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Since the invention of the first biosensors 70 years ago, they have turned into valuable and versatile tools for various applications, ranging from disease diagnosis to environmental monitoring. Traditionally, antibodies have been employed as the capture probes in most biosensors, owing to their innate ability to bind their target with high affinity and specificity, and are still considered as the gold standard. Yet, the resulting immunosensors often suffer from considerable limitations, which are mainly ascribed to the antibody size, conjugation chemistry, stability, and costs. Over the past decade, aptamers have emerged as promising alternative capture probes presenting some advantages over existing constraints of immunosensors, as well as new biosensing concepts. Herein, we review the employment of antibodies and aptamers as capture probes in biosensing platforms, addressing the main aspects of biosensor design and mechanism. We also aim to compare both capture probe classes from theoretical and experimental perspectives. Yet, we highlight that such comparisons are not straightforward, and these two families of capture probes should not be necessarily perceived as competing but rather as complementary. We, thus, elaborate on their combined use in hybrid biosensing schemes benefiting from the advantages of each biorecognition element.

Nanostructured porous silicon (PSi) films have been widely studied for the past two decades as optical transducers for the detection of various molecules, with advantages of simple fabrication, high internal surface, well-established surface chemistry and unique optical properties. Despite these significant advantages, the clinical implementation of label-free PSi-based biosensors has been impaired by their insufficient sensitivity, usually in the micromolar range for protein and DNA targets. In this work, we investigate the limiting factors of PSi-based optical biosensors and design methods for their improvement. As a model system, we study PSi Fabry-Pérot thin films and utilize reflective interferometric Fourier transform spectroscopy for real-time and label-free detection of different target proteins. The selectivity of the biosensors is achieved by functionalization of the porous layer with DNA aptamers, as capture probes. We investigate the advantages of these emerging synthetic capture probes in comparison to the corresponding gold-standard antibodies. We demonstrate that a similar biosensing performance, in terms of dynamic detection range, sensitivity and selectivity, is achieved when the respective capture probe is carefully immobilized onto the PSi transducer surface, considering orientation and surface density. Nevertheless, the stability and low cost of DNA aptamers in comparison to antibodies facilitate the production, shelf-life storage, and potential reusability of these aptamer-based biosensors. To decipher the limiting factors of PSi biosensors, we derive a comprehensive mathematical model, which considers all mass transport and reaction kinetics phenomena in these biosensors. We solve the model numerically and demonstrate that the model successfully captures target binding rate in these biosensors, contrary to the conventional model used in the literature. The model is used to elucidate the orders of magnitude deviations between experimental and th

Porous silicon (PSi) nanomaterials have been widely studied as label-free optical biosensors for protein detection. However, these biosensors’ performance, specifically in terms of their sensitivity (which is typically in the micromolar range), is insufficient for many applications. Herein, we present a proof-of-concept application of the electrokinetic isotachophoresis (ITP) technique for real-time preconcentration of a target protein on a PSi biosensor. With ITP, a highly concentrated target zone is delivered to the sensing area, where the protein target is captured by immobilized aptamers. The detection of the binding events is conducted in a label-free manner by reflective interferometric Fourier transformation spectroscopy (RIFTS). Up to 1000-fold enhancement in local concentration of the protein target and the biosensor’s sensitivity are achieved, with a measured limit of detection of 7.5 nM. Furthermore, the assay is successfully performed in complex media, such as bacteria lysate samples, while the selectivity of the biosensor is retained. The presented assay could be further utilized for other protein targets, and to promote the development of clinically useful PSi biosensors.