SARS-CoV-2 detection enabled by a portable and label-free photoelectrochemical genosensor using graphitic carbon nitride and gold nanoparticles.

Fast, sensitive, simple, and cheap sensors are highly desirable to be applied in the health system because they improve point-of-care diagnostics, which can reduce the number of cases of infection or even deaths. In this context, here we report the development of a label-free genosensor using a screen-printed electrode modified with 2D-carbonylated graphitic carbon nitride (c-g-C3N4), poly(diallyldimethylammonium) chloride (PDDA), and glutathione-protected gold nanoparticles (GSH-AuNPs) for photoelectrochemical (PEC) detection of SARS-CoV-2. We also made use of Arduino and 3D printing to miniaturize the sensor device. The electrode surface was characterized by AFM and SEM techniques, and the gold nanoparticles by UV-Vis spectrophotometry. For SARS-CoV-2 detection, capture probe DNA was immobilized on the electrode surface. The hybridization of the final genosensor was tested with a synthetic single-strand DNA target and with natural saliva samples using the photoelectrochemistry method. The device presented a linear range from 1 to 10,000 fmol L-1 and a limit of detection of 2.2 and 3.4 fmol L-1 using cpDNA 1A and 3A respectively. The sensibility and accuracy found for the genosensor using cpDNA 1A using biological samples were 93.3 and 80% respectively, indicating the potential of the label-free and portable genosensor to detect SARS-CoV-2 RNA in saliva samples.

An electrochemical genomagnetic assay for detection of SARS-CoV-2 and Influenza A viruses in saliva.

Viral respiratory infections represent a major threat to the population's health globally. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes COVID-19 disease and in some cases the symptoms can be confused with Influenza disease caused by the Influenza A viruses. A simple, fast, and selective assay capable of identifying the etiological agent and differentiating the diseases is essential to provide the correct clinical management to the patient. Herein, we described the development of a genomagnetic assay for the selective capture of viral RNA from SARS-CoV-2 and Influenza A viruses in saliva samples and employing a simple disposable electrochemical device for gene detection and quantification. The proposed method showed excellent performance detecting RNA of SARS-CoV-2 and Influenza A viruses, with a limit of detection (LoD) and limit of quantification (LoQ) of 5.0 fmol L-1 and 8.6 fmol L-1 for SARS-CoV-2, and 1.0 fmol L-1 and 108.9 fmol L-1 for Influenza, respectively. The genomagnetic assay was employed to evaluate the presence of the viruses in 36 saliva samples and the results presented similar responses to those obtained by the real-time reverse transcription-polymerase chain reaction (RT-PCR), demonstrating the reliability and capability of a method as an alternative for the diagnosis of COVID-19 and Influenza with point-of-care capabilities.

Molecular test for COVID-19 diagnosis based on a colorimetric genomagnetic assay.

The world is in a long pandemic period caused by the SARS-CoV-2 virus and massive diagnostic tests to assist efforts to control the spread of the disease and also to avoid new coronavirus variants are still needed. Herein, we propose a simple and accurate saliva-based colorimetric test for the diagnosis of COVID-19. Magnetic beads (MBs) modified with a sequence of single-strand DNA (ssDNA) complementary to the N gene of the SARS-CoV-2 RNA were developed and used for magnetic capture and separation from a complex saliva sample. A second biotinylated ssDNA sequence was applied, and the colorimetric detection was carried out by adding streptavidin-horseradish peroxidase conjugate, H2O2, and tetramethylbenzidine (TMB) as chromogenic substrate. The test does not require viral RNA isolation, transcription, or amplification steps and can be performed at room temperature. The molecular assay test can be run using 96-well microplates, allowing the diagnosis of a large number of samples in 90 min. A simple support for magnets was designed and constructed using a 3D printer that allows the magnetic separations directly in the 96-well microplate. The colorimetric test showed an excellent ability to discriminate between healthy individuals and patients infected with SARS-CoV-2, with 92% and 100% of clinical sensitivity and specificity, respectively. This performance was similar to that achieved using the gold standard RT-PCR technique. The proposed genomagnetic assay offers an opportunity to greatly increase population testing, contribute to controlling the spread of the virus, and improve health equity in testing for COVID-19.

A sensitive electrochemical detection of metronidazole in synthetic serum and urine samples using low-cost screen-printed electrodes modified with reduced graphene oxide and C60.

Monitoring the concentration of antibiotics in body fluids is essential to optimizing the therapy and minimizing the risk of bacteria resistance, which can be made with electrochemical sensors tailored with appropriate materials. In this paper, we report on sensors made with screen-printed electrodes (SPE) coated with fullerene (C60), reduced graphene oxide (rGO) and Nafion (NF) (C60-rGO-NF/SPE) to determine the antibiotic metronidazole (MTZ). Under optimized conditions, the C60-rGO-NF/SPE sensor exhibited a linear response in square wave voltammetry for MTZ concentrations from 2.5 × 10-7 to 34 × 10-6 mol/L, with a detection limit of 2.1 × 10-7 mol/L. This sensor was also capable of detecting MTZ in serum and urine, with recovery between 94% and 100%, which are similar to those of the standard chromatographic method (HPLC-UV). Because the C60-rGO-NF/SPE sensor is amenable to mass production and allows for MTZ determination with simple principles of detection, it fulfills the requirements of therapeutic drug monitoring programs.

Disposable electrochemical microfluidic device for ultrasensitive detection of egg allergen in wine samples.

Food allergies have been increasing all over the world. Egg is an important component in the food industries and the second most common cause of food allergy, shortly after milk. In the wine industry, egg white is applied as a fining agent for tannin removal. In this study, a sandwich-based immunoassay for ultrasensitive detection of ovalbumin (OVA) in wine samples was developed. The assay involves the use of magnetic beads (MBs) decorated with a polyclonal anti-OVA antibody (Ab2) and horseradish peroxidase (HRP), used as label for the quantification in a disposable electrochemical microfluidic device (DEµD) here developed. The Ab2-MB-HRP prepared was applied to capture, separate, and pre-concentrate OVA from wine samples. In the DEµD, OVA was immune-magnetically captured (OVA-Ab2-MB-HRP), producing a sandwich structure (GO-Ab1-OVA-Ab2-MB-HRP) on the electrode's surface. This arrangement results in an ultrasensitive device, achieving the ultralow limit of detection of 0.2 fg mL-1 OVA. Five samples of wines were analyzed by using the immuno-magneto-assay which presents excellent accuracy compared with enzyme-linked immunosorbent assay (ELISA).

A compact miniaturized continuous flow system for the determination of urea content in milk.

A multicommutation-based flow system with photometric detection was developed, employing an analytical microsystem constructed with low temperature co-fired ceramics (LTCC) technology, a solid-phase reactor containing particles of Canavalia ensiformis DC (urease source) immobilized with glutaraldehyde, and a mini-photometer coupled directly to the microsystem which monolithically integrates a continuous flow cell. The determination of urea in milk was based on the hydrolysis of urea in the solid-phase reactor and the ammonium ions produced were monitored using the Berthelot reaction. The analytical curve was linear in the urea concentration range from 1.0 x 10(-4) to 5.0 x 10(-3) mol L(-1) with a limit of detection of 8.0 x 10(-6) mol L(-1). The relative standard deviation (RSD) for a 2.0 x 10(-3) mol L(-1) urea solution was lower than 0.4% (n = 10) and the sample throughput was 13 h(-1). To check the reproducibility of the flow system, calibration curves were obtained with freshly prepared solutions on different days and the RSD obtained was 4.7% (n = 6). Accuracy was assessed by comparing the results of the proposed method with those from the official procedure and the data are in close agreement, at a 95% confidence level.

Non-enzymatic electrochemical determination of creatinine using a novel screen-printed microcell.

A low-cost and disposable microcell was constructed with a screen-printed electrode for the non-enzymatic electrochemical determination of creatinine. The working electrode was modified with carbon black and maintained in contact with paper-adsorbed iron (III) ions. A small sample volume of 3 µL was required for the device operation. Then, iron (III) ions were complexed in the presence of creatinine in a chemical step, followed by an electrochemical reduction of non-complexed metallic ions in excess. Cyclic voltammetry and differential-pulse voltammetry experiments were employed for the electrochemical characterizations and analytical performance evaluation of the microcell. The working electrode modification with carbon black provided a significant increase of analytical signal. The sensor presented a linear response for creatinine concentrations ranging from 0.10 to 6.5 mmol L-1, with a limit of detection of 0.043 mmol L-1. Experiments for creatinine determination in real samples were successful performed through of standard recovery in urine.

Fast and flexible strategy to produce electrochemical paper-based analytical devices using a craft cutter printer to create wax barrier and screen-printed electrodes.

This paper describes a simple, low-cost, and highly flexible rapid prototyping method to construct electrochemical paper-based analytical device (ePAD) for multiplexed analyte determinations. The ePAD was composed of two electrochemical cell (EC) compartments, separated by hydrophobic barriers of wax, and screen-printed electrodes (SPEs) deposited directly over the surface of the filter paper. The ePAD was entirely constructed using an inexpensive craft cutter printer with no needed of a wax printer. The rapid prototyping method involves two steps: the deposition of the SPEs and the creation of the wax barriers. In this case, the SPEs were screen-printed on filter paper by using adhesive tape as mask by cutting the electrodes pattern with the cutter printer. Following, the wax barriers were created using stamps made of filter paper also cut with the printer and impregnated with wax. In the ePAD, each ECs containing an array of 4-working electrodes, allowing up to 4 replicates in a single measurement. Both ECs shared one counter and one reference electrodes, permitting the simultaneous multianalysis. The ePAD was successfully applied to simultaneous detection of paracetamol (PAR), caffeine (CAF), and ascorbic acid (AA) in drugs. PAR and CAF were detected in a sample using one EC and AA was detected, in a different sample, on the other EC, both with no chemical modifications in the working electrodes. Limits of detection of 0.04 mmol L-1 for PAR, 0.22 mmol L-1 for CAF, and 0.40 mmol L-1 for AA were obtained. The construction process proposed provide an easy way to implement screen-printing electrodes and wax barriers in filter paper to create electrochemical devices for fast and simultaneous multianalysis.

Electrochemical paper-based microfluidic device for high throughput multiplexed analysis.

A disposable microfluidic electrochemical paper-based device for multiplexed analysis based on sixteen independent microfluidic channels with electrochemical detection is proposed. A major advantage of this work was the non-necessary use of a wax printer for devices manufacturing which has a high cost of operation. In addition, a commercial multiplexing module was used that has the multiplexing capability of 8-16 channels and, for the first time using this module, the strategy of multiplexing both the working and reference electrodes were used. These sixteen channels with the respective sensors can be operated employing one or multiple electrochemical techniques with good repeatability and reproducibility for high throughput analysis. As a proof of concept, the electrochemical performance of device was tested with ferrocenecarboxylic acid solution employing cyclic voltammetry, square-wave voltammetry, differential-pulse voltammetry and chronoamperometry. This innovative sensing platform presented capacity of production in large scale and application for clinical tests with safety and short time of assays. A biosensor was constructed using glucose oxidase on the platform for the glucose determination in urine as a non-invasive strategy. The analytical curve was linear in the glucose concentration range from 1.0 × 10-4 mol L-1 to 4 × 10-2 mol L-1, with a limit of detection of 3 × 10-5 mol L-1.

A simple method to produce 2D and 3D microfluidic paper-based analytical devices for clinical analysis.

This paper describes the fabrication of 2D and 3D microfluidic paper-based analytical devices (µPADs) for monitoring glucose, total protein, and nitrite in blood serum and artificial urine. A new method of cutting and sealing filter paper to construct µPADs was demonstrated. Using an inexpensive home cutter printer soft cellulose-based filter paper was easily and precisely cut to produce pattern hydrophilic microchannels. 2D and 3D µPADs were designed with three detection zones each for the colorimetric detection of the analytes. A small volume of samples was added to the µPADs, which was photographed after 15 min using a digital camera. Both µPADs presented an excellent analytical performance for all analytes. The 2D device was applied in artificial urine samples and reached limits of detection (LODs) of 0.54 mM, 5.19 µM, and 2.34 µM for glucose, protein, and nitrite, respectively. The corresponding LODs of the 3D device applied for detecting the same analytes in artificial blood serum were 0.44 mM, 1.26 µM, and 4.35 µM.