Biosensors are defined as devices that integrate a specific biomolecule (or, a bioreceptor),

binding specifically to a target analyte, to a transducing element, which then converts the

biological signal into another energy form (conductivity/resistivity/voltage/wavelength

change, etc.). Since their conception in the 1960s, biosensors have come a long way from the

traditional membrane-based enzyme electrodes. In the current times, technological advances

have allowed the development of biosensing devices like dermal tattoos, paperfluidics, and

thread-based devices that can monitor analyte concentrations in different body fluids

including sweat/interstitial fluid, tears and, saliva [36, 69-72]. Different biosensing

architectures have emerged that cater to different kinds of applications, e. g., microelectrodes

for ion detection, microfluidic biosensors for health care applications, and microbial fuel cells

for environmental monitoring [24, 73-76]. The various applications of biosensors are shown

in Figure 1.7. The reasons for their wide success and applicability are quantitative responses,

ease of miniaturization, low power requirements, small volume requirements, and ease of use

[73]. They are designed to be used outside of a laboratory, having wide implications in field

of health care and emergency medicine. Further, the utilization of nanomaterials in biosensors

has enabled faster response and improved sensitivity and specificity [77] [78]. Many

biosensing modalities even allow real time monitoring of target analytes/parameters. These

advantages have made biosensors an attractive alternative to the conventional diagnostic

technologies.

Typically, a biosensor is fabricated by integrating the following elements: i) bioreceptor

(antibodies, enzymes, DNA, live cells, etc. depending upon the target), ii) immobilization

matrix for immobilization of bioreceptors (nanomaterials, metallic electrodes, polymer films,

etc.), and iii) transducer that transforms the biological recognition signal to an observable

electrochemical/electrical/optical/piezoelectric signal (Figure 1.8). These are discussed in

detail in the following sections.

Figure 1.7. Flow chart depicting the various applications of biosensors.
Figure 1.8. Basic components of a biosensor.

Bioreceptors

Bioreceptors refer to biomolecules that possess binding affinity to specific analyte(s).

All cellular processes are based upon the principle of ligand -receptor binding, wherein

receptors are molecules that bind specifically and with high affinity to a single (or, a family

of) biomolecule, referred to as the ligand [79]. This binding/recognition principle is utilized

by biosensors. The primary factors that are involved in the choice of a particular bioreceptor

include the type of target analyte to be detected, degree of specificity required in the

biosensing device, and the application of biosensor. Enzymes and antibodies are the most

utilized bioreceptor in biosensing assays. Enzymes bind to specific substrates and process

them into products, which is often accompanied by exchange of electrons (current flow). This
current generated can be easily detected by an electrode. Thus, enzymatic biosensors are

highly specific and sensitive in nature. The integration of nanomaterials with enzymes has

further led to an increase in biosensor performance in terms of sensitivity [80, 81]. However,

enzymes are not very thermostable and can degrade readily with slight fluctuations in

temperature or ion concentration [80]. Antibodies are immune-mediating molecules with high

binding affinity for specific target analytes. Monoclonal antibodies can bind to only one

specific analyte, while polyclonal antibodies bind to analogous but different molecules.

Contrary to enzymes, antibody-target interaction does not lead to any change in current,

hence antibody-based biosensors (immunosensors) can often use labels for signal

amplification [82]. Like enzymes, antibodies also suffer from the limitations of low

thermostability and degradation with repeated thawing cycles. Antibodies are also very

expensive to produce and purify. Whole living cells-based biosensors possess applications in

environmental monitoring, food analysis, pharmacology, heavy metals, pesticides, detection

of organic contaminants, and drug screening [83]. This is because living cells produce

stimuli-specific responses and are inexpensive to obtain. However, they are not suitable for

every application due to complexities involved in signal analysis.

Nucleic acids (NA) (like DNA, RNA, peptide nucleic acids (PNA), aptamers, DNA

nanostructures, etc.) have increasingly become the prominent choice as bioreceptors. Unlike

enzymes and antibodies, NAs are highly thermostable and have a long shelf life. They do not

lose their binding/recognition ability even after immobilization to a substrate, which is often a

requirement in biosensors. NA biosensors have found applications in various fields including

infectious agent detection, pollution monitoring, sequence specific information gene ration,

mutation analysis, etc. [84, 85]. All types of NA molecules consist of nitrogenous bases

((adenine (A), guanine (G), cytosine (C), thymine (T) or uracil (U)) linked to sugar molecules

(ribose in RNA and deoxyribose in DNA). The sugar molecules are, in turn, linked to
phosphate groups. These three building blocks make up the monomeric unit of NAs known as

nucleotides. The nitrogenous bases are highly specific in binding to each other; A always

forms two H-bonds with T (or, U) and G always forms three H-bonds with C. This specific

base pairing (also known as complementary base pairing) is the basic principle of the

simplest NA sensors.

In the simplest mechanism of a DNA biosensor, a single stranded DNA (ssDNA) is

immobilized onto the biosensing substrate/transducer and allowed to bind to the target DNA

strand via complementary base pairing. The ssDNA utilized as the bioreceptor is often

referred to as the ‘capture’ probe. To enhance the sensitivity and specificity of the biosensors,

different hybridization strategies have been developed over the years.

Transducers

Transducers can be roughly classified into the following groups: electrochemical,

optical, thermal, electronic, and gravimetric. Electrochemical transducers are the most

extensively studied and utilized modality of biosensors. They produce detectable

electrochemical signals in the form of voltage (potentiometric), current

(amperometric/voltammetric), and impedance (impedimetric) as a result of interactions

between the bioreceptor and specific analyte on the transducer surface [97].

a) Electrochemical transducers: Electrochemical transducers have become a

cornerstone in the development of biosensors, offering numerous advantages that make them

highly attractive for various applications. These transducers play a pivotal role in converting

a biological interaction between the bioreceptor and target analyte into an electrical signal,

enabling accurate and sensitive detection in biosensing devices [97]. Electrochemical

transducers are known to be highly sensitive to minute changes in target concentration ,

making them invaluable tools in fields like medical diagnostics, environmental monitoring,
and food safety [97-99]. They can be applied to detect a wide range of analytes, including

ions, small molecules, proteins, and nucleic acids. Electrochemical biosensors offer the

advantage of real-time monitoring. Additionally, the rapid response times allow for

continuous measurements, making them suitable for point-of-care (PoC) applications and

situations where immediate detection is essential, such as glucose monitoring in diabetes

management [98, 100]. Electrochemical transducers can be easily integrated into electronic

devices using cost-effective methods, enabling sensor networking and addition of other

integrative technologies such as Internet of things (IoT) [101, 102]. An electrochemical

biosensor consists of a three-electrode system, with the biosensing platform referred to as the

working electrode (WE). This WE is connected to a counter (CE) (mostly Platinum, Pt) and a

reference electrode (RE) (Silver-silver chloride, Ag/AgCl) to complete the circuit and keep

potential at the WE constant at any particular instant, respectively (Figure 1.11(A-C)).

Figure 1.11. (A) Schematic of the 3-electrode assembly showing the WE, RE, and CE
connected to a potentiostat, (B) the corresponding circuit diagram, and (C) The redox
reaction taking place at the WE surface [103]. (D) Image of a commercial screen printed
electrode (SPE) [104].