The ability of a diagnostic test to detect multiple pathogens simultaneously is useful to obtain meaningful information for clinical treatment and preventive measures. We report a highly sensitive and specific electrochemical biosensor assay for simultaneous detection of three gene targets using quantum dots (QDs). The targets are novel non-protein coding RNA (npcRNA) sequences of Vibrio cholerae, Salmonella sp. and Shigella sp., which cause diarrheal diseases. QDs (PbS, CdS, ZnS) were synthesized and functionalized with DNA probes that were specific to each pathogen. Electrochemical detection of QDs was performed using square wave anodic stripping voltammetry (SWASV). The QDs gave distinct peaks at 0.5 V (PbS), 0.75 V (CdS) and 1.1 V (ZnS). There was no interference in signal response when all three QDs were mixed and detected simultaneously. The detection limits of single and multiplex assays with linear targets and PCR products were in the attomolar ranges. The high assay sensitivity, in combination with specific npcRNA sequences as novel diagnostic targets, makes it a viable tool for detecting pathogens from food, environment and clinical samples.
The aim of this study was to develop a highly specific electrochemical DNA sensor using functionalized lead sulphide (PbS) quantum dots for hepatitis E virus genotype 3 (HEV3) DNA target detection. Functionalized-PbS quantum dots (QDs) were used as an electrochemical label for the detection of HEV3-DNA target by the technique of square wave anodic stripping voltammetry (SWASV). The functionalized-PbS quantum dots were characterized by UV-vis, FTIR, XRD, TEM and zeta potential techniques. As-prepared, functionalized-PbS quantum dots have an average size of 4.15 ± 1.35 nm. The detection platform exhibited LOD and LOQ values of 1.23 fM and 2.11 fM, respectively. HEV3-DNA target spiked serum is also reported.Graphical abstract.
Epizootic ulcerative syndrome (EUS) is a devastating fish disease caused by the fungus, Aphanomyces invadans. Rapid diagnosis of EUS is needed to control and treat this highly invasive disease. The current diagnostic methods for EUS are labor intensive. We have developed a highly sensitive and specific electrochemical genosensor towards the 18S rRNA and internal transcribed spacer regions of A. invadans. Multiple layers of latex were synthesized with the help of polyelectrolytes, and labeled with gold nanoparticles to enhance sensitivity. The gold-latex spheres were functionalized with specific DNA probes. We describe here the novel application of this improved platform for detection of PCR product from real sample of A. invadans using a premix sandwich hybridization assay. The premix assay was easier, more specific and gave higher sensitivity of one log unit when compared to the conventional method of step-by-step hybridization. The limit of detection was 0.5 fM (4.99 zmol) of linear target DNA and 1 fM (10 amol) of PCR product. The binding positions of the probes to the PCR amplicons were optimized for efficient hybridization. Probes that hybridized close to the 5' or 3' terminus of the PCR amplicons gave the highest signal due to minimal steric hindrance for hybridization. The genosensor is highly suitable as a surveillance and diagnostic tool for EUS in the aquaculture industry.
Vibrio cholerae is a Gram-negative bacterium that causes cholera, a diarrheal disease. Cholera is widespread in poor, under-developed or disaster-hit countries that have poor water sanitation. Hence, a rapid detection method for V. cholerae in the field under these resource-limited settings is required. In this paper, we describe the development of an electrochemical genosensor assay using lyophilized gold nanoparticles/latex microsphere (AuNPs-PSA) reporter label. The reporter label mixture was prepared by lyophilization of AuNPs-PSA-avidin conjugate with different types of stabilizers. The best stabilizer was 5% sorbitol, which was able to preserve the dried conjugate for up to 30 days. Three methods of DNA hybridization were compared and the one-step sandwich hybridization method was chosen as it was fastest and highly specific. The performance of the assay using the lyophilized reagents was comparable to the wet form for detection of 1aM to 1fM of linear target DNA. The assay was highly specific for V. cholerae, with a detection limit of 1fM of PCR products. The ability of the sensor is to detect LAMP products as low as 50ngµl(-1). The novel lyophilized AuNPs-PSA-avidin reporter label with electrochemical genosensor detection could facilitate the rapid on-site detection of V. cholerae.
DNA strand displacement is an attractive, enzyme-free target hybridization strategy for nano-biosensing. The target DNA induces a strand displacement reaction by replacing the pre-hybridized strand that is labeled with gold nanoparticles (AuNPs). Thus, the amount of displaced-AuNP-labeled strand is proportional to the amount of target DNA in the sample. The use of a magnetogenosensing technique to isolate the target DNA allows for a simple, one-pot detection approach, which minimizes possible carry-over contamination and pipetting errors. We sought a proof-of-concept for this technology in its ability to detect DNA-equivalent of hepatitis E virus (HEV), which causes acute viral hepatitis for which rapid and simple diagnostic methods remain limited. Signal detection was done via visual observation, spectrophotometry, and electrochemistry. The sensor demonstrated good sensitivity with detection limits of 10 pM (visual), 10 pM (spectrophotometry) and 1 fM (electrochemical). This sensor also exhibited high specificity for real target amplicons and could discriminate between perfect and mismatched sequences. Lyophilized biosensor reagents stored at 4 °C, 25 °C, and outdoor ambient temperature, were stable for up to 90, 50, and 40 days, respectively. The integration of magnetic separation and target DNA-induced strand displacement reaction in a dry reagent form makes the sensing platform easy-to-use and suitable for field settings.
In view of the presence of pathogenic Vibrio cholerae (V. cholerae) bacteria in environmental waters, including drinking water, which may pose a potential health risk to humans, an ultrasensitive electrochemical DNA biosensor for rapid detection of V. cholerae DNA in the environmental sample was developed. Silica nanospheres were functionalized with 3-aminopropyltriethoxysilane (APTS) for effective immobilization of the capture probe, and gold nanoparticles were used for acceleration of electron transfer to the electrode surface. The aminated capture probe was immobilized onto the Si-Au nanocomposite-modified carbon screen printed electrode (Si-Au-SPE) via an imine covalent bond with glutaraldehyde (GA), which served as the bifunctional cross-linking agent. The targeted DNA sequence of V. cholerae was monitored via a sandwich DNA hybridization strategy with a pair of DNA probes, which included the capture probe and reporter probe that flanked the complementary DNA (cDNA), and evaluated by differential pulse voltammetry (DPV) in the presence of an anthraquninone redox label. Under optimum sandwich hybridization conditions, the voltammetric genosensor could detect the targeted V. cholerae gene from 1.0 × 10-17-1.0 × 10-7 M cDNA with a limit of detection (LOD) of 1.25 × 10-18 M (i.e., 1.1513 × 10-13 µg/µL) and long-term stability of the DNA biosensor up to 55 days. The electrochemical DNA biosensor was capable of giving a reproducible DPV signal with a relative standard deviation (RSD) of <5.0% (n = 5). Satisfactory recoveries of V. cholerae cDNA concentration from different bacterial strains, river water, and cabbage samples were obtained between 96.5% and 101.6% with the proposed DNA sandwich biosensing procedure. The V. cholerae DNA concentrations determined by the sandwich-type electrochemical genosensor in the environmental samples were correlated to the number of bacterial colonies obtained from standard microbiological procedures (bacterial colony count reference method).
Cervical cancer emerges as the third most prevalent types of malignancy among women on a global scale. Cervical cancer is significantly associated with the persistent infection of human papillomavirus (HPV) type 16. The process of diagnosing is crucial in order to prevent the progression of a condition into a malignant state. The early detection of cervical cancer through initial stage screening is of the utmost significance in both the prevention and effective management of this disease. The present detection methodology is dependent on quantitative polymerase chain reaction (qPCR), which necessitates the use of a costly heat cycler instrument. In this study, we report the development of an electrochemical DNA biosensor integrated with an isothermal recombinase polymerase amplification (RPA) reaction for the detection and identification of the high-risk HPV-16 genotype. The electrochemical biosensor exhibited a high degree of specificity and sensitivity, as evidenced by its limit of detection (LOD) of 0.23 copies/μL of HPV-16 DNA. The validity of this electrochemical platform was confirmed through the analysis of 40 cervical tissues samples, and the findings were consistent with those obtained through polymerase chain reaction (PCR) testing. Our straightforward electrochemical detection technology and quick turnaround time at 75 min make the assay suitable for point-of-care testing in low-resource settings.
Since the declaration of COVID-19 as a pandemic in early 2020, multiple variants of the severe acute respiratory syndrome-related coronavirus (SARS-CoV-2) have been detected. The emergence of multiple variants has raised concerns due to their impact on public health. Therefore, it is crucial to distinguish between different viral variants. Here, we developed a machine learning web-based application for SARS-CoV-2 variant identification via duplex real-time polymerase chain reaction (PCR) coupled with high-resolution melt (qPCR-HRM) analysis. As a proof-of-concept, we investigated the platform's ability to identify the Alpha, Delta, and wild-type strains using two sets of primers. The duplex qPCR-HRM could identify the two variants reliably in as low as 100 copies/μL. Finally, the platform was validated with 167 nasopharyngeal swab samples, which gave a sensitivity of 95.2%. This work demonstrates the potential for use as automated, cost-effective, and large-scale viral variant surveillance.
We report a highly sensitive and selective multiplex assay by empowering an electrochemical DNA sensor with isothermal rolling circle amplification. The assay could simultaneously detect and discriminate three common entero-pathogens in a single reaction, with femtomolar sensitivity. It is useful for field- or resource-limited settings.
Coronavirus disease 2019 (COVID-19) is a highly contagious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Diagnosis of COVID-19 depends on quantitative reverse transcription PCR (qRT-PCR), which is time-consuming and requires expensive instrumentation. Here, we report an ultrasensitive electrochemical biosensor based on isothermal rolling circle amplification (RCA) for rapid detection of SARS-CoV-2. The assay involves the hybridization of the RCA amplicons with probes that were functionalized with redox active labels that are detectable by an electrochemical biosensor. The one-step sandwich hybridization assay could detect as low as 1 copy/μL of N and S genes, in less than 2 h. Sensor evaluation with 106 clinical samples, including 41 SARS-CoV-2 positive and 9 samples positive for other respiratory viruses, gave a 100% concordance result with qRT-PCR, with complete correlation between the biosensor current signals and quantitation cycle (Cq) values. In summary, this biosensor could be used as an on-site, real-time diagnostic test for COVID-19.