Biosensors (Part 2)

Continuation. The beginning of the article is here.

Technical strategies

The technical strategies used to develop biosensors are based on the identification of biomarkers with and without the use of labels. Detection using tags is based primarily on the specific properties of labeling compounds used for targeted detection. Biosensors of this type are reliable, but often require a combination of specific sensing elements manufactured using an immobilized target protein. On the other hand, a method that does not use labels allows you to identify target molecules that are not suitable for labeling. Recent interdisciplinary approaches in the field of biotechnology and bioengineering, electrical engineering and electronics have paved the way for the development of non-label biosensors for various detection methods with a wide range of applications in the field of medicine and environmental science.

Electrochemical biosensors

The creation of a glucose meter based on glucose oxidase biosensors, which has already become a classic, is the first step in the history of the development of electrochemical biosensors. Glucose biosensors are very popular in clinics and diagnostic institutions, as they are necessary for periodic monitoring of blood glucose levels in patients with diabetes mellitus. However, these biosensors have disadvantages due to the unstable activity or inhomogeneity of the enzyme, which determines the importance of additional calibration. In fact, these potential drawbacks led to the development of a spectrum of biomolecules with different electrochemical properties, which led to the emergence of more stable glucose biosensors. Recently, electrochemical biosensors are typically manufactured by modifying the surface of metal and carbon electrodes using biomaterials such as enzymes, antibodies or DNA. The biosensor output signal is usually generated as a result of specific binding reactions or catalytic reactions between materials on the electrode surface. The need to develop electrochemical sensors has become especially relevant for the clinical diagnosis of diseases in which early detection or monitoring is of great importance. In this context, synthetic materials are considered for the development of non-enzymatic biosensors instead of proteins. An interesting fact is that different types of biomolecules have different stability and selectivity, which ultimately allows the development of new types of electrochemical biosensors for different purposes. Depending on the scope of application, different types of electrochemical biosensors have been developed, and glucose biosensors have undergone rapid evolution.

Another modern invention is a biochemical sensor for assessing the levels of reactive oxygen species in physiological systems. An important area of application of this direction is the identification of uric acid as the most important end product of purine metabolism in body fluids, which can act as a tool for the diagnosis of various clinical anomalies and diseases. However, an extremely important task is to develop a cost-effective and sensitive method. Similar to the situation with the quantitative measurement of glucose levels, the electrochemical approach to assessing the level of uric acid oxidation looks like an ideal candidate. However, the similarity in terms of oxidative processes of uric acid with ascorbic acid seriously complicates the development of a highly sensitive electrochemical biosensor. To overcome this problem, the authors have developed a biosensor based on amperometric detection, which allows measuring both reducing and oxidative potentials. Considering the cost and reproducibility of this procedure, an important point is the immobilization or application of enzymes to the surface of conventional electrodes or electrodes based on nanomaterials, ideal for the development of single-use selective, cost-effective and sensitive biosensors for routine measurement of uric acid levels. In this regard, recent advances in the field of three-dimensional bioprinting look very promising, the purpose of which is to create biosensors based on living cells encapsulated in a three-dimensional microenvironment. The newly developed wireless biosensor-capa for continuous real-time determination of the concentration of uric acid in saliva belongs to the same direction. This technology can be used in the development of wearable devices on the body, for monitoring various health parameters. Electrochemical sensors have already been successfully used to measure hormone levels, but the prospects for using this approach still require detailed consideration. Another potential direction of technological developments in the field of biosensors is based on targeted effects on nucleic acids. It is well known that cellular expression of small interfering RNAs (miRNAs) is an ideal biomarker for the diagnosis of disease manifestation and exposure to it increases the effectiveness of gene therapy for genetic diseases. miRNAs are usually detected by northern blotting, microchips, and polymerase chain reaction (PCR). Modern technologies make it possible to create ideal electrochemical biosensors for detecting miRNAs, the principle of operation of which is based on the tagless identification of the guanine oxidation reaction triggered by the formation of a hybrid between miRNAs and the corresponding capture probe based on an inosine analog. All these inventions appeared thanks to modern approaches used to promote the technology of electrochemical biosensors in biomedicine.

Environmental monitoring is another important area in which biosensor technology is in demand for the rapid detection of residual amounts of pesticides to eliminate threats to human health. Traditional approaches, such as high-performance liquid chromatography, capillary electrophoresis and mass spectrometry, are effective as methods for detecting pesticides in the environment, but they have limitations due to the complexity of implementation, large time costs, the need for high-tech tools and operational capabilities. Therefore, simple biosensors have a huge advantage, but the development of a unified device for the analysis of various classes of pesticides is a very difficult task. To solve this problem, enzyme-based biosensors, namely acetylcholinesterase inhibitors, have been developed to determine the physiological contribution of pesticides to the environment, food safety and quality control. Over the past 10-20 years, this technology has been further refined for rapid analysis using immobilization techniques and other manufacturing strategies. Similarly, piezoelectric biosensors have been developed to detect the environmental effects of organophosphate and carbamic acid pesticides. Organochlorine pesticides are known to have an adverse effect on the environment, while such as endosulfan can cause serious damage. Pesticides of this class have different effects on the reproductive system of male and female fish. Considering this fact, as well as the phenomenon of biological accumulation (biomagnification), the invention of biosensors for the analysis of aquatic ecosystems would be very useful. In order to meet this need, a revolution has taken place in the field of biosensors, within the framework of rapid progress in the methods of manufacturing and using nanomaterials, quartz crystals or silicon dioxide. It is very important to pay special attention to the selection of receptors for the development of biosensors, the use of various signal conversion technologies and rapid screening strategies for use in the fields of food and environmental safety, as well as monitoring. To achieve this, the manufacture of biosensors is of great importance; achievements in this field are clearly described below.

Optical/visual biosensors

As described above, biomedical and environmental areas require the development of simple, fast-working and highly sensitive biosensors. This can be realized with the help of immobilizers, which can be made of gold, carbon-based materials, quartz or glass. In fact, the incorporation of gold nanoparticles or quantum dots using the microtechnology method is a new technology for the development of highly sensitive and portable biosensors based on the cytochrome P450 enzyme for use for certain purposes. Moreover, fiber-optic chemical sensors are very relevant for various fields, such as the search for new drugs, biosonding and biomedicine. Recently, hydrogels used as DNA-based sensors have become popular as materials for immobilization in fiber-optic chemistry. Compared to other materials, immobilization in a hydrogel occurs in three dimensions, which ensures the loading of a large number of sensitive molecules. Hydrogels (polyacrylamide) are hydrophilic polymers with cross-links, which can be given different shapes for immobilization, ranging from thin films to nanoparticles. Hydrogels are considered to be a simple substrate for DNA immobilization, having a number of advantages, such as the ability to retain molecules, their controlled release, enrichment of analytes and protection of DNA. These characteristics are unique for hydrogels compared to other materials suitable for biomolecular immobilization. Moreover, the good optical transparency of hydrogels makes it possible to apply a convenient visual detection strategy. Methods of immobilization of DNA biosensors in monolithic polyacrylamide gels and gel microparticles are often considered as a technical achievement in the field of biosensor technologies. The identification of single molecules for DNA identification has also become possible with the help of electrochemical oxidation of hydrazine.

Biosensors based on silicon oxide, quartz and glass

The search for new methods for the development of biosensors has led to the use of materials with unique properties of silicon oxide, quartz and glass. Among these materials, a special place is occupied by nanomaterials based on silicon oxide, which have the highest potential for use in the production of biosensors due to their biocompatibility, accessibility, as well as electronic, optical and mechanical properties. Moreover, such materials are non-toxic, which is a very important condition for biomedical and biological applications. Silicon oxide-based materials can be used for bio-imaging, biosensory analysis and in cancer therapy. In addition, fluorescent materials based on silicon oxide have long been used in bio-imaging. An interesting fact is that silicon oxide nanowires in combination with gold nanoparticles are hydride structures used as part of revolutionary approaches to cancer treatment. Covalent attachment of thiol-modified DNA oligomers to silicon oxide or glass ensures the formation of DNA films that increase the efficiency of UV spectroscopy and hybridization methods. Despite the many advantages of using silicon oxide nanoparticles, there are a number of difficulties that need to be solved, such as the development of methods for large-scale low-cost production, as well as biocompatibility after biomolecular contact. The solution of these issues will provide the possibility of converting silicon oxide-based nanomaterials into components of modern biosensors. Having no wires and electrodes, biosensors based on microweights on a quartz crystal represent another platform for analyzing interactions between biomolecules with high sensitivity. Pulsations of quartz oscillators are excited and recorded using antennas through electromagnetic waves without wired connections. This precise non-contact change is a key moment for the development of ultra-high-sensitivity identification of proteins in a liquid using measuring devices based on biosensors on quartz crystals. The unique characteristics of materials based on silicon oxide, quartz or glass have allowed the development of several new high-tech biosensors to improve measuring instruments used in the field of biomedical technologies, but their economic feasibility and biological safety require attention.

Biosensors based on nanomaterials

A wide range of nanomaterials are used to immobilize biosensors, including gold, silver, silicon oxide and copper nanoparticles, as well as carbon-based materials such as graphite, graphene and carbon nanotubes. In the development of electrochemical and other biosensors, nanoparticle-based materials provide high sensitivity and specificity. Among metal nanoparticles, gold nanoparticles that are resistant to oxidation and practically non-toxic are the most suitable for practical use. At the same time, nanoparticles from other metals, such as silver, when introduced into the body, for example, for the delivery of drugs, are oxidized and have a toxic effect. In general, the use of nanomaterials as part of biomedical biosensors is associated with potential difficulties. Moreover, signal amplification strategies using nanoparticles have potential advantages and disadvantages. Nevertheless, nanomaterials are considered important components of bioanalytical devices due to their ability to increase sensitivity and detection thresholds when detecting single molecules. In this context, it is worth mentioning the invention of platinum-based nanoparticles for electrochemical amplification with a single-level reaction to detect a low concentration of DNA. Similarly, semiconductor quantum dots and iron oxide nanocrystals with both optical and magnetic properties can be effectively bound to tumor-specific ligands, such as monoclonal antibodies, peptides or small molecules, for targeting tumor antigens with high affinity and specificity. Quantum dot technology can be used in the study of the tumor microenvironment during therapy, as well as for the delivery of nanopreparations.

Genetically encoded or synthetic fluorescent biosensors

The development of labeled biosensors using genetically encoded or synthetic fluorescence has provided an opportunity to study biological processes, including various molecular pathways inside the cell. In fact, the method of detecting fluorescence-labeled antibodies was first developed to obtain images of fixed cells. This strategy has provided new opportunities for the development of such biosensors using biological proteins and small molecules that bind to analytes and secondary messengers. Subsequently, fluorescent biosensors were developed for the analysis of motor proteins using the method of detecting single molecules at a certain concentration of analyte. Despite these advantages, the methodology for identifying and analyzing the label looks complicated. The invention of green fluorescent protein and other fluorescent proteins has provided a number of advantages in terms of design and efficiency of the optical probe. Over the past decade, genetically encoded biosensors specific to molecules involved in energy synthesis, reactive oxygen species, and cAMP have made it possible to better understand the physiology of mitochondria. cAMP is an important signaling molecule and a therapeutic target for the cardiovascular system. With this in mind, biosensors operating on the basis of the method of resonant fluorescence energy transfer have been developed to visualize cGMP, cAMP and calcium ions inside the cell. Some of these sensors are effectively used for in vivo imaging in primary cultures and living cells. To date, quite a lot of aspects of the development of sensors for visualization in a living organism have been worked out. Small-angle X-ray scattering for the development of calcium channels and resonant fluorescence energy transfer for the detection of kinases, which appeared as a result of the optimization of such approaches, are recognized as the best biosensory techniques in modern physiology. Thus, a number of biosensors specific to specific targets based on microorganisms (microbes) and cellular organelles have been developed. As explained earlier, electrochemical, electromechanical and optical biosensors are being developed for more efficient detection of miRNA compared to other molecular techniques. The advent of in vivo imaging using small molecule biosensors has provided an opportunity to better understand the cellular activity and mechanisms of action of many other molecules, including DNA, RNA and miRNA. To date, transformation in this area requires a genome-wide approach, implying the use of more effective genetic biosensors based on optics. At the present stage, it is believed that optical biosensors combining fluorescence and small molecule/nanomaterial technologies allow achieving the best results in terms of applicability and sensitivity.

Microbial biosensors developed using synthetic biology and genetic/protein engineering

A more recent trend in environmental monitoring and bioremediation (biological purification) implies the use of the latest innovative technologies based on genetic/protein engineering and synthetic biology to program microorganisms, endowing them with specific output signals, sensitivity and selectivity. For example, living cells with enzymatic activity that ensures the degradation of xenobiotic compounds will be widely used in bioremediation. Microbial fuel biosensors have also been developed to monitor biochemical oxygen demand and environmental toxicity. Bacteria have the potential to degrade the organic substrate and generate electricity for fermentation. In essence, the technology consists in using a bioelectrochemical device that regulates the strength of microbial respiration to convert organic substrates directly into electrical energy. Despite these possibilities, the limitations of the use of microbial biosensors are due to the low specific power in terms of cost and operating costs. The specialists managed to significantly improve productivity and reduce costs with the help of new system approaches, which made it possible to create a platform based on these technologies for the development of microbial biosensors with the desired properties with an autonomous power source. Another area of use of microbial biosensors is their use for the detection of pesticides and heavy metals, in which eukaryotic microorganisms have an advantage over prokaryotes. This is mainly due to the advantage of developing whole-cell biosensors for detecting the toxicity of pesticides and heavy metals with high selectivity and sensitivity. In addition, more complex eukaryotic microorganisms may have a wider range of sensitivity to various toxic molecules and have a greater affinity for higher animals. Microbial biosensors have a wide range of applications, ranging from environmental monitoring to energy production. In the future, such microbial biosensors will be more widely used in monitoring environmental pollution by heavy metals and eco-efficient power generation.

Continuation: Technological comparison of biosensors.

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15.06.2017