Optical biosensors can be grouped into two categories, as follows: Bio-optrodes and evanescent field-based ones. Bio-optrodes are based on the interaction between an analyte and an immobilized reagent in the exit of a fiber, which produces a quantifiable change in the transductor’s optical properties. This change is optically evidenced by active groups like dyes, fluorescent molecules, and bio- or chemo-luminescents [
128]. Evanescent field-based biosensors are based on electromagnetic waveguides that transmit light by multiple internal reflections in total reflection conditions, thereby making an evanescent field capable of penetrating internal reflections at a determined distance from the waveguide surface, modified by the receptor [
118]. Optical evanescent wave biosensors are the most numerous and are characterized by involving the use of some type of electromagnetic field and the principle of ideal evanescent field detection for measuring any biochemical reaction taking place within it, thus making them indispensable tools for analyzing and identifying chemical or biological substances with a high degree of sensitivity and selectivity [
129]. Fibre-optic devices, therefore, belong within the bio-optrode biosensor category, while evanescent field devices include SPR-based, surface-enhanced Raman scattering (SERS), total internal reflection fluorescence (TIRF), optical waveguide interferometer, and elipsometric and reflectrometric interference spectroscopy (RlfS) biosensors.
4.1.1. Fibre-Optic Biosensors
Fibre-optic biosensors (optrodes) are devices in which a biocatalyzer is immobilized at the distal end/tip of a fibre-optic detection device. A biocatalyzer mediates between a sensor and an analyte, forming a detectable compound from a sample of interest [
130]. This type of biosensor has been studied for monitoring cells in clinical samples, endotoxins produced by Staphylococcus aureus, Clostridium botulinum [
131,
132], and proteins with clinical relevance, such as cardiac markers and anticoagulants [
133].
Using this type of biosensor to quantify drugs has gradually increased in prevalence. An example of this would be the reports in the pertinent literature concerning molecules such as phenytoin, an anticonvulsant that is widely used in clinical practice. A high level of efficiency was found regarding its sensitivity compared to the reference method (gas chromatography), which has a 4.45 µM detection limit at 37 °C. It is worth stressing that variables like temperature and pH were evaluated in the study as they affect equipment functionality [
134]. This type of device has also been used to measure theophylline, a drug used for treating respiratory symptoms associated with diseases like asthma, chronic bronchitis, and emphysema. Abs have been used to determine analyte concentrations, leading to a change in the Ab binding equilibrium/balance between labelled theophylline and unlabeled theophylline, provoking an increase in fluorescence. Detection ranged from 55 to 110 µM in human serum [
135].
Optrodes have been used in sectors such as agriculture to quantify organophosphorus pesticides using the principle of conical-shaped optical fiber detection. This form of fiber facilitates better evanescent field interaction, providing greater sensitivity and specificity. A 2.4 × 10
−10 M limit for detecting methyl-parathion (a prohibited organophosphate due to its high toxicity indices for human health and the environment) makes these devices extremely useful for determining pesticide concentrations worldwide, which have caused intoxication and death in animals, plants, and human beings [
136].
Scientists have modified this technique to obtain better results, calling this biosensor a “bioluminescent-based fibre-optic biosensor” based on its cells’ capacity for continuous monitoring of their microenvironment and responses to environmental changes when expressing specific genes [
8]. Cellular responses thus become signals that are detectable by a sensor. Genetically modified live cells are used for this as they can emit a bioluminescent signal that is detectable by an analyte. Such cells have previously been immobilized in optical fibers arranged in a series of high-density microwells [
118]. This type of biosensor has mainly been used for detecting genes; for example, Biran et al. demonstrated the efficacy and sensitivity of this type of sensor in determining the presence of genotoxic agents using a genetically modified E coli strain, using a luminescent signal as their experimental model [
118,
137].
4.1.2. Surface Plasmon Resonance (SPR)-Based Optical Biosensors
SPR biosensors are new technologies that are widely used due to their greater sensitivity and simplicity and ability to provide real-time results [
138]. This phenomenon was observed for the first time and reported in the pertinent literature by RH Ritchie (1957), who demonstrated that there was a loss of energy when the electrons penetrated metal, giving rise to the concept of “metallic plasmon” for describing fluctuations in the internal density of metal electrons [
139]. The term plasmon was derived from the concept of plasma, due to them both being constituted by charged particles collectively responding to a stimulus. Plasmons are defined as a quantum of energy (i.e., plasma oscillation) associated with wave propagation in material via the collective movement of a large amount of electrons, phonons, or photons [
140], in which free electrons respond by collectively oscillating in resonance at the same frequency as the incident light. Such oscillations are known as plasmon surface (PS) resonance [
117].
The SPR phenomenon is based on an optical measurement of refractive index changes using monochromatic light that excites the plasmons. The measurement involves immobilizing the recognition element over the surface of a metal and placing it against a prism from the equipment’s optical system. The light from a polarized infrared light emitting diode (LED) is focused via the prism onto the metal surface, such that the incident light beam becomes dispersed, giving a range of incident angles [
141]. The light reflected from the metal is detected by the photodiode matrix covering an appropriate interval of refraction angles. The plasmons are excited at a determined incident angle, and the corresponding loss of beam potential reflected at such an angle is recorded [
140]. The incident angle depends on many factors, such as the refractive index close to the back part of the metal film, where the molecules were previously immobilized, and the chemical nature of the molecules [
139].
These types of biosensors are considered universal detectors, as they can detect a large amount of molecules. However, the reduced size of some molecules in therapeutic drugs does not affect the refraction index, meaning that they cannot be detected on some occasions. Indirect techniques, such as competition assays and secondary detection with functionalized Abs and nanoparticles (NPs) have thus been introduced to increase the sensitivity of these biosensors.
SPR approaches have been at the forefront of clinical research, mainly in quantifying drugs requiring TDM, such as antibiotics, anticancer drugs, and anticoagulants. Regarding antibiotics, vancomycin and chloroeremomycin have been quantified by covalently coupling bacterial cell wall peptides to an HS(CH
2)
15CO
2H self-assembled monolayer (SAM) on a gold film. The vancomycin detection limit was found to be 20 ± 0.31 mM and 2.5 ± 0.04 mM for chloroeremomycin, thereby showing that these antibiotics (especially chloroeremomycin) are related to bacterial cell wall peptides, enabling better binding to the monolayer and improving quantification [
142,
143]. Ciprofloxacin has been monitored by an SPR biosensor based on a molecularly imprinted polymer (MIP); this modification has been of great use in in the food industry and the medical field, facilitating the selective detection of antibiotics. It had a ~0.08 μg/L detection limit, which is lower than that reported in the literature, thus making it a more sensitive technique for quantifying molecules of this type in different matrices [
144].
Tomassetti et al. worked on the direct detection of ampicillin using an SPR operating in flow conditions. This proved to be more selective than other biosensors when compared to antibiotics with similar structures, but less sensitive than biosensors lacking such modifications (10
−3 M to 10
−1 M detection range) [
145]. Another modification providing advantages for this type of biosensor is based on Laser Doppler Micro-electrophoresis with UV-Visible (UV-vis) spectroscopy, used to detect gentamycin. This enabled the researchers to determine a 0.05 ng/mL detection limit, which is lower than that for ELISA techniques (0.1 ng/mL limit), making it one of the best alternatives for detecting this type of antibiotic [
146].
Neomycin B is another antibiotic that has been monitored with aptamer-based biosensors. High sensitivity has been found, with a detection range from 10 nM to 100 µM, thus highlighting the feasibility of the high sensitivity detection of small molecules using RNA fragments [
147]. Amikacin has been one of the most reported molecules. Amikacin is an antibiotic that is mainly used in neonates but is suggested for TDM due to its high toxicity, which is inherent in its use. The forgoing has meant that adult and neonate sera have been evaluated by SPR-based indirect competition immunoassays, finding high levels of specificity and sensitivity (1.4 ng/mL 50 CI) and a 0.13 ng/mL detection limit, thereby allowing this drug to be quantified in real-time [
120,
121].
Regarding anticancer molecules, levels of a highly cytotoxic drug called metotrexate (MTX) have been recently measured in the serum of chemotherapy patients using folic acid-functionalized gold nanoparticles (FA-AuNPs) in assays with MTX. It had a 28 nM detection limit [
125], which is lower than that previously reported by the author (155 nM), quantified by an LSPR (localized surface plasmon resonance) biosensor [
127]. This makes SPR a highly sensitive technique for detecting certain antineoplastics. Simultaneously, studies have been carried out using the interaction of doxorubicin (DOX) with electrodes in different types of biosensors, including SPR. DOX is an antineoplastic, which is commonly used in chemotherapy, despite being cytotoxic. It was found that the monolayer’s hydrophilic and hydrophobic properties are fundamental for proper functioning of the device [
148]. Such studies enable procedures to be standardized, thereby facilitating correct functioning when quantifying a target drug (i.e., for TDM purposes).
Some anticoagulants are characterized by having an NTI, and their pharmacokinetics and pharmacodynamics depend on a particular patient’s conditions where high or low doses could cause death or irreparable damage to some organs. An example of this would be quantifying heparin in plasma samples with a 0.2 U/mL detection limit and using protamine and polyethyleneimine (PEI) as heparin affinity surfaces [
5,
124,
149]. Studies have also been carried out to quantify opioids, such as morphine-3-glucuronide, in urine (i.e., M3G is the main metabolite in heroin and morphine). This process involved immunoassays using polyclonal Abs from New Zealand rabbits (Enterprise Ireland and Science and Technology Against Drug Initiative, Dublin, Ireland). Two types of Abs were obtained in the following detection ranges, as follows: From 762–24,400 pg/mL (Ab 1) and 976–62,500 pg/mL (Ab 2). It was concluded that using biosensors is a sensitive technique for detecting opioid analgesic drugs [
150].
In addition to this type of biosensor’s advantages and numerous applications, there are some variations, such as SPR imaging (SPRi) and localized surface plasmon resonance (LSPR). SPR imaging (SPRi) combines SPR sensitivity with spatial imaging, thereby enabling multiple interactions to be studied simultaneously. It is characterized by having high performance, high sensitivity, and the ability to obtain images of biointeractions [
151]. The pertinent literature contains few reports about TDM and this type of biosensor. These types of sensors have mainly been used for quantifying metaloproteinase-2, a relevant enzyme in angiogenesis, wound healing, and tumor cell metastasis [
152]. This type of biosensor offers so many advantages that recent studies have incorporated a smartphone into the system to obtain real-time results when taking measurements, without the need for sophisticated or large volume-occupying equipment, such as a computer [
153].
Localized surface plasmon resonance (LSPR) is based on the collective oscillation of free electrons within metallic NPs (gold and silver), where the energy essentially depends on the form and size of the NPs [
85]. This technique is highly sensitive, especially in the field of diagnosis, for detecting diseases identified by biomarkers, such as proteins [
154]. It has also been used in measuring MTX, an anticancer drug, by using NPs or FA-AuNPs. This device was made for detecting nanomolar to micromolar concentrations of a target drug in plasma (a 155 nM detection limit was eventually found). Measuring this drug enabled the monitoring of MTX levels in patients undergoing chemotherapy [
127].
Tobramycin is one of the antibiotics being monitored by this type of biosensor. It is a molecule with harmful secondary effects that cause nephrotoxicity, cochlear and vestibular toxicity, ototoxicity, and neuromuscular blocking. Studies reported in the literature have quantified this drug by transmission localized surface plasmon resonance (T-LSPR). This process includes antibiotic-specific DNA-aptamers. Its high sensitivity and specificity were determined, with a detection limit of 0.5 μM [
155]. Caglayan and Onur made another type of modification to this type of biosensor, which involved a colorimetric detection technique using silver NPs showing the interaction between negatively-charged particles and cationic aminoglycoside antibiotics and visually indicating a change from yellow to red in the presence of gentamycin, tobramycin, and amikacin. The detection ranges were 20 to 60 ng/mL for gentamycin, 23 to 60 ng/mL for tobramycin, and 60 to 100 ng/mL for amikacin [
156,
157].
Other types of drugs have been quantified, such as an anticoagulant called megalatran. This drug was monitored by LSPR integrated into a microfluidic lab-on-a-chip device. This process involved immobilizing human α-thrombin on the biosensor’s gold surface for the enantioselective detection of the drug’s enantiomers (0.9 nM detection limit), this being one of the pioneering studies regarding the use of enantioselective biosensors [
158]. Studies are currently being advanced for quantifying acenocoumarol (Sintrom), an oral anticoagulant. These studies involve using an LSPR-based nanoplasmonic biosensor alongside highly specific polyclonal antibodies with a 0.66 nM detection limit, which has been catalogued as being a relevant limit for quantifying these drugs [
159].
This method has also been used for detecting, in serum, drugs that have been used to control arrhythmias and cardiac problems; DOX is one such example, as it has an NTI, but few studies have been made on its dose/administration and high toxicity. This technique consists of LSPR quantification using gold NPs (2 ng/mL detection limit), thus making it sensitive and effective for quantifying this type of molecule [
160].
4.1.4. Total Internal Reflection Fluorescence (TIRF) Biosensors
TIRF biosensors are based on using fluorescent molecular markers where evanescent field radiation is absorbed by a probe immobilized on the waveguide’s surface, thereby inducing its fluorescence. Such emission intensity is measured and related to the concentration of the analyte in a particular sample [
164]. TIRF techniques give better results than direct detection techniques. Some of their advantages are related to having greater specificity regarding a molecule of interest, as their response is not affected by a sample’s components, meaning that they have been used on liquid samples such as wastewater, sewage, and/or plasma. Fluorescent probes have greater stability than the enzymatic components used in other biosensors and usually have a longer shelf-life and stability than radioactive probes, making them safer [
165].
These biosensors have thus been widely used in different areas, especially for the environmental monitoring of wastewater from industries, which could affect human and animal health [
166]. Ehrentreich-Forster et al. have described using fluorescence biosensors for detecting explosives, toxins, narcotics, and other compounds prohibited by law, which is of great help in controlling illegal substances [
167].
This type of biosensor has been of great use in quantifying immunosuppressant drugs due to their narrow therapeutic ranges, where high levels can cause secondary effects and low levels can increase the risk of rejection. Mycophenolic acid (MPA) or mycophenolate mofetil (MMF) using sheep and donkey polyclonal Abs have been reported in the literature. It has been found that this device can detect the drug, thereby providing a great advancement in personalized medicine for patients undergoing transplants [
168]. They have also been used for quantifying antithrombin using immobilized heparin [
169].
Other types of optical biosensor-related devices have been used for quantifying other types of molecules that are important in medicine and industry, such as optical waveguide interferometer biosensors, which are based on combining evanescent field detection with methods for measuring phase difference [
170]. This technique has been useful for detecting cell content redistribution, taking cell responses and processes into account, such as detecting the avian flu virus [
171]. Elipsometric biosensors are based on changes in the polarization of light when reflected off a surface. These are mainly used in detecting tumor biomarkers or the influenza virus [
172]. Reflectometric interference spectroscopy biosensors are based on changes in the phase and amplitude of polarized light, thereby providing information about a protein’s refraction index [
173]. This method is used for detecting cancer cells and quantifying contaminants in milk [
174].
The use of optical biosensors for TDM at the patient’s bedside poses a challenge for researchers because it requires portable devices that guarantee high specificity, sensitivity, speed, and low cost, with the transmission of new materials and technologies that monitor in real time [
175]. This type of biosensor uses different bodily fluids, among which are mainly sweat, tears, saliva, and urine.
Among the portable optical biosensors used are biosensors based on contact lenses for the quantification of glucose in tears. These devices are made of a selective glucose hydrogel film functionalized with phenylboronic acid. Evidence shows a sensitivity of 12 nm/mM and a saturation response time of fewer than 30 min. This biosensor is compatible with smartphones, so patients can see the results in real time [
176]. On other hand, using dermal biosensors to control glucose levels is a novel technique that allows the measurement of blood levels to be a non-intrusive technology for patients. This new method is based on a band that is placed on the wrist; through combined near-visible infrared spectroscopy (Vis-NIR), it allows the measurement of glucose found mainly in arterial blood [
177].
This new technology applied to optical biosensors has allowed the quantification of some drugs, such as phenytoin, a salivary antiepileptic using a portable handheld SPRi with a detection limit of 50 nM; obtaining results in less than 5 min. The measurements of this device were approximately 25 × 10 × 5 cm
3 [
178]. Another example of portable biosensors is those used for the measurement of antineoplastic agents, such as tamoxifen (TMX), using a four-channel portable LSRP. This biosensor allows for high sensitivity (5 nM) because it uses gold nanoparticles and allows quick reading in less than 5 min [
125]. Cappi et al. have shown that the measurements of serum tobramycin levels with LSPR are the size of the palm of one hand. The biological components were aptamers functionalized with gold nanoislands (NI) deposited on a glass slide covered with fluorine-doped tin oxide. The detection limit was 3.4 µM. [
155].
However, the use of portable biosensors in TDM is very low, as these biosensors are mainly used to determine the concentrations of different pollutants in environmental samples. An example of this is the research conducted by Shriver et al, which determined the presence of a trinitrotoluene (TNT) explosive using a portable fiber optic biosensor [
179] or the use of biosensors for the quantification of organic contaminants in water and food by optical immunosensors [
180,
181]. On the other hand, research has been carried out focused on the determination of antibiotics in milk to preserve the quality of the food, as is the case of the quantification of fluoroquinolone residues using SPR [
182].
Considering the studies described above, a new window of possibilities for the implementation of portable biosensors in TDM has been opened. This will allow us to find new methodologies to determine and quantify different molecules in body fluids. It is important to keep in mind that there may be different variants according to the nature of the molecule and the matrix used so they can guarantee the development of optical biosensors.
Optical biosensors provide a great tool that has enabled new technologies to be advanced in the area of personalized medication using nanotechnology in different fields of medicine.
Table 2 lists the most relevant studies that have involved using different types of optical biosensors.