Label-free Electrochemical Biosensor Research Articles

A label-free electrochemical biosensor for highly sensitive detection of GM2A based on gold nanoparticles/conducting amino-functionalized thiophene polymer layer

In this study, a new immunosensor based on an indium tin oxide (ITO) substrate functionalized with gold nanoparticles (GNPs)/amino-functionalized thiophene polymer P(ThiAmn) multilayer was fabricated for impedimetric determination of GM2 activator protein (GM2A). To engineer the biosensor, a relatively simple approach composed of GNPs electrodeposition and ThiAmn electropolymerization was utilized. The use of GNPs and P(ThiAmn) increased the substrate surface area, which was beneficial to immobilize a large amount of anti-GM2A biorecognition elements. The designed bifunctional layer served as a promising matrix material and provided an innovative sensor fabrication. Electrochemical techniques were employed to investigate the specific immuno-interaction process between biorecognition anti-GM2A antibodies and GM2A antigens. Apart from these techniques, spectral techniques were utilized for the characterization of different modified electrode surfaces. Under optimum conditions, GM2A was determined in a linear concentration range from 0.0185 to 111 pg/mL with a detection limit (LOD) of 5.8 fg/mL by electrochemical impedance spectroscopy (EIS) technique. This biosensor exhibited good reproducibility, long storage-stability, and excellent specificity for GM2A antigens. Additionally, this immunosensor was applied to quantify GM2A in commercial serum samples, and satisfactory results were obtained.

A Label-Free Electrochemical Biosensor for Homocysteine Detection Using Molecularly Imprinted Polymer and Nanocomposite-Modified Electrodes.

An essential biomarker for the early detection of cardiovascular diseases is serum homocysteine (Hcy). In this study, a molecularly imprinted polymer (MIP) and nanocomposite were used to create a label-free electrochemical biosensor for reliable Hcy detection. A novel Hcy-specific MIP (Hcy-MIP) was synthesized using methacrylic acid (MAA) in the presence of trimethylolpropane trimethacrylate (TRIM). The Hcy-MIP biosensor was fabricated by overlaying the mixture of Hcy-MIP and the carbon nanotube/chitosan/ionic liquid compound (CNT/CS/IL) nanocomposite on the surface of a screen-printed carbon electrode (SPCE). It showed high sensitivity, with a linear response of 5.0 to 150 µM (R2 of 0.9753) and with a limit of detection (LOD) at 1.2 µM. It demonstrated low cross-reactivity with ascorbic acid, cysteine, and methionine. Recoveries of 91.10-95.83% were achieved when the Hcy-MIP biosensor was used for Hcy at 50-150 µM concentrations. The repeatability and reproducibility of the biosensor at the Hcy concentrations of 5.0 and 150 µM were very good, with coefficients of variation at 2.27-3.50% and 3.42-4.22%, respectively. This novel biosensor offers a new and effective method for Hcy assay compared with the chemiluminescent microparticle immunoassay at the correlation coefficient (R2) of 0.9946.

Highly sensitive and label-free electrochemical detection of C-reactive protein on a peptide receptor−gold nanoparticle−black phosphorous nanocomposite modified electrode

C-reactive protein (CRP) is a phylogenetically highly conserved plasma protein found in blood serum, and an enhanced CRP level is indicative of inflammatory conditions such as infection and cancer, among others. In this work, we developed a novel high CRP-affinity peptide-functionalized label-free electrochemical biosensor for the highly sensitive and selective detection of CRP. Throughout biopanning with random peptide libraries, high affinity peptides for CRP was successfully identified, and then a series of synthetic peptide receptor, of which C-terminus was incorporated to gold binding peptide (GBP) as an anchoring motif was covalently immobilized onto gold nanoparticle (AuNPs) tethered polydopamine (PDA)‒black phosphorus (BP) (AuNPs@BP@PDA) nanocomposite electrode. Interaction between the CRP-binding peptide and CRP was confirmed via enzyme-linked immunosorbent assay along with various physicochemical and electrochemical analyses. Under the optimized experimental conditions, the proposed peptide-based biosensor detects CRP in the range of 0–0.036 μg/mL with a detection limit (LOD) of 0.7 ng/mL. The developed sensor effectively detects CRP in the real samples of serum and plasma of Crohn's disease patients. Thus, the fabricated peptide-based biosensor has potential applications in clinical diagnosis and medical applications.

Specific Adsorption of Magnetic Fe3O4@hydroxyapatite Nanocomposites to a Hybridized Duplex for Immobilization and Label-Free Electrochemical Biosensing of MicroRNA

The unique discriminating ability of hydroxyapatite (HAP) to single- and double-stranded nucleic acid implies its potential as a biosensing material for nucleic acid analysis. Herein, a novel immobilization- and label-free electrochemical strategy has been developed for miRNA let-7a detection based on the specific adsorption of magnetic Fe3O4@hydroxyapatite (Fe3O4@HAP) to a duplex structure. Typically, the highly efficient homogeneous reaction between probe DNA (pDNA) and target miRNA let-7a produces the nucleic acid duplex, which is specifically adsorbed on the surface of Fe3O4@HAP. Thereafter, the electroactive intercalator Nile blue (NB) is introduced as the label-free signal molecule and inserts between the stacked base pairs of the pDNA–miRNA duplex. Thus, the Fe3O4@HAP bearing pDNA–miRNA and numerous intercalated NBs are adsorbed by the magnetic glassy carbon electrode, giving rise to an intense sensing signal without the need of nuclease-assisted cyclic amplification. With this advantage, the sensing system shows a wide linear range from 1.0 fM to 100 nM and a low detection limit of 0.051 fM for miRNA let-7a analysis. What is more, the synergy of the base-pairing-driven hybridization, the specific intercalation of NB to the duplex structure, and the selective adsorption of Fe3O4@HAP contribute to a high sensing specificity of the biosensing strategy. The sensing method provides a state-of-the-art strategy for the sensitive and facile bioanalysis and clinical diagnosis of miRNA.

Label-free immunosensing of telomerase using bio-conjugation of biotinylated antibody to poly(chitosan) gold nanoparticles.

Aim: This study aimed to establish a label-free electrochemical biosensor for telomerase detection in human biofluid. Method: Synthesized green nanocomposite (poly[chitosan] decorated by gold nanoparticles) was used for the efficient immobilization of biotinylated antibody of telomerase and immunocomplex of antigen-antibody. Poly(chitosan) was decorated by gold nanoparticles on the surface of a glassy carbon electrode using an electrochemical coating technique. Results: The constructedimmunosensor exhibited widedynamic range (0.078-160IU/ml-1) with a low limit of quantification of 0.078IU/ml-1, which present a unique manner for telomerase assays in early prognosis for cancers. Conclusion: This study encourages scientists and scholars to design and develop new biosensor platforms for point-of-care diagnostics for telomerase management, an interesting reference for future research.

11-(triethoxysilyl) undecanal agent-based biosensor system using disposable ITO-PET electrode for tumour necrosis factor-alpha detection

In this study, a label-free electrochemical biosensor system based on a disposable indium tin oxide polyethylene terephthalate (ITO-PET) electrode modified with the 11-(triethoxysilyl) undecanal (11-TESU) agent was developed for the detection of tumour necrosis factor-alpha (TNF-α) in serum. The developed biosensor was observed with electrochemical impedance spectroscopy (EIS), cyclic voltammetry (CV) techniques, square wave voltammetry (SWV) and single frequency impedance (SFI) technique which is utilized for the specific interaction between anti-TNF-α and TNF-α antigen. In addition, scanning electron microscopy was used to look at how the morphology of each ITO-PET surface changed (SEM). All parameters such as 11-TESU concentration, anti-TNF-α concentration and anti-TNF-α incubation time, were optimized. The biosensor system was characterized by measuring its linear determination range, repeatability, reproducibility, reusability, storage stability, and surface coverage. The TNF-αelectrochemical biosensor showed high levels of repeatability and reproducibility as well as a large dynamic range of detection (from 0.03 pg mL-1 to 3 pg mL-1). The LOD and LOQ for the biosensor were extremely low at 1x10-4 pg mL-1 and 5x10-4 pg mL-1, respectively. It was applied to real samples to determine whether the proposed biosensor would be useful in clinical settings.

Recent Advances in CMOS Electrochemical Biosensor Design for Microbial Monitoring: Review and Design Methodology.

Rapid, high-sensitivity, and real-time characterization of microorganisms plays a significant role in several areas, including clinical diagnosis, human healthcare, early detection of outbreaks, and the protection of living beings. Integrating microbiology and electrical engineering promises the development of low-cost, miniaturized, autonomous, and high-sensitivity sensors to quantify and characterize bacterial strains at various concentrations. Electrochemical-based biosensors are receiving particular attention in microbiological applications among the different biosensing devices. Several approaches have been adopted to design and fabricate cutting-edge, miniaturized, and portable electrochemical biosensors to track and monitor bacterial cultures in real time. These techniques differ in their sensing interface circuits and microelectrode fabrication. The goals of this review are (i) to summarize the current state of CMOS sensing circuit designs in label-free electrochemical biosensors for bacteria monitoring and (ii) to discuss the material and size of the electrodes used in electrochemical biosensors in microbiological applications. In this paper, we reviewed the latest and most advanced CMOS integrated interface circuits that have recently been used in electrochemical biosensors to identify and characterize bacteria species, such as impedance spectroscopy, capacitive, amperometry, and voltammetry, etc. In addition to the interface circuit design, other crucial factors, such as the material and scale of the electrodes, must be considered to increase the sensitivity of electrochemical biosensors. Surveying the literature in this field improves our knowledge about the impact of electrode designs and materials on sensing precision and will help future designers adapt, design, and fabricate appropriate electrode configurations based on their application. Thus, we summarized the conventional microelectrode designs and materials mainly employed in microbial sensors, including interdigitated electrodes (IDEs), microelectrode arrays (MEAs), paper, and carbon-based electrodes, etc.

A label-free ultrasensitive microRNA-21 electrochemical biosensor based on MXene (Ti3C2)-reduced graphene oxide-Au nanocomposites

The detection of microRNA is widely used in the early diagnosis of diseases such as cancer and acute kidney injury. In this work, we successfully constructed a label-free ultrasensitive biosensor for the instant detection of miRNA-21. The MXene-reduced graphene oxide (rGO)-Au was synthesized as electrode materials to immobilize DNA probes and MXene-AuPd was used as signal amplification materials. The highly sensitive biosensor had a wide range of linearity from 1 fM to 1 nM, and the limit of detection of as-fabricated biosensor was lower as 0.42 fM under optimized conditions. The biosensor also showed high specificity, outstanding stability and desirable reproducibility, which could also realize the accurate detection of miRNA-21 in real urine samples of both mouse and human with high sensitivity. The label-free electrochemical biosensor based on MXene nanocomposites is a promising candidate for potential applications on the early diagnosis of cancers and acute kidney injury.

Detection of plasma miR-223 by a novel label-free graphene oxide/gold nanocomposite immunosensor in colorectal cancer patients: An electrochemical biosensor approach

MicroRNAs (miRNAs) are one group of the pertinent molecular biomarkers in the early detection of cancers, especially colorectal cancer. A simple and label-free nanostructured electrochemical biosensor was fabricated to detect miR-223, a colorectal cancer miRNA, by a vertical arrangement of graphene oxide (GO) decorated with gold (Au) Nano-flower nanostructures (GO@Au-NS). The use of high surface-to-volume aspect ratio nanostructures with excellent electroactivity that contain thiolated DNA probes (Cap-223) could increase the accessibility, chance, and recognition performance of miR-223. The substrate characteristics are scanned by HRSEM, EDX, and electrochemical techniques. Furthermore, the successful immobilization of Cap-223 on GO@Au-NS and their hybridization with targeted miR-223 macro-molecules were investigated by electrochemical techniques (CV, EIS, and DPV). The electroactivity of the working electrode before and after GO@Au-NS fabrication showed a significant difference. A wide range of linear detection of miR-223 by a fabricated biosensor was from zM to nM, with a lower detection limit (LOD) of 0.012 aM. The selectivity of the nanostructured miR-223 biosensor was also investigated by studying the discrimination ability of mismatched miRNAs sequences, which resulted in high selectivity for the biosensor. In addition, its efficiency in the detection of miR-223 in a real sample (human serum) showed a remarkable ability of the biosensor to detect it with a low percent of relative standard deviation (RSD = 5.7%). The developed electrochemical biosensor exhibited great potential for miRNA-based early detection of colorectal cancer and had excellent sensitivity, and selectivity, making it helpful for use in translational medicine research and clinical applications.

Label-Free Electrochemical Biosensor Platforms for Cancer Diagnosis: Recent Achievements and Challenges.

With its fatal effects, cancer is still one of the most important diseases of today's world. The underlying fact behind this scenario is most probably due to its late diagnosis. That is why the necessity for the detection of different cancer types is obvious. Cancer studies including cancer diagnosis and therapy have been one of the most laborious tasks. Since its early detection significantly affects the following therapy steps, cancer diagnosis is very important. Despite researchers' best efforts, the accurate and rapid diagnosis of cancer is still challenging and difficult to investigate. It is known that electrochemical techniques have been successfully adapted into the cancer diagnosis field. Electrochemical sensor platforms that are brought together with the excellent selectivity of biosensing elements, such as nucleic acids, aptamers or antibodies, have put forth very successful outputs. One of the remarkable achievements of these biomolecule-attached sensors is their lack of need for additional labeling steps, which bring extra burdens such as interference effects or demanding modification protocols. In this review, we aim to outline label-free cancer diagnosis platforms that use electrochemical methods to acquire signals. The classification of the sensing platforms is generally presented according to their recognition element, and the most recent achievements by using these attractive sensing substrates are described in detail. In addition, the current challenges are discussed.

Label-Free Electrochemical Methods for Disease Detection.

Label-free electrochemical biosensing leverages the advantages of label-free techniques, low cost, and fewer user steps, with the sensitivity and portability of electrochemical analysis. In this review, we identify four label-free electrochemical biosensing mechanisms: (a) blocking the electrode surface, (b) allowing greater access to the electrode surface, (c) changing the intercalation or electrostatic affinity of a redox probe to a biorecognition unit, and (d) modulating ion or electron transport properties due to conformational and surface charge changes. Each mechanism is described, recent advancements are summarized, and relative advantages and disadvantages of the techniques are discussed. Furthermore, two avenues for gaining further diagnostic information from label-free electrochemical biosensors, through multiplex analysis and incorporating machine learning, are examined.

A novel spin-based label-free electrochemical dna hybridization biosensor and its applications for dengue virus detection

In this study, we have rationally constructed a spin-based label-free DNA hybridization sensor employing the spin-polarized electrons as a tool of detection. Recent research, that takes into account chiral-induced spin selectivity, has demonstrated colossal spin filtering by DNA at room temperature. Traditional DNA hybridization sensors use electronic(charge) current to probe the hybridization of double-strand (ds)-DNA. The benefit of the chiral-induced spin selectivity (CISS)-based DNA sensor lies in the direct probe of hybridization through its symmetry present in the secondary structure of ds-DNA. To this end, a self-assembled monolayer of DNA was prepared on Au-coated Ni devices and hybridization phenomena were investigated using differential pulse voltammetry. The accuracy of the CISS-based hybridization sensor was tested using additional complementary techniques such as cyclic voltammetry, electrochemical impedance spectroscopy, FTIR, and XPS, and the same method was successfully employed to detect the Dengue virus as well. With a limit of detection of up to 0.12 picomolar (pM), the results are reliable and repeatable. The current approach opens up new possibilities for developing spin-based point-of-care devices in the future.

Aptasensor for the Detection of Moraxella catarrhalis Adhesin UspA2

Innovative point-of-care (PoC) diagnostic platforms are desirable to surpass the deficiencies of conventional laboratory diagnostic methods for bacterial infections and to tackle the growing antimicrobial resistance crisis. In this study, a workflow was implemented, comprising the identification of new aptamers with high affinity for the ubiquitous surface protein A2 (UspA2) of the bacterial pathogen Moraxella catarrhalis and the development of an electrochemical biosensor functionalized with the best-performing aptamer as a bioreceptor to detect UspA2. After cell-systematic evolution of ligands by exponential enrichment (cell-SELEX) was performed, next-generation sequencing was used to sequence the final aptamer pool. The most frequent aptamer sequences were further evaluated using bioinformatic tools. The two most promising aptamer candidates, Apt1 and Apt1_RC (Apt1 reverse complement), had Kd values of 214.4 and 3.4 nM, respectively. Finally, a simple and label-free electrochemical biosensor was functionalized with Apt1_RC. The aptasensor surface modifications were confirmed by impedance spectroscopy and cyclic voltammetry. The ability to detect UspA2 was evaluated by square wave voltammetry, exhibiting a linear detection range of 4.0 × 104-7.0 × 107 CFU mL-1, a square correlation coefficient superior to 0.99 and a limit of detection of 4.0 × 104 CFU mL-1 at pH 5.0. The workflow described has the potential to be part of a sensitive PoC diagnostic platform to detect and quantify M. catarrhalis from biological samples.

A laser-assisted electrochemical biosensor based on folic acid-functionalized tungsten disulfide nanosheets for label-free cancer cell detection

This study reports a label-free and high-efficiency electrochemical biosensor based on functionalized tungsten disulfide (WS2) nanosheets with cysteine (CYS) and folic acid (FA) to diagnose cancer cells. The large surface area of WS2-CYS-FA nanosheets, combined with its strong binding affinity toward cancer cells, makes it an ideal material for modifying the working electrode to enhance the performance of cytosensors. The WS2 nanosheets were prepared using a liquid exfoliation method and decorated with FA through CYS. The successful preparation of WS2-CYS-FA was confirmed through various characterization techniques. The gold electrode was then modified with WS2-CYS-FA for the electrochemical detection of two breast cancer cell lines, MDA-MB-231 and MCF-7, classified as FR-positive cells, and the HUVEC cell line as FR-negative control cells. During electrochemical measurements, the WS2-CYS-FA@Au working electrode was irradiated with laser light. Electron-hole pairs generated by laser photons boosted interfacial electron transfer and improved electrochemical efficiency. The electrochemical results showed that the proposed cytosensor has great sensitivity and selectivity with a detection limit of 10 cells/mL of MDA-MB-231 cells in a linear concentration range from 20 to 2 × 105 cells/mL (S/N = 3). The fabricated biosensors were successfully tested with whole blood samples and had a high recovery rate.

Label-free electrochemical detection of cancer biomarkers DNA and anti-p53 at tin oxide quantum dot-gold-DNA nanoparticle modified electrode

Lung cancer is one of the deadliest types of cancer and accounts for 8.1% of all cancer related deaths. To prevent a growing death rate, it is crucial to identify lung cancer at an early stage by single polynucleotide morphism detection. In this paper, we present a novel label-free electrochemical biosensor based on composites of tin oxide quantum dots and gold nanoparticles (SnO2-QD-Au) for the sensitive and precise detection of lung cancer DNA. The SnO2-QD and SnO2-QD-Au nanoparticles were characterized using Scanning and Transmission Electron Microscopes (SEM and TEM), X-ray diffractometer (XRD), X-ray photoelectron spectroscopy (XPS), UV–Vis spectroscopy (UV), Fourier transmission infrared spectroscopy (FTIR), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) techniques. Gold thiol covalent bonding was used for immobilising probe DNA on the surface of SnO2-QD-Au nanoparticles followed by target DNA hybridization and detected electrochemically in presence of 1 mM [Fe(CN6)]3−/4−as a redox couple probe. Under ideal circumstances, the sensor showed the lowest detection limit of 3.2 × 10−20 M with a linear range of 1 × 10−6 − 1 × 10−20 M. Additionally, the sensing method was applied to find a cancer biomarker, Anti-P53 antibody.

A label-free electrochemical DNA biosensor used a printed circuit board gold electrode (PCBGE) to detect SARS-CoV-2 without amplification.

The emergence of coronavirus disease 2019 (COVID-19) motivates continuous efforts to develop robust and accurate diagnostic tests to detect severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Detection of viral nucleic acids provides the highest sensitivity and selectivity for diagnosing early and asymptomatic infection because the human immune system may not be active at this stage. Therefore, this work aims to develop a label-free electrochemical DNA biosensor for SARS-CoV-2 detection using a printed circuit board-based gold substrate (PCBGE). The developed sensor used the nucleocapsid phosphoprotein (N) gene as a biomarker. The DNA sensor-based PCBGE was fabricated by self-assembling a thiolated single-stranded DNA (ssDNA) probe onto an Au surface, which performed as the working electrode (WE). The Au surface was then treated with 6-mercapto-1-hexanol (MCH) before detecting the target N gene to produce a well-oriented arrangement of the immobilized ssDNA chains. The successful fabrication of the biosensor was characterized using cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and atomic force microscopy (AFM). The DNA biosensor performances were evaluated using a synthetic SARS-CoV-2 genome and 20 clinical RNA samples from healthy and infected individuals through EIS. The developed DNA biosensor can detect as low as 1 copy per μL of the N gene within 5 minutes with a LOD of 0.50 μM. Interestingly, the proposed DNA sensor could distinguish the expression of SARS-CoV-2 RNA in a patient diagnosed with COVID-19 without any amplification technique. We believe that the proposed DNA sensor platform is a promising point-of-care (POC) device for COVID-19 viral infection since it offers a rapid detection time with a simple design and workflow detection system, as well as an affordable diagnostic assay.

Activity-enhanced DNAzyme for design of label-free copper(II) biosensor.

Metal ion-driven, DNA-cleaving DNAzymes are characterised by high selectivity and specificity. However, their use for metal ion sensing remains largely unexplored due to long reaction times and poor reaction yields relative to RNA-cleaving DNAzymes and other sensing strategies. Herein we present a study demonstrating a significant rate enhancement of a copper-selective DNA cleaving DNAzyme by both polydopamine (PDA) and gold (Au) nanoparticles (NPs). PDA NPs enhance the reaction through the production of hydrogen peroxide, while for AuNPs the enhancement is aided by the presence of citrate surface moeities, both of which drive the oxidative cleavage of the substrate. A 50-fold enhancement for PDA NPs makes the combination of PDA and DNAzyme suitable for a practical application as a sensitive biosensor for Cu(II) ions. Using DNAzyme deposition onto a gold electrode followed by Polydopamine Assisted DNA Immobilisation (PADI), we achieve a cost-effective, label-free and fast (within 15 min) electrochemical biosensor with a limit of detection of 180 nmol (11 ppm), thus opening a route for the rational design of a new generation of hybrid DNAzyme-based biosensors.

Recent advances in label-free optical, electrochemical, and electronic biosensors for glioma biomarkers.

Gliomas are the most commonly occurring primary brain tumor with poor prognosis and high mortality rate. Currently, the diagnostic and monitoring options for glioma mainly revolve around imaging techniques, which often provide limited information and require supervisory expertise. Liquid biopsy is a great alternative or complementary monitoring protocol that can be implemented along with other standard diagnosis protocols. However, standard detection schemes for sampling and monitoring biomarkers in different biological fluids lack the necessary sensitivity and ability for real-time analysis. Lately, biosensor-based diagnostic and monitoring technology has attracted significant attention due to several advantageous features, including high sensitivity and specificity, high-throughput analysis, minimally invasive, and multiplexing ability. In this review article, we have focused our attention on glioma and presented a literature survey summarizing the diagnostic, prognostic, and predictive biomarkers associated with glioma. Further, we discussed different biosensory approaches reported to date for the detection of specific glioma biomarkers. Current biosensors demonstrate high sensitivity and specificity, which can be used for point-of-care devices or liquid biopsies. However, for real clinical applications, these biosensors lack high-throughput and multiplexed analysis, which can be achieved via integration with microfluidic systems. We shared our perspective on the current state-of-the-art different biosensor-based diagnostic and monitoring technologies reported and the future research scopes. To the best of our knowledge, this is the first review focusing on biosensors for glioma detection, and it is anticipated that the review will offer a new pathway for the development of such biosensors and related diagnostic platforms.

A Rapid Label-Free Disposable Electrochemical Salivary Point-of-Care Sensor for SARS-CoV-2 Detection and Quantification.

The coronavirus disease 2019 (COVID-19) pandemic has created an urgent need for accurate early diagnosis and monitoring. A label-free rapid electrochemical point-of-care (POC) biosensor for SARS-CoV-2 detection in human saliva is reported here to help address the shortcomings of traditional nucleic acid amplification methods and give a quantitative assessment of the viral load to track infection status anywhere, using disposable electrochemical sensor chips. A new chemical construct of gold nanoparticles (GNp) and thionine (Th) are immobilized on carboxylic acid functionalized carbon nanotubes (SWCNT-COOH) for high-performance biosensing. The sensor uses saliva with a one-step pretreatment and simple testing procedure as an analytical medium due to the user-friendly and non-invasive nature of its procurement from patients. The sensor has a response time of 5 min with a limit of detection (LOD) reaching 200 and 500 pM for the freely suspended spike (S) protein in phosphate buffer saline (PBS) and human saliva, respectively. The sensor's performance was also proven for detecting a COVID-19 pseudovirus in an electrolyte solution with a LOD of 106 copies/mL. The results demonstrate that the optimized POC sensor developed in this work is a promising device for the label-free electrochemical biosensing detection of SARS-CoV-2 and different species of viruses.

Analytical Perspectives in the Study of Polyvalent Interactions of Free and Surface-Bound Oligonucleotides and Their Implications in Affinity Biosensing

The high affinity and/or selectivity of oligonucleotide-mediated binding offers a myriad of therapeutical and analytical applications, whose rational design implies an accurate knowledge of the involved molecular mechanisms, concurring equilibrium processes and key affinity parameters. Oligonucleotide-functionalized gold surfaces or nanostructures are regularly employed analytical platforms for the development of label-free optical or electrochemical biosensors, and recently, novel detection platform designs have been increasingly considering the synergistic effect of polyvalent binding, involving the simultaneous interaction of two or several oligonucleotide strands. Considering the general lack of studies involving ternary single-stranded DNA (ssDNA) interactions, a complementary analytical workflow involving capillary gel electrophoretic (CGE) mobility shift assay, microcalorimetry and computational modeling has been deployed for the characterization of a series of free and surface-bound binary and ternary oligonucleotide interactions. As a proof of concept, the DNA analogue of MicroRNA 21 (miR21), a well-known oncogenic short MicroRNA (miRNA) sequence, has been chosen as a target molecule, simulating limiting-case scenarios involved in dual molecular recognition models exploited in affinity (bio)sensing. Novel data for the characterization of oligonucleotide interacting modules is revealed, offering a fast and complete mapping of the specific or non-specific, often competing, binary and ternary order interactions in dynamic equilibria, occurring between various free and metal surface-bound oligonucleotides.