Aptasensors versus immunosensors—Which will prevail?

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

Mass Transfer Limitations of Porous Silicon-Based Biosensors for Protein Detection

Porous silicon (PSi) thin films have been widely studied for biosensing applications, enabling label-free optical detection of numerous targets. The large surface area of these biosensors has been commonly recognized as one of the main advantages of the PSi nanostructure. However, in practice, without application of signal amplification strategies, PSi-based biosensors suffer from limited sensitivity, compared to planar counterparts. Using a theoretical model, which describes the complex mass transport phenomena and reaction kinetics in these porous nanomaterials, we reveal that the interrelated effect of bulk and hindered diffusion is the main limiting factor of PSi-based biosensors. Thus, without significantly accelerating the mass transport to and within the nanostructure, the target capture performance of these biosensors would be comparable, regardless of the nature of the capture probe-target pair. We use our model to investigate the effect of various structural and biosensor characteristics on the capture performance of such biosensors and suggest rules of thumb for their optimization.

3D-printed microfluidics integrated with optical nanostructured porous aptasensors for protein detection

Microfluidic integration of biosensors enables improved biosensing performance and sophisticated lab-on-a-chip platform design for numerous applications. While soft lithography and polydimethylsiloxane (PDMS)-based microfluidics are still considered the gold standard, 3D-printing has emerged as a promising fabrication alternative for microfluidic systems. Herein, a 3D-printed polyacrylate-based microfluidic platform is integrated for the first time with a label-free porous silicon (PSi)–based optical aptasensor via a facile bonding method. The latter utilizes a UV-curable adhesive as an intermediate layer, while preserving the delicate nanostructure of the porous regions within the microchannels. As a proof-of-concept, a generic model aptasensor for label-free detection of his-tagged proteins is constructed, characterized, and compared to non-microfluidic and PDMS-based microfluidic setups. Detection of the target protein is carried out by real-time monitoring reflectivity changes of the PSi, induced by the target binding to the immobilized aptamers within the porous nanostructure. The microfluidic integrated aptasensor has been successfully used for detection of a model target protein, in the range 0.25 to 18 μM, with a good selectivity and an improved limit of detection, when compared to a non-microfluidic biosensing platform (0.04 μM vs. 2.7 μM, respectively). Furthermore, a superior performance of the 3D-printed microfluidic aptasensor is obtained, compared to a conventional PDMS-based microfluidic platform with similar dimensions. Graphical abstract: [Figure not available: see fulltext.]. © 2021, The Author(s)

3D-printed microfluidics integrated with optical nanostructured porous aptasensors for protein detection

Microfluidic integration of biosensors enables improved biosensing performance and sophisticated lab-on-a-chip platform design for numerous applications. While soft lithography and polydimethylsiloxane (PDMS)-based microfluidics are still considered the gold standard, 3D-printing has emerged as a promising fabrication alternative for microfluidic systems. Herein, a 3D-printed polyacrylate-based microfluidic platform is integrated for the first time with a label-free porous silicon (PSi)–based optical aptasensor via a facile bonding method. The latter utilizes a UV-curable adhesive as an intermediate layer, while preserving the delicate nanostructure of the porous regions within the microchannels. As a proof-of-concept, a generic model aptasensor for label-free detection of his-tagged proteins is constructed, characterized, and compared to non-microfluidic and PDMS-based microfluidic setups. Detection of the target protein is carried out by real-time monitoring reflectivity changes of the PSi, induced by the target binding to the immobilized aptamers within the porous nanostructure. The microfluidic integrated aptasensor has been successfully used for detection of a model target protein, in the range 0.25 to 18 μM, with a good selectivity and an improved limit of detection, when compared to a non-microfluidic biosensing platform (0.04 μM vs. 2.7 μM, respectively). Furthermore, a superior performance of the 3D-printed microfluidic aptasensor is obtained, compared to a conventional PDMS-based microfluidic platform with similar dimensions. Graphical abstract: [Figure not available: see fulltext.]. © 2021, The Author(s)

Porous silicon biosensors for protein targets: modelling and sensitivity enhancement

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 theoretical affinities between the capture probes and the target proteins observed in these biosensors and to develop rule of thumbs for their optimization. To enhance the performance of PSi-based biosensors, we design methods for mass transfer acceleration. These include application of isotachophoresis (ITP) method for on-chip protein concentration, target mixing on top of the biosensor or simple microfluidic integration, with up to 1000-fold enhancement in sensitivity. To allow flexible study of different microfluidic designs, we integrate for the first time PSi-based biosensor in 3D-printed polyacrylate microfluidic devices by a simple bonding method and demonstrate an improved performance of the 3D-printed microfluidics, compared to the gold-standard polydimethylsiloxane (PDMS) polymer used for microfluidic fabrication. Finally, we develop a PSi-based biosensor for detection of a relevant protein cancer biomarker and present a selective target detection in a highly complex fluid of pancreatic juice. By application of the methods described above, we were able to improve the sensitivity of the biosensor to the nanomolar range. This work paves the way towards clinical application of PSi-based biosensors and their translation to point of care settings

On Chip Protein Pre-Concentration for Enhancing the Sensitivity of Porous Silicon Biosensors

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

Accurate Prediction of Antimicrobial Susceptibility for Point‐of‐Care Testing of Urine in Less than 90 Minutes via iPRISM Cassettes

Abstract The extensive and improper use of antibiotics has led to a dramatic increase in the frequency of antibiotic resistance among human pathogens, complicating infectious disease treatments. In this work, a method for rapid antimicrobial susceptibility testing (AST) is presented using microstructured silicon diffraction gratings integrated into prototype devices, which enhance bacteria‐surface interactions and promote bacterial colonization. The silicon microstructures act also as optical sensors for monitoring bacterial growth upon exposure to antibiotics in a real‐time and label‐free manner via intensity‐based phase‐shift reflectometric interference spectroscopic measurements (iPRISM). Rapid AST using clinical isolates of Escherichia coli (E. coli) from urine is established and the assay is applied directly on unprocessed urine samples from urinary tract infection patients. When coupled with a machine learning algorithm trained on clinical samples, the iPRISM AST is able to predict the resistance or susceptibility of a new clinical sample with an Area Under the Receiver Operating Characteristic curve (AUC) of ∼ 0.85 in 1 h, and AUC > 0.9 in 90 min, when compared to state‐of‐the‐art automated AST methods used in the clinic while being an order of magnitude faster