Biosensors Based on Aptamers and Enzymes

Biosensors representing the technological counterpart of living senses have found routine application in amperometric enzyme electrodes for decentralized blood glucose measurement, interaction analysis by surface plasmon resonance in drug development, and to some extent DNA chips for expression analysis and enzyme polymorphisms. These technologies have already reached a highly advanced level and need minor improvement at most. The dream of the “100-dollar” personal genome may come true in the next few years provided that the technological hurdles of nanopore technology or of polymerase-based single molecule sequencing can be overcome. Tailor-made recognition elements for biosensors including membrane-bound enzymes and receptors will be prepared by cell-free protein synthesis. As alternatives for biological recognition elements, molecularly imprinted polymers (MIPs) have been created. They have the potential to substitute antibodies in biosensors and biochips for the measurement of low-molecular-weight substances, proteins, viruses, and living cells. They are more stable than proteins and can be produced in large amounts by chemical synthesis. Integration of nanomaterials, especially of graphene, could lead to new miniaturized biosensors with high sensitivity and ultrafast response. In the future individual therapy will include genetic profiling of isoenzymes and polymorphic forms of drug-metabolizing enzymes especially of the cytochrome P450 family. For defining the pharmacokinetics including the clearance of a given genotype enzyme electrodes will be a useful tool. For decentralized online patient control or the integration into everyday “consumables” such as drinking water, foods, hygienic articles, clothing, or for control of air conditioners in buildings and cars and swimming pools, a new generation of “autonomous” biosensors will emerge.

It has been 20 years since aptamer and SELEX (systematic evolution of ligands by exponential enrichment) were described independently by Andrew Ellington and Larry Gold. Based on the great advantages of aptamers, there have been numerous isolated aptamers for various targets that have actively been applied as therapeutic and analytical tools. Over 2,000 papers related to aptamers or SELEX have been published, attesting to their wide usefulness and the applicability of aptamers. SELEX methods have been modified or re-created over the years to enable aptamer isolation with higher affinity and selectivity in more labor- and time-efficient manners, including automation. Initially, most of the studies about aptamers have focused on the protein targets, which have physiological functions in the body, and their applications as therapeutic agents or receptors for diagnostics. However, aptamers for small molecules such as organic or inorganic compounds, drugs, antibiotics, or metabolites have not been studied sufficiently, despite the ever-increasing need for rapid and simple analytical methods for various chemical targets in the fields of medical diagnostics, environmental monitoring, food safety, and national defense against targets including chemical warfare. This review focuses on not only recent advances in aptamer screening methods but also its analytical application for small molecules.

The process of “structure-switching” enables biomolecular switches to function as effective biosensing tools. Biomolecular switches can be activated or inactivated by binding to a specific target that triggers a precise conformational change in the biomolecules involved. Although many examples of aptamer-based biomolecular switches can be found in nature, substantial effort has been made in the last decade to engineer structure-switching aptamer sensors by coupling aptamers to a signal transduction method to generate a readout signal upon target binding to the aptamer domain. This chapter focuses on the progress of research on engineered structure-switching aptamer sensors. We begin by discussing the origin of the structure-switching aptamer design, highlight the key developments of structure-switching DNA aptamers for fluorescence-, electrochemistry-, and colorimetry-based detection, and introduce our recent efforts in exploring RNA aptamers to create structure-switching molecular sensors.

Recent progress in using DNAzyme-functionalized gold nanoparticles (AuNPs) for biosensing is summarized in this chapter. A variety of methods, including those for attaching DNA on AuNPs, detecting metal ions and small molecules by DNAzyme-functionalized AuNPs, and intracellular applications of DNAzyme-functionalized AuNPs are discussed. DNAzyme-functionalized AuNPs will increasingly play more important roles in biosensing and many other multidisciplinary applications.

This chapter covers the recent advancement in biosensing applications of DNAzyme-functionalized gold nanoparticles, including the detection of metal ions, small molecules, and intracellular imaging.

Aptamers are short oligonucleotides that are capable of selectively binding to their corresponding target. Therefore, they can be thought of as a nucleic acid-based alternative to antibodies and can substitute for their amino acid-based counterparts in analytical applications, including as receptors in biosensors. Here they offer several advantages because their nucleic acid nature and their binding via an induced fit mechanism enable novel sensing strategies. In this article, the utilization of aptamers as novel bio-receptors in combination with nanoparticles as transducer elements is reviewed. In addition to these analytical applications, the medical relevance of aptamer-modified nanoparticles is described.

Aptamers are DNA and RNA oligonucleotides that can bind to a variety of nonnucleic acid targets with high affinity and specificity. Pathogen detection is a promising area in aptamer research. One of its major advantages is the ability of the aptamers to target and specifically differentiate microbial and viral strains without previous knowledge of the membrane-associated antigenic determinants or molecular biomarkers present in that particular microorganism. Electrochemical sensors emerged as a promising field in the area of aptamer research and pathogen detection. An electrochemical sensor is a device that combines a recognition element and an electrochemical transduction unit, where aptamers represent the latest addition to the large catalog of recognition elements. This chapter summarizes and evaluates recent developments of electrochemical aptamer-based sensors for microbial and viral pathogen detection, viability assessment of microorganisms, bacterial typing, identification of epitope-specific aptamers, affinity measurement between aptamers and their respective targets, and estimation of the degree of aptamer protection of oncolytic viruses for therapeutic purposes.

Theranostics, a new term consisting of the words “therapy” and “diagnostics,” represents the concept of selecting specific patients for appropriate drug administration using diagnostics. For the development of a molecular targeting drug, the theranostics approach is effective. Therefore, the market for molecular diagnostics is likely to grow at an extraordinary rate over the next 10 years. In this review, we focus on aptamer-based electrochemical biosensors for theranostics. Aptamers are molecular recognition elements that can bind to various target molecules from small compounds to proteins with affinities and specificities comparable to those of antibodies. Inasmuch as various molecules would be targeted for analysis using theranostics, aptamer-based biosensors would be an attractive format because they can be developed for various molecules using the same sensing format. Although a diverse sensing system can be constructed, we focus on electrochemical biosensors in this review because they can measure biomarkers rapidly in a miniaturized sensing system with low cost, such as blood glucose sensors. We summarize the sensing systems of aptamer-based electrochemical biosensors and discuss their advantages for theranostics.

Glucose biosensors have an important place in the diagnosis of diabetes as well as in various food and biotechnological processes. Recent advances in nanomaterials have directly improved enzymatic glucose biosensors owing to their distinguished structural and physiochemical properties. Here, we review the recent developments in electrochemical and fluorescent glucose biosensors based on nanomaterials. New technologies that combine nanomaterials with glucose-sensing enzymes will result in promising glucose biosensors with high specificity and sensitivity.

Since the first glucose biosensor was developed by Clark and Lyons, there have been great efforts to develop effective enzyme biosensors for wide applications. Those efforts are closely related to the enhancement of biosensor performance, including sensitivity improvement, elevation of selectivity, and extension of the range of analytes that may be determined. Introduction of a cascadic multienzyme reaction to the electrochemical biosensor is one of those efforts. By employing more than two enzymes to the biosensor, its sensitivity and accuracy can be enhanced. Also, the narrow application range that is a typical limitation of single enzyme-based biosensor can be overcome. This chapter will discuss the fundamental principles for the development of cascadic multienzyme reaction-based electrochemical biosensors and their applications in clinical and environmental fields.

This chapter provides an overview of different assembly methodologies used for the construction of multilayer architectures with biomolecules for application in sensors. Besides the use of bioaffinity interactions and covalent strategies, special attention will be paid to the electrostatic layer-by-layer technique. Different building blocks can be used for the formation of multilayers with a clear preference for polymers and nanoparticles. Among the biomolecules, enzymes and redox proteins are in the focus. Because of the high importance of multilayers formed on electrodes, the chapter will concentrate on sensor systems with electrochemical transduction. Particularly advantageous are schemes that can avoid diffusible shuttling molecules between the biomolecule and the electrode and that represent artificial signal chains by exploiting direct protein–protein communication in the immobilized state.

The present chapter describes the use of biosensors based on enzyme inhibition as analytical tools. The parameters that affect biosensor sensitivity, such as the amount of immobilized enzyme, incubation time, and immobilization type, were critically evaluated, highlighting how the knowledge of enzymatic kinetics can help researchers optimize the biosensor in an easy and fast manner. The applications of these biosensors demonstrating their wide application have been reported. The objective of this survey is to give a critical description of biosensors based on enzyme inhibition, of their assembly, and their application in the environmental, food, and pharmaceutical fields.