The molecular imprinting of proteins is the process of forming biomimetics with entailed protein-recognition by means of a template-assisted synthesis. Protein-imprinted polymers (pMIPs) have been ...successfully employed in separations, assays, sensors, and imaging. From a technical point of view, imprinting a protein is both costly, for protein expression and purification, and challenging, for the preservation of the protein’s structural properties. In fact, the imprinting process needs to guarantee the preservation of the same protein three-dimensional conformation that later would be recognized. So far, the captivating idea to imprint just a portion of the protein, i.e., an epitope, instead of the whole, proved successful, offering reduced costs, compatibility with many synthetic conditions (solvents, pH, temperatures), and fine-tuning of the peptide sequence so to target specific physiological and functional conditions of the protein, such as post-translational modifications. Here, protein-protein interactions and the biochemical features of the epitopes are inspected, deriving lessons to prepare more effective pMIPs. Epitopes are categorized in linear or structured, immunogenic or not, located at the protein’s surface or buried in its core and the imprinting strategies are discussed. Moreover, attention is given to freely available online bioinformatics resources that might offer key tools to gain further rationale amid the selection process of suitable epitopes templates.
Optical sensing, taking advantage of the variety of available optical structures, is a rapidly expanding area. Over recent years, whispering gallery mode resonators, photonic crystals, optical ...waveguides, optical fibers and surface plasmon resonance have been exploited to devise different optical sensing configurations. In the present review, we report on the state of the art of optical sensing devices based on the aforementioned optical structures and on synthetic receptors prepared by means of the molecular imprinting technology. Molecularly imprinted polymers (MIPs) are polymeric receptors, cheap and robust, with high affinity and selectivity, prepared by a template assisted synthesis. The state of the art of the MIP functionalized optical structures is critically discussed, highlighting the key progresses that enabled the achievement of improved sensing performances, the merits and the limits both in MIP synthetic strategies and in MIP coupling.
In several biomedical applications, the detection of biomarkers demands high sensitivity, selectivity and easy-to-use devices. Organic electrochemical transistors (OECTs) represent a promising class ...of devices combining a minimal invasiveness and good signal transduction. However, OECTs lack of intrinsic selectivity that should be implemented by specific approaches to make them well suitable for biomedical applications. Here, we report on a biosensor in which selectivity and a high sensitivity are achieved by interfacing, in an OECT architecture, a novel gate electrode based on aptamers, Au nanoparticles and graphene hierarchically organized to optimize the final response. The fabricated biosensor performs state of the art limit of detection monitoring biomolecules, such as thrombin-with a limit of detection in the picomolar range (≤ 5 pM) and a very good selectivity even in presence of supraphysiological concentrations of Bovine Serum Albumin (BSA-1mM). These accomplishments are the final result of the gate hierarchic structure that reduces sterich indrance that could contrast the recognition events and minimizes false positive, because of the low affinity of graphene towards the physiological environment. Since our approach can be easily applied to a large variety of different biomarkers, we envisage a relevant potential for a large series of different biomedical applications.
Liquid biopsy is expected to become widespread in the coming years thanks to point of care devices, which can include label-free biosensors. The surface functionalization of biosensors is a crucial ...aspect that influences their overall performance, resulting in the accurate, sensitive, and specific detection of target molecules. Here, the surface of a microring resonator (MRR)-based biosensor was functionalized for the detection of protein biomarkers. Among the several existing functionalization methods, a strategy based on aptamers and mercaptosilanes was selected as the most highly performing approach. All steps of the functionalization protocol were carefully characterized and optimized to obtain a suitable protocol to be transferred to the final biosensor. The functionalization protocol comprised a preliminary plasma treatment aimed at cleaning and activating the surface for the subsequent silanization step. Different plasma treatments as well as different silanes were tested in order to covalently bind aptamers specific to different biomarker targets, i.e., C-reactive protein, SARS-CoV-2 spike protein, and thrombin. Argon plasma and 1%
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mercaptosilane were found as the most suitable for obtaining a homogeneous layer apt to aptamer conjugation. The aptamer concentration and time for immobilization were optimized, resulting in 1 µM and 3 h, respectively. A final passivation step based on mercaptohexanol was also implemented. The functionalization protocol was then evaluated for the detection of thrombin with a photonic biosensor based on microring resonators. The preliminary results identified the successful recognition of the correct target as well as some limitations of the developed protocol in real measurement conditions.
Postoperative pancreatic fistula (POPF), the major driver of morbidity and mortality following pancreatectomy, is caused by an abnormal communication between the pancreatic ductal epithelium and ...another epithelial surface containing pancreas-derived, enzyme-rich fluid. There is a strong correlation between the amylase content in surgically-placed drains early in the postoperative course and the development of POPF. A simple and cheap method to determine the amylase content from the drain effluent has been eagerly advocated. Here, we developed an amylase optical biosensor, based on a surface plasmon resonance (SPR) plastic optical fiber (POF), metallized with a 60 nm layer of gold and interrogated with white light. The sensor was made specific by coupling it with an anti-amylase antibody. Each surface derivatization step was optimized and studied by XPS, contact angle, and fluorescence. The POF-biosensor was tested for its response to amylase in diluted drain effluents. The volume of sample required was 50 µL and the measurement time was 8 min. The POF-biosensor showed selectivity for amylase, a calibration curve log-linear in the range of 0.8-25.8 U/L and a limit of detection (LOD) of ~0.5 U/L. In preliminary tests, the POF-biosensor allowed for the measurement of the amylase content of diluted surgically-placed drain effluents with an accuracy of >92% with respect to the gold standard. The POF-biosensor allows for reliable measurement and could be implemented to allow for a rapid bedside assessment of amylase value in drains following pancreatectomy.
A proof of principle biosensor for the Brucella abortus recognition onsite is presented. The system is based on a plasmonic optical fiber probe functionalized with an oriented antibody layer ...immobilized on a short polyethyleneglycol (PEG) interface through carbodiimide chemistry and protein G as an intermediate layer. The biosensor is inserted in a holder built in 3D printing technology, obtaining a custom holder useful for housing the sample to be measured and the equipment. The removable sensor chip is a low-cost Surface Plasmon Resonance (SPR) platform based on D-shaped plastic optical fibers (POFs), built-in in 3D printed connectors, used here for the first time to detect bacteria via a bio-receptor layer specific for its membrane protein. The performances of the biosensor in Brucella abortus recognition are tested by using two different SPR-POF probes combined with the same bio-receptor layer. The best sensor configuration has presented a sensitivity at low concentrations of one order of magnitude greater than the other. A limit of detection (LoD) of 2.8 bacteria/mL is achieved well competitive with other systems but without the need for amplification or special sample treatments. Specificity has been tested using Salmonella bacteria, and reproducibility, regenerability and stability are moreover evaluated. These experimental results pave the way for building an efficient and specific biosensor system for Brucella abortus detection onsite and in a few minutes. Moreover, the proposed POF-based SPR biosensor device, with respect to the already available technologies, could be a Point-of-care-test (POCT), simple to use, small-size and portable, low-cost, don't necessary of a microfluidic system, and can be connected to the Internet (IoT).
The functionalization of inorganic surfaces by organic functional molecules is a viable and promising method towards the realization of novel classes of biosensing devices. The proper comprehension ...of the chemical properties of the interface, as well as of the number of active binding sites for bioreceptor molecules are characteristics that will determine the interaction of the sensor with the analyte, and thus its final efficiency. We present a new and reliable surface functionalization route based on supersonic molecular beam deposition (SuMBD) using 2,6-naphthalene dicarboxylic acid as a bi-functional molecular linker on the chemically inert silicon nitride surface to further allow for stable and homogeneous attachment of biomolecules. The kinetically activated binding of the molecular layer to silicon nitride and the growth as a function of deposition time was studied by X-ray photoelectron spectroscopy, and the properties of films with different thicknesses were investigated by optical and vibrational spectroscopies. After subsequent attachment of a biological probe, fluorescence analysis was used to estimate the molecular layer's surface density. The successful functionalization of silicon nitride surface via SuMBD and the detailed growth and interface analysis paves the way for reliably attaching bioreceptor molecules onto the silicon nitride surface.
In this work, Fe2O3 was investigated as a doping agent for poly(methyl methacrylate) (PMMA) in order to enhance the plasmonic effect in sensors based on D-shaped plastic optical fibers (POFs). The ...doping procedure consists of immerging a premanufactured POF sensor chip in an iron (III) solution, avoiding repolymerization and its related disadvantages. After treatment, a sputtering process was used to deposit a gold nanofilm on the doped PMMA in order to obtain the surface plasmon resonance (SPR). More specifically, the doping procedure increases the refractive index of the POF's PMMA in contact with the gold nanofilm, improving the SPR phenomena. The doping of the PMMA was characterized by different analyses in order to determine the effectiveness of the doping procedure. Moreover, experimental results obtained by exploiting different water-glycerin solutions have been used to test the different SPR responses. The achieved bulk sensitivities confirmed the improvement of the plasmonic phenomenon with respect to a similar sensor configuration based on a not-doped PMMA SPR-POF chip. Finally, doped and non-doped SPR-POF platforms were functionalized with a molecularly imprinted polymer (MIP), specific for the bovine serum albumin (BSA) detection, to obtain dose-response curves. These experimental results confirmed an increase in binding sensitivity for the doped PMMA sensor. Therefore, a lower limit of detection (LOD), equal to 0.04 μM, has been obtained in the case of the doped PMMA sensor when compared to the one calculated for the not-doped sensor configuration equal to about 0.09 μM.
Aptamers represent the next frontier as biorecognition elements in biosensors thanks to a smaller size and lower molecular weight with respect to antibodies, more structural flexibility with the ...possibility to be regenerated, reduced batch-to-batch variation, and a potentially lower cost. Their high specificity and small size are particularly interesting for their application in optical biosensors since the perturbation of the evanescent field are low. Apart from the conventional plasmonic optical sensors, platforms based on silica and plastic optical fibers represent an interesting class of devices for point-of-care testing (POCT) in different applications. The first example of the coupling between aptamers and silica optical fibers was reported by Pollet in 2009 for the detection of IgE molecules. Six years later, the first example was published using a plastic optical fiber (POF) for the detection of Vascular Endothelial Growth Factor (VEGF). The excellent flexibility, great numerical aperture, and the large diameter make POFs extremely promising to be coupled to aptamers for the development of a sensitive platform easily integrable in portable, small-size, and simple devices. Starting from silica fiber-based surface plasmon resonance devices, here, a focus on significant biological applications based on aptamers, combined with plasmonic-POF probes, is reported.
In this article, we designed and studied silicon oxynitride (SiON) microring-based photonic structures for biosensing applications. We designed waveguides, directional couplers, and racetrack ...resonators in order to measure refractive index changes smaller than 10-6 refractive index units (RIU). We tested various samples with different SiON refractive indexes as well as the waveguide dimensions for selecting the sensor with the best performance. Propagation losses and bending losses have been measured on test structures, along with a complete characterization of the resonator's performances. Sensitivities and limit of detection (LOD) were also measured using glucose-water solutions and compared with expected results from simulations. Finally, we functionalized the resonator and performed sensing experiments with Aflatoxin M1 (AFM1). We were able to detect the binding of aflatoxin for concentrations as low as 12.5 nm. The results open up the path for designing cost-effective biosensors for a fast and reliable sensitive analysis of AFM1 in milk.
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