Head: Assoc. Prof. Nina DIMCHEVA, PhD, CV
Assoc. Prof. Maria Stoyanova, PhD, CV
Assoc. Prof. Stela Statkova-Abeghe, PhD, CV
Assoc. Prof. Dimitar Tokmakov, PhD, CV
Assoc. Prof. Sotir Sotirov, PhD, CV
Chief. Asst. Prof. Vanina Kolcheva, PhD, CV
Mariya Pimpilova, Researcher, CV
The planned within the work pack research activities fall into the following scientific areas: chemical sciences, biotechnology and pharmacy.
The design of sensing/biosensing devices for quantification of biologically important compounds in pharmaceutical formulations, blood, urine, and other objects of clinical analysis provides the opportunity for rapid and selective monitoring of various analytes, such as the active ingredients of medications, metabolites or pathogens. Biosensing devices have the potential to considerably shorten the duration of the laboratory analyses of a number of clinical indicators, thereby contributing to the reduction of the time necessary for diagnosis and the choice of appropriate treatment, and therefore result in more rational spending of the funds for the treatment of patients.
On the other hand, the biosensors can be easily integrated into the production lines of the pharmaceutical industries to monitor the active ingredients of medications produced, which in turn minimize the likelihood of the production of defective or dangerous products.
In this context, the research activities planned in the present work package, refer to the development of biosensing systems, in which biochemical receptors (biological elements for molecular recognition-enzymes, proteins, etc.) are integrated with an electrochemical sensor/actuator, which correlates with thematic area 3 of the ISSS of the Republic of Bulgaria: “Industry for healthy living and biotechnology” in the following priority sub-areas: “Personalized medicine, diagnostics and individual therapy , healing and medicinal forms and means”and” Biotechnologies applicable in a healthy life”.
Electrochemical methods play an important role in the pharmaceutical and biomedical research [C. Cristea & B. Ciui, World J Pharm Sci; 3(4): 682-686, 2015]. From analytical viewpoint, their capabilities for automation and miniaturization (allowing for apparatus mobility and portability) far exceed the ones of conventional analytical equipment. They can significantly reduce the response time, the economic costs, as well as to improve the selectivity of the determination, to drastically lower the detection limits especially in the cases of complex matrices, such as biological samples. The continuous progress in this area requires the development of novel types of biosensors which comply with the requirements of the market and can be applied both in centralized and mobile laboratories for biomedical analysis.
This work package aims to develop nanomaterials- based electrochemical biosensors for sensitive and selective determination of active ingredients in pharmaceutical formulations, metabolites or clinical indicators. Nanomaterials offer a convenient platform for immobilization of biologically active substances. Among the available variety of nanostructures, the gold nanoparticles and carbon nanotubes, show a number of advantages that are useful for the development of biosensors, among these the opportunity to immobilize enzymes, using supramolecular chemistry is the most advantageous. Finally, the nano-structuring of polymers using a variety of methods allows to control both the electrode surface area and its porosity, and offer the opportunity to immobilize a large variety of biological elements (enzymes and aptamers, single-stranded DNA- pieces with high specificity towards the target molecule) that leads to an overall improvement of the operational characteristics of the biosensor. An alternative to enzyme-electrochemical methods for analysis of metabolites is the use of biomimetic catalysts. As a rule, their use is cheaper and gives greater sensor stability with time, as well as upon changing pH and temperature of the operating medium. The term “enzyme-less biosensor” has been adopted by some researchers to define this particular type of sensors.
The innovative character of the activities that shall be implemented within the framework of this work package are related to finding a fundamentally new approach in:
- The analysis of active ingredients of pharmaceutical products – e.g. vitamins, antioxidants, catecholamines (L-DOPA, dopamine, epinephrine, serotonin, etc.);
- The determination of clinical indices in bodily fluids – e.g. blood sugar, blood lactate, uric acid;
- The miniaturization of biosensor devices.
Despite the progress in biosensor technology on a global scale (the first biosensor has been constructed some 50 years ago) the only type of biosensing devices with wide practical applications are the commercially available blood glucose monitors for diabetic care (http://www.news-medical.net/health/Biosensor-Applications.aspx), whilst their application in clinical analyses for rapid assessment of other clinical indicators is still potential. In this context, all the research activities related to the development of biosensing systems scheduled in this package include innovative elements and to date have not found wide practical application (only the stages of testing are reached so far). Moreover, in order to develop cheaper, faster and more selective biosensing methods for analysis of active ingredients of medications or for assaying the concentrations of metabolites (e.g., in emergency medicine or in chronic metabolic disorders) than the widely known traditional methods, there are no universal approaches and each individual practical application shall be further optimized for a better performance. The expected impacts from the successful implementation of the planned research activities relates to the economic effect (since the control of pharmaceutical production of certain medications may become less expensive) and the rapidity of the analysis – will help to react faster in emergency, urgent and lifesaving situations.
The specific practical aim that we have set in this work package is to developing a series of biosensor devices applicable in biomedical analysis and pharmaceutical industries. In this connection, we identified the following specific research objectives:
- Creation of enzyme-based biosensors with electrochemical detection for the quantitative analysis of active ingredients of drugs (injectable forms, tablets);
- Design of biosensor systems for the quantitative analysis of clinical indicators (blood lactate, xanthine, bilirubin, glucose, neurotransmitters, etc.);
- Scaling-down of the biosensor system for monitoring metabolites.
To achieve the above-mentioned practical goals and objectives, the following investigations are planned to be implemented within this work package:
- Design and characterisation of modified electrode materials with prospective application in the development of electrochemical sensors/biosensors;
- Immobilization (binding) of a particular enzyme to the modified electrode materials, whilst selecting the most suitable method for attachment to the electrode surface in order to construct a biosensor with high sensitivity;
- Optimizing the operation parameters of the resulting biosensors: elucidating the detection principle, adjusting the composition of the working medium, assessing the impact of potentially interfering substances on the response, etc.;
- Validation of the biosensor systems: comparison of the results obtained from the biosensor analysis of real samples with those from a reference analytical method (used as standard)
The planned research is expected to have an immediate effect on the following processes/products of contemporary technological development:
- Development of novel materials for sensor technology, especially when using nanocomposite materials, nano-structured polymers and modified with nano-particles sensor surfaces;
- Development of protocols for the immobilization of enzymes and proteins on electrode surfaces – despite the progress of the biotechnological and chemical sciences there is no universal approach to attach a bioreceptor (biological component for molecular recognition) to the transducer, which might be applied in all cases. An individual approach consistent with the particular application of the designed device is needed for that purpose;
- Elucidation of the detection principle of the biosensor devices: the measurement of electrical current or potential are applicable only in cases where the substances involved in the biochemical transformations are electrochemically active (could be oxidised or reduced), however it’s possible that biochemical transformation being accompanied with electrostatic interactions (the surface charge of sensor changes) or with the formation of complexes (which is detectable through the measurement of the optical or other physical characteristics of the sensor); The innovative element here is the identification of the physical (or chemical) specifications, which vary upon the interaction of bioreceptor with the target analyte molecule that shall be used as the basis for designing biosensors.
- Optimization of the performance of biosensors in model environment with subsequent validation of biosensing devices when working with real samples – blood serum, plasma, urine. In this case, potentially interfering interactions with the components normally attending real samples are to be established and a way they can be eliminated or minimized is to be found. Another option is through the development of approaches for removing them (chemical elimination) or through a differentiated assessment of the effect of the interfering substances on the biosensor response.
It is possible that the planned research result in the development of new concepts for immobilization of biological catalysts (receptors) onto nanostructures (further development of Nano-Biotechnology), since at the moment there is no clarity about the essence/nature of the processes in the interaction of a biomolecule with a nano-particle. The development of this research area is forthcoming. Part of the research activities are alongside this direction that will result in further development of nano-biotechnology and the sensor/biosensor technologies. The experience of the research team gained in the field of nanotechnology, the production and characterisation of nano-structured materials, as well as the recent progress in the development of third (last) generation biosensor devices are promising prerequisites for the achievement of a high-quality research in the field of biosensor technologies on international scale.
The objective criteria that will be applied to the measurement of the expected level of these research activities are the following:
- Articles in journals with a high impact factor – at least one publication per year (after the first year of the project) or up to 9 high impact factor publications for the entire period of the project.
- Publications in scientific journals among the top 10% in the field – expected up to 3. The scientific team publishes regularly in high-impact factor scientific journals, such as Bioelectrochemistry, Analytical & Bioanalytical Chemistry, Microchimica Acta, Journal of the Electrochemical Society, etc., which are leading in the field of electrochemistry and bio-analytical methods.
- Intellectual Property Rights- applied developments (prototyps of electrochemical biosensor systems developed methodologies or algorithms for biosensor analysis of antioxidants, neurotransmitters, metabolites, etc.) will be subjected to intellectual property suvey and in case of the irrefutable proof of their originality will be protected by a utility model or a patent for an invention – expected up to 3.
- International interest – broad international impact and international prominence by attracting attention of international research networks and groups, ensuring the specialization/training of at least one researcher in a leading scientific organization in the EU in order to increase the capacity of the Centre in the field of sensor technology and biosensors; attending international scientific events (congresses and international conferences on physical chemistry, electrochemistry and bioelectrochemistry) at least once a year (after the second year of the project) or total for the period up to 8 scientific forums
- Publications on the project results in prestigious scientific journals, reports/presentations on international scientific forums; the project achievements will be disseminated electronically among topic – related teams from research and educational institutions of the European Union
A wide spectrum of electrochemical, microscopic, spectroscopic, chromatographic and other instrumental methods for characterization of the newly developed materials, for monitoring the kinetics of the studied biochemical/chemical processes, or as reference methods to validate the newly developed biosensor systems will be used. All these methods have proven their reliability in both scientific research and routine analyses. The reliability of newly developed methodologies shall be verified in the process of validation, and for this purpose approuved as standards analytical methods will be used as reference.
Methods for preparation: to be used in the development of electrocatalytic materials with prospective application as transducers in biosensors. Research on surface modification of different compact materials with precious metals (Au, Pt, Pd, Rh, Ir), metal oxides, enzymes and proteins, are traditional for scientific team and have proven their reliability. As the most accessible and feasible technique for surface dispersion of the modifying metal phase electrochemical deposition of metal clusters is proposed, but techniques such as impregnation with nano-dispersed phase and surface coverage with layers of composite material, have also proved their reliability as surface-modifying approaches.
Enzyme immobilization Certain approaches for attachment of biocatalysts on the surface of the transducing element are expected to have a higher potential to retain their activity when used in the construction of biosensor devices. As the most reliable approaches for immobilization are identified two groups of methods: physical (physical adsorption or entrapment of the biocatalyst within polymer layer) and chemical (chemical bonding between the electrode surface and the biocatalyst).
This type of studies are focused on the optimization of biosensor performance and stabilization of biocatalysts.
- A wide range of electrochemical techniques will be used to implement the experimental tasks planned in the work package. These electrochemical methods will be used for exploring the main factors possessing a direct impact on the operational characteristics of electrochemical biosensors, namely: the electrode material; its nature, as well as the composition, concentration and ionic strength of the reaction medium; the method of immobilization of the biocatalyst, its nature and origin; the structure of the enzyme substrate, temperature, etc.
- Electrochemical methodologies using forced convection, as rotating disk electrode (RDE) or flow-injection analysis are commonly used for the quantitative characterization of the studied systems, due to the opportunity to eliminating the diffusion limitations – and as basic techniques for the study of electrode kinetics.
- Electrochemical impedance spectroscopy (EIS): Electrochemical impedance spectroscopy is a flexible tool for electrochemical characterisation of redox properties of any intrinsic electrode material and its surface (see: A Bard and L. Faulkner: Electrochemical methods: Fundamentals and applications, 2001, Willey Interscience). EIS is based on analyzing the impedance (resistance when passing AC) of the studied system as a function of the frequency of the current and the excitation signal. This analysis provides quantitative information about conductivity, dielectric constants, static characteristics of the surfaces in the system and their dynamic change as a result of transfer of electric charge or adsorption of electroactive species. In EIS an alternating current with a low amplitude, which facilitates the non-invasive study of any kind of sample without a noticeable impact on its electrochemical characteristics, is used.-