Are Aptamers Really Promising as Receptors for Analytical Purposes? Insights into Anti-Lysozyme DNA Aptamers through a Multitechnique Study.

Aptamers are recognition elements increasingly used for the development of biosensing strategies, especially in the detection of proteins or small molecule targets. Lysozyme, which is recognized as an important biomarker for various diseases and a major allergenic protein found in egg whites, is one of the main analytical targets of aptamer-based biosensors. However, since aptamer-based strategies can be prone to artifacts and data misinterpretation, rigorous strategies for multifaceted characterization of the aptamer-target interaction are needed. In this work, a multitechnique approach has been devised to get further insights into the binding performance of the anti-lysozyme DNA aptamers commonly used in the literature. To study molecular interactions between lysozyme and different anti-lysozyme DNA aptamers, measurements based on a magneto-electrochemical apta-assay, circular dichroism spectroscopy, fluorescence spectroscopy, and asymmetrical flow field-flow fractionation were performed. The reliability and versatility of the approach were proved by investigating a SELEX-selected RNA aptamer reported in the literature, that acts as a positive control. The results confirmed that an interaction in the low micromolar range is present in the investigated binding buffers, and the binding is not associated with a conformational change of either the protein or the DNA aptamer. The similar behavior of the anti-lysozyme DNA aptamers compared to that of randomized sequences and polythymine, used as negative controls, showed nonsequence-specific interactions. This study demonstrates that severe testing of aptamers resulting from SELEX selection is the unique way to push these biorecognition elements toward reliable and reproducible results in the analytical field.

Evaluation of a Voltametric E-Tongue Combined with Data Preprocessing for Fast and Effective Machine Learning-Based Classification of Tomato Purées by Cultivar.

The potential of a voltametric E-tongue coupled with a custom data pre-processing stage to improve the performance of machine learning techniques for rapid discrimination of tomato purées between cultivars of different economic value has been investigated. To this aim, a sensor array with screen-printed carbon electrodes modified with gold nanoparticles (GNP), copper nanoparticles (CNP) and bulk gold subsequently modified with poly(3,4-ethylenedioxythiophene) (PEDOT), was developed to acquire data to be transformed by a custom pre-processing pipeline and then processed by a set of commonly used classifiers. The GNP and CNP-modified electrodes, selected based on their sensitivity to soluble monosaccharides, demonstrated good ability in discriminating samples of different cultivars. Among the different data analysis methods tested, Linear Discriminant Analysis (LDA) proved to be particularly suitable, obtaining an average F1 score of 99.26%. The pre-processing stage was beneficial in reducing the number of input features, decreasing the computational cost, i.e., the number of computing operations to be performed, of the entire method and aiding future cost-efficient hardware implementation. These findings proved that coupling the multi-sensing platform featuring properly modified sensors with the custom pre-processing method developed and LDA provided an optimal tradeoff between analytical problem solving and reliable chemical information, as well as accuracy and computational complexity. These results can be preliminary to the design of hardware solutions that could be embedded into low-cost portable devices.

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.

Smart Immunosensors for Point-of-Care Serological Tests Aimed at Assessing Natural or Vaccine-Induced SARS-CoV-2 Immunity.

Innovative and highly performing smart voltammetric immunosensors for rapid and effective serological tests aimed at the determination of SARS-CoV-2 antibodies were developed and validated in human serum matrix. Two immunosensors were developed for the determination of immunoglobulins directed against either the nucleocapsid or the spike viral antigen proteins. The immunosensors were realized using disposable screen-printed electrodes modified with nanostructured materials for the immobilization of the antigens. Fast quantitative detection was achieved, with analysis duration being around 1 h. Signal readout was carried out through a smart, compact and battery-powered potentiostat, based on a Wi-Fi protocol and devised for the Internet of Things (IoT) paradigm. This device is used for the acquisition, storage and sharing of clinical data. Outstanding immunosensors' sensitivity, specificity and accuracy (100%) were assessed, according to the diagnostic guidelines for epidemiological data. The overall performance of the sensing devices, combined with the portability of the IoT-based device, enables their suitability as a high-throughput diagnostic tool. Both of the immunosensors were validated using clinical human serum specimens from SARS-CoV-2 infected patients, provided by IRCCS Ospedale San Raffaele.

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 novel magnetic ligand-based assay for the electrochemical determination of BRD4.

The first magnetic ligand-based electrochemical assay aimed at the determination of BRD4 was developed and validated. BRD4 is an epigenetic regulator of great interest in oncology in relation to its overexpression observed in the pathogenesis of several cancer diseases. BRD4 also represents a major target for the development of innovative treatments aimed at protein inhibition or degradation. Despite the relevance of BRD4 both for diagnostics and therapeutic purposes, current methodologies for its determination are limited to commercial ELISA kits. We present a novel magnetic ligand-based assay for the electrochemical determination of BRD4. The developed assay is based on the use of a small synthetic fragment of the natural protein ligand for BRD4 as receptor, thus exploiting the intrinsic biological protein-protein recognition mechanism. In addition, the assay features the use of magnetic beads as immobilization platforms and peroxidase-conjugated monoclonal anti-BRD4 antibody for the generation of the electrochemical signal. The ligand-based assay shows outstanding performance in terms of rapidity, with results achievable in less than 20 min, no matrix effect when applied to human plasma or cell lysate samples, and excellent specificity. The proposed method exhibits a limit of detection of 2.66 nM and a response range tunable as a function of the amount of immobilized receptor. The developed ligand-based assay was successfully applied to the accurate determination of BRD4 in untreated cell lysates, as proven by the ELISA reference method. The good performance of the proposed bioassay for determination of BRD4 showed potential application of this strategy in convenient point-of-care testing.

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 (KD) 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.

Rapid Quantification of SARS-Cov-2 Spike Protein Enhanced with a Machine Learning Technique Integrated in a Smart and Portable Immunosensor.

An IoT-WiFi smart and portable electrochemical immunosensor for the quantification of SARS-CoV-2 spike protein was developed with integrated machine learning features. The immunoenzymatic sensor is based on the immobilization of monoclonal antibodies directed at the SARS-CoV-2 S1 subunit on Screen-Printed Electrodes functionalized with gold nanoparticles. The analytical protocol involves a single-step sample incubation. Immunosensor performance was validated in a viral transfer medium which is commonly used for the desorption of nasopharyngeal swabs. Remarkable specificity of the response was demonstrated by testing H1N1 Hemagglutinin from swine-origin influenza A virus and Spike Protein S1 from Middle East respiratory syndrome coronavirus. Machine learning was successfully used for data processing and analysis. Different support vector machine classifiers were evaluated, proving that algorithms affect the classifier accuracy. The test accuracy of the best classification model in terms of true positive/true negative sample classification was 97.3%. In addition, the ML algorithm can be easily integrated into cloud-based portable Wi-Fi devices. Finally, the immunosensor was successfully tested using a third generation replicating incompetent lentiviral vector pseudotyped with SARS-CoV-2 spike glycoprotein, thus proving the applicability of the immunosensor to whole virus detection.

PNA-functionalized magnetic microbeads as substrates for enzyme-labelled voltammetric genoassay for DNA sensing applied to identification of GMO in food.

A novel enzyme-labelled voltammetric magnetogenoassay for DNA sensing based on the use of carboxyl-surface coated magnetic microbeads functionalized with PNA probes and subsequent read-out on screen-printed electrode (SPE) substrates was developed. The assay was validated for determination of non-amplified genomic DNA from genetically modified Roundup Ready soy. Outstanding performance with respect to other genoassays requiring preliminary amplification of target DNA via PCR was demonstrated. The analytical performance was also improved compared to previous methods based on the immobilization of the same PNA probes on SPE substrates, since the method was found capable of achieving LOD and LOQ of 415 fM and 995 fM, respectively. The ability of the magnetogenoassay to detect the presence of Roundup Ready soy DNA sequence was tested on genomic DNA extract from European Reference Material soy flours, demonstrating the capability of the method to match the European Union regulation for labelling of food containing a percentage of GM products greater than 0,9%.

Novel amperometric genosensor based on peptide nucleic acid (PNA) probes immobilized on carbon nanotubes-screen printed electrodes for the determination of trace levels of non-amplified DNA in genetically modified (GM) soy.

A novel amperometric genosensor based on PNA probes covalently bound on the surface of Single Walled Carbon Nanotubes - Screen Printed Electrodes (SWCNT-SPEs) was developed and validated in samples of non-amplified genomic DNA extracted from genetically modified (GM)-Soy. The sandwich assay is based on a first recognition of a 20-mer portion of the target DNA by a complementary PNA Capture Probe (CP) and a second hybridization with a PNA Signalling Probe (SP), with a complementary sequence to a different portion of the target DNA. The SP was labelled with biotin to measure current signal by means of a final incubation of an Alkaline Phosphatase-streptavidin conjugate (ALP-Strp). The electrochemical detection was carried out using hydroquinone diphosphate (HQDP) as enzymatic substrate. The genoassay provided a linear range from 250 pM to 2.5 nM, LOD of 64 pM and LOQ of 215 pM Excellent selectivity towards one base mismatch (1-MM) or scrambled (SCR) sequences was obtained. A simple protocol for extraction and analysis of non-amplified soybean genomic DNA without sample treatment was developed and validated. Our study provides insight into how the outstanding recognition efficiency of PNAs can be combined with the unique properties of CNTs in terms of signal response enhancement for direct detection of genomic DNA samples at the level of interest without previous amplification.