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

Abstract

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.

Fast and Accurate Determination of Minute Ochratoxin A Levels in Cereal Flours and Wine with the Label-Free White Light Reflectance Spectroscopy Biosensing Platform

“…The immunosensor proposed herein is the first optical sensor using “compact” instrumentation that is based on the white light reflectance spectroscopy and has, therefore, high potential to evolve to a point-of-need analytical tool. The same detection principle with less integrated instrumentation has been previously applied for the determination of other mycotoxins in different matrices, including aflatoxin B1, fumonisins B and deoxynivalenol in cereals and aflatoxin M1 in milk [ 20 ], but not to the detection of OTA. In terms of analytical sensitivity, the LoD of the proposed method (30 pg/mL in buffer, 60 pg/mL in cereal flour extracts, which corresponds to 0.3 μg/kg in the initial cereal sample and 300 pg/mL in wine) is 10 times lower than the current EU regulatory limit for OTA in cereals (3 μg/kg) and 7 times lower than the one in wines (2 ng/mL).…”

“…In the current work, a newly developed polyclonal antibody against OTA was employed for the development of an immunosensor for the accurate, rapid, low-cost and laborious-free determination of OTA in cereal flours and wines based on the white light reflectance spectroscopy (WLRS) principle. The WLRS sensing platform has been successfully applied to the quantitative determination of both high- and low-molecular-weight analytes [ 20 ] after proper biofunctionalization of the sensing surface with immunoreagents (antigen conjugates or antibodies depending on the assay format). WLRS biosensing principle relies on the real-time monitoring of the reflectance spectrum spectral shifts in the VIS/NIR range and associates them with the effective thickness of the biomolecular layer that grows on the biochip surface during the bioreaction.…”

Covalent and Non-covalent In-Flow Biofunctionalization for Capture Assays on Silicon Chips: White Light Reflectance Spectroscopy Immunosensor Combined with TOF-SIMS Resolves Immobilization Stability and Binding Stoichiometry

“…Spectra were recorded with a 15 ms integration time and fitted in the spectral range 400–560 nm using the Levenberg–Marquardt algorithm considering the refractive indices n (λ) of all layers of a multilayer stack of water/protein and silane layer/SiO 2 /silicon substrates. The algorithm was applied to evaluate the initial thickness of the SiO 2 /APTES adlayer and to transform in real time the spectral shift in effective biomolecular layer thickness d Γ …”

“…Light emitted from the light source is directed vertically to the chip surface by six fibers arranged at the circumference of the reflection probe. The reflected light from the chip is collected and guided to the spectrometer by the seventh central fiber to be continuously recorded and analyzed . For in-flow protein immobilization and assay, transparent microfluidic modules (ThetaMetrisis S.A., Athens, Greece) were placed on top of functionalized Si/SiO 2 chips. , A single chip assembled with its microfluidic cell was then placed in the WLRS instrument docking station.…”

“…8 The deposition of biomolecules on the sensor surface from solution could be realized by the in-flow strategy or under static conditions involving immersion in solution or bioprinting techniques. Despite the fact that in most biosensors a microfluidic module is typically involved to allow flow of the sample solution and real-time monitoring of the layer formation during the assay, immobilization of the capture molecules is achieved frequently under static conditions before assembling the microfluidic module 25 rather than using an in situ in-flow strategy. The advantages of the latter are the opportunity to monitor the formation of the capture molecule layer and also the spatial limitation of their immobilization to the array covered by the fluidic channels.…”