Utilizing COVID-19 as a Model for Diagnostics Using an Electrochemical Sensor.

This paper reports a rapid and sensitive sensor for the detection and quantification of the COVID-19 N-protein (N-PROT) via an electrochemical mechanism. Single-frequency electrochemical impedance spectroscopy was used as a transduction method for real-time measurement of the N-PROT in an immunosensor system based on gold-conjugate-modified carbon screen-printed electrodes (Cov-Ag-SPE). The system presents high selectivity attained through an optimal stimulation signal composed of a 0.0 V DC potential and 10 mV RMS-1 AC signal at 100 Hz over 300 s. The Cov-Ag-SPE showed a log response toward N-PROT detection at concentrations from 1.0 ng mL-1 to 10.0 µg mL-1, with a 0.977 correlation coefficient for the phase (θ) variation. An ML-based approach could be created using some aspects observed from the positive and negative samples; hence, it was possible to classify 252 samples, reaching 83.0, 96.2 and 91.3% sensitivity, specificity, and accuracy, respectively, with confidence intervals (CI) ranging from 73.0 to 100.0%. Because impedance spectroscopy measurements can be performed with low-cost portable instruments, the immunosensor proposed here can be applied in point-of-care diagnostics for mass testing, even in places with limited resources, as an alternative to the common diagnostics methods.

Simultaneous detection of dopamine and ascorbic acid by using a thread-based microfluidic device and multiple pulse amperometry.

This study presents a novel approach for the simultaneous detection of ascorbic acid (AA) and dopamine (DA) using an affordable and user-friendly microfluidic device. Microfluidic devices, when combined with electrochemical detectors like screen-printed electrodes (SPEs), offer numerous advantages such as portability, high sample throughput, and low reagent consumption. In this study, a 3D-printed microfluidic device called a µTED was developed, utilizing textile threads as microfluidic channels and an unmodified SPE as the amperometric detector. The method employed multiple pulse amperometry (MPA) with carefully selected potential values (+0.65 V and -0.10 V). The reduction current signals generated by dopamine o-quinone were used to calculate a correction factor for the oxidation signals of ascorbic acid, enabling simultaneous quantification. The developed microfluidic device ensured a stable flow rate of the carrier solution at 1.19 µL s-1, minimizing the consumption of samples and reagents (injection volume of 2.0 µL). Under the optimized experimental conditions, a linear range from 50 to 900 µmol L-1 was achieved for both DA and AA. The obtained sensitivities were 2.24 µA L mmol-1 for AA and 5.09 µA L mmol-1 for DA, with corresponding limits of detection (LOD) of 2.60 µmol L-1 and 1.54 µmol L-1, respectively. To confirm the effectiveness of the proposed method, it was successfully applied to analyze AA and DA in a commercial blood serum sample spiked at three different concentration levels, with a medium recovery rate of 70%. Furthermore, the MPA technique demonstrated its simplicity by enabling the simultaneous determination of AA and DA without the need for prior separation steps or the use of chemically modified electrodes.

Gold-binding peptide as a selective layer for electrochemical detection of SARS-CoV-2 antibodies.

Electrochemical immunosensors are excellent alternatives to prepare portable platforms used for rapid and inexpensive diagnostic of infectious diseases such as the recently emerged COVID-19. Incorporating synthetic peptides as selective recognition layers combined with nanomaterials such as gold nanoparticles (AuNPs) can significantly enhance the analytical performance of immunosensors. In the present study, an electrochemical immunosensor based on solid-binding peptide was built and evaluated towards SARS-CoV-2 Anti-S antibodies detection. The peptide used as recognition site has two important portions: one based on the viral receptor binding domain (RBD), capable of recognizing antibodies of the spike protein (Anti-S), and another suitable for interacting with gold nanoparticles. Gold-binding peptide (Pept/AuNP) dispersion was used directly to modify a screen-printed carbon electrode (SPE). The voltammetric behavior of the [Fe(CN)6]3-/4- probe after every construction and detection step was recorded using cyclic voltammetry by assessing the stability of the Pept/AuNP as a recognition layer onto the electrode surface. Differential pulse voltammetry was used as a detection technique, and a linear working range from 75 ng mL-1 to 15 µg mL-1 was established, with 1.059 µA dec-1 of sensitivity and R2 = 0.984. The response selectivity against SARS-CoV-2 Anti-S antibodies was investigated in presence of concomitant species. The immunosensor was used to detect SARS-CoV-2 Anti-spike protein (Anti-S) antibodies in human serum samples, successfully differentiating between negative and positive responses of samples at a 95% confidence level. Therefore, the gold-binding peptide is a promising tool to be applied as a selective layer for antibody detection.

Novel approach based on GQD-PHB as anchoring platform for the development of SARS-CoV-2 electrochemical immunosensor.

In the present work, we report an innovative approach for immunosensors construction. The experimental strategy is based on the anchoring of biological material at screen-printed carbon electrode (SPE) modified with electrodeposited Graphene Quantum Dots (GQD) and polyhydroxybutyric acid (PHB). It was used as functional substract basis for the recognition site receptor-binding domain (RBD) from coronavirus spike protein (SARS-CoV-2), for the detection of Anti-S antibodies (AbS). SEM images and EDS spectra suggest an interaction of the protein with GQD-PHB sites at the electrode surface. Differential pulse voltametric (DPV) measurements were performed before and after incubation, in presence of the target, shown a decrease in voltametric signal of an electrochemical probe ([Fe(CN)6]3/4-). Using the optimal experimental conditions, analytical curves were performed in PBS and human serum spiked with AbS showing a slight matrix effect and a relationship between voltametric signal and AbS concentration in the range of 100 ng mL-1 and 10 µg mL-1. The selectivity of the proposed sensor was tested against yellow fever antibodies (YF) and the selective layer on the electrode surface did not interact with these unspecific antibodies. Eight samples of blood serum were analyzed and 87.5% of these total investigated provided adequate results. In addition, the present approach showed better results against traditional EDC/NHS reaction with enhancements in time and the possibility to develop an immunosensor in a single drop, since the proteins can be anchored prior to the electrode modification step.

Label-free aptasensor for p24-HIV protein detection based on graphene quantum dots as an electrochemical signal amplifier.

Human immunodeficiency virus (HIV) is still considered a pandemic, and the detection of p24-HIV protein has an important role in the early diagnosis of HIV in adults and newborns. The accessibility of these trials depends on the price and execution difficulty of the method, which can be reduced using electrochemical methods by using enzymeless approaches, disposable and accurate devices. In this work, graphene quantum dots were acquired by a simple synthesis and employed as an electrochemical signal amplifier and support for the aptamer immobilization through a feasible and stable modification of disposable screen-printed electrodes. The device has been easily assembled and used to detect p24-HIV protein without the interference of similar proteins or sample matrix. Using the best set of experimental conditions, a linear correlation between analytical signal and log of p24-HIV concentration from 0.93 ng mL-1 to 93 µg mL-1 and a limit of detection of 51.7 pg mL-1 were observed. The developed device was applied to p24 determination in spiked human serum and provided distinct levels of signal for positive and negative samples, successfully identifying real samples with the target protein. This sensor is a step towards the development of point-of-care devices and the popularization of electrochemical methods for trials and diagnostics of relevant diseases.

3D-printed electrode as a new platform for electrochemical immunosensors for virus detection.

Simple, low-cost, and sensitive new platforms for electrochemical immunosensors for virus detection have been attracted attention due to the recent pandemic caused by a new type of coronavirus (SARS-CoV-2). In the present work, we report for the first time the construction of an immunosensor using a commercial 3D conductive filament of carbon black and polylactic acid (PLA) to detect Hantavirus Araucaria nucleoprotein (Np) as a proof-of-concept. The recognition biomolecule was anchored directly at the filament surface by using N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and N-Hydroxysuccinimide (EDC/NHS). Conductive and non-conductive composites of PLA were characterized using thermal gravimetric analysis (TGA), revealing around 30% w/w of carbon in the filament. Morphological features of composites were obtained from SEM and TEM measurements. FTIR measurement revealed that crosslinking agents were covalently bonded at the filament surface. Electrochemical techniques such as cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were used for the evaluation of each step involved in the construction of the proposed immunosensor. The results showed the potentiality of the device for the quantitative detection of Hantavirus Araucaria nucleoprotein (Np) from 30 µg mL-1 to 240 µg mL-1 with a limit of detection of 22 µg mL-1. Also, the proposed immunosensor was applied with success for virus detection in 100x diluted human serum samples. Therefore, the PLA conductive filament with carbon black is a simple and excellent platform for immunosensing, which offers naturally carboxylic groups able to anchor covalently biomolecules.

Quick electrochemical immunoassay for hantavirus detection based on biochar platform.

This work describes the first method using biochar (BC) as carbonaceous platform for immunoassay application. BC is a highly functionalized material obtained through biomass pyrolysis under controlled conditions. Due to the highly functionalized surface, covalent binding between BC and biomolecules can be performed by EDC/NHS conjugation. The application of the modified electrode was done with Hantavirus, that are etiologic agents mainly transmitted by wild rodents. Among its pathologies Hantavirus Cardiopulmonary Syndrome (HCPS) arises at Americas, caused by Hantavirus Araucária and reaches 40% lethality. The diagnostic is based on the presence of specific hantavirus nucleoprotein (Np), under viremic condition or IgG2b antibodies (Ab), during first symptoms. The results presented a device sensitivity of 5.28 µA dec-1 and a LOD of 0.14 ng mL-1 to the Np detection, ranging from 5.0 ng mL-1 to 1.0 µg mL-1, the Ab detection works as qualitative type sensor above 200 ng mL-1. Both sensors were evaluated its selectivity and serum samples; selectivity against Gumboro disease, VP2 protein, and antibody IgG2a against Yellow fever disease (YF), respectively. So, the devices here proposed are promising tool suitable for both rodent and human hantavirus clinical surveys.