible nanocomposite consisting of single-walled carbon nanotubes (CNTs) dispersed in a hyaluronic acid (HA) was investigated as a sensing platform for a mediatorless electrochemical detection of NADH. The device was characterised by scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR) and extensively by electrochemistry. CNT-HA bionanocomposite showed more reversible electrochemistry, higher short-term stability of NADH sensing and higher selectivity of NADH detection compared to frequently used CNT-CHI (chitosan) modified GCE. Finally the performance of the sensor modified by CNT-HA was tested in a batch and flow injection analysis (FIA) mode of operation with basic characteristics revealed. The NADH sensor exhibits a good long-term operational stability (95% of the original sensitivity after 22 h of continuous operation). Subsequently a d-sorbitol biosensor based on such a nanoscale built interface was prepared and characterised with a d-sorbitol dehydrogenase used as a biocatalyst.

Off-line FIA monitoring of D-sorbitol consumption during L-sorbose production using a sorbitol biosensor.

A ferricyanide mediated amperometric biosensor system implementing D-sorbitol dehydrogenase together with diaphorase for sensitive detection of D-sorbitol was used. The biosensor system was successfully integrated into an off-line FIA system with a throughput of detection of 10 h(-1). The device exhibited limit of detection of 20 microM with an average relative standard deviation of analysis of samples of 2.2%. The signal of the biosensor was linear up to 1.1 mM for D-sorbitol with sensitivity of (72 +/- 2) nA mM(-1), while a dynamic range was much wider up to 18 mM. The sorbitol biosensor gave reliable results even in the presence of a high molar excess of L-sorbose, a product of the biotransformation process, as judged from an excellent agreement with HPLC and GC.

Whole-cell Gluconobacter oxydans biosensor for 2-phenylethanol biooxidation monitoring

A microbial biosensor for 2-phenylethanol (2-PE) based on the bacteria Gluconobacter oxydans was developed and applied in monitoring of a biotechnological process. The cells of G. oxydans were immobilized within a disposable polyelectrolyte complex gel membrane consisting of sodium alginate, cellulose sulphate and poly(methylene-co-guanidine) attached onto a miniaturized Clark oxygen electrode, forming whole cell amperometric biosensor. Measured changes in oxygen concentration were proportional to changes in 2-PE concentration. The biosensor sensitivity was 864 nA mM(-1) (RSD=6%), a detection limit of 1 μM, and the biosensor response towards 2-PE was linear in the range 0.02-0.70 mM. The biosensor preserved 93% of its initial sensitivity after 7h of continuous operation and exhibited excellent storage stability with loss of only 6% of initial sensitivity within two months, when stored at 4°C. The developed system was designed and successfully used for an off-line monitoring of whole course of 2-PE biooxidation process producing phenylacetic acid (PA) as industrially valuable aromatic compound. The biosensor measurement did not require the use of hazardous organic solvent. The biosensor response to 2-PE was not affected by interferences from PA and phenylacetaldehyde at concentrations present in real samples during the biotransformation and the results were in a very good agreement with those obtained via gas chromatography.

Application of nanomaterials in microbial-cell biosensor constructions

Microbial cell biosensors, where cells are in direct connection with a transducer enabling quantitative and qualitative detection of an analyte, are very promising analytical tools applied mainly for assays in the environmental field, food industry or biomedicine. Microbial cell biosensors are an excellent alternative to conventional analytical methods due to their specificity, rapid detection and low cost of analysis. Nowadays, nanomaterials are often used in the construction of biosensors to improve their sensitivity and stability. In this review, the combination of microbial and other individual cells with different nanomaterials (carbon nanotubes, graphene, gold nanoparticles, etc.) for the construction of biosensors is described and their applications are provided as well.

Electrochemical Features of Bilirubin Oxidase Immobilized on Different Carbon Nanostructures

An electrode interface was prepared using a mixture of a cheap carbon nanomaterial KetjenBlack (KB) and carbon nanotubes (CNT) dispersed in a biopolymer chitosan. Bilirubin oxidase (BOD) was proved to adsorb effectively on such a nanointerface, retaining its catalytic activity for reduction of dioxygen to water, which was proved by cyclic voltammetry. Moreover, three distinct cathodic redox reactions were determined in the absence of oxygen, suggesting that KB/CNT template provides a suitable micro and nanoporosity for direct electron transfer between BOD and the modified electrodes revealing all three known active sites of BOD. Furthermore, BOD was adsorbed on graphene oxide with subsequent electrochemical reduction of graphene oxide into a conductive graphene film with BOD trapped within the matrix. Two active sites of BOD were observed on the electrode modified by graphene suggesting the enzyme is oriented in a different way compared to the KB/CNT nanointerface due to changes in the nature of functional groups within the nanocomposite, changed porosity of the nanointerface or as a result of electrochemical perturbation of the matrix during reduction of graphene oxide. A more detailed fundamental investigation of the influence of the nanointerface matrix on an adsorption and orientation of BOD will without any doubt allow us to tailor ability of such composites to reduce dioxygen to water with high efficiency, what is a feature important for construction of robust and effective biocathodes of enzymatic biofuel cells.

Interfacing of microbial cells with nanoparticles: Simple and cost-effective preparation of a highly sensitive microbial ethanol biosensor

Various types of carbon nanoparticles were directly mixed with microbial cells of Gluconobacter oxydans within a 3-D bionanocomposite in order to prepare a highly sensitive ethanol biosensor with a short response time. From all carbonaceous nanomaterials tested, single- or multi-walled carbon nanotubes provided the highest sensitivity of detection (117–121 µA cm−2 mM−1), but from a practical point of view, Ketjen black 300 and 600 provide very low detection limit (2–6 µM) and high sensitivity for the ethanol analysis (84–88 µA cm−2 mM−1)with a shortresponse time (14–33 s). Moreover, the price of Ketjen black is a few orders of magnitude lower compared to that of carbon nanotubes. Finally, the study showed that the morphology of nanoparticles rather than their surface modification is the key element in achieving high sensitivity of ethanol detection.

A mediatorless electrochemical detection of NADH on a biopolymer dispersed carbon nanotube layer

A mediatorless sensor device based on a glassy carbon electrode modified by a hyaluronic acid (HA) dispersed carbon nanotubes (CNTs) for detection of NADH in a flow injection mode was prepared with good operational stability.