1
General Introduction to Biosensors and Recognition Receptors

Frank Davis1 and Zeynep Altintas2

1 Department of Engineering and Applied Design, University of Chichester, Chichester, UK

2 Technical University of Berlin, Berlin, Germany

1.1 Introduction to Biosensors

There are laboratory tests and protocols for the detection of various biomarkers, which can be used to diagnose heart attack, stroke, cancer, multiple sclerosis, or any other conditions. However, these laboratory protocols often require costly equipment, and skilled technical staff, and hospital attendance and have time constraints. Much cheaper methods can provide cost-effective analysis at home, in a doctor's surgery, or in an ambulance. Rapid diagnosis will also aid in the treatment of many conditions. Biosensors generically offer simplified reagentless analyses for a range of biomedical [1-8] and industrial applications [9, 10]. Due to this, biosensor technology has continued to develop into an ever-expanding and multidisciplinary field during the last few decades.

The IUPAC definition of a biosensor is "a device that uses specific biochemical reactions mediated by isolated enzymes, immunosystems, tissues, organelles or whole cells to detect chemical compounds usually by electrical, thermal or optical signals." From this definition, we can gain an understanding of what a biosensor requires.

Most sensors consist of three principal components:

1. Firstly there must be a component, which will selectively recognize the analyte of interest. Usually this requires a binding event to occur between the recognition element and target.
2. Secondly some form of transducing element is needed, which converts the biochemical binding event into an easily measurable signal. This can be a generation of an electrochemically measurable species such as protons or H2O2, a change in conductivity, a change in mass, or a change in optical properties such as refractive index.
3. Thirdly there must be some method for detecting and quantifying the physical change such as measuring an electrical current or a mass or optical change and converting this into useful information.

There exist many methods for detecting binding events such as electrochemical methods including potentiometry, amperometry, and AC impedance; optical methods such as surface plasmon resonance; and piezoelectric methods that measure mass changes such as quartz crystal microbalance (QCM) and surface acoustic wave techniques. A detailed description of these would be outside the remit of this introduction, but they are described in many reviews and elsewhere in this book. Instead this chapter focuses on introducing the recognition receptors used in biosensors.

1.2 Enzyme-Based Biosensors

Leyland Clark coated an oxygen electrode with a film containing the enzyme glucose oxidase and a dialysis membrane to develop one of the earliest biosensors [11]. This could be used to measure levels of glucose in blood; the enzyme converted the glucose to gluconolactone and hydrogen peroxide with a concurrent consumption of oxygen. The drop in dissolved oxygen could be measured at the electrode and, with careful calibration, levels of blood glucose calculated. This led to the widespread use of enzymes in biosensors, mainly driven by the desire to provide detection of blood glucose. Diabetes is one of the major health issues in the world today and is predicted to affect an estimated 300 million people by 2045 [12]. The world market for biosensors was approximately $15-16 billion in 2016. In 2009 approximately half of the world biosensor market was for point-of-care applications and about 32% of the world commercial market for blood glucose monitoring [13].

Enzymes are excellent candidates for use in biosensors, for example, they have high selectivities; glucose oxidase will only interact with glucose and is unaffected by other sugars. Being highly catalytic, enzymes display rapid substrate turnovers, which is important since otherwise they could rapidly become saturated or fail to generate sufficient active species to be detected. However, they demonstrate some disadvantages: for instance, a suitable enzyme for the target of interest may simply not exist. Also enzymes can be difficult and expensive to extract in sufficient quantities and can also be unstable, rapidly denaturing, and becoming useless. They can also be subject to poisoning by a variety of species. Moreover, detection of enzyme turnover may be an issue, for instance, in the glucose oxidase reaction; it is possible to directly electrochemically detect either consumption of oxygen [11] or production of hydrogen peroxide. However in samples such as blood and saliva, there can be other electroactive substances such as ascorbate, which also undergo a redox reaction and lead to false readings. These types of biosensors are often called "first-generation biosensors." To address this issue of interference, a second generation of glucose biosensors was developed where a small redox-active mediating molecule such as a ferrocene derivative was used to shuttle electrons between the enzyme and an electrode [14]. The mediator readily reacts with the enzyme, thereby avoiding competition by ambient oxygen. This allowed much lower potentials to be used in the detection of glucose, thereby reducing the problem of oxidation of interferents and increasing signal accuracy and reliability. Figure 1.1 shows a schematic of a second-generation glucose biosensor.

Figure 1.1 Schematic of a second-generation biosensor.

Third-generation biosensors have also been developed where the enzyme is directly wired to the electrode, using such materials as osmium-containing redox polymers [15] or conductive polymers such as polyaniline [16]. More recently nanostructured materials such as metal nanoparticles, carbon nanotubes, and graphene have been used to facilitate direct electron transfer between the enzyme and the electrode as described in later chapters. As an alternative to glucose oxidase, sensors based on glucose dehydrogenase have also been developed.

The techniques for glucose sensing using glucose oxidase can be applied to almost any oxidase enzymes, allowing sensors to be developed based on cholesterol oxidase, lactate oxidase, peroxidase enzymes, and many others. Sensors have also been constructed using urease, which converts urea to ammonia, causing a change in local pH that can be detected potentiometrically or optically by combining the enzyme with a suitable optical dye. Enzyme cascades have also been developed; for example, cholesterol esters can be determined using electrodes containing cholesterol esterase and cholesterol oxidase. Applications of enzyme-containing biosensors have been widely reviewed [16-18].

1.3 DNA- and RNA-Based Biosensors

DNA is contained within all living cells as a blueprint for making proteins, and it can be thought of as a molecular information storage device. RNA also has a wide number of applications in living things, including acting as a messenger between DNA and the ribosomes that synthesize proteins and as a regulator of gene expression. Both DNA and RNA are polymeric species based on a sugar-phosphate backbone with nucleic bases as side chains, in DNA, namely, adenine, cytosine, guanine, and thymine. In RNA uracil is utilized instead of thymine. It is the specific binding between base pairs, that is, guanine to cytosine or adenine to thymine (uracil), that determine the structure of these polymers, in the case of DNA leading to a double helix structure (Figure 1.2) [19].

Figure 1.2 Schematic of interstrand binding in DNA.

DNA sensors are usually of a format where one oligonucleotide chain is bound to a suitable transducer, that is, an electrode, surface plasmon resonance (SPR) chip, quartz crystal microbalance (QCM), and so on, and is exposed to a solution containing an oligonucleotide strand of interest [20]. The surface-bound oligonucleotide is selected to be complementary to the oligonucleotide of interest, and the bound and solution strands will undergo sequence-specific hybridization as the recognition event.

An in-depth review of DNA sensing is outside the scope of this introduction and has been reviewed elsewhere [20-24]; however, a few examples are given here. A method based on ruthenium-mediated guanine oxidation allowed selective electrochemical detection of messenger RNA from tumors at 500?zmol?L-1 levels [25]. A sandwich-type assay using magnetic beads and fluorescence analysis utilized a complementary nucleotide to dengue fever virus RNA to allow detection at levels as low as 50?pmol?L-1 [26]. Five different probe DNAs could be immobilized onto an SPR-imaging chip and simultaneously used to determine binding of RNA sequences found in several pathogenic bacteria such as Brucella abortus, Escherichia coli, and Staphylococcus aureus [27] for use in food safety.

1.4 Antibody-Based Biosensors

Antibodies are natural Y-shaped proteins produced by living systems, usually as a defense mechanism against invading bacteria or viruses. They bind to specific species (antigens) with an extremely high degree of specificity by a mixture of hydrogen bonds and other non-covalent interactions, with the binding taking place in the cleft of the protein molecule...