Single-Response Duplexing of Electrochemical Label-Free Biosensor from the Same Tag.

Multiplexing is a valuable strategy to boost throughput and improve clinical accuracy. Exploiting the vertical, meshed design of reproducible and low-cost ultra-dense electrochemical chips, the unprecedented single-response multiplexing of typical label-free biosensors is reported. Using a cheap, handheld one-channel workstation and a single redox probe, that is, ferro/ferricyanide, the recognition events taking place on two spatially resolved locations of the same working electrode can be tracked along a single voltammetry scan by collecting the electrochemical signatures of the probe in relation to different quasi-reference electrodes, Au (0 V) and Ag/AgCl ink (+0.2 V). This spatial isolation prevents crosstalk between the redox tags and interferences over functionalization and binding steps, representing an advantage over the existing non-spatially resolved single-response multiplex strategies. As proof of concept, peptide-tethered immunosensors are demonstrated to provide the duplex detection of COVID-19 antibodies, thereby doubling the throughput while achieving 100% accuracy in serum samples. The approach is envisioned to enable broad applications in high-throughput and multi-analyte platforms, as it can be tailored to other biosensing devices and formats.

ZnO-Based Electrochemical Immunosensor to Assess Vaccine-Induced Antibody-Mediated Immunity against Wild-Type and Gamma SARS-CoV-2 Strains.

The evaluation of serological responses to COVID-19 is crucial for population-level surveillance, developing new vaccines, and evaluating the efficacy of different immunization programs. Research and development of point-of-care test technologies remain essential to improving immunity assessment, especially for SARS-CoV-2 variants that partially evade vaccine-induced immune responses. In this work, an impedimetric biosensor based on the immobilization of the recombinant trimeric wild-type spike protein (S protein) on zinc oxide nanorods (ZnONRs) was employed for serological evaluation. We successfully assessed its applicability using serum samples from spike-based COVID-19 vaccines: ChAdOx1-S (Oxford-AstraZeneca) and BNT162b2 (Pfizer-BioNTech). Overall, the ZnONRs/ spike-modified electrode displayed accurate results for both vaccines, showing excellent potential as a tool for assessing and monitoring seroprevalence in the population. A refined outcome of this technology was achieved when the ZnO immunosensor was functionalized with the S protein from the P.1 linage (Gamma variant). Serological responses against samples from vaccinated individuals were acquired with excellent performance. Following studies based on traditional serological tests, the ZnONRs/spike immunosensor data reveal that ChAdOx1-S vaccinated individuals present significantly less antibody-mediated immunity against the Gamma variant than the BNT162b2 vaccine, highlighting the great potential of this point-of-care technology for evaluating vaccine-induced humoral immunity against different SARS-CoV-2 strains.

Electrochemical Immunosensors Based on Zinc Oxide Nanorods for Detection of Antibodies Against SARS-CoV-2 Spike Protein in Convalescent and Vaccinated Individuals.

Even after over 2 years of the COVID-19 pandemic, research on rapid, inexpensive, and accurate tests remains essential for controlling and avoiding the global spread of SARS-CoV-2 across the planet during a potential reappearance in future global waves or regional outbreaks. Assessment of serological responses for COVID-19 can be beneficial for population-level surveillance purposes, supporting the development of novel vaccines and evaluating the efficacy of different immunization programs. This can be especially relevant for broadly used inactivated whole virus vaccines, such as CoronaVac, which produced lower titers of neutralizing antibodies. and showed lower efficacy for specific groups such as the elderly and immunocompromised. We developed an impedimetric biosensor based on the immobilization of SARS-CoV-2 recombinant trimeric spike protein (S protein) on zinc oxide nanorod (ZnONR)-modified fluorine-doped tin oxide substrates for COVID-19 serology testing. Due to electrostatic interactions, the negatively charged S protein was immobilized via physical adsorption. The electrochemical response of the immunosensor was measured at each modification step and characterized by scanning electron microscopy and electrochemical techniques. We successfully evaluated the applicability of the modified ZnONR electrodes using serum samples from COVID-19 convalescent individuals, CoronaVac-vaccinated with or without positive results for SARS-CoV-2 infection, and pre-pandemic samples from healthy volunteers as controls. ELISA for IgG anti-SARS-CoV-2 spike protein was performed for comparison, and ELISA for IgG anti-RBDs of seasonal coronavirus (HCoVs) was used to test the specificity of immunosensor detection. No cross-reactivity with HCoVs was detected using the ZnONR immunosensor, and more interestingly, the sensor presented higher sensitivity when compared to negative ELISA results. The results demonstrate that the ZnONRs/spike-modified electrode displayed sensitive results for convalescents and vaccinated samples and shows excellent potential as a tool for the population's assessment and monitoring of seroconversion and seroprevalence.

Modular Label-Free Electrochemical Biosensor Loading Nature-Inspired Peptide toward the Widespread Use of COVID-19 Antibody Tests.

Limitations of the recognition elements in terms of synthesis, cost, availability, and stability have impaired the translation of biosensors into practical use. Inspired by nature to mimic the molecular recognition of the anti-SARS-CoV-2 S protein antibody (AbS) by the S protein binding site, we synthesized the peptide sequence of Asn-Asn-Ala-Thr-Asn-COOH (abbreviated as PEP2003) to create COVID-19 screening label-free (LF) biosensors based on a carbon electrode, gold nanoparticles (AuNPs), and electrochemical impedance spectroscopy. The PEP2003 is easily obtained by chemical synthesis, and it can be adsorbed on electrodes while maintaining its ability for AbS recognition, further leading to a sensitivity 3.4-fold higher than the full-length S protein, which is in agreement with the increase in the target-to-receptor size ratio. Peptide-loaded LF devices based on noncovalent immobilization were developed by affording fast and simple analyses, along with a modular functionalization. From studies by molecular docking, the peptide-AbS binding was found to be driven by hydrogen bonds and hydrophobic interactions. Moreover, the peptide is not amenable to denaturation, thus addressing the trade-off between scalability, cost, and robustness. The biosensor preserves 95.1% of the initial signal for 20 days when stored dry at 4 °C. With the aid of two simple equations fitted by machine learning (ML), the method was able to make the COVID-19 screening of 39 biological samples into healthy and infected groups with 100.0% accuracy. By taking advantage of peptide-related merits combined with advances in surface chemistry and ML-aided accuracy, this platform is promising to bring COVID-19 biosensors into mainstream use toward straightforward, fast, and accurate analyses at the point of care, with social and economic impacts being achieved.

In Situ Nanocoating on Porous Pyrolyzed Paper Enables Antibiofouling and Sensitive Electrochemical Analyses in Biological Fluids.

Electrochemical detection in complex biofluids is a long-standing challenge as electrode biofouling hampers its sensing performance and commercial translation. To overcome this drawback, pyrolyzed paper as porous electrode coupled with the drop casting of an off-the-shelf polysorbate, that is, Tween 20 (T20), is described here by taking advantage of the in situ formation of a hydrophilic nanocoating (2 nm layer of T20). The latter prevents biofouling while providing the capillarity of samples through paper pores, leveraging redox reactions across both only partially fouled and fresh electrodic surfaces with increasing detection areas. The nanometric thickness of this blocking layer is also essential by not significantly impairing the electron-transfer kinetics. These phenomena behave synergistically to enhance the sensibility that further increases over long-term exposures (4 h) in biological fluids. While the state-of-the-art antibiofouling strategies compromise the sensibility, this approach leads to peak currents that are up to 12.5-fold higher than the original currents after 1 h exposure to unprocessed human plasma. Label-free impedimetric immunoassays through modular bioconjugation by directly anchoring spike protein on gold nanoparticles are also allowed, as demonstrated for the COVID-19 screening of patient sera. The scalability and simplicity of the platform combined with its unique ability to operate in biofluids with enhanced sensibility provide the generation of promising biosensing technologies toward real-world applications in point-of-care diagnostics, mass testing, and in-home monitoring of chronic diseases.

Application of nanomaterials for the electrical and optical detection of the hepatitis B virus.

This work describes different approaches for the detection of hepatitis B virus (HBV) genomic DNA, using electrochemical and optical techniques. The platforms consisted of a single-stranded DNA probe (HEPB1S), specific to HBV, grafted on a gold electrode modified with reduced graphene oxide or gold nanoparticles. Differential pulse voltammetry analysis indicates that the addition of HBV genomic DNA caused an increase of about 1.4 times in the current peak value, when compared to the negative control. It was observed a linear dependence with the log HBV-genomic DNA concentration and the electrochemical biosensor detected until 7.65 pg µL-1 of the target. Electrochemical impedance spectroscopy measurements showed an increase of about 2 times in the charge transfer resistance, after the addition of HBV genomic DNA. Assays using colloidal suspension of gold nanoparticles showed a shift of the peak wavelength, linearly proportional to the HBV-genomic DNA concentration, with a detection limit of 0.15 ng µL-1. The applicability of the gold nanoparticles for clinical samples was tested with success in the blood plasma. All the approaches used in this work were effective in detecting genomic DNA or blood plasma in positive samples for HBV.

Development of direct assays for Toxoplasma gondii and its use in genomic DNA sample.

This work describes an approach for the selection and detection of specific DNA probes related to Toxoplasma gondii, a protozoan parasite responsible for toxoplasmosis. The detection system was developed on graphite carbon electrode modified with poly(3-hydroxybenzoic acid) sensitized with ToxG1 probe. The hybridization of the specific genomic DNA related to T. gondii showed good response by direct detection of guanine residue oxidation using differential pulse voltammetry (DPV). The biosensor was able to distinguish both the complementary and non-complementary targets and detect up to 100ngµL-1 of the T. gondii genomic DNA. The hybridization (ToxG1: T. gondii genomic DNA) was confirmed by optical measurement. Optical assays using gold nanoparticles:ToxG1 probe showed a significant change in the absorbance peak in the presence of the T. gondii genomic DNA according to the electrochemical results. This novel biosensor shows potential as electrochemical transducer and was successfully applied in the biological sample.

Detection of a specific biomarker for Epstein-Barr virus using a polymer-based genosensor.

This paper describes methodology for direct and indirect detections of a specific oligonucleotide for Epstein-Barr virus (EBV) using electrochemical techniques. The sequence of oligonucleotide probe (EBV1) revealed a high sequence identity (100%) with the EBV genome. For the development of the genosensor, EBV1 was grafted to the platform sensitized with poly(4-aminothiophenol). After that, the hybridization reaction was carried out with the complementary target (EBV2) on the modified electrode surface using ethidium bromide as DNA intercalator. The oxidation peak currents of ethidium bromide increased linearly with the values of the concentration of the complementary sequences in the range from 3.78 to 756 µmol·L⁻¹. In nonstringent experimental conditions, this genosensor can detect 17.32 nmol·L⁻¹ (three independent experiments) of oligonucleotide target, discriminating between complementary and non-complementary oligonucleotides, as well as differentiating one-base mismatch, as required for detection of genetic diseases caused by point mutations. The biosensor also displayed high specificity to the EBV target with elimination of interference from mix (alanine, glucose, uric acid, ascorbic acid, bovine serum albumin (BSA), glutamate and glycine) and good stability (120 days). In addition, it was possible to observe differences between hybridized and non-hybridized surfaces through atomic force microscopy.