Recent Developments in the Field of Optical Immunosensors Focusing on a Label-Free, White Light Reflectance Spectroscopy-Based Immunosensing Platform

Optical immunosensors represent a research field of continuously increasing interest due to their unique features, which can mainly be attributed to the high-affinity and specific antibodies they use as biorecognition elements, combined with the advantageous characteristics of the optical transducing systems these sensors employ. The present work describes new developments in the field, focusing on recent bioanalytical applications (2021–2022) of labeled and label-free optical immunosensors. Special attention is paid to a specific immunosensing platform based on White Light Reflectance Spectroscopy, in which our labs have gained specific expertise; this platform is presented in detail so as to include developments, improvements, and bioanalytical applications since the mid-2000s. Perspectives on the field are been briefly discussed as well, highlighting the potential of optical immunosensors to eventually reach the state of a reliable, highly versatile, and widely applicable analytical tool suitable for use at the Point-of-Care.


Introduction
Immunosensors can be described as devices capable of detecting/quantifying certain analytes in various samples. As the "immuno"-part of the term denotes, immunosensors are based on specific antibodies that recognize the analyte and allow its consequent assay; on the other hand, they are based on appropriate transducers that can detect/transform the assay signal and reliably "translate" it into detection/quantification of the analyte present in the sample of interest.
Depending on the transducer systems they employ, immunosensors can be further divided into groups, such as electrochemical, piezoelectric, or optical ones [1,2]. Among the above-mentioned groups, optical immunosensors have proven very popular; while quite similar in principle, for a number of reasons they represent the next technological step compared to conventional immunoassays, the vast majority of which are based on optical signal/detection. More specifically, optical immunosensors offer short analysis times and artificial cerebrospinal fluid [30], beta-lactoglobulin and human IgE in unprocessed human serum and whole blood [31], interleukin-6 [32], and intact virions of vesicular stomatitis virus [33]. In addition, IgG has been recently determined in aqueous solutions with an IRIS sensor employing protein A as a specific binder instead of an antibody [34]; moreover, IRIS sensors have been applied to studies of antigen-antibody interactions [35], including determination of affinity constants [36] and binding kinetics [37].
White-light reflectance spectroscopy (WLRS) immunosensors may be considered as a special type of label-free optical immunosensors based on reflectometric interference spectroscopy. Our research group has developed a WLRS-based biosensing platform [38]; initially introduced for conducting kinetic studies of interactions between biomolecules [38] or determining the thickness of polymeric layers [39,40], the WLRS system has several bioanalytical applications nowadays, as will be presented in more detail below.

Newest Developments in Optical Immunosensors: Bioanalytical Applications
Several recent review articles (2021-2022) refer to optical immunosensors, often among other topics of interest in the field of biosensing. Thus, some of these articles describe optical immunosensors that have been developed for various (groups of) biomolecules [1,41,42], such as COVID-19-related biomarkers [43][44][45][46]. Immunosensing based on optical fiber technology [47,48] and application of spectroscopic ellipsometry to immunosensing [49] have been presented in recent reviews. Multiplexed optical immunosensors that are capable of simultaneously detecting/quantifying more than one analyte of diagnostic interest in the same sample and can be used for Point-of-Care applications have been recently presented as well [2,50]. Surface-Enhanced Raman Spectroscopy lateral flow immunoassays (SERSbased LFIAs) performed on properly functionalized membranes, which may themselves be considered as a type of optical immunosensor, have been described in a review article presenting Raman Scattering-based biosensors [51]. Optical immunosensors employing appropriate dendrimers [52] or various nanomaterials [53] as a means of improving sensing efficiency/sensitivity have been presented in recent reviews.
Most of the latest reports (2021-2022) regarding bioanalytical applications of both labeled and label-free optical immunosensors are summarized in Table 1. Starting with the former group, a simple yet highly useful screening assay for methicillin-resistant Staphylococcus aureus that may be considered as a type of colorimetric immunosensor was described; the assay was based on a pair of antibodies against penicillin binding protein 2a (anti-PBP 2a) and employed capture antibodies immobilized on activated cotton swabs along with detection antibodies immobilized on colored polymeric nanoparticles [54]. Moreover, a lately reported immunochromatographic strip (lateral flow immunoassay) for zearalenone can be considered as a type of dual colorimetric/fluorescent sensor; the key reagent of the strip consisted of Staphylococcus aureus biosynthesized polymer dots that could generate a colorimetric/fluorescent signal, onto which anti-zearalenone antibodies were coupled through their Fc region [55]. In addition, a fluorescence immunosensor was reported in [56] based on graphene quantum dots functionalized through covalent linking with an anti-neuron-specific enolase antibody (energy donor) along with a hybrid of Ti 3 C 2 -MXene nanosheets and silver nanoparticles (energy acceptor), which exhibited high quenching and energy-transfer efficiency. MXene is a new material with the general formula of M n+1 X n T x , where M represents a transition metal, X can be nitrogen and/or carbon, and T x stands for denoting surface functionalization [56]. Moreover, a fluorescence immunosensor based on the optical properties of carbon dots and the well-known metal-enhanced fluorescence effect of silver nanoparticles, both covalently "decorated" with anti-analyte antibodies, was reported for human epididymis protein 4 [57], while a strip fluorescence immunosensor was reported for aflatoxin B1 based on anti-aflatoxin B1 antibodies covalently coupled to quantum dot nanobeads as fluorescent label [58]. In addition, a fluorescence immunosensor for Salmonella typhi VI antigen was described based on iron porphyrin-like graphene quantum dots (Fe-N-GQDs) as a novel fluorescent label on which the anti-analyte antibody was covalently immobilized [59], while a fluorescence immunosensor for Escherichia coli O157 was reported which employed cell-based (i.e., inactivated Staphylococcus aureus-based) fluorescent microspheres labeled with carbon dots and loaded with monoclonal antibodies against E. coli O157 through the protein A present on the surface of the inactivated cells [60]. A novel fluorescent immunosensor for microcystin-LR (a toxin produced by Cyanobacteria) was reported which employed a Cy5.5-labeled anti-analyte antibody in an elegantly designed light-sheet skew rays-enhanced U-shaped fiber-optic evanescent wave immunosensing platform [61]. In a recently published article, an immunosensor based on an antibody-analyte-aptamer structure and a CRISPR/Cas12a fluorescence detection system was developed on a glass fiber and applied to the detection of small proteins such as INF-γ and insulin [62]. A phosphorescence-based displacement assay was designed for small molecules, especially estrone (E1) and estradiol (E2); phosphorescent labels based on palladium tetrabenzoporphyrin were conjugated to E1 and E2 directly or through a linker moiety, and the labeled molecules thus formed were used in a competitive-type assay for E1 and E2 and measured with an optical device developed for that purpose; the same approach could be used for other small molecules of interest [63]. On the other hand, a portable, fiber-based chemiluminescence immunosensor was developed for methylamphetamine, which was based on biotinylated anti-analyte antibodies along with streptavidin-biotinylated HRP nanocomposites and a chemiluminescence-emitting HRP substrate [64]. A dual chemiluminescence/colorimetric lateral flow immunoassay (LFIA) sensor was described for detecting immunoglobulin A (IgA) against SARS-CoV-2 proteins in serum and saliva of patients with COVID-19. The sensor employed a recombinant nucleocapsid antigen, which specifically bound the anti-SARS-CoV-2 antibodies, and anti-human IgA antibody labeled with either gold nanoparticles or HRP; detection was achieved either through a smartphone camera-based device measuring the color signal emitted by nanogold-labeled anti-human IgA or by means of a contact imaging portable device based on cooled CCD that could measure the chemiluminescence emitted by HRP-labeled anti-human IgA upon reaction with a H 2 O 2 /luminol/enhancer substrate [65].
Many of the optical immunosensors included in Table 1 are label-free, and a brief presentation of their basic principle/reagents has been considered of interest. Thus, a label-free sensor was reported which employed dynamic light scattering to detect the binding of anti-SARS-CoV-2 spike glycoprotein antibodies conjugated with gold nanoparticles to spike glycoprotein through the increase in the conjugates' size [66]. Moreover, a photoluminescence label-free immunosensor for aflatoxin B1 was developed and integrated with a microfluidic cell. The sensor was based on polyacrylonitrile/zinc oxide (PAN/ZnO) nanofibers, which were suitably treated and then loaded with an anti-analyte antibody; the photoluminescence of PAN/ZnO/antibody nanocomposites changed upon binding of aflatoxin B1 in a concentration-dependent manner [67]. A long-period fiber grating (LPFG) immunosensor that employed an avian antibody (IgY) recognizing Staphylococcus aureus was reported; upon bacteria-antibody interaction, an increase in the bioconjugate thickness and density was created and was tracked as a change of resonance wavelength in the LPFG transmission spectrum [68]. An all-fiber-optic immunosensor based on elliptical core helical intermediate fiber grating (E-HIPFG) was developed and evaluated through the detection of human IgG after functionalizing the sensor surface with a goat anti-human IgG antibody [69]. Two immunosensors based on specific antibodies against the Cor a 14-hazelnut allergen and electrochemical or SPR-based optical detection were reported and proposed as a platform for hazelnut allergen analysis in food; mammalian IgG and avian IgY anti-analyte antibodies were immobilized on the gold-coated sensors' surface, with the latter leading to a better outcome regarding mainly assay specificity [70]. Moreover, an immunosensor for COVID-19 diagnosis was reported based on antibodies against the SARS-CoV-2 nucleocapsid protein; the capture antibodies were conjugated to light scattering particles, while the detection ones were immobilized on an optical waveguide film [71]. In addition, an immunosensor for collagen I was described which employed a half-reduced monoclonal antibody, i.e., an anti-collagen I antibody the intramolecular S-S bridges of which were enzymatically reduced; subsequent covalent immobilization of the so-treated antibody on a monolayer of self-assembled gold nanoparticles led to a properly oriented and fully-functional antibody layer. Then, either electrochemical impedance spectroscopy or SPR were combined with the antibody-gold nanoparticles and the relevant immunosensors were developed [72]. A SPR immunosensor for programmed death ligand 1 (PD-L1) was described based on both an aptamer conjugated to magnetite nanorods containing ordered mesocages and silver nanoclusters (MNOM@AgNPs) and an anti-analyte antibody immobilized on the surface of a gold chip properly treated with para-sulfonatocalix [4]arene, thus leading to highly specific recognition of PD-L1 [73]. A SPR immunosensor for cortisol was reported which employed an unclad plastic optical fiber coated with a gold/palladium alloy on which an anti-analyte antibody was covalently linked [74]. A recently published paper has described a SPR-immunosensor for ricin and abrin; the sensor uses monoclonal antibodies for the biotoxins, which have been immobilized onto the SPR-chip surface through protein G [75]. Another recently published paper has reported a SPR-immunosensor with a highly enhanced signal for the detection of CD5 cancer biomarkers; the sensor employed capture anti-CD5 antibodies immobilized on the gold surface of the SPR-disc along with detection anti-CD5 antibodies coupled onto gold-coated magnetic nanoparticles [76]. A SERS immunosensor employing 4-mercaptobenzonitrile as a Raman probe was developed for imidaclopir; the sensor was based on Fe 3 O 4 magnetic nanoparticles coated with an anti-imidaclopir antibody along with bimetallic nanocuboid particles, containing gold in the core and silver in the shell and linked with the antigen and 4-mercaptobenzonitrile [77]. A harmonic microfiber Bragg grating (H-mFBG) immunosensor for cardiac troponin I was reported based on anti-analyte antibody immobilized onto the fiber, and was designed to operate also in vivo [78]. Novel immunosensors based on optical waveguide light mode spectroscopy (OWLS) were developed and applied to the detection of Fusarium mycotoxin zearalenone; both immobilized antibody and immobilized antigen assay formats were set up, with the immunoreagents' immobilization being performed on epoxy-, amino-, or carboxy-functionalized sensing surfaces [79]. A micro-sized non-spectroscopic optical reflector gadget was fabricated that was based on anti-analyte antibodies conjugated to retroreflective particles; the gadget was integrated with a commercial smartphone and applied to the detection of the creatine kinase-myocardial band [80]. Moreover, a Fabry-Pérot interferometric surface stress sensor was developed and applied to detection of prostate-specific antigen (PSA); the sensor employed anti-analyte antibodies covalently immobilized on a deformable biomembrane constructed on a parylene-C nanosheet support/silicon substrate, and the reflection spectra shifts created upon antibody-analyte binding due to membrane deformation were recorded [81]. Moreover, a planar waveguide immunosensor for zearalenone was described: the waveguide was composed of a thin silicon nitride layer between two thicker silicon dioxide layers, and the sensor worked as a polarization interferometer; the anti-analyte antibody was immobilized on the waveguide via a polyelectrolyte layer on which protein A was adsorbed [82]. In a very recent article, an optical immunosensor for COVID-19 diagnosis was described based on a new polymer-type imprinted photonic crystal film (IPCF) that diffracts light in an angle-dependent way; diffraction intensity decreases when a biomolecule, e.g., a specific antibody, is adsorbed on the film surface, and continues to decrease upon immunoadsorption of the analyte. The sensor employed an antibody against the SARS-CoV-2 spike protein immobilized on the IPCF surface, through which spike proteins present in artificial saliva could be detected with the aid of a smartphone-equipped optical setup [83]. In another recently published article, an optical immunosensor based on total internal reflection ellipsometry was reported; the sensor was applied to the detection and study of polyclonal antibodies circulating in the serum of human individuals after vaccination against COVID-19, which could recognize the spike proteins of three SARS-CoV-2 variants [84]. Finally, four immunosensors that relied on WLRS were reported [85][86][87][88]; these immunosensors will be presented in detail below along with further information concerning the WLRS immunosensing platform.
Bioanalytical applications of optical immunosensors reported in the last couple of years (2021-2022) cover a wide range of areas, from disease diagnosis to food analysis (Table 1). Thus, several of the relevant articles present new optical immunosensors for the detection/quantification of proteins of the SARS-CoV-2 virus or human antibodies developed against them [65,66,71,84]. Moreover, due to the COVID-19 pandemic outbreak and the consequent high interest in new tools for COVID-19 diagnosis, several recent review articles have presented, among other analytical approaches, research efforts toward development of optical immunosensors associated with COVID-19 [43][44][45][46]83]. Several optical immunosensors have been developed for the detection of whole bacteria, such as Staphylococcus aureus [68], especially methicillin-resistant Staphylococcus aureus [54], Salmonella typhi VI [59], Escherichia coli O157 [60], and Salmonella typhimurium [88]. Other optical immunosensors have been applied to and/or evaluated for the detection/quantification of specific disease biomarkers, such as neuron-specific enolase [56], myocardial creatine-kinase [80], prostate-specific antigen [81], human epididymis protein 4 [57], programmed death ligand 1 [73], cardiac troponin I [78], and C-reactive protein [87]. Another group includes optical immunosensors that have been applied to and/or evaluated through the detection/quantification of basic and important biomolecules, such as human INF-γ or insulin [62], immunoglobulin G [69], CD5 [76], collagen I [72], cortisol [74], and estrone and estradiol [63]. On the other hand, other optical immunosensors have been applied to the detection/quantification of exogenous substances, especially natural or synthetic toxic compounds, such as methylamphetamine [64], ochratoxin A [85], aflatoxin B1 [58,67], microcystin-LR [61], zearalenone [55,79,82], hazelnut Cor a14 allergen [70], imidacloprid [77], carbendazim [86], and ricin and abrin [75]. Zearalenone in millet and corn samples/Food analysis [55]   a : non-competitive format (direct-type): In this, one primary anti-analyte antibody is used that is usually immobilized on the transducer surface and the binding of the analyte is directly recorded. b : non-competitive format (sandwich-type): In this, a pair of primary anti-analyte antibodies is used, consisting of the so-called capture and detection antibodies. The capture antibodies are usually immobilized on the transducer surface and bind the analyte, while the detection antibodies, which are often appropriately labeled, bind to the analyte molecule through a different epitope before the "sandwich-type" binding of the analyte is finally recorded.

WLRS-Based Optical Immunosensors
As already mentioned, WLRS-sensors are a special type of label-free optical sensors. A review article concerning WLRS-based biosensing platforms was published by our group few years ago, providing information on both the operating principles and specific applications in various areas [89].
In its current form, the WLRS immunosensing platform employs a Si chip with a transparent SiO 2 layer on it, which operates as a sensing surface through immobilization of a suitable molecule, e.g., a primary or secondary antibody or a suitable analyte-protein bioconjugate. The biofunctionalized Si chip is coupled with a tailor-made microfluidic cell through which the assay solutions are continuously delivered, forming a biochip. The reader of the WLRS biochip includes a stabilized broadband light source, a high resolution in terms of both intensity and spectral content spectrometer, and a dedicated reflection probe consisting of seven optical fibers: six at the periphery of the probe, which direct the light from the source to the biochip surface, and one at the center of the probe, which collects the light reflected by the biochip and directs it to the spectrometer. The light passes through the transparent microfluidic cell and is directed to the sensing surface vertically, and is reflected at the various interfaces (the sample under analysis/functionalized layer, biofunctionalized layer/SiO 2 layer, and SiO 2 layer/Si substrate) due to the difference in the refractive index between adjacent layers (Figure 1a). Thus, interference occurs at each wavelength, resulting in an interference spectrum which can be collected by the central fiber of the reflection probe (Figure 1b). The increase in thickness of the biomolecular adlayer caused by the biomolecular interactions on the sensing surface leads to red-shifting of the interference spectrum. The WLRS reader is combined with specially developed software which can evaluate the initial thickness of the SiO 2 /biomolecular adlayer and transform the shift of the interference spectrum into the effective thickness of the biomolecular adlayer (nm), which is actually the signal of the WLRS sensor. More specifically, a reference [Ref(λ)] and a dark spectrum [D(λ)] are obtained prior to real-time continuous recording of the reflectance spectrum [S(λ)] (Figure 1c), and the absolute reflectance spectrum is calculated by Equation (1): The normalized spectrum is further processed through the Levenberg-Marquart algorithm [70] to calculate the thickness of the biomolecular adlayer (Figure 1d), d 1 , from the shift in the interference spectrum wavelength, δλ, according to Equation (2): where r 1 and r 2 and n 1 and n 2 are the Fresnel coefficients and refractive indices of the biomolecular and the SiO 2 layer, respectively, d 1 and d 2 are the thickness of the two layers, and λ is the wavelength, where λ 0m is the reflectance extremum. In this format, the WLRS-sensing platform is suitable for label-free real-time monitoring of the biomolecular interactions taking place on the Si/SiO 2 chip, with a detectable change in effective adlayer thickness < 0.1 nm.
It should be noted that various improvements have been incorporated into WLRSimmunosensing devices over the years ( Figure 2). As an example, a single channel spectrometer was introduced [90] to replace the beam splitter and double spectrometer previously employed to receive the reference and reflectance spectrum at the same time [91]. The most recent improvements include the following: integration of all optical and electronic components, along with the peristaltic pump, a computer-controlled carousel for the reagents' solutions, and a sampler into a compact instrument of rather small size (L × W × H 32 × 36 × 38 cm); stabilization of the light source to allow for long-term operation; selection of spectral range concerning the spectrometer employed; and enrichment of the software to enhance automation from assay performance up to data acquisition.
The first bioanalytical applications of the WLRS sensing platform [91][92][93][94][95][96] were previously described in [89]. Both these and later-developed optical immunosensors based on White Light Reflectance Spectroscopy have been included in Table 2, which summarizes the critical features of the sensors developed.
W × H 32 × 36 × 38 cm); stabilization of the light source to allow for long-term operation; selection of spectral range concerning the spectrometer employed; and enrichment of the software to enhance automation from assay performance up to data acquisition.
The first bioanalytical applications of the WLRS sensing platform [91][92][93][94][95][96] were previously described in [89]. Both these and later-developed optical immunosensors based on White Light Reflectance Spectroscopy have been included in Table 2, which summarizes the critical features of the sensors developed.  All WLRS immunosensors employ proper biochips. More specifically, Si chips (5 × 15 mm 2 ) covered with a thin (~1000 nm) SiO2 layer were suitably treated with O2 plasma or Piranha solution and subsequently with 2%, v/v, aqueous 3-aminopropyl triethoxysilane (APTES) solution, as previously described in detail [86,93], while biofunctionalization of the chip surface was achieved through immobilization of a suitable biomolecule depending on the immunoassay format/design, either by adsorption or covalent binding. Biofunctionalization of distinct areas of the surface with different biomolecules has led to the development of multiplexed-type immunosensors [93,94,97]. On the other hand, as described in a recent application, 3D structuring of areas with different thickness on the SiO2 layer, which were biofunctionalized with different biomolecules and then analyzed with a single reflection probe (Multi Area Reflecting Spectroscopy, MARS) and suitable software, has led to a multiplexed immunosensor as well [98]. In all cases, the WLRSsensors were based on the immobilization of a suitable biomolecule, e.g., a primary antibody, on the Si/SiO2 chip, with no need to use any type additional specific materials, which are often developed in-house and can be expensive.
A variety of antibodies (mammalian IgGs or avian IgYs, monoclonal or polyclonal, in-house developed or commercially available) and assay formats (competitive and noncompetitive) have been employed by the WLRS platform. Concerning primary antibodies, it is worth mentioning rabbit polyclonal ones recognizing benzimidazole-type pesticides, which have been developed in-house starting with low-cost commercially available chemicals. These antibodies, which were preliminarily integrated into a cell-based electrochemical sensor [99,100], have proven well suited to the WLRS platform and been applied to the determination of carbendazim in fruit juices [86]. Moreover, biotinylated antibodies, either primary or secondary, were often used in the WLRS platform in combination with streptavidin, thus taking advantage of the well-established advantages of the (strept)avidin-biotin system [101] to increase sensitivity and improve the detection limit of the immunosensor.
In all cases, the WLRS-based immunosensors exhibited very good analytical characteristics, i.e., high sensitivity and precision, along with the practical advantages of high economic impact, such as short analysis time and re-usage ability. Thus, almost all of the WLRS immunosensors developed to date have been extensively tested for their ability to perform multiple sequential analyses onto a single biochip. All tested sensors exhibited All WLRS immunosensors employ proper biochips. More specifically, Si chips (5 × 15 mm 2 ) covered with a thin (~1000 nm) SiO 2 layer were suitably treated with O 2 plasma or Piranha solution and subsequently with 2%, v/v, aqueous 3-aminopropyl triethoxysilane (APTES) solution, as previously described in detail [86,93], while biofunctionalization of the chip surface was achieved through immobilization of a suitable biomolecule depending on the immunoassay format/design, either by adsorption or covalent binding. Biofunctionalization of distinct areas of the surface with different biomolecules has led to the development of multiplexed-type immunosensors [93,94,97]. On the other hand, as described in a recent application, 3D structuring of areas with different thickness on the SiO 2 layer, which were biofunctionalized with different biomolecules and then analyzed with a single reflection probe (Multi Area Reflecting Spectroscopy, MARS) and suitable software, has led to a multiplexed immunosensor as well [98]. In all cases, the WLRS-sensors were based on the immobilization of a suitable biomolecule, e.g., a primary antibody, on the Si/SiO 2 chip, with no need to use any type additional specific materials, which are often developed in-house and can be expensive.
A variety of antibodies (mammalian IgGs or avian IgYs, monoclonal or polyclonal, in-house developed or commercially available) and assay formats (competitive and noncompetitive) have been employed by the WLRS platform. Concerning primary antibodies, it is worth mentioning rabbit polyclonal ones recognizing benzimidazole-type pesticides, which have been developed in-house starting with low-cost commercially available chemicals. These antibodies, which were preliminarily integrated into a cell-based electrochemical sensor [99,100], have proven well suited to the WLRS platform and been applied to the determination of carbendazim in fruit juices [86]. Moreover, biotinylated antibodies, either primary or secondary, were often used in the WLRS platform in combination with streptavidin, thus taking advantage of the well-established advantages of the (strept)avidin-biotin system [101] to increase sensitivity and improve the detection limit of the immunosensor.  In all cases, the WLRS-based immunosensors exhibited very good analytical characteristics, i.e., high sensitivity and precision, along with the practical advantages of high economic impact, such as short analysis time and re-usage ability. Thus, almost all of the WLRS immunosensors developed to date have been extensively tested for their ability to perform multiple sequential analyses onto a single biochip. All tested sensors exhibited considerably high stability, with regeneration cycles ranging from 12 up to 30 (Table 2). Particular attention has been paid to keeping total analysis time as short as possible through optimization studies; thus, in most cases the time needed to analyze a sample was less than 30 min, while direct-type assays with analysis time as short as 60 sec have been described [90,91].
The WLRS platform has been well suited to the immunodetection of various entities, ranging from whole microorganisms, i.e., bacteria cells [88] and high molecular weight biomolecules [87,[90][91][92][93]95,96], to low molecular weight compounds [85,86,94,97,[102][103][104], which proves the high versatility and wide applicability of WLRS immunosensors. Consequently, the WLRS immunosensors developed thus far have been applied to various areas of interest and various types of samples, from disease diagnosis (determination of C-reactive protein and/or D-dimer as inflammation biomarkers in human serum/whole blood, detection of the complement activation product C3b and its metabolites in human serum as autoimmunity indicators) [87,91,93,96] and determination of total and free PSA as cancer biomarkers [92] to food analysis and monitoring of environmental pollution (detection of Salmonella typhimurium in water samples [88], determination of the natural toxins, ochratoxin A in flour [85], deoxynivalenol in cereals [102], aflatoxin B1 and fumonisin B1 in wheat and maize [98], and aflatoxin M1 in milk [104], and the determination of the pesticides carbendazim in fruit juices [86], glyphosate in drinking water [103], paraquat and atrazine in water [97], and chlorpyrifos, imazalil, and thiabendazole in water and wine [94]). Moreover, a WLRS-based immunosensor has been developed and applied to the field of forensic sciences as well [95] (determination of PSA as semen indicator).

Discussion and Future Perspectives
Recent research efforts in the field of optical immunosensors have mainly focused on two directions. First, the improvement of the analytical assay characteristics, e.g., sensitivity and specificity, which are predominantly, although not exclusively, dependent on the biomolecules used for biorecognition (in our case, anti-analyte antibodies) along with the overall assay format/design. Second, efforts have been directed towards building up transduction systems of improved characteristics, such as fluorescence-quenching systems with high sensitivity/low background at the detection level; moreover, intense effort has been invested in the development of label-free systems, such as SPR-and WLRS-based platforms, with high analytical performance, high versatility, robustness, low cost, and the ability to be used at the Point-of-Care. Research on signal enhancement systems is a topic of continuous interest and high importance, although it has been considered outside the main scope of this article.
Concerning efforts to improve biorecognition in optical immunosensors, tailor-made antibodies with desirable characteristics have been employed in several of the most recently reported optical immunosensing platforms (Tables 1 and 2), although commercially available mouse monoclonals remain the antibodies most widely used. Alternative approaches include antibodies isolated from the egg yolk of avian species (IgYs). Avian antibodies are considered to offer better specificity and overall efficiency in assays targeting biomolecules of mammalian origin due to higher phylogenetic distance between mammalian and avian species [70,105]. Other features of avian IgYs, such as higher molecular weight in comparison with that of their mammalian counterparts (180 kDa vs. 150 kDa), may prove to be advantageous for application to certain types of sensors, e.g., label-free reflectrometric immunosensors, in which the thickness of the analyte-antibody adlayer is of critical importance. Other qualities of avian IgYs, such as high robustness and low cost, as well as avoidance of animal bleeding, which offers better compliance with the "3Rs" ethical prin-ciple governing research with animals (Replacement-Reduction-Refinement) [68,106], may help IgYs to become more widely applied as biorecognition molecules in optical immunosensors. Moreover, in accordance with the recommendation of the European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM) [107], nonanimal-derived antibodies might be eventually applied to immunosensors along with other products of synthetic biology, such as aptamers or molecular imprinted polymers [108,109], provided that current problems related to high cost, low availability, and often poor analytical features of the latter can be solved. Alternatively, combined use of totally-synthetic biorecognition molecules and traditional antibodies might prevail [110,111]. Last but not least, it should be noted that chemical functionalization of sensing surfaces so as to enable/facilitate efficient immobilization and better orientation of antibodies or other suitable biomolecules onto the sensing surface is an issue of special interest.
Concerning the development/improvement of label-free transducer systems, several relevant recent research efforts have been presented in this work (Tables 1 and 2). Among label-free optical immunosensors, the WLRS-based sensors ( Table 2) show a series of advantageous inherent features, such as simple and robust instrumentation, use of inexpensive sensing material(s) (i.e., Si/SiO 2 ), low interference from sample matrix, and high versatility concerning suitable analytes (ranging from whole bacteria cells and high molecular weight disease biomarkers to low molecular weight food toxins). The above advantages, along with the high potential of adopting/integrating further improvements, especially toward construction of a small-size, low weight, ideal for point-of-need applications, "all-in-one" functional device, may eventually render the WLRS-based immunosensing platform an invaluable analytical tool with high applicability.

Conclusions
New optical immunosensors are continuously being developed in an attempt to eventually achieve the construction of an "ideal" bioanalytical device, overcoming current problems and pitfalls. In this review paper, new developments in the field have been presented, including recent bioanalytical applications of optical immunosensors, while future perspectives have been briefly discussed. Special focus has been directed to the label-free WLRS-based immunosensing platform, which is considered a very promising tool for conducting bioanalytical studies in many areas of interest.
Funding: This research received no external funding.
Institutional Review Board Statement: Not applicable.