Biosensing Basics

Abstract

The aim of this chapter is to understand biosensor basics. A biosensor is a sophisticated analytical device that uses a biological sensing component to detect biological or chemical reactions. It combines an electronic component with a biological element, producing quantifiable signals and detects physiological changes, environmental components, diseases, harmful chemicals, and pH values in various sizes and designs. Biosensors detect substances by detecting an analyte, such as glucose, creatinine, lactate, L-phenylalanine, L-alanine, pyruvate, salicylate, and urea. Biosensors, including DNA, are crucial in medical and environmental monitoring due to their sensitivity, selectivity, reproducibility, linearity, and stability. They are immobilized using physical and chemical methods, with chemical immobilization involving chemical interactions between biorecognition elements and transducer surfaces. Physical immobilization involves affixing enzymes to the transducer’s surface without chemical bonds, such as entrapment, microencapsulation, electropolymerization, and adsorption. Biosensors are essential for managing human health, identifying diseases, rehabilitating patients, and monitoring their health. They detect bacteria, viruses, and pathogens, and can enhance healthy behavior through step and activity trackers. They are used in various medical sciences, including post-surgery activities, glucose monitoring, biological abnormalities, inpatient detection, biomolecular detection, heart rate tracking, body chemistry, diet monitoring, air quality tracking, accurate results, patient status, and disease management.

1. Introduction to biosensor

A biosensor is an advanced analytical tool that detects biological or chemical reactions by combining an electronic component with a biological element, such as an enzyme or antibody. This produces a quantifiable signal proportional to the analyte’s concentration. Biosensors come in various shapes and sizes, capable of measuring and detecting even small amounts of diseases, dangerous substances, and pH levels [1, 2].

2. Working of biosensors

There are the following elements of a conventional biosensor (Figure 1).

Figure 1.

Biosensor elements/steps.

2.1 Analyte

The detection in a biosensor of the required substance is known as an “analyte.” Glucose and lactate continue to be the key analytes of interest among those reported in the literature. A viable commercial solution will likely require significant improvements to sensor life and performance with untreated blood samples of varied viscosity and hematocrit. However, many of the sensors would require dilution of samples for clinical applications. The common analytes are glucose, creatinine, lactate, L-phenylalanine, L-alanine, pyruvate, pyruvate, salicylate, and urea [2, 3, 4].

2.2 Bioreceptor/biorecognition

A bioreceptor is a molecule that identifies the analyte in a specific manner. A small number of examples include cells, DNA, enzymes, aptamers, and antibodies. The best bioreceptors, which are used in the formation of electrochemical DNA biosensors for the purpose of food-borne detection of illnesses, are aptamers or single-stranded nucleic acids. The bioreceptor and the target react to one another on the electrode surface. DNA is a biosensor. Two different types of DNA exist: aptamer DNA, which is synthetic DNA produced intentionally in vitro using a known base sequence, and naturally occurring recognition element DNA. “Genosensors” are biosensors that use naturally occurring DNA as a bioreceptor. Such sensors typically target the DNA of infections. By pairing complementary bases, DNA probes immobilized on electrode surfaces can identify and hybridize with targets’ DNA. Aptamers made of tiny molecules, entire cells, and high-molecular-weight substances can also recognize and capture targets. Aptasensors are biosensors that use aptamers as bioreceptors. We credit the strong affinity between the target and the single-stranded DNA (ssDNA) or aptamer for the high selectivity of electrochemical DNA biosensors.

Biorecognition is the procedure of signal development following the association of the bioreceptor with an analyte. For instance, the conversion of heat, pH, light, charge, etc. A biorecognition element’s main objective is to give a biosensor analyte specificity. Biorecognition components fall into a number of categories, including both naturally occurring and artificial substances. Antibodies and enzymes are examples of naturally occurring biorecognition elements. These biologically derived constructions make use of physiological interactions that have evolved over time. To serve as an example of each category, prominent biorecognition components from each will be succinctly outlined. There are several distinct types of recognition structures that make up each class of biorecognition element [2, 3, 4, 5].

2.2.1 Antibody

Antibody biorecognition elements are affinity based, and the binding event is monitored using colorimetric or piezometric transduction methods. Covalent bonding between antibodies and a sensor surface immobilizes them, generating a brush-like array [6].

2.2.2 Enzymes

The target bioanalyte is captured and catalyzed by enzymes, which act as biocatalytic biosensors to produce a quantifiable end product. Since they are frequently embedded inside surface structures, there are very brief diffusion paths between the transducer and the biorecognition element. Since enzymes frequently reside in surface structures, there are little barriers to diffusion between the biorecognition component and the transducer [6].

2.2.3 Nucleic acid

In order to obtain bioanalyte selectivity, the complementary DNA-binding motif is being used by nucleic acid biosensors (genosensors). Locked nucleic acids (LNAs) and peptide nucleic acids (PNAs) in the usage of nucleic acid recognition elements are recently known to take part in developments. The 3′-endo conformation of the ribose is locked by LNA, which decreases conformational flexibility and enhances binding to the target of corresponding nucleic acid. A repeating aminoethyl-glycine unit connected by a peptide makes up the synthetic oligonucleotide known as PNA. Overall, because their utilization is best suited for biosensor applications that target nucleic acids, nucleic acid biorecognition elements have a very narrow range of applications [6].

2.2.4 Aptamer

The SELEX (Systemic Evolution of Ligands by Exponential Enrichment) method is useful for obtaining single-stranded oligonucleotides named aptamers that are shaped. SELEX is an iterative procedure for significant binding affinities, which lies between the target analyte and oligonucleotide sequences in a collection of randomly produced oligonucleotide sequences. The majority of aptamers are 100 base pairs in length, keeping both ends with consistent primer binding regions and a randomly chosen base pair binding region in the middle [6].

2.2.5 Molecularly imprinted polymers (MIPs)

In order to attain analyte specificity, templated polymer matrix is being used by synthetic biorecognition elements that are known as MIPs. MIPs create artificial patterns of recognition between the polymer matrix and the bioanalyte. MIPs are made to enclose the target bioanalyte. The choice of the target bioanalyte, cross-linker, functional monomer, and solvent affects tunability. Thus, biochemical identification of a particular biorecognition element-bioanalyte pairing is not necessary [6].

2.3 Transducer

Transducer is basically an energy transformer device, that is, it transforms one form of the energy into another. In a biosensor, it quantifies the signal by converting biorecognition activity. Such a technique for converting the energy is referred to as signalization. As an illustration, electrical or optical signals produced by transducers are normally proportional to the interactions’ quantity between the bioreceptor and the analyte.

The five main classes of transducers in biosensors are magnetometric, thermometric, electrochemical, piezoelectric, and optical. These transducers are influenced by the material used, sensor features, and signal conversion technique. Materials can be classified as inorganic, organic, conductor, insulator, semiconductor, or biological. The device’s design also significantly impacts the specifications. The transducer mechanism categorizes biosensors such as electrochemical biosensors. The market offers a wide range of transducing mechanisms for wearable biosensors, with user uptake and accuracy being crucial for developing user-friendly and sustainable technologies. Transducing mechanisms must be built to transform biosensing technology into wearable gadgets [3, 7].

2.4 Electronics

The transducer output signal is directed to the electronics section. Such a signal is analog, which is then transformed into a digital signal by a dedicated electronics circuit. This digital signal is subsequently measured based on its magnitude.

Flexible biosensors have become more common due to advancements in artificial intelligence, human-machine interfaces, and prosthetic skin. These systems consist of a sensor array, low-noise amplifier, analog-to-digital conversion (ADC), digital signal processing (DSP), and wireless transmitter. Integrated circuit development has been influenced by the need for low-noise and low-power sensing signal processing. Sigma-delta and successive approximation register (SAR) ADCs have performance limitations due to comparator and quantization noise. A more quantized capacitive DAC (CDAC) with a greater effective number of bits (ENOBs) is needed, expanding the core size and increasing power consumption [3, 8].

2.5 Display

According to the needs of the end user, this component generates the biosensor’s results as an output signal on the display that may be graphical, tabular, numerical, or an image (Table 1) [3].

Output signal of Biosensors
Sr.#	Measureable outputs	Signal to be read out
1	Formation of Hg^2+-thymine or Ag^+-cytosine complexes and release of fluorophore-labeled aptamers from graphene oxide (GO) surface	Fluorescence
2	Configuration switch of DNA tetrahedron	Fluorescence change
3	RNA amplification and the assembly of a four-way junction structure	Current trace
4	Configuration switch of DNA triangular prism	Intracellular fluorescence resonance energy transfer (FRET) signal
5	Release of G-quadruplex sequence	Blue color of oxidized 3, 3′, 5, 5′-tetramethylbenzidine
6	Hybridization of biotinylated ssDNA on DNA origami and release of fluorophore-labeled aptamers from the GO surface	Nanoscale symbols of Streptavidin on DNA origami
7	Binding of thrombin to its aptamer	Electrical signals
8	Release of DNA from gold nanoparticles (AuNPs)	Aggregation of AuNPs
9	Release of ssDNA from DNA structures with a Y or an X form	Photodynamic therapy effect (PTE) and cell membrane fluorescence
10	Single-stranded DNA (ssDNA) release from DNA triangular prism	Cell membrane-based fluorescence
11	Fluorophore release or quencher modified ssDNA and occurrence of four-way strand exchange reactions	Intracellular fluorescence
12	Release of AS1411 and dimeric assembly of DNA tetrahedron	FRET on cell membrane

Table 1.

Output signals’ shape/mode of biosensors [9].

3. Working principles of biosensors

There are the following working principles for biosensors [10, 11, 12, 13, 14, 15].

• Electrochemistry

• Optics/Fluorescence

• Electromagnetic

4. Features of biosensors

There are the following features of biosensors (Figure 2).

Figure 2.

Features of biosensors.

4.1 Sensitivity

The lowest concentration of analyte that a biosensor can detect is basically its limit of detection (LOD) or sensitivity. To check the occurrence of analyte traces in a sample at analyte concentrations, a biosensor is needed in several environmental tracking and medical applications for dealing with analyte concentrations with a sensitivity as low as nanograms per milliliter (ng/ml), and in some cases, it reaches femtograms per milliliter (fg/ml) [10].

4.2 Selectivity

Selectivity is arguably the most significant element of a biosensor. The bioreceptor’s capability to identify a particular analyte in a sample that contains different contaminants and admixtures is referred to as selectivity. The selectivity in the example is the relationship between an antibody and an antigen. Traditionally, antibodies are fixed to the surface of the transducer to act as bioreceptors. A solution, which is normally a salt containing buffer, is then introduced to the transducer, where there is only an interaction of antibodies with the antigens [10].

4.3 Reproducibility

Reproducibility is the capacity of the biosensor to deliver similar results under the same analysis conditions. The precise and accurate transducer and biosensor electronics define reproducibility. After a sample is checked more than once, precision denotes the sensor’s ability to consistently produce results, whereas capacity deals with accuracy to provide a mean value that is close to the right value. The inference made from a biosensor’s response is particularly robust and reliable while there are repeatable signals [10].

4.4 Linearity

The term linearity in biosensors means a characteristic that shows the accuracy of the measured response for a set of measurements to a straight line with various analyte concentrations in the mathematical equation y = mc, where m is the sensitivity of the biosensor, c is the analyte concentration, and y is the signal at output. Both the range of analyte concentrations and the biosensor’s resolution being tested impacted the biosensor’s linearity. The biosensor resolution is the lowest change in concentration of an analyte that is required to alter the biosensor’s response [10].

4.5 Stability

The word “linear range,” which is linked to linearity, denotes the analyte concentration range for which the response of the biosensor differs linearly with the change in concentration.

By considering application, a strong resolution may be necessary because most biosensor applications require analyte concentration monitoring and not only analyte detection across a wide working range [10].

5. Immobilization techniques in biosensors

To create a biosensor, biological elements must be immobilized on the transducer’s surface. Depending on the component of biorecognition, the physical-chemical environment, the transducer, and the features of the analyte, two well-known ways are physical and chemical immobilization strategies. One of the most crucial steps in sensor preparation is enzyme immobilization, which guarantees that the biomolecules can retain their biological activity, structure, and usefulness [2].

5.1 Chemical immobilization

This technique involves creating powerful chemical interactions between the functional groups of the biorecognition element and the transducer surface, such as covalent binding or covalent connection. Chemical immobilization techniques, on the basis of chemical bonding type, are divided into two groups: (a) direct covalent binding and (b) covalent cross-linking [2].

5.1.1 Direct covalent bonding

The biorecognition component is directly covalently bonded, a technique frequently used to immobilize enzymes, to either the electrode or transducer surface, the inert matrix of the membrane, or both. The production of the functional polymer and covalent immobilization are the two approachable steps. Excellent environmental resilience, less leakage of the biorecognition element (enzyme), and strong bond formation between the biorecognition element (enzyme) and matrix are benefits of direct covalent binding. The inability to replenish the formed matrix after use and the use of harsh chemicals are two downsides of this method [2].

5.1.2 Covalent cross-linking

Cross-linking is a technique where biorecognition elements and inactive proteins create intermolecular covalent cross-linkages, such as enzymes. It uses multifunctional chemicals as linkers to connect enzyme molecules in three-dimensional (3D) aggregates to the transducer surface. The optimal conditions for cross-linking include pH, temperature, and ionic strength. It offers advantages like faster reaction times, stronger adhesion, and higher enzyme catalytic activity. However, partial protein denaturation limits cross-linking immobilization and results in covalent cross-links between protein molecules rather than between the protein and matrix [2].

5.2 Physical immobilization

This method relies on affixing enzymes to the transducer’s surface without using chemical bonds. These immobilizations consist of (a) entrapment and (b) adsorption.

5.2.1 Entrapment

This method involves using 3D matrices to physically entrap biorecognition components through covalent or noncovalent bonds. Enzymes are added to a monomer solution, polymerized by a chemical reaction, or modified experimental conditions. Biorecognition components are connected to organic or inorganic materials in a 3D network. Organic materials include polydimethylsiloxane, photopolymer, gelatin, alginate, cellulose, and acetate phthalate, while inorganic materials include activated carbon and porous ceramic materials. Sol-gel methods, microencapsulation, and electropolymerization are used for this process [2].

5.2.1.1 Sol-gel method

At low temperatures, the sol-gel method is a method used to capture enzymes. Metal alkoxides are hydrolyzed and condensed to produce a nanoporous material that contains bioelements in a three-dimensional matrix. It is more easily synthesized, thermally and chemically stable, and capable of encapsulating large concentrations of biomolecules under mild immobilizing conditions [2].

5.2.1.2 Microencapsulation

A semipermeable membrane is enclosed by biorecognition components (such as enzymes) by using the microencapsulation technique. The two processes that are preferred are the polymerization of a monomer at the interface of two immiscible substances and the phase separation of enzyme microdroplets in water-immiscible liquid phases. As a result, the polymeric membrane’s internal enzyme is obscured [2].

5.2.1.3 Electropolymerization

In electropolymerization, a polymer that traps enzymes near the electrode develops when a current or voltage is functionalized in an electrolyte or aqueous solution that contains monomer molecules and biomolecules. Thiophene, pyrrole, and aniline are the films that are utilized for enzyme immobilization [2].

5.2.2 Adsorption

A biorecognition component is immobilized on the outer surface of an passive solid material using the adsorption approach. This is done by attractive forces but weak like the ionic bonding, van der Waals force, electrostatic force, or hydrogen bonding. It is nondestructive and does not call for the creation of a matrix or the modification of biological components. However, it has weak connections and is susceptible to deviations in ionic strength, pH, and temperature. It has poor operational and storage stability [2].

6. Types of biosensors

6.1 Glucose oxidase-based biosensor

Clark and Lyons discovered the first electrochemical biosensor in 1962 using a glucose oxidase-based glucometer. Electrochemical glucose biosensors dominate the market due to their superior sensitivity, repeatability, maintenance simplicity, and low price. They come in conductometric, amperometric, and potentiometric varieties [16, 17, 18, 19]. The D-glucose oxidation is catalyzed by immobilized glucose oxidase (GOx) in a biosensor of glucose that uses molecular oxygen, resulting in the production of hydrogen peroxide and gluconic acid [20]. A typical enzyme used in biosensors, GOx, has a slightly better selectivity for glucose. Less strict production conditions are achievable since GOx can survive more pH, ionic strength, and temperature fluctuations than many other enzymes [20, 21]. The glucose biosensor consists of an oxygen electrode, an inner semipermeable membrane, a thin layer of GOx, and an outside dialysis membrane. Enzymes can be immobilized to create an enzyme electrode for electrochemical detection. Updike and Hicks improved the electrochemical glucose assay by immobilizing and stabilizing GOx, measuring glucose levels in body fluids for the first time [16, 22]. Due to its high sensitivity and selectivity, cheap cost, and improved patient compliance, glucose oxidase-based biosensor technology is growing to be a significant competitor in the field of glucose-level monitoring [17].

6.2 Glycated hemoglobin (HbA1c) biosensor

In 1976, HbA1c was identified as a helpful tool for assessing diabetic patients’ glycemic management. In the 1980s, the HbA1c test became widely used in clinical settings [23]. The system relies on electrochemistry, with glycated hemoglobin (HbA1c) indicating blood glucose levels during 2 to 3 months, similar to erythrocyte lifespans of 100–120 days [19]. Hemoglobin glycosylation involves a two-step, sequential, nonenzymatic process, creating reversible Schiff base intermediates when neutral amino groups from Hb N-terminal or Lys residues interact with sugar molecules’ aldehyde or ketone groups [24]. Recognition elements in electrochemical HbA1c biosensors include boronic acid derivatives, antibodies, and enzymes [25]. Clinicians may monitor a patient’s long-term glucose management and determine a patient’s prospective risk for developing diabetes issues using the HbA1c level in a method that is unaffected by changes in blood glucose levels [19].

6.3 Uric acid biosensor

Uric acid biosensor works on electrochemistry. In serum or urine generated by the kidney, uric acid is a naturally occurring antioxidant. It is produced as a byproduct of the body’s metabolism of purine nucleotides and their derivatives, and it reacts to various biological changes. Unhealthy bodily fluid concentrations are associated with a variety of medical conditions. Because of this, it’s important to frequently evaluate uric acid levels. Clinics routinely utilize high-performance liquid chromatography (HPLC), spectrophotometry, uric acid biosensors, chemiluminescence analysis, and other methods to detect uric acid. The uric acid biosensor has drawn increasing attention due to its advantages of being simple to create, highly sensitive, inexpensive, and selective. It is used for the clinical abnormalities’ or diseases’ detection [1, 25, 26, 27].

6.4 Piezoelectric effect biosensor

The term “piezoelectricity,” sometimes known as “the piezoelectric effect,” refers to a material’s ability to produce voltage when physically stretched. The result also applies in the opposite circumstance. When applied to a piezoelectric material’s surface, alternating voltage results in mechanical stress or oscillation. Piezoelectric materials typically have anisotropic crystals, or crystals without a center of symmetry. It does not require the application of any particular reagents and may easily capture affinity interactions, so the development of biosensors appears to be best suited to the piezoelectric platform. On the other hand, certain specific factors like fragility and the sensitivity in micrograms required to cause a detectable shift in oscillations should be taken into account.

Piezoelectricity is a powerful technique for building biosensors in analytical chemistry. Two electrodes deliver alternating voltage to excite the biosensor’s surface, causing mechanical oscillations in a crystal. The frequency of these oscillations is measured when an analyte or mass is attached to the crystal’s surface, specifically to the electrodes. This process allows for accurate measurement of the oscillation frequency.

Piezoelectric immunosensors are analytical tools that may be used to distinguish between various bacteria and macromolecules. Piezoelectric immunosensors use an antibody as a biorecognition component, with the specificity of the antibody determining the overall immunosensor’s specificity. This ensures that the electrode and other sensitive parts of the piezoelectric material remain sensitive to unspecific interactions with chemicals. Although the reverse reaction is also conceivable, immunosensors typically include immobilized antibodies and can detect antigens. It means that the immunosensor may be used to identify an antibody and that the only molecule being examined is an antibody. Immobilized antigens may also be included in the immunosensor [28, 29].

Piezoelectric immunosensors are appropriate for analyte measurements that have large molecular weights as they lead to a significant reduction in oscillation frequency. The challenge of utilizing antibodies mounted on a piezoelectric substrate to directly recognize small molecular weight analytes is another disadvantage of this method. Microorganisms are one type of analyte that piezoelectric immunosensors can specifically assess directly, and new models of piezoelectric immunosensors are frequently used to test these analytes [30, 31, 32].

Genetic data can be used as a biorecognition component in many biosensors. For the purpose of building biosensors, single-stranded, brief strains of DNA or RNA can be written down as typical instances of genetic information formats. The whole chromosomes, however, are also useful for tracking particular interactions. These biosensors frequently analyze human tissue or blood samples as well as genetic information from pathogens. The analyte and the biorecognition component of the biosensor, however, can be suggested as a potential pair since they interact with double-stranded DNA chains or immobilized chromosomes [33, 34, 35, 36].

The diagnosis of disorders with a genetic basis is well optimized for the use of DNA biosensors. Promising studies in this area can be introduced through a few examples. Pang and colleagues made the decision to examine codon DC17 of the beta-thalassemia gene for point mutations. The hybridization of a DNA probe on gold nanoparticles and DNA from the sample served as the basis for the detection. The evaluated oligonucleotides had an assay limit of detection of 2.6 nmol/L, and the hybridization was carried out using a quartz crystal microbalance (QCM) sensor. Ye and coworkers disclosed using a combination of quantum dots and magnetic nanoparticles to identify DNA in an experiment [37, 38].

Lectins, a group of proteins with high carbohydrate content, are potential piezoelectric biosensors with high specificity to sugars. These proteins work in immunoassays like antibodies, requiring only the carbohydrate moiety for interactions. The D-mannose-binding lectin from jackfruit Artocarpus heterophyllus was used to recognize N-glycosylated receptors on leukemia-related hematopoietic cells as a biosensor [39, 40, 41].

6.5 Silicon biosensor

Silicon photonic integrated circuit (PIC) technology is a promising method for producing photonic biosensors. It can be produced efficiently and volumetrically using complementary metal-oxide-semiconductor (CMOS) foundry techniques. The high refractive index difference between silicon and surrounding media allows for the fabrication of multiple sensors on a single chip, creating small, miniature sensor devices. Silicon photonics offer superior transducers for continuous and quantitative label-free biosensing, responding quickly to affinities between analyte and receptor molecules [42, 43, 44, 45, 46].

Because of their short response times and ability to solve the deficiency in sensitivity of flow-over porous silicon (Psi)-based biosensors, porous silicon membranes (PSiMs) are prospective biosensors. Also, they do not need any action to get the analyte on to the transducer and keep it there. In this study, we recapitulate the potential of these platforms for detection and emphasize their application to the detection of bacteria. Standard microfabrication methods and electrochemical etching were used to create PSiMs. A large number of samples could be manufactured in one try, and the entire manufacturing process took less than a week. The sensors were not functionalized since endolysins were used to provide specificity [47, 48, 49, 50, 51, 52, 53].

6.6 Fluorescent biosensor

Fluorescence-based biosensors are used in various applications, including medical diagnostics, pharmaceutical administration, drug discovery, environmental monitoring, and food safety. Techniques for detecting various analytes include fluorescence intensity, anisotropy, decay time, energy transfer, quenching efficacy, and quantum yield. These sensors consider various variables to ensure accurate and reliable detection of various analytes.

Many compounds spontaneously fluoresce or seem luminous in one condition but nonfluorescent in another. By using this characteristic, a relatively straightforward fluorescence biosensor can be created; for instance, nicotinamide adenine dinucleotide + hydrogen (NADH) is fluorescent, whereas nicotinamide adenine dinucleotide (NAD+) is not. Therefore, fluorescence-based detection is a viable option for all enzymatic processes reliant on NAD/NADH. Analytical chemists frequently employ this method to identify and measure diverse analytes. The format employed here is direct fluorescence. Many proteins and other macromolecules, including nucleic acids, NADH, green fluorescent proteins, and flavin nucleotides, have inherent fluorescence capabilities. However, these molecules’ fluorescent characteristics, such as their emission intensity or polarization, alter when these proteins bind to ligands or these ligands bind to these proteins [53, 54, 55, 56, 57, 58].

6.7 Microbial fuel-based biosensor

Microbial fuel-based biosensors monitor environmental toxicity and biochemical oxygen demand by converting organic substrates into electrical energy. These biosensors use bioelectrochemical devices to control microbial respiration, but are limited by low-power density and high production and operating expenses. Technological advancements are aiming to increase performance and save costs, enabling self-powered microbial biosensors.

Another use for microbial biosensors is the detection of heavy metals and pesticides, where eukaryotic bacteria have an advantage over prokaryotic cells. By creating whole-cell biosensors, it is helpful for the selective and sensitive detection of pesticide and heavy metal toxicity. In a way that is applicable to higher animals, higher eukaryotic bacteria can likewise be more susceptible to a number of toxic substances. The fact that microbial biosensors may be used for everything from energy production to environmental monitoring is noteworthy. Innovative techniques will enable the development of novel biosensors with selectivity and high sensitivity, from modified prokaryotes to eukaryotes of microbial origin [59, 60, 61].

6.8 Magnetic biosensor

Recent research supports efforts in magnetic sensors for biomedicine applications, detecting nanoparticles. Examples of extremely effective scientific and clinical techniques include the use of nanoparticles with magnetic properties in the treatment of hyperthermia, guided medication administration, and the usage of magnetic particles as magnetic resonance imaging (MRI) contrast agents. Because of their exceptional benefits, magnetic biosensors have garnered more attention than other types of biosensors. For instance, magnetic biosensors have four advantages over fluorescent-based techniques.

First, magnetic probes may be employed for long-term labeling tests because they are more stable over time in culture. Magnetic nanotags are not influenced by their reliability over time like fluorescent tags are since they are chemical compounds, as opposed to fluorescent tags. Such a feature may be used for long-term labeling tests while fabricating tissues and organs. Second, unlike fluorescently tagged samples, magnetic materials do not produce background noise effects. In biological samples, background fluorescence is a frequent occurrence that results from the tissue’s natural characteristics. Third, applying regulated magnetic fields to the outside surface offers a method for monitoring and controlling the biological environment from a distance. Finally, magnetic assays have been found to have greater sensitivity than fluorescence tests. Compared to fluorescent-based methods, the great sensitivity permits detection at much lower protein concentrations [62].

7. Limit of detection (LOD) and limit of quantity (LOQ)

Validation is crucial in analytical procedures as it assesses the method’s sensitivity. The limit of detection (LOD) is the lowest concentration or quantity of a substance that can be detected with sufficient probability using a specific analytical technique. LOD is the mean blank value plus three times the standard deviation, while LOQ is the lowest concentration or quantity that can be quantified with adequate accuracy and precision. There are no studies on the sensitivity of thin-layer chromatography (TLC) in terms of the effect of mobile and stationary phases on LOD and LOQ of fluoxetine and sertraline.

$$\mathrm{LOD}=\frac{3.3\sigma }{S.}$$E1

The limit of quantification (LOQ) is calculated using the following formula:

$$\mathrm{LOQ}=\frac{\mathbf{10}\mathit{\sigma }}{\mathit{S}}$$E2

Where: σ = standard deviation and S = slope of the calibration curve. Knowing the LOD allows for a rapid calculation of the LOQ value, which is given as follows:

$$\mathrm{LOD}=3·\mathrm{LOQ}$$E3

Correctly estimated limits of detection must adhere to the following assumptions:

$$10·\mathrm{LOD}>\mathrm{C}\text{and}\mathrm{LOD}<\mathrm{C}.$$E4

Where C = analyte concentration in the standard samples (Figure 3).

Figure 3.

Limit of detection (LOD) and limit of quantity (LOQ) graph as a sample, where signal versus concentration are discussed.

Linear calibration curves have well-defined limits, which are essential for sensor measurements. So to determine concentrations from signals, one must discriminate between the signal axis (Y) and the concentration axis (X). The blank and calibration curves represent distribution functions for limit of decision and sample measurements, respectively. They overlap within a statistically defined limit, resulting in the lowest value on the calibration curve with LOD, which is the minimal detectable value by projection to the X-axis in concentration [63, 64, 65].

8. Applications of biosensors

Biosensors aid in managing human health by detecting bacteria, viruses, and other pathogens, preventing diseases, rehabilitating patients, and monitoring their well-being. People may create and enhance healthy behaviors by using step and activity trackers. For those working in clinical science, these tools provide new avenues. Without extracting blood, these sensors pick up substances. Biosensors have several uses in the medical sciences [14, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83].

• Glucose monitoring

• Tracking biological abnormalities

• Detecting signs in a patient

• Tracking biological data

• Tracking cell protein

• Biomolecular detection and measurement

• Tracking heart rhythms of a cardiac patient

• Tracking body chemistry

• Diet monitoring

• Tracking air quality

• Accurate results and decision-making

• Patient status in the healthcare unit

• Management of disease

9. Scope for biosensors

Marine biosensors detect eutrophication using nitrite and nitrate sensors, while nucleic acid hybridization-based sensors detect organisms. Monterey Bay Aquarium Research Institute (MBARI) is developing an “environmental sample processor” sensor. Ribosomal RNA probes detect hazardous algae on moorings, with biosensors aiming to detect pesticides, heavy metals, and pollution. Nanomaterials offer opportunities for new biosensor technology, improving mechanical, electrochemical, optical, and magnetic properties, enabling single-molecule biosensors and high-throughput arrays.

Utilizing biomolecules and nanomaterials for single-molecule, multifunctional nanocomposites, nanofilms, and nanoelectrodes remains a challenge due to their unique structures and functions. Key obstacles include processing, characterization, interface problems, high-quality nanomaterial availability, customization, and electrode behavior. Strategies to increase signal-to-noise ratio and transduction and amplification are also crucial [84].

10. Achievements and challenges in biosensors

The growing demand for biosensors has led to the development of biofabrication for high-precision detection of cellular and animal activity. This requires multiplexed settings and complex transducers for both two-dimensional (2D) and 3D detection. Research methodologies, such as aptamers, affibodies, peptide arrays, and molecularly imprinted polymers, have been employed. However, promising compounds for medicinal, antibacterial, and drug delivery have had limited success. Electrochemical biosensors have emerged as reliable analytical tools for pathogen detection of the avian influenza virus in complex matrices. A recent study discovered potential applications for affinity-based biosensors in sports medicine and doping control analyses. The primary factors affecting biosensor development include sensitivity, specificity, nontoxicity, small compound detection capacity, and cost-effectiveness. These characteristics will address both biosensor technology limitations and fundamental requirements. Recent advancements in electrochemical sensor improvements and nanomaterials have led to new types of biosensors, such as printed temporary tattoo electrochemical biosensors for physiological and security detection of chemical components. For pandemics like COVID-19, it can be used for remarkable changes.

Modern era biosensors require a combination of biosensing, biofabrication, and synthetic biology techniques, utilizing electrochemical, optical, or bioelectronic principles or a combination of all three [1, 85, 86, 87].