Single-Walled Carbon Nanotubes as Enhancing Substrates for PNA-Based Amperometric Genosensors

A new amperometric sandwich-format genosensor has been implemented on single-walled carbon nanotubes screen printed electrodes (SWCNT-SPEs) and compared in terms of performance with analogous genoassays developed using the same methodology on non-nanostructured glassy carbon platforms (GC-SPE). The working principle of the genosensors is based on the covalent immobilization of Peptide Nucleic Acid (PNA) capture probes (CP) on the electrode surface, carried out through the carboxylic functions present on SWCNT-SPEs (carboxylated SWCNT) or electrochemically induced on GC-SPEs. The sequence of the CP was complementary to a 20-mer portion of the target DNA; a second biotin-tagged PNA signalling probe (SP), with sequence complementary to a different contiguous portion of the target DNA, was used to obtain a sandwich hybrid with an Alkaline Phosphatase-streptavidin conjugate (ALP-Strp). Comparison of the responses obtained from the SWCNT-SPEs with those produced from the non-nanostructured substrates evidenced the remarkable enhancement effect given by the nanostructured electrode platforms, achieved both in terms of loading capability of PNA probes and amplification of the electron transfer phenomena exploited for the signal transduction, giving rise to more than four-fold higher sensitivity when using SWCNT-SPEs. The nanostructured substrate allowed to reach limit of detection (LOD) of 71 pM and limit of quantitation (LOQ) of 256 pM, while the corresponding values obtained with GC-SPEs were 430 pM and 1.43 nM, respectively

A Wi-Fi cloud-based portable potentiostat for electrochemical biosensors

The measurement of the analyte concentration in electrochemical biosensors traditionally requires costly laboratory equipment to obtain accurate results. Innovative portable solutions have recently been proposed, but usually, they lean on personal computers (PCs) or smartphones for data elaboration and they exhibit poor resolution or portability and proprietary software. This paper presents a low-cost portable system, assembling an ad hoc -designed analog front end (AFE) and a development board equipped with a system on chip integrating a microcontroller and a Wi-Fi network processor. The wireless module enables the transmission of measurements directly to a cloud service for sharing device outcome with users (physicians, caregivers, and so on). In doing so, the system does not require neither the customized software nor other devices involved in data acquisition. Furthermore, when any Internet connection is lost, the data are stored on board for subsequent transmission when a Wi-Fi connection is available. The noise output voltage spectrum has been characterized. Since the designed device is intended to be battery-powered to enhance portability, investigations about battery lifetime were carried out. Finally, data acquired with a conventional benchtop Autolab PGSTAT-204 electrochemical workstation are compared with the outcome of our developed device to validate the effectiveness of our proposal. To this end, we selected ferri/ferrocyanide as redox probe, obtaining the calibration curves for both the platforms. The final outcomes are shown to be feasible, accurate, and repeatable

A self-calibrating IoT portable electrochemical immunosensor for serum human epididymis protein 4 as a tumor biomarker for ovarian cancer

Nowadays analytical techniques are moving towards the development of smart biosensing strategies for point-of-care accurate screening of disease biomarkers, such as human epididymis protein 4 (HE4), a recently discovered serum markers for early ovarian cancer diagnosis. In this context, the present work represents the first implementation of a competitive enzyme-labelled magneto-immunoassay exploiting a homemade IoT Wi-Fi cloud-based portable potentiostat for differential pulse voltammetry readout. The electrochemical device was specifically designed capable of autonomous calibration and data processing, switching between calibration and measurement modes: in particular, firstly a baseline estimation algorithm is applied for correct peak computation, then calibration function is built by interpolating data with a four-parameter logistic function. The calibration function parameters are stored on the cloud for inverse prediction to determine the concentration of unknown samples. Interpolation function calibration and concentration evaluation are performed directly on-board, reducing the power consumption. The analytical device was validated in human serum, demonstrating good sensing performance for analysis of HE4 with detection and quantitation limits in human serum of 3.5 and 29.2 pM, respectively, reaching the sensitivity required for diagnostic purposes, with high potential for applications as portable and smart diagnostic tool for point-of-care testing

Optimization of a rapid QuEChERS sample treatment method for HILIC-MS2 analysis of paralytic shellfish poisoning (PSP) toxins in mussels

A rapid and simple QuEChERS sample treatment was proposed for the development of a selective hydrophilic interaction liquid chromatography-ESI-MS2-based method for the determination of saxitoxins (STXs) in mussel samples. Among different sorbents, ABS Elut-NEXUS phase, composed of polystyrene cross-linked with 50 % divinyl benzene and poly(methyl methacrylate), provided the best results. The effects of experimental parameters, including sorbent amount, vortexing time and centrifugation time were investigated and optimized by experimental design. In particular, regression models and desirability functions were applied to find the experimental conditions providing the highest global extraction response. The method was validated under the optimized conditions; detection and quantification limits in the 3-159 μg/kg and 7-436 μg/kg ranges respectively were obtained, except for C2 for which highest values were calculated due to its low ESI ionization efficiency. Finally, the analysis of twenty-eight mussel samples permitted to detect and quantify some of the investigated STXs, proving the applicability of the devised method

A Folding-Based Electrochemical Aptasensor for the Single-Step Detection of the SARS-CoV-2 Spike Protein

Efficient and timely testing has taken center stage in the management, control, and monitoring of the current COVID-19 pandemic. Simple, rapid, cost-effective diagnostics are needed that can complement current polymerase chain reaction-based methods and lateral flow immunoassays. Here, we report the development of an electrochemical sensing platform based on single-walled carbon nanotube screen-printed electrodes (SWCNT-SPEs) functionalized with a redox-tagged DNA aptamer that specifically binds to the receptor binding domain of the SARS-CoV-2 spike protein S1 subunit. Single-step, reagentless detection of the S1 protein is achieved through a binding-induced, concentration-dependent folding of the DNA aptamer that reduces the efficiency of the electron transfer process between the redox tag and the electrode surface and causes a suppression of the resulting amperometric signal. This aptasensor is specific for the target S1 protein with a dissociation constant (K-D) value of 43 +/- 4 nM and a limit of detection of 7 nM. We demonstrate that the target S1 protein can be detected both in a buffer solution and in an artificial viral transport medium widely used for the collection of nasopharyngeal swabs, and that no cross-reactivity is observed in the presence of different, non-target viral proteins. We expect that this SWCNT-SPE-based format of electrochemical aptasensor will prove useful for the detection of other protein targets for which nucleic acid aptamer ligands are made available

Evaluation of Fructooligosaccharides and Inulins as Potentially Health Benefiting Food Ingredients by HPAEC-PED and MALDI-TOF MS

This paper describes the complementarity of high-performance anion exchange chromatography coupled with pulsed electrochemical detection (HPAEC-PED) and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF-MS) to evaluate commercial available fructans (fructooligosaccharides (FOS) and inulins), having different degrees of polymerization (DP) which are usually employed by food industry as functional ingredients either for their prebiotic properties or as a fat replacer, giving a fat-like mouth feel and texture. The developed HPAEC-PED methods are able to analyze FOS (fructans with DP 3–10) and inulins (DP ranging from 3 to 80) with a good resolution and relatively short retention times to evaluate structural differences between fructooligosaccharide and inulins and the possible presence of inulooligosaccharides as well as of branching. To characterize FOS and inulin at different degrees of polymerization and to assure correct molecular assignment, MALDI-TOF MS analysis was also investigated. The 2,5-dihydroxy benzoic acid (2,5-DHB) was found to be the best matrix for FOS analysis as Actilight and Raftilose P95 products, while 3-aminoquinoline (3-AQ) seems to be the best matrix for inulin with higher DP. The applicability of the optimized methods to the identification and determination of FOS contained in a symbiotic milk as well as a type of inulin added as functional ingredient to a cooked ham is demonstrated

Controlling Dynamic DNA Reactions at the Surface of Single-Walled Carbon Nanotube Electrodes to Design Hybridization Platforms with a Specific Amperometric Readout

: Carbon nanotube (CNT)-based electrodes are cheap, highly performing, and robust platforms for the fabrication of electrochemical sensors. Engineering programmable DNA nanotechnologies on the CNT surface can support the construction of new electrochemical DNA sensors providing an amperometric output in response to biomolecular recognition. This is a significant challenge, since it requires gaining control of specific hybridization processes and functional DNA systems at the interface, while limiting DNA physisorption on the electrode surface, which contributes to nonspecific signal. In this study, we provide design rules to program dynamic DNA structures at the surface of single-walled carbon nanotubes electrodes, showing that specific DNA interactions can be monitored through measurement of the current signal provided by redox-tagged DNA strands. We propose the use of pyrene as a backfilling agent to reduce nonspecific adsorption of reporter DNA strands and demonstrate the controlled formation of DNA duplexes on the electrode surface, which we then apply in the design and conduction of programmable DNA strand displacement reactions. Expanding on this aspect, we report the development of novel amperometric hybridization platforms based on artificial DNA structures templated by the small molecule melamine. These platforms enable dynamic strand exchange reactions orthogonal to conventional toehold-mediated strand displacement and may support new strategies in electrochemical sensing of biomolecular targets, combining the physicochemical properties of nanostructured carbon-based materials with programmable nucleic acid hybridization

IoT and Biosensors: A Smart PortablePotentiostat With AdvancedCloud-Enabled Features

Recent advances in Internet-of-Things technology have opened the doors to new scenariosfor biosensor applications. Flexibility, portability, and remote control and access are of utmost importanceto move these devices to people’s homes or in a Point-of-Care context and rapidly share the results withusers and their physicians. In this paper, an innovative portable device for both quantitative and semi-quantitative electrochemical analysis is presented. This device can operate autonomously without the needof relying on other devices (e.g., PC, tablets, or smartphones) thanks to built-in Wi-Fi connectivity. Thedeveloped hardware is integrated into a cloud-based platform, exploiting the cloud computational powerto perform innovative algorithms for calibration (e.g., Machine Learning tools). Results and configurationscan be accessed through a web page without the installation of dedicated APPs or software. The electricalinput/output characteristic was measured with a dummy cell as a load, achieving excellent linearity.Furthermore, the device response to five different concentrations of potassium ferri/ferrocyanide redox probewas compared with a bench-top laboratory instrument. No difference in analytical sensitivity was found.Also, some examples of application-specific tests were set up to demonstrate the use in real-case scenarios.In addition, Support Vector Machine algorithm was applied to semi-quantitative analyses to classify theinput samples into four classes, achieving an average accuracy of 98.23%. Finally, COVID-19 related testsare presented and discussed (PDF) IoT and Biosensors: A Smart Portable Potentiostat With Advanced Cloud-Enabled Features. Available from: https://www.researchgate.net/publication/355214115_IoT_and_Biosensors_A_Smart_Portable_Potentiostat_With_Advanced_Cloud-Enabled_Features [accessed Oct 25 2021]

FFF-based high-throughput sequence shortlisting to support the development of aptamer-based analytical strategies

Aptamers are biomimetic receptors that are increasingly exploited for the development of optical and electrochemical aptasensors. They are selected in vitro by the SELEX (Systematic Evolution of Ligands by Exponential Enrichment) procedure, but although they are promising recognition elements, for their reliable applicability for analytical purposes, one cannot ignore sample components that cause matrix effects. This particularly applies when different SELEX-selected aptamers and related truncated sequences are available for a certain target, and the choice of the aptamer should be driven by the specific downstream application. In this context, the present work aimed at investigating the potentialities of asymmetrical flow field-flow fractionation (AF4) with UV detection for the development of a screening method of a large number of anti-lysozyme aptamers towards lysozyme, including randomized sequences and an interfering agent (serum albumin). The possibility to work in native conditions and selectively monitor the evolution of untagged aptamer signal as a result of aptamer-protein binding makes the devised method effective as a strategy for shortlisting the most promising aptamers both in terms of affinity and in terms of selectivity, to support subsequent development of aptamer-based analytical devices