Unleashing inkjet-printed nanostructured electrodes and battery-free potentiostat for the DNA-based multiplexed detection of SARS-CoV-2 genes.

Following the global COVID-19 pandemic triggered by SARS-CoV-2, the need for rapid, specific and cost-effective point-of-care diagnostic solutions remains paramount. Even though COVID-19 is no longer a public health emergency, the disease still poses a global threat leading to deaths, and it continues to change with the risk of new variants emerging causing a new surge in cases and deaths. Here, we address the urgent need for rapid, cost-effective and point-of-care diagnostic solutions for SARS-CoV-2. We propose a multiplexed DNA-based sensing platform that utilizes inkjet-printed nanostructured gold electrodes and an inkjet-printed battery-free near-field communication (NFC) potentiostat for the simultaneous quantitative detection of two SARS-CoV-2 genes, the ORF1ab and the N gene. The detection strategy based on the formation of an RNA-DNA sandwich structure leads to a highly specific electrochemical output. The inkjet-printed nanostructured gold electrodes providing a large surface area enable efficient binding and increase the sensitivity. The inkjet-printed battery-free NFC potentiostat enables rapid measurements and real-time data analysis via a smartphone application, making the platform accessible and portable. With the advantages of speed (5 min), simplicity, sensitivity (low pM range, ∼450% signal gain) and cost-effectiveness, the proposed platform is a promising alternative for point-of-care diagnostics and high-throughput analysis that complements the COVID-19 diagnostic toolkit.

Graphene Pseudoreference Electrode for the Development of a Practical Paper-Based Electrochemical Heavy Metal Sensor.

Paper-based electrochemical devices (PEDs) have emerged as versatile platforms that bridge analytical chemistry and materials science, demonstrating advantages of portability, cost-effectiveness, and environmental sustainability. This study investigates the integration of a graphene pseudoreference electrode (GPRE) into a PED, and it exhibits potential advantages over the traditional Ag/AgCl pseudoreference electrode (PRE). In addition, the electrochemical properties and stability of GPRE are compared with those of the traditional Ag/AgCl PRE. The results demonstrate that GPRE exhibits a stable and reproducible potential during electrochemical measurement throughout 180 days, demonstrating its suitability as an alternative to an expensive metal PRE. Furthermore, a GPRE-incorporated paper-based device is designed and evaluated for use in the electrochemical detection of cadmium (Cd) and lead (Pb) using an in situ bismuth-modified electrode. The GPRE-incorporated PED exhibited good analytical performance, with a low limit of detection of 0.69 and 5.77 ng mL-1 and electrochemical sensitivities of 70.16 and 38.34 µA·mL·µg-1·cm-2 for Cd(II) and Pb(II), respectively. More than 99.9% accuracy of the sensor was obtained for both ions with respect to conventional inductively coupled plasma-mass spectrometry. The results highlight the effectiveness and suitability of the GPRE-incorporated PED as a sensor for various applications, such as environmental monitoring, food quality control, and medical diagnostics.

Sequential Flow Controllable Microfluidic Device for G-Quadruplex DNAzyme-Based Electrochemical Detection of SARS-CoV-2 Using a Pyrrolidinyl Peptide Nucleic Acid.

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been a significant health issue globally. Point-of-care (POC) testing that can offer a rapid and accurate diagnosis of SARS-CoV-2 at the early stage of infection is highly desirable to constrain this outbreak, especially in resource-limited settings. Herein, we present a G-quadruplex DNAzyme-based electrochemical assay that is integrated with a sequential flow controllable microfluidic device for the detection of SARS-CoV-2 cDNA. According to the detection principle, a pyrrolidinyl peptide nucleic acid probe is immobilized on a screen-printed graphene electrode for capturing SARS-CoV-2 DNA. The captured DNA subsequently hybridizes with another DNA probe that carries a G-quadruplex DNAzyme as the signaling unit. The G-quadruplex DNAzyme catalyzes the H2O2-mediated oxidation of hydroquinone to benzoquinone that can be detected using square-wave voltammetry to give a signal that corresponds to the target DNA concentration. The assay exhibited high selectivity for SARS-CoV-2 DNA and showed a good experimental detection limit at 30 pM. To enable automation, the DNAzyme-based assay was combined with a capillary-driven microfluidic device featuring a burst valve technology to allow sequential sample and reagent delivery as well as the DNA target hybridization and enzymatic reaction to be operated in a precisely controlled fashion. The developed microfluidic device was successfully applied for the detection of SARS-CoV-2 from nasopharyngeal swab samples. The results were in good agreement with the standard RT-PCR method and could be performed within 20 min. Thus, this platform offers desirable characteristics that make it an alternative POC tool for COVID-19 diagnosis.

An alternative label-free DNA sensor based on the alternating-current electroluminescent device for simultaneous detection of human immunodeficiency virus and hepatitis C co-infection.

Coinfection of HIV/HCV is a significant public health issue globally, as it increases the risk of liver cancer in co-infected individuals. The point-of-care testing (POCT) device for HIV/HCV DNA detection is promptly needed for diagnosis and monitoring of the disease progression. Here, the alternating-current electroluminescence (ACEL) technique is proposed as a sensitive POCT sensing platform for HIV/HCV cDNA detection. A conductance-based light emission modulated by the hybridization between a pyrrolidinyl PNA probe and the DNA target enabled the DNA detection in a label-free format. Enhanced electroluminescence was observed in the presence of the target DNA due to the increased proton conductivity. Under the optimal conditions, the linearity range from 1 nM to 1 µM was achieved for HIV and HCV cDNA with LODs of 1.86 pM (HIV cDNA) and 1.96 pM (HCV cDNA). The spiked HIV/HCV cDNA in healthy human serum was successfully detected, demonstrating the feasibility of the developed device for the detection of cDNA in real biological samples. Additionally, simultaneous HIV/HCV cDNA detection on a single ACEL device employing a 2x2-array detection zone design. The cross-reactivity with other viral DNA was shown to be minimal due to the high specificity of the PNA probes used. Finally, the negative and positive samples from the patient's serum were tested and the results were in 100% agreement with the commercial kit based-on real-time PCR method, thus illustrating the high sensitivity and specificity of the developed sensor.

Amplification-free DNA Sensor for the One-Step Detection of the Hepatitis B Virus Using an Automated Paper-Based Lateral Flow Electrochemical Device.

Until now, an electrochemical lateral flow assay (eLFA) capable of detecting nucleic acids has remained a challenge and has been scarcely explored because of its complicated multistep nature. Here, we report an automated paper-based eLFA device for the quantitative detection of the hepatitis B virus (HBV)-the major cause of liver cirrhosis and hepatocellular carcinoma (HCC). Using a time-delayed microfluidic strategy fabricated on paper, an automated and precisely sequenced solution transfer was enabled by single sample loading. A gold metallization strategy was employed for the signal-on electrochemical detection of the target DNA. Furthermore, a pyrrolidinyl peptide nucleic acid (so-called "acpcPNA") was used as a probe in this study because it offers higher specificity and yields lower background currents than those of traditional probes. Under optimal conditions, a broad dynamic range (10 pM to 2 µM) with an excellent detection limit (down to 7.23 pM) was achieved. The overall operation can be completed within 7 min of sample loading. The proposed sensor was successfully applied in HBV DNA detection in sera from patients without any amplification step (e.g., PCR) required, thus simplifying the operation further. Additionally, the results obtained from this present device are in accordance with the standard real-time PCR, thus supporting the accuracy of the method.