Recent Advances in Biosensing in Tissue Engineering and Regenerative Medicine

Abstract

In tissue engineering and regenerative medicine, biosensors act as analytical devices that combine biological elements with electrical components to generate a measurable signal. The application of biosensing in the nearest future may need high performance, incorporation of biosensors into feedback-based devices, advanced diagnostics as well as detection of toxins. These functionalities will aid the biosensors with increased sensitivity, specificity, and the ability to detect multiple analytes. With the newly improved strategies in fabrication, sensors may develop high spatial sensitivity and draw us near actualizing capable devices. Although biosensors have been produced in past years, there are still pending challenges such as scale-up process and long-term stability of commercial products that should be addressed. This review will also involve the application of additive manufacturing techniques such as 3D bioprinting to produce world-recognized biosensors. We will focus on some bioprinting techniques including laser direct-write and also consider microfluidic tissue engineering which can sense biomolecules in the miniaturized tissue constructs in real time at quite low concentration through different sensing systems. We also review its advances in mobile Health (mhealth) technologies for detection and monitoring as biosensors are produced with living cells encapsulated in 3D microenvironments. These advances and many more will, however, grow the community of biosensors and their availability in tissue engineering and regenerative medicine.

1. Introduction

The biosensor is an analytical device or probe that combines biological elements (enzymes or antibodies) with an electronic component to produce signals that can easily be measured. It can also be defined as an integrated single device with the capacity to provide results by recognizing a biological element that is in direct contact with a transducer [1]. This electronic device identifies, processes, and communicates data about the physiological changes of an analyte and the presence of various chemical or biological materials in the environment.

All these biosensors are produced in a variety of sizes and shapes. They can detect and measure even low levels of infections, harmful chemicals, and pH values. On the other hand, biosensing is the act of measuring or detecting the presence of particular chemicals in a physiological activity with the aid of a biosensor device. The major components of the biosensor including the transducer are displayed in Figure 1, and they are briefly defined in the following section.

Figure 1.

The main components of a biosensor arranged in chronological order. It begins with the analyte and ends with the end user.

Analyte: This biosensor component is the biochemical substance of interest to be identified by the biosensor. A typical example of an analyte in blood glucose can be detected by the glucometer (biosensor) [2].

Bioreceptor: This is a biological molecule that recognizes the analytes. Some examples include enzymes, cells, aptamers, deoxyribonucleic acid (DNA), and antibodies. The biological receptor generates a signal in the form of light, heat, pH, charge, or mass change when it interacts with a target analyte in the process known as biorecognition [2].

Transducer: This component converts the biochemical signal received from the biological receptor into a measurable and quantifiable signal in a process known as signalization [2].

Electronics: This part processes the transduced signals and prepares them for display. Its electronic circuitry is complex, and it also performs signal conditioning such as amplification and conversion of an analog signal into a digital form [2].

Display: The display unit consists of a user interpretation system such as the liquid crystal display (LCD) of a computer or a direct printer that generates numbers or curves understandable by the user. It usually consists of a combination of hardware and software that generates results of the biosensor in a user-friendly manner. The output signal on the display can be numeric, graphic, tabular, or an image, depending on the end user’s requirements [2].

1.1 History of biosensors

The concept of biosensors has gone through a series of evolution in terms of what is referred to as a “biosensor.” Accordingly, biosensing devices have metamorphosed into complex systems since their first invention.

The premier reported idea of biosensing rather than its “term” began in 1906 by M. Cremer. He emphasized that the concentration of an acid suspended in an aqueous solution is equal to the electric potential produced between sections of the solution when separated by a glass membrane [2]. Cremer’s discovery led to the introduction of pH by Soren Peder Lauritz Sorensen in 1909. After the invention of an electrode to measure the pH was achieved by Hughes in 1922, 34 years later, an oxygen probe was developed by Leland C. Clark who eventually became the father of biosensors after building what is described as a “real biosensor” in 1959 [3]. Based on this study, he described how “to make electrochemical sensors (pH, polarographic, potentiometric, or conductometric) more intelligent ‘by incorporating’ enzyme transducers as membrane-enclosed sandwiches” at a conference in New York in 1962 [4].

The term “enzyme electrode” which was originally used to describe the first biosensor was adopted by Updike and Hicks to describe a similar device in 1967 [5]. Guilbault & Montalvo [6] used glass electrodes coupled with urease to measure urea concentration by potentiometric measurement instead of the amperometric method.

In the electrochemical community during that period, the research on ion ion-selective electrodes (ISEs) was very active, and the idea of extending the range of sensors to non-electrochemical active compounds had been widely accepted, even for nonionic substances like glucose [5, 7]. Since then, great strides have been made in developing highly sensitive and selective biosensing devices where biological elements are combined with electrochemical sensors [5, 8]. Some of these changes are listed as follows:

• The first change took place by Clemen’s team where they developed a “bedside artificial pancreas” that included an electrochemical glucose biosensor. This was performed in 1976 and was sold by Miles (Elkhart) as the Biostator Glucose-Controlled Insulin Infusion System shortly after [5].

• The second change that occurred was performed by Pharmacia researchers. They began collaborating with physics and biochemistry academics at Linkoping University in 1982 to develop a novel bioanalytical device capable of monitoring biomolecule interactions. Pharmacia biosensor was founded in 1984 and in 1990, the business launched BIA core, a new instrument [5].

• In 1984, Cass and his colleagues published a scientific paper demonstrating the use of ferrocene and its derivatives as mediators for amperometric biosensors. A few years later, the Medisense Exac Tech Glucose Meter was launched on the market and became the world’s bestselling biosensor product. The initial product was a pen-shaped meter with a disposable screen-printed electrode [5].

• From 1999 till the present, research in biosensing has led to the development of a nanoelectromechanical biosensor (BioNMES), quantum dots, nanoparticles, nanocantilever, nanowire, and nanotube. The biosensor’s “driving force” exploits the selectivity of the biological element [4, 7].

1.2 Features of a biosensor: the basic features of a biosensor are as follows

1.2.1 Selectivity

This is usually the most important feature of a biosensor. A bioreceptor detects a specific analyte in a sample containing other admixtures and contaminants. The interaction of an antigen with the antibody depicts an example of this selectivity of a biosensor. Antibodies act as bioreceptors and are immobilized on the surface of the transducer. A solution (usually a buffer containing salts) containing the antigen is then exposed to the transducer where antibodies interact only with the antigens [2].

1.2.2 Reproducibility

This is the ability of the biosensor to produce identical results in different experimental setups. Reproducibility is characterized by the precision and accuracy of the transducer and electronics in a biosensor. Precision is the ability of the sensor to provide reproducible results every time a sample is measured and accuracy indicates the sensor’s capacity to provide a mean value close to the true value when a sample is measured more than once [2]. Reproducible signals provide high reliability and robustness to the inference made on the response of a biosensor.

1.2.3 Stability

This is the degree of susceptibility to ambient disturbances in and around the biosensing system [2]. These disturbances can cause a drift in the output signals of a biosensor under measurement. An error can occur in the measured concentration and can affect the precision and accuracy of the biosensor, and stability is the most crucial feature in applications where a biosensor requires long incubation steps or continuous monitoring [2]. The response of transducers and electronics can be temperature-sensitive, and this may likely influence the stability of a biosensor. Therefore, appropriate tuning of electronics is required to ensure a stable response of the sensor. Another factor that can affect the stability is the affinity of the bioreceptor, which is the degree to which the analyte binds to the bioreceptor. Bioreceptors with high affinities encourage either strong electrostatic bonding or covalent linkage of the analyte that fortifies the stability of a biosensor. Also, the degradation of the bioreceptor over some time is another factor that affects the stability of measurement [2].

1.2.4 Sensitivity

The minimum amount of analyte that can be detected by a biosensor defines its limit of detection (LOD) or sensitivity. In several medical and environmental monitoring applications, a biosensor is required to detect analyte concentrations as low as nanogram/milliliter (ng/ml) or even femtogram/milliliter (fg/ml) to confirm the presence of traces of analytes in a sample [2]. For instance, a prostate-specific antigen (PSA) concentration of 4 ng/ml in the blood is associated with prostate cancer for which doctors suggest biopsy tests. Hence, sensitivity is considered to be an important property of a biosensor [2].

1.2.5 Linearity

Linearity is the feature that shows the accuracy of the measured response (for a set of measurements with different concentrations of the analyte) to a straight line, mathematically represented as y = mc, where c is the concentration of the analyte, y is the output signal, and m is the sensitivity of the biosensor [2]. Linearity of the biosensor can be associated with the resolution of the biosensor and the range of analyte concentrations under test. The resolution of the biosensor is defined as the smallest change in the concentration of an analyte that is required to bring a change in the response of the biosensor. Depending on the application, a good resolution is required as most biosensor applications require not only analyte detection but also the measurement of concentrations of the analyte over a wide working range. Another term associated with linearity is a linear range, which is defined as the range of analyte concentrations for which the biosensor response changes linearly with the concentration [2]. These features are essential for the biosensors’ proper functioning, which can be used for various applications.

1.3 Applications of biosensors

The use of biosensors aims to improve the quality of life, for environmental monitoring, disease detection, food safety, defense, drug discovery, and many more. One of the main applications of biosensors is the detection of biomolecules that are either indicators of a disease or targets of a drug. For example, electrochemical biosensing techniques can be used as clinical tools to detect protein cancer biomarkers [9, 10].

Biosensors can also be used as platforms for monitoring food traceability, quality, safety, and nutritional value [11, 12]. Furthermore, an application such as pollution monitoring [12, 13] requires a biosensor to function from a few hours to several days. Such biosensors can be termed as “long-term monitoring” analysis tools. Long-term monitoring biosensors find their use as technologically advanced devices both in resource-limited settings and sophisticated medical setups. Some examples are as follows:

• Applications in drug discovery [14, 15];

• For the detection of several chemical and biological agents that are considered to be toxic materials of defense interest [16];

• For use in artificial implantable devices such as pacemakers [17];

• Used in prosthetic devices [18];

• Sewage epidemiology [19].

A range of electrochemical, optical, and acoustic sensing techniques have been utilized, along with their integration into analytical devices for various applications. Figure 2 depicts the various applications of biosensors. This book chapter will focus on tissue engineering, regenerative medicine, and mobile health (mHealth) technologies.

Figure 2.

Applications of Biosensors in different areas of specialization.

1.4 Types of biosensors

Biosensors are grouped based on the type of transducer deployed. They are as follows:-

• Electrochemical biosensors;

• Calorimetric/thermal detection biosensors;

• Optical biosensors;

• Piezoelectric biosensors.

1.4.1 Electrochemical biosensors

Electrochemical biosensors are simple devices that use bioelectrodes to measure electric current, ionic, or conductance changes. These biosensors have different types according to the transducer deployed and also based on the measurements of electrical parameters including potentiometric, amperometric, and voltammetric biosensors. The electrochemical biosensor has three electrodes namely reference, working, and counter electrodes [1]. A typical example of the electrochemical biosensor is shown in Figure 3.

Figure 3.

A schematic representation of an electrochemical biosensor illustrating its application in enzyme, antibody, or aptamer measurements [20].

1.4.2 Piezoelectric biosensors

They produce an electrical signal based on the principle of acoustics (sound vibrations) when mechanical force is applied. Quartz crystals are a common piezoelectric material used in biosensors. Figure 4 displays a commonly used piezoelectric biosensor.

Figure 4.

A schematic representation of a piezoelectric sensor. (a) Target antigen and antibody on a piezoelectric material before and after binding, (b) voltage-time curve before and after binding in a piezoelectric sensor and (c) amplitude of a piezoelectric sensor before and after binding concerning frequency [21].

1.4.3 Optical biosensors

Optical biosensors are used for analyte detection by absorption, fluorescence, or light scattering. They can also detect microscopic changes when cells bind to receptors immobilized on the transducer surface. They utilize the changes that occur in mass, concentration, or several molecules to direct changes in the characteristics of light [22, 23]. Here, both catalytic and affinity reactions can both be assessed. Figure 5 depicts a type of optical biosensor.

Figure 5.

A schematic representation of an optoelectrical arrangement of an optical biosensor [24].

1.4.4 Thermometric biosensors

They are made up of a heat-insulated box with a heat exchanger (calorimetric cylinder), and the reaction takes place in a tiny enzyme-packed bed reactor. The substrate is transformed into a product and heat is created as it enters the bed. Figure 6 shows a typical example of an optical biosensor.

Figure 6.

A schematic diagram of an optical biosensor showing its components [25].

A summary of types of biosensors and some of their applications that can be used in tissue engineering, regenerative medicine, or mhealth technology are shown in Table 1.

S/N	Types of biosensors		Some applications	References
1	Electrochemical biosensors	Potentiometric biosensor	Detection of urea in blood serum	[26]
		Amperometric biosensor	Detection of ethanol, glucose, and lactate	[27]
		Impedimetric biosensor	Detection, identification, and quantification of bacteria in the field of microbiology	[28]
		Immuno-sensor	Detection of several pathogens such as viruses like COVID-19 and influenza.	[29]
		Voltammetric biosensor	It can be used for analyzing paracetamol	[30]
2	Piezoelectric biosensor; Example is micromembrane biosensor		They are able to detect the presence of cells and their masses, used in the rapid detection of HIV in biological fluids	[31, 32]
3	Thermal biosensor; Examples are micro-electromechanical systems (MEMSs) biosensors		Low-cost integration of miniaturized devices, allow-cost batch fabrication, and measurement of multiple samples in parallel. It is used to measure enzyme activity, clinical monitoring, environmental monitoring, etc.	[33, 34]
4	Optical biosensor; Examples are evanescent wave fluorescence biosensors and bioluminescent optical fiber biosensors		For the rapid, sensitive, and highly selective detection of 17β-estradiol, an endocrine-disrupting compound is frequently detected in environmental water samples.; It enables the multi-detection of genotoxins and is used in the study of transferrin-binding proteins, lipooligosaccharide; (LOS)-antibody interactions, and serum responses to experimental vaccines	[35, 36, 37]

Table 1.

The various types of biosensors, some of their applications, and references.

2. Design and fabrication of a biosensor

There are many types of biosensors including electrochemical biosensors (potentiometric biosensors, amperometric biosensors, and conductometric biosensors). These biosensors are uniquely designed and fabricated based on their applications. This book chapter will explore the design and fabrication of two biosensors such as an amperometric electrochemical biosensor and a surface plasmon resonance (SPR) optical biosensor used for glucose level detection or measurement and bioprinting, respectively.

2.1 Amperometric electrochemical biosensor

The design of the electrodes and chip is a crucial aspect in the development of an amperometric electrochemical biosensor. In general, designing electrochemical biosensor electrodes necessitate a thorough understanding of fluid flow, particularly its behavior in microscales. Furthermore, it also requires a detailed understanding of mass transport phenomena and microflow mass transport foundations.

Biosensors have been improved using a variety of designs. The design of electrodes in a microfluidic system is a crucial pillar for improved performance. Electrode design necessitates a thorough understanding of electron diffusion phenomena. In most current flows, electron diffusion is the limiting step. Diffusion is generally hampered by crucial parameters such as the electrode surface and the number of active sites available for the target. When a device with a microchannel is utilized as an analytical platform, the analyte is injected into the channel using two alternative methods namely pressure-driven flow and electrokinetic flow [38]. A pressure gradient induces flow in pressure-driven flow, and the nature of the flow is influenced by the channel geometry and flow rates. The Reynolds number is commonly used to express the ratio of inertial and viscous forces:

$$R_e=\frac{\rho VD}{\mu }$$

where R_e is the Reynolds number, V is the characteristic velocity for the flow, D is the characteristic distance, ρ is the density of the fluid, and $\mu$ is the fluid viscosity. Laminar flow occurs at Reynolds numbers below 2300 [38]. Flow in the microchannel is laminar because of the micron-scale size of microchannels, and the low velocity requires fluids to move across the channels. For laminar flow, fluid travels in a steady and time-independent manner at each location. The flow profile is parabolic, in which the velocity of the flow is negligible at the wall surfaces.

Materials used in the design of amperometric electrochemical biosensors are classified as: (1) materials for the electrode and supporting substrate; (2) materials for the immobilization of biological recognition elements; (3) materials for the fabrication of the outer membrane; and (4) biological elements, such as enzymes, antibodies, antigens, mediators, and cofactors.

Solid electrode systems and supporting substrates are frequently constructed with metals and carbon. Due to their superior electrical and mechanical qualities, metals such as platinum, gold, silver, and stainless steel have long been employed as electrochemical electrodes [39]. Figure 7 depicts the various techniques used for the production of conductive supporting substrates.

Figure 7.

A schematic diagram of the techniques used for the production of the conductive supporting substrate.

The basic elements of biosensors are the bioelement and the sensing element. Any organic organism that can detect specific analytes from the medium of interest while remaining unresponsive to any other potentially inquisitive/interfering species is referred to as a bioelement. The signal transducing section of the biosensor is known as the sensing element, and it can take the shape of any magnetic, optical, electrical, or electrochemical transducing mechanism [40].

2.2 Design and fabrication of a surface plasmon resonance (SPR) biosensor

A surface plasmon resonance biosensor can be designed and manufactured using a variety of periodic structural patterns. One of the structural patterns employed in the design and fabrication of a surface plasmon resonance biosensor is the nanohole creation procedure using thermal nanoimprint lithography which is discussed in this book chapter.

The stamps for the nanohole array are made by thermal nanoimprint lithography, residual layer etching, (titanium/gold) Ti/Au deposition, and lift-off procedures. For example, if a 10-cm thick glass wafer is utilized for the imprinting process and is coated with a 100-nm thermoplastic polymer layer, it has to be spin-coated at 3000 rpm for 30s to obtain this layer. A hot embossing system can be used to perform nanoimprint lithography. The leftover layer is then etched with oxygen (O₂) plasma [41].

The polymer is etched uniformly in this procedure until the residual layer is completely removed and the pattern is transferred to the substrate. A metallic titanium (Ti) (adhesion layer, 5 nm)/gold (Au) (50 nm) layer is deposited using electron beam evaporation. Finally, the resist lift-off operation is carried out in an ultrasonic hot acetone bath to obtain the nanohole array structure [42].

2.2.1 Design and fabrication considerations for biosensors

Studying the target analyte and identifying how it reacts with biological molecules is the first phase in constructing a biosensing device.

Other phases include are as follows:

2.2.1.1 Biological receptor selection

The sensitivity and selectivity of a biosensor to the analyte of interest are decided by the biological receptor used. As a result, a receptor with a high affinity for the analyte is suggested. It is critical to understand the benefits and drawbacks of various biological receptors in diverse biosensor applications when selecting an appropriate receptor [41, 43, 44].

2.2.1.2 Selection of an appropriate immobilization method

Biological molecule must be attached to the surface of a transducer to function consistently as a biological receptor. Immobilization is the term for this procedure. This goal has been accomplished using a variety of techniques including adsorption, entrapment, covalent attachment, microencapsulation, and crosslinking [45, 46].

2.2.1.3 Transducer element selection

The efficiency of the biosensor device is heavily influenced by the transducer element. The use of an effective transducer will result in a device with greater efficiency, whereas the use of an ineffective transducer will result in a device with reduced efficiency [45, 47].

2.3 Recent advances in biosensing

2.3.1 Tissue engineering

Biosensors are particularly useful in tissue engineering applications, such as maintaining three-dimensional (3D)-printed cell cultures [48] and developing “organs-on-chips” models, where biomolecule concentrations such as glucose, adenosines, and hydrogen peroxide levels play a key role in determining the fate of cells and tissues. Changes in oxygen consumption, pH, membrane potentials, ion concentrations, and the release of numerous metabolic chemicals and proteins are all well-known physical and chemical signals that living cells communicate [49]. Monitoring these analytes in real time can provide insight into cellular activity.

2.3.1.1 Application of biosensors in tissue engineering

2.3.1.1.1 3D-bioprinted sensing devices

The deposition of a bioink (living cells and biomaterials) onto a printing surface is described as bioprinting, and it is a new approach for fabricating tissues and organs by accurately controlling the periodic arrangement of diverse biological materials, such as biomolecules and biocells. It has a wide range of characteristics that can be used in biosensing applications, such as fast deposition and patterning of proteins and other biomolecules [50]. A typical illustration of a 3D-printed tissue construct can be seen in Figure 8.

Figure 8.

Stages in submerged bioprinting of a 3D tissue construct. A) The cell-laden hydrogel bioink is printed in droplets, layer by layer following the provided model. The printing nozzle is submerged in high-density perfluorocarbons that are immiscible in water and oil. Perfluorocarbons are suitable for submerged cells due to the presence of oxygen and carbon dioxide transport capability. B) The hydrogel droplets are printed in a vertical or lateral dimension to produce branching constructs without solid support [51].

There are a variety of bioprinting technologies that can be used to make biosensors, and they are basically grouped into two methods, namely contact-based and noncontact-based printing. Both biomaterials and bioinks are essential for biological signal transduction. For advanced extrusion-based bioprinting such as coaxial or triaxial, optimization of the bioink viscosity is a major consideration to prevent clogging. Other properties including pore size and cellular behavior may influence biosensing [52]. Using an electric field, some printing processes, such as electrodeposition, may be able to transfer thin films of metal nanoparticles [50] or nanowires [53] to a substrate. Creating circuits that could be an intrinsic part of a biosensor, as well as some immunoassays or microarrays, can be done by printing thin metal sheets [54, 55]. Even thin films of biological material, such as proteins, enzymes, nucleic acids, polysaccharides, and bacterial cells, have been printed using electrodeposition [56, 57, 58]. More work needs to be done on bioprinting techniques that may be utilized to deposit a wide range of biologics and mammalian cells in precise spatial positions, rather than thin films, which have been used for biosensing applications.

2.3.1.1.2 Biosensors for diabetes

Diabetes is a serious chronic metabolic illness that affects over 400 million people globally. Uncontrolled chronic hyperglycemia damages and destroys various organs, resulting in significant morbidity and mortality [59]. Blood glucose control can help to reduce the frequency and severity of these problems [60]. By putting the glucose oxidase enzyme on an oxygen electrode, Clark and Lyons created the first biosensor for monitoring glucose levels in 1962 [61]. The care of diabetic patients was transformed when the first self-monitoring blood glucose (SMBG) gadget based on the glucose dehydrogenase enzyme was introduced in 1987 [62]. SMBG in Figure 9 is now widely used in the treatment of diabetes, particularly type I [64, 65].

Figure 9.

Various parts of an electrochemical glucose biosensor for diabetes care [63].

2.3.1.1.3 Biosensors for wound healing

Wound healing is a multistep process that necessitates the collaboration of numerous tissues and biochemical pathways [66]. Chronic wounds result from the failure of these processes to proceed in a timely and organized manner, possibly putting a huge financial strain on healthcare systems [67]. Uncontrolled inflammatory processes, bacterial infections, alterations in the acidic pH of the skin, oxygen levels, and matrix metalloproteinases (MMPs) are all involved in such failures [67, 68]. Biosensors are being researched to allow doctors to closely monitor the healing process, as regular monitoring is crucial in chronic wound management [67]. Screen-printing electrodes with Ag/AgCl-conductive ink were used to create a wearable pH sensor [69]. Wearable sensors for biomarkers detection for wound infections can be shown in Figure 10.

Figure 10.

Wearable Sensors for the detection of biomarkers for wound infection [70].

2.3.1.1.4 Biosensors for cancer applications

The use of biosensors in cancer diagnostics has a lot of promise. Cancer is the second biggest cause of mortality [71], and because biomarker concentrations in the early stages of tumor formation are relatively low, biosensor sensitivities or their LODs are critical for early diagnosis [72]. Early diagnosis of malignant cells before they spread has been shown to improve treatment outcomes and save lives. As a result, specialized, accurate, and rapid-response biosensors are in high demand in oncology, some of which can be seen in Figure 11. Recent biosensor advancements have greatly improved breast cancer diagnosis [74]. Breast cancer is the second most frequent cancer in women in the United States, after skin cancer, and the second most lethal, after lung cancer [75]. Traditional breast cancer diagnostic methods such as mammography, magnetic resonance imaging, and enzyme-linked immunosorbent assays (ELISAs) have produced impressive results; however, many false-negative or false-positive results continue to occur, and the adverse effects of some invasive techniques necessitate the development of new highly sensitive, reliable, and noninvasive methods for detection and prognosis [74].

Figure 11.

Common biosensors and biomarkers used in the detection of cancer [73].

2.3.1.1.5 Biosensors for cardiovascular applications

Cardiovascular illnesses are the leading cause of death worldwide, and early identification could save tens of thousands of lives each year. Biosensors are being utilized to measure cardiac troponin (both T and I), C-reactive protein (CRP), creatine kinase (CK), myoglobin, and other cardiac indicators. One of the most significant indicators for detecting myocardial infarction is cardiac troponin [76]. Some implantable biosensors can be used for cardiovascular applications. A typical example can be seen in Figure 12.

Figure 12.

Implantable biosensor: (a) head implant (in vivo); (b) head implant (in vitro); and (c) heart implant for recording electrocardiogram [77].

2.3.1.1.6 Biosensors in artificial limbs (prostheses)

The potential of prostheses to restore human skin’s sensory capabilities would provide users of mechanical limbs with a more natural feeling [78]. The use of a pressure sensor on an artificial hand, for example, might change the amount of force applied by the fingers when gripping objects. This could protect the object from falling due to an underapplied force or breaking due to an overapplied force. A system with sensors for electromyography, temperature, and strain incorporated into stimulation electrodes was developed [79], and its practical use for prosthesis control with sensory input as well as electrical muscle stimulation was reported [80].

2.3.2 Regenerative medicine

Biosensors serve as a control platform for other technologies, allowing for real-time monitoring of system behavior for improved efficiency. Biosensing technologies are used in regenerative medicine for a variety of purposes, including biomanufacturing (for example, product release requirements), organ-on-a-chip technologies, and therapeutic efficacy indicators.

2.3.2.1 Application of biosensors in regenerative medicine

2.3.2.1.1 Biomanufacturing

Biomanufacturing is a relatively recent industrial strategy to produce economically relevant biological goods such as human tissues by leveraging biological systems. Industrial-scale bioproducts are made using additive manufacturing techniques such as 3D printing and other biofabrication technologies Figure 13 [81]. shows DNA biosensing with 3D printing technology.

Figure 13.

DNA biosensing with 3D printing technology [82].

Biomanufacturing facilities may also use altered cells to manufacture chemical or molecular products, as well as mass culture cells for organ fabrication. Biomanufacturing may be used in a variety of industries, including healthcare, food production, and even agriculture. Controlling the quality and condition of the biological structure is crucial for producing trustworthy goods, and biosensing technologies can help with this. Electrochemical enzyme-based biosensors, for example, have been utilized to monitor metabolites in cell culture medium in real time [81].

2.3.2.1.2 Organ-on-a-chip technologies

Using microfluidic technology and organoids, organ-on-a-chip technologies have opened up a new biomedical research field. Organoids are tiny cell clusters of a certain tissue type that can mimic the behavior of regular tissues and organs more accurately. Organ-on-a-chip technology is utilized for a variety of purposes, including evaluating the response of organoids to medications and other external stimuli [83]. The use of biosensors for real-time monitoring of the behavior of microtissues and organoids has progressed the technique significantly. Damage to cardiac organoids was monitored using a new microfluidic aptamer-based electrochemical biosensor Figure 14 [84]. shows the use of biosensors to develop organs-on-a-chip technology.

Figure 14.

Diagram showing the use of biosensors in organ-on-a-chip integration [85].

2.3.2.1.3 As indicators for therapeutic efficacy

Given that most outcomes are observed visually (e.g. a regenerated tissue or a healed wound) or functionally (e.g. improved sensory ability), biosensors for detecting the efficacy of regenerative medicine-related therapies remain relatively unexplored. Biosensors, on the other hand, may play an increasingly essential role in therapeutic evaluation in the future. For example, with glucose sensors, patients undergoing treatment can make use of biosensors to self-monitor the efficacy of the treatment (for instance, the presence of the required growth factors in their bloodstream after undergoing treatment).

Also, biosensors that monitor stem cell differentiation status before transplantation for therapeutic purposes can be made with nanotechnology [86]. Small cellular surface proteins and neurotransmitters, for example, can be measured to validate the differentiation of stem cells into dopamine-producing brain cells before their implantation into Parkinson’s disease patients [87].

Future applications of biosensing can be seen in the monitoring of regenerative medicine therapies in patients, such as biosynthesized tissue preparation and posttreatment self-monitoring. With the advancement of technology and stem cell-related applications, physicians and patients will be able to use biosensors in new ways.

2.3.3 Mobile health (mHealth)

The pathbreaking spread of mobile technologies together with innovative application advancements has brought up deliberate attempts to address health-related matters using mobile devices. This has led to the evolution of a new pathway of electronic health (eHealth), known as mHealth. According to the International Telecommunication Union, there are about 5 billion mobile phone subscriptions in the world, with over 85% of the world’s population now covered by a commercial wireless signal [88]. Mobile phones have penetrated most low-income countries more than other infrastructures such as paved roads and electricity. The increasing quality of these networks which involves providing higher speeds of data transmission alongside cheaper and more powerful handsets is transforming the way health services and information are accessed, delivered, and managed. With increased accessibility comes a greater possibility of personalization and adoption in healthcare delivery [89].

The term “mobile” in mHealth connotes a sense of freedom and flexibility to function anywhere and at any time [90]. There is no one generally accepted definition for the term – mobile health, and how it is defined keeps changing with time, and as you move from one field to the other. However, World Health Organization Global Observatory for eHealth (WHO, GOe) has defined mHealth as a subdivision of eHealth (electronic health). This subdivision is referred to as medical and public health practice supported by mobile devices. The mobile devices include the following:

• Mobile phones

• Patient monitoring devices

• Personal digital assistants (PDAs)

• Other wireless devices

mHealth capitalizes on a mobile phone’s core utility of voice and short messaging service (SMS) as well as more complex functionalities and applications including general packet radio service (GPRS), third- and fourth-generation mobile telecommunications (3G and 4G systems), a global positioning system (GPS), and Bluetooth technology [89].

On the other hand, [91] it has described mHealth as wireless devices and sensors (which include mobile phones) which are meant to be carried or accessed by an individual throughout regular activities that are performed daily. This definition tells us that an important component of mHealth is the sensor that can monitor and measure physiological data; hence, the sensors can be used for various applications including monitoring and measuring physiological data in mHealth.

2.3.3.1 Application of biosensors in mHealth

There are many types of biosensors employed in mHealth for telecare. For biosensors to fit into mobile devices, they have to be of high quality and miniaturized, and consume low power. This has been better achieved through innovation in materials and instrumentation [92, 93, 94, 95]. As biosensors gain more and more attachments with smart devices for mHealth, they become necessary for researchers to design biosensors with suitable functionalities and specifications to work flawlessly with accompanying hardware and software [96].

Two features will remain immutable with mHealth devices: a sensing technology for sensing health parameters and processing software to transform the sensor data into useful information. Hence, biosensors will remain invaluable components of mHealth. In designing a biosensor for mHealth, the biosensor can be built as a distinct microfluidic chip to communicate with the smartphone via wired or wireless connectivity. Alternatively, the biosensing chip with computing features can be incorporated directly into the design of smartphones, and this will eliminate additional hardware, thereby improving portability and possibly bringing about overall cost reduction [97].

Regarding smartphone-based mHealth, recent smartphones lack some key health sensor modalities. An integrated smartphone biosensor has limitations in the types of health data it can collect. Yet, the presence of connectivity technology such as USB, Bluetooth, and WiFi that enable them to interface with a large number of external biosensors to expand their range of signal acquisition is a great advantage. In mHealth, data processing can either be local processing (on the smartphone or a standalone biosensing accessory) or server processing (taking place on the cloud or on a nearby computer that communicates with the smartphone or a standalone biosensing accessory) [97].

Smartphones are not very suitable for data processing that requires high computing power as they may take a long time to process the data into useful information. However, smartphones can take advantage of their built-in connectivity features to transfer sensor data to a more powerful server. After the processing is completed, the server can transmit the results back to the smartphone to be accessible to the user.

2.3.3.1.1 Some of the specific applications of biosensors in mHealth

2.3.3.1.1.1 Detection of melanoma

The biosensor has been employed in the detection of melanoma using a fully integrated smartphone application [98]. A 10x detachable lens is used to capture the image of the target site of the skin, and the image is then passed through a series of processing steps such as preprocessing, segmentation, and feature extraction. A support vector machine classifier is then used to determine whether the image is an indication of a malignant or benign lesion [98].

2.3.3.1.1.2 Phone microscopy

Orth et al. developed a dual-mode smartphone microscope. The system uses a camera flash or ambient light for brightfield and darkfield imaging [99]. They devised a clip-on 3D-printed attachment that easily attaches to the smartphone as shown in Figure 15. A cell nuclei imaging of unlabeled cells, cattle sperm, and zooplankton were demonstrated with this system. Another phone microscopy device called MoleScope is a commercially available smartphone attachment for dermoscopy that allows the user to obtain magnified images of the skin with controlled lighting. The images can be stored and viewed on a computer using a web platform and can be shared with a dermatologist, thereby facilitating teledermatology [97].

Figure 15.

Dual-mode microscope attachment (left) designed for the smartphone [99].

2.3.3.1.1.3 Semen analysis

Kanakasabapathy et al. developed a smartphone-based semen analyzer for point-of-care (POC) male infertility screening as shown in Figure 16. The system is composed of a disposable microfluidic device that handles the semen samples and an optical attachment for the smartphone that enhances image magnification and device alignment [100].

Figure 16.

Semen analyzer for point-of-care male fertility screening [100].

2.3.3.1.1.4 Lung function test

An acoustic-based diagnostic biosensor used for lung function tests has been developed by researchers as shown in Figure 17. It uses the audio signal from the in-built microphone of the smartphone with a mouthpiece attachment. The mechanism is based on a variable frequency complex demodulation using lung function parameter estimation [101].

Figure 17.

Smartphone device with mouthpiece attachment for lung function testing [101].

2.3.3.1.1.5 Enzyme-linked immunosorbent assays at the point of care

Ozcan and the group developed a smartphone-based microplate reader for performing enzyme-linked immunosorbent assays at the point of care [102]. The system is composed of a 96-well plate held by a 3D-printed attachment and an LED array for the illumination of the plate. The smartphone camera is mounted on the same attachment, connected with the optical fibers that carry the light to the camera as shown in Figure 18. The image taken is sent to online servers for analysis, and the results are sent back to the phone in about 1-minute time. [103] also developed injectable dermal tattoo biosensors for measuring pH, glucose, and albumin concentrations. The biosensors undergo a colorimetric change upon exposure to varying levels of these analytes, such as pH, glucose, and albumin concentrations [103].

Figure 18.

Smartphone-based microplate reader for point-of-care enzyme-linked immunosorbent assays [102].

2.3.3.1.1.6 Amperometric electrochemical analysis

Guo devised a system for measuring blood ketones in order to detect diabetic ketoacidosis early. The concentration of blood ketone is detected using disposable test strips from fingerstick whole blood analysis on a smartphone-powered electrochemical analyzer. The -hydroxybutyrate dehydrogenase integrated with the test strip converts -hydroxybutyrate to acetyl acetic acid after a drop of whole blood is added. The oxidation of NADH into NAD+ is then triggered by this cascade, which may be monitored amperometrically using an electrochemical analyzer. The results of mapping the current produced to the concentration of hydroxybutyrate are then sent to the smartphone through USB as shown in Figure 19 [104].

Figure 19.

Amperometric-based system for blood ketone monitoring [104].

3. Advantages of biosensing in tissue engineering and regenerative medicine

• It can be used in real-time monitoring of bioanalytes [52, 81].

• Development of “organs-on-chips” models in which concentrations of biomolecules such as glucose, adenosines, and hydrogen peroxide levels play important roles in determining the fate of the cells and tissues [48].

• Biosensing can be employed for the early detection of cancer biomarkers from blood samples in a noninvasive manner. Surface plasmon resonance (SPR) and electrochemical biosensors have been successfully used for the detection of carcinoembryonic antigen (CEA) biomarkers in the early diagnosis of lung cancer in serum [105, 106, 107].

4. Challenges

There are many challenges researchers face in biosensing research. These include the following:

• Difficulties in translating academic research into commercially viable prototypes by industries.

• Complex regulatory issues in clinical applications.

• Difficulties in finding researchers with a background in biosensor technology or engaging researchers from different disciplines of science and engineering to work together.

• Identifying a market that is interested in a biosensor for a specific analyte of interest.

• Clear-cut advantages over existing methods for the analysis of that analyte.

• Testing the performance of the biosensor both in use and after storage. Response of a biosensor after 6 months of storage is the absolute minimum for any practical commercial application.

• Stability, costs, and ease of manufacturing of each component of the biosensor; hazards and ethics associated with the use of the developed biosensor [2].

5. Conclusion and future perspectives

The application of biosensing in tissue engineering, regenerative medicine, and mHealth has been fast growing. However, the growth has been limited even though some sensors including piezoelectric sensors have been described in previous research works and are already present in the market depicting high sensitivity and sensibility. The popularly known and successful ones among all are the electrochemical and mHealth, whereas some others cannot be used practically.

In tissue engineering and regenerative medicine, real-time monitoring of analytes is still at its early stage, and further research can bring enormous possibilities in the field. Future studies should focus on overcoming the challenges of miniaturization and integration of biosensors in microfluidic systems. Microfluidic technology with automated, sensitive, and real-time monitoring capabilities will play significant roles in translating to clinics. The use of microfluidic technology and many other mentioned technologies (methods) for global biosensing applications will need the utmost high standard of the systems and the whole process.

Incorporating biofabrication techniques into biosensing fields is important. For multiplexing signals and evaluating cellular responses in 2D and 3D, high-quality transducers could be used to separate and quantify analytes of interest. Future studies could be carried out by joining the recent biofabrication techniques (contact-based and noncontact-based), to yield better advances in biosensing technology, more particularly, in advanced extrusion-based bioprinting (noncontact-based printing method), to print living biosensing structures (as implantable therapeutics) with the use of coaxial and triaxial nozzles for various healthcare-related issues. The synergy of biofabrication and sensing will generate the next generation of biosensors possessing a high degree of sensitivity, throughput, and dynamic range in one sensor. In the end, the synergetic effect will yield a great impact on future sensing, monitoring of diseases, research, diagnostics, and therapeutic applications.

Acknowledgments

The authors would like to thank Mr. Ekwebelem George and Ms. Nwaigwe Ogochukwu for their great contributions to the areas of design and fabrication of a biosensor, and recent advances in biosensing.

Conflict of interest

The authors declare no conflict of interest.

Authors’ contributions

This book chapter was written by all authors. All authors have approved the final version of the book chapter. A.T.B (corresponding author): conceptualization, writing, editing, supervision, and review. C.A.N: co-ordination, writing, and editing. E.M.B: conceptualization and writing.