Capture Probe Research Articles

Ultrasensitive Electrochemiluminescence Immunosensor for Bladder Marker Human Complement Factor H-Related Protein Detection.

The development of noninvasive and sensitive detection methods for the early diagnosis and monitoring of bladder cancer is critical but challenging. Herein, an ultrasensitive electrochemiluminescence (ECL) immunosensor that uses Ru(bpy)32+-metal-organic framework (Ru-MOF) nanospheres and a DNA tetrahedral (TDN) probe was established for bladder cancer marker complement factor H-related protein (CFHR1) detection. The synthesized Ru(bpy)32+-metal-organic frameworks (Ru-MOFs) served as a linked substrate for immobilization of AuNPs and antibody (Ab2) to prepare the ECL signal probe (Ru-MOF@AuNPs-Ab2), exhibiting a stable and strengthened ECL emission. At the same time, the inherent advantages of TDN probes on the electrode as the capture probe (TDN-Ab1) improve the accessibility of targets to probes. In the presence of CFHR1, the signal probe Ru-MOF@AuNPs-Ab2 was modified on the electrode through immune binding, thereby obtaining an outstanding ECL signal. As expected, the developed ECL immunosensor exhibited splendid performance for CFHR1 detection in the range of 0.1 fg/mL to 10 pg/mL with a quite low detection limit of 0.069 fg/mL. By using the proposed strategy to detect CFHR1 from urine, it showed acceptable accuracy, which can effectively distinguish between bladder cancer patients and healthy samples. This work contributes to a novel, noninvasive, and accurate method for early clinical diagnosis of bladder cancer.

Two birds with one stone: A universal design and application of signal-on labeled fluorescent/electrochemical dual-signal mode biosensor for the detection of tetracycline residues in tap water, milk and chicken

An ingenious and universal design of fluorescent/electrochemical dual-signal mode sensing platform was constructed for the sensitive, selective and accurate detection of tetracycline (TET). Apt-functionalized nano-magnetic beads (Fe3O4-Apt) as capture probe, Apt-complementary short-chain functionalized fluorescent MOF loaded with methylene blue (MB) (cDNA-MOF-MB) as dual-signal tag were prepared. The sensing platform (Fe3O4-Apt/cDNA-MOF-MB) was formed based on the base complementary pairing of Apt and cDNA. With the help of Apt for target recognition, together with magnetic separation technology, a dual-signal mode biosensor was constructed. The dual-signal mode biosensor exhibited a wide linear concentration range from 1.00 × 10−9 g/mL to 1.00 × 10−4 g/mL with a low limit of detection (LOD) of 1.69 × 10−10 g/mL (fluorescence mode assay) and 1.15 × 10−10 g/mL (electrochemical mode assay). The proposed biosensor had been successfully applied to the determination of TET content in real samples with satisfactory recoveries (94.99–101.30%).

3D DNAzyme walker based electrochemical biosensor for attomolar level microRNA-155 detection

Herein, an ultrasensitive electrochemical biosensor for microRNA-155 (miR-155) detection based on the powerful catalytic and continuous walking signal amplification capability of 3D DNAzyme walker and the gold nanoparticles/graphene aerogels carbon fiber paper-based (AuNPs/GAs/CFP) flexible sensing electrode with excellent electrochemical performance was successfully constructed. In a proof-of-concept experiment, in the presence of miR-155, the DNAzyme strands anchored on the streptavidin-modified magnetic beads (MBs) silenced by locked strands can be activated, thus generating the walking arm of the 3D DNAzyme walker. Meanwhile, the substrate strands modified with Fe-MOF-NH2 nanoparticles were evenly distributed on the surface of MBs and served as tracks of the 3D DNAzyme walker. Once the DNAzyme strand was activated, the catalytic site in the substrate strand can be cleaved in the presence of Mn2+, and a large number of stumps modified with Fe-MOF-NH2 nanoparticles (output@Fe-MOF-NH2) will be generated during the continuous and efficient walking cleavage of the DNAzyme walker, driving the recognition-catalysis-release cycle process for signal amplification. Immediately afterwards, the signal was read out through the base complementary pairing of capture probe (PS) immobilized on the surface of the paper-based flexible sensing electrode AuNPs/GAs/CFP and signal probes output@Fe-MOF-NH2, thus achieving the quantitative detection of miR-155. Under optimal experimental conditions, the designed 3D DNAzyme walker-based biosensor exhibited a relatively lower limit of detection (LOD) of 56.23 aM, with a linear range of 100 aM to 100 nM. Overall, the proposed 3D DNAzyme walker biosensor exhibited good interference and reproducibility, demonstrating a promising future in the field of clinical disease diagnosis.

Gold nanoparticles engineered nanoporous membrane with sandwich structure for sensitive electrochemical quantification of exosome

Exosome has been identified as an excellent potential biomarker for tumor diagnosis. Herein, electrochemical aptasensor for exosome detection was developed via a sandwich-structured nanoporous membrane which was fabricated by assembling multilayer gold nanoparticles (mulAu) and monolayer gold nanoparticles (monAu) on the opposite side of a nanoporous alumina membrane (NAM). The tumor-derived exosomes can be efficiently captured by the specific probes confined on the monAu side of the membrane. And the mulAu side that was tightly attached with a Si wafer allowed the electrochemical exosome detection by monitoring the amperometric signal of ferricyanide that transported across the nanochannel array of the membrane. After optimization of pore diameter of the NAM, base sequence of the capture probe and incubation temperature as well as incubation time for exosomes, this NAM-based electrochemical aptasensor was applied to the quantification of the MCF-7 cells-derived exosomes. The steady state current displayed linearity relationship with the concentration of the exosomes in a broad range of 103 to 107 particles μL−1, along with a detection limit of 2.8 × 102 particles μL−1. By changing capture probe, this sandwich-structured membrane could be further extended to label-free, sensitive and rapid quantification of other tumor-associated vesicles.

Enzyme-free dual amplification biosensor based on functional nucleic acid and CDs/CoOOH for detection of leukemia fusion gene

Acute promyelocytic leukemia (APL) is an acute myeloid leukemia (AML) with a specific fusion gene target, PML/RARα fusion gene (PML/RARα), which is formed by the translocation of chromosomes 15 and 17. Detection of PML/RARα is the most reliable parameter for the diagnosis, treatment adjustment, efficacy evaluation, prognosis analysis and relapse prediction of APL. In this study, a novel biosensor was constructed for rapid enzyme-free detection of PML/RARα using DNAzyme and carbon dots/cobalt oxhydroxide nanosheet complexs (CDs/CoOOH). In the detection system, the separated DNAzymes could specifically recognize and bind together by the PML/RARα to form a complete DNAzyme for shearing hairpin probe (HP), then generated trigger, which was the first signal amplification. Then, trigger could hybridize with the capture probe (CP) anchored to streptavidin (SA) modified microplate as well as fluorescence quenching signal probe (SP@CDs/CoOOH). Finally, ascorbic acid (AA) was added to decompose CoOOH and the fluorescence of CDs was released, which was the second signal amplification. Through the dual signal amplification of DNAzyme and CDs/CoOOH, PML/RARα could be detected quickly and sensitively, which overcame the limitation of protein enzyme in traditional fluorescence methods, showing potential clinical application value in the diagnosis and treatment of leukemia.

Signal-on electrogenerated chemiluminescence detection of gonyautoxin 1/4 based on proximity ligation-induced an electrode-bound pseudoknot DNA

A “signal on” electrogenerated chemiluminescence (electrochemiluminescence, ECL) aptasensor based on proximity ligation-induced an electrode-bound pseudoknot DNA for sensitive detection of gonyautoxin 1/4 (GTX1/4) was developed on basis of the competitive type reaction mode. Aptamer was adopted as recognition element. Ru(bpy)32+ as ECL signal, was attached on the glassy carbon electrode (GCE) surface modified with nafion and gold nanoparticles (AuNPs) by electrostatic attraction to obtain the ECL platform. The pseudoknot DNA as capture probe, was immobilized onto the ECL platform via Au–S bond to obtain the ECL aptasensor. In the absence of GTX1/4, Y-shape proximate cooperative complex among aptamer, pseudoknot DNA and DNA1 was formed, drawing the ferrocene groups Fc, as ECL quencher) of both pseudoknot DNA and DNA1 near the electrode surface and resulting in low ECL signal. In the presence of GTX1/4, GTX1/4 competed with pseudoknot DNA and DNA1 for aptamer in homogeneous solution, preventing the formation of proximate cooperative complex and keeping the capture DNA in the pseudoknot conformation with Fc groups far away from the electrode surface, generating a high ECL signal. The recovery of ECL intensity increased with the GTX1/4 concentration and allowed the detection of GTX1/4 in the range of 0.01 ng/mL to 10 ng/mL with a detection of limit as low as 6.56 pg/mL. Additionally, the accuracy of this method was validated for analysis of spiked sea water samples with good recoveries, which indicates great potential in commercial application.

A Novel Ochratoxin A Aptasensor Based on Three-Dimensionally Ordered Macroporous RGO-AuNPs-MoS2 Enhanced Electrocatalysis of Methylene Blue and AuNPs-Fe3O4@C Composite as Signal Probe Carrier

In this work, a novel electrochemical aptasensor for the detection of ochratoxin A (OTA) was developed using a three-dimensionally ordered macroporous (3DOM) reduced graphene oxide–Au nanoparticles–molybdenum disulfide (RGO-AuNPs-MoS2) composite material as the sensing interface and Au nanoparticles–carbon-coated ferroferric oxide (AuNPs-Fe3O4@C) composite as the signal amplifier. The 3DOM RGO-AuNPs-MoS2 composite exhibited excellent conductivity and catalytic activity. The 3DOM RGO-AuNPs-MoS2 film was electrodeposited onto the Au electrode surface to immobilize DNA capture probe (CP), which was then hybridized with DNA helper strands (HS) and OTA aptamer (OPT) to form a Y-shaped structure. In the presence of OTA, the OPT was released from the electrode and then combined with AuNPs-Fe3O4@C containing the signal DNA1-metheylene blue (S1-MB) and DNA2-metheylene blue (S2-MB). The current response coming from MB was proportional with the OTA concentration. Under optimal conditions, the linear range of the aptasensor was 1 fg/mL to 0.1 μg/mL, with a detection limit as low as 0.56 fg/mL. The aptasensor was also used to detect OTA in rice and wheat samples, and the results were in agreement with those obtained by liquid chromatography-mass spectrometry (HPLC-MS).

Engineering DNA tetrahedron as a sensing surface of lateral flow test strips and ratiometric visual detection of exosomal microRNA-150–5p

We here report that DNA tetrahedral probe-based sensing surfaces are capable of efficiently capturing gold nanoparticles (AuNPs) on lateral flow test strips, and we have developed a ratiometric biosensor for exosomal microRNAs by integrating a tetrahedral probe and a catalytic hairpin assembly (CHA). DNA tetrahedrons were devised with barcodes and labeled with biotin as capture probes on the test and control lines of the strip. The CHA system consists of two hairpin substrates that can form stable duplexes via a programmable assembly reaction triggered by exosomal miRNAs. The duplexes hybridized with barcoded tetrahedra via complementary sequences and bound to streptavidin-modified AuNPs (SA-AuNPs) via biotin. Thus, a red line was observed on the test line owing to the immobilized SA-AuNPs. The remaining SA-AuNPs were captured by using a biotinylated tetrahedron on the control line. Because the barcoded and biotinylated tetrahedra consume SA-AuNPs simultaneously, a stronger signal in the test line results in a weaker signal in the control line, thus forming a ratiometric result. Tetrahedral probes avoid movement along with sample flow on sensing zones like single-stranded probes. The strip can have a detection limit of 58.90 fM with high selectivity due to the use of ratiometric color changing response, CHA signal amplification and tetrahedral capture probes. This system is able to rapidly detect exosomal microRNA-150–5p and may have a potential utility in point-of-care diagnosis of incipient diabetic nephropathy.

A flash signal amplification approach for ultrasensitive and rapid detection of single nucleotide polymorphisms in tuberculosis

In recent years, the demand for rapid, sensitive, and simple methods for diagnosing deoxyribonucleic acid (DNA) has grown due to the increase in the variation of infectious diseases. This work aimed to develop a flash signal amplification method coupled with electrochemical detection for polymerase chain reaction (PCR)-free tuberculosis (TB) molecular diagnosis. We exploited the slightly miscible properties of butanol and water to instantly concentrate a capture probe DNA, a single-stranded mismatch DNA, and gold nanoparticles (AuNPs) to a small volume to reduce the diffusion and reaction time in the solution. In addition, the electrochemical signal was enhanced once two strands of DNA were hybridized and bound to the surface of the gold nanoparticle at an ultra-high density. To eliminate non-specific adsorption and identify mismatched DNA, the self-assembled monolayers (SAMs) and Muts proteins were sequentially modified on the working electrode. This sensitive and specific approach can detect as low as attomolar levels of DNA targets (18 aM) and is successfully applied to detecting tuberculosis-associated single nucleotide polymorphisms (SNPs) in synovial fluid. More importantly, as this biosensing strategy can amplify the signal in only a few seconds, it possesses a great potential for point-of-care and molecular diagnosis applications.

Heating promoted super sensitive electrochemical detection of p53 gene based on alkaline phosphatase and nicking endonuclease Nt.BstNBI-assisted target recycling amplification strategy at heated gold disk electrode

An ultrasensitive electrochemical biosensor for detecting p53 gene was fabricated based on heated gold disk electrode coupling with endonuclease Nt.BstNBI-assisted target recycle amplification and alkaline phosphatase (ALP)-based electrocatalytic signal amplification. For biosensor assembling, biotinylated ssDNA capture probes were first immobilized on heated Au disk electrode (HAuDE), then combined with streptavidin-alkaline phosphatase (SA-ALP) by biotin-SA interaction. ALP could catalyze the hydrolysis of ascorbic acid 2-phosphate (AAP) to produce ascorbic acid (AA). While AA could induce the redox cycling to generate electrocatalytic oxidation current in the presence of ferrocene methanol (FcM). When capture probes hybridized with p53, Nt.BstNBI would recognize and cleave the duplexes and p53 was released for recycling. Meanwhile, the biotin group dropt from the electrode surface and subsequently SA-ALP could not adhere to the electrode. The signal difference before and after cleavage was proportional to the p53 gene concentration. Furthermore, with electrode temperature elevated, the Nt.BstNBI and ALP activities could be increased, greatly improving the sensitivity and efficiency for p53 detection. A detection limit of 9.5 × 10−17 M could be obtained (S/N = 3) with an electrode temperature of 40 °C, ca. four magnitudes lower than that at 25 °C.

The first case of Echinococcus canadensis found in a moose in Norway: What does it entail?

The first case of Echinococcus canadensis found in a moose in Norway: What does it entail?

Development of an electrochemiluminescent oligonucleotide-based assay for the quantification of prostate cancer associated miR-141-3p in human serum

MicroRNAs (miRNAs) are small oligonucleotides (18–25 bases), biologically relevant for epigenetic regulation of key processes, particularly in association with cancer. Research effort has therefore been directed towards the monitoring and detection of miRNAs to progress (early) cancer diagnoses. Traditional detection strategies for miRNAs are expensive, with a lengthy time-to-result. In this study we develop an oligonucleotide-based assay using electrochemistry for the specific, selective and sensitive detection of a circulating miRNA (miR-141) associated with prostate cancer. In the assay, the excitation and readout of the signal are independent: an electrochemical stimulation followed by an optical readout. A ‘sandwich’ approach is incorporated, consisting of a biotinylated capture probe immobilised on streptavidin-functionalised surfaces and a detection probe labelled with digoxigenin. We show that the assay allows the detection of miR-141 in human serum, even in the presence of other miRNAs, with a LOD of 0.25 pM. The developed electrochemiluminescent assay has, therefore, the potential for efficient universal oligonucleotide target detection via the redesign of capture and detection probes.

Application of colorimetric sensor array coupled with machine-learning approaches for the discrimination of grains based on freshness.

Volatile organic compounds (VOCs) in grain fluctuate depending on the degree of grain freshness. A new colorimetric sensor array (CSA) was developed as capture probes for the quantification of VOCs in grains in this work, and it was designed to monitor the variation of grain VOCs. CSA spectral data acquisition using visible-near-infrared spectroscopy and image processing of CSA's image imformation by computer were used comparatively. Then, machine-learning-based models - for example, synergistic interval partial least squares, genetic algorithm, competitive adaptive reweighted sampling (CARS) algorithm, and ant colony optimization (ACO) algorithm - were introduced to optimize variables. Moreover, principal component analysis, and linear discriminant analysis (LDA), and K-nearest neighbors (KNN) were used for the classification. Ultimately, quantitative models for detecting grain freshness are developed using various variable selection strategies. Compared with the pattern recognition results of image processing, visible-near-infrared spectroscopy could better separate the grains with different freshness from principal component analysis, and the prediction set of LDA models could correctly identify 100% of rice, 96.88% of paddy, and 97.9% of soybeans. In addition, compared with CARS and ACO, the LDA model and KNN model based on genetic algorithms show the best prediction performance. The prediction set could correctly identify 100% of rice and paddy samples and 95.83% of soybean samples. The method developed could be used for non-destructive detection of grain freshness. © 2023 Society of Chemical Industry.

Near-Infrared Fluorescent Biosensors Based on Covalent DNA Anchors.

Semiconducting single-walled carbon nanotubes (SWCNTs) are versatile near-infrared (NIR) fluorophores. They are noncovalently modified to create sensors that change their fluorescence when interacting with biomolecules. However, noncovalent chemistry has several limitations and prevents a consistent way to molecular recognition and reliable signal transduction. Here, we introduce a widely applicable covalent approach to create molecular sensors without impairing the fluorescence in the NIR (>1000 nm). For this purpose, we attach single-stranded DNA (ssDNA) via guanine quantum defects as anchors to the SWCNT surface. A connected sequence without guanines acts as flexible capture probe allowing hybridization with complementary nucleic acids. Hybridization modulates the SWCNT fluorescence and the magnitude increases with the length of the capture sequence (20 > 10 ≫ 6 bases). The incorporation of additional recognition units via this sequence enables a generic route to NIR fluorescent biosensors with improved stability. To demonstrate the potential, we design sensors for bacterial siderophores and the SARS CoV-2 spike protein. In summary, we introduce covalent guanine quantum defect chemistry as rational design concept for biosensors.

Electrochemical aptamer biosensor for DNA detection based on label-free aptamers

Electrochemical aptasensor has been broadly advanced for nucleic acid detection. However, it is a long-term goal to design an aptasensor with high specificity, flexibility, and simplicity. In this work, we develop a strategy of triblock DNA probe, which consists of two DNA probes at both ends and ployA fragments in the middle as probe-polyA-probe. PolyA fragment has high affinity to the surface of gold electrode, so it can be assembled on the electrode surface via polyA instead of traditional Au-S bonds. When the target DNA is simultaneously hybridized with the two capture probes, the hybridization stability can be improved due to the strong base stacking effect. [Ru(NH3)6]3+, as signal probe, can be electrostatically adsorbed on the negatively charged DNA skeleton. A wide linear range (10 pM-10 μM) is obtained with a detection limit of 2.9 pM. Our electrochemical aptasensor has good repeatability, stability, and specificity. More importantly, the electrochemical sensor can successfully detect DNA in human serum samples, which proves its practical value and extensive applicability in complex environment.

Goldenrod herbariomics: Hybrid-sequence capture reveals the phylogeny of Solidago.

The phylogenetic relationships among the ca. 138 species of goldenrods (Solidago; Asteraceae) have been difficult to infer due to species richness, and shallow interspecific genetic divergences. This study aims to overcome these obstacles by combining extensive sampling of goldenrod herbarium specimens with the use of a custom Solidago hybrid-sequence capture probe set. A set of tissues from herbarium samples comprising ca. 90% of Solidago species was assembled and DNA was extracted. A custom hybrid-sequence capture probe set was designed, and data from 854 nuclear regions were obtained and analyzed from 209 specimens. Maximum likelihood and coalescent approaches were used to estimate the genus phylogeny for 157 diploid samples. Although DNAs from older specimens were both more fragmented and produced fewer sequencing reads, there was no relationship between specimen age and our ability to obtain sufficient data at the target loci. The Solidago phylogeny was generally well-supported, with 88/155 (57%) nodes receiving ≥95% bootstrap support. Solidago was supported as monophyletic, with Chrysoma pauciflosculosa identified as sister. A clade comprising Solidago ericameriodes, Solidago odora, and Solidago chapmanii was identified as the earliest diverging Solidago lineage. The previously segregated genera Brintonia and Oligoneuron were identified as placed well within Solidago. These and other phylogenetic results were used to establish four subgenera and fifteen sections within the genus. The combination of expansive herbarium sampling and hybrid-sequence capture data allowed us to quickly and rigorously establish the evolutionary relationships within this difficult, species-rich group.

Exploring magnetic capture to improve the detection of human norovirus in strawberries

AbstractHuman norovirus is a leading cause of foodborne illness worldwide, and its detection in foods remains a challenge due to the difficulty of eluting and enriching trace amounts of viral particles. In this study, we developed porcine gastric mucin–labeled magnetic capture probes and optimized the conditions for their binding with norovirus, elution of viral particles, and RNA isolation based on the previous research. We evaluated the magnetic capture‐based pretreatment method in artificially contaminated strawberry samples and compared it with the conventional polyethylene glycol precipitation–based method using reverse transcriptional‐quantitative PCR (RT‐qPCR). The results revealed that Tris·HCl (pH 9.5) was the optimal buffer for the binding of magnetic probes with viral particles, and RNA recovery rate obtained by heating‐based release was significantly higher than that obtained by a commercial kit, with only active virus being captured. This buffer was similar to the RT‐qPCR buffer, which facilitated the sensitivity of RT‐qPCR. Furthermore, the buffer was an effective solution for eluting viral particles from foods in previous research and was optimized to ensure maximum elution and no inhibitory effect on viral capture. We explored multiple factors or steps to ensure that the sensitivity of our method was three orders of magnitude higher than that of the conventional method. Our findings suggest that magnetic capture‐based detection has great potential for the sensitive detection of human norovirus.

Differential expression of genes controlling lymphocyte differentiation and migration in two distinct endotypes of type 1 diabetes.

Morphological studies of pancreas samples obtained from young people with recent-onset type 1 diabetes have revealed distinct patterns of immune cell infiltration of the pancreatic islets suggestive of two age-associated type 1 diabetes endotypes that differ by inflammatory responses and rates of disease progression. The objective of this study was to investigate whether these proposed disease endotypes are associated with pathological differences in immune cell activation and cytokine secretion by applying multiplexed gene expression analysis to pancreatic tissue from recent-onset type 1 diabetes cases. RNA was extracted from samples of fixed, paraffin-embedded pancreas tissue from type 1 diabetes cases characterised by endotype and from controls without diabetes. Expression levels of 750 genes associated with autoimmune inflammation were determined by hybridisation to a panel of capture and reporter probes and these were counted as a measure of gene expression. Normalised counts were analysed for differences in expression between 29 type 1 diabetes cases and 7 controls without diabetes, and between the two type 1 diabetes endotypes. Ten inflammation-associated genes, including INS, were significantly under-expressed in both endotypes and 48 genes were more highly expressed. A different set of 13 genes associated with the development, activation and migration of lymphocytes was uniquely overexpressed in the pancreas of people developing diabetes at younger age. The results provide evidence that histologically defined type 1 diabetes endotypes differ in their immunopathology and identify inflammatory pathways specifically involved in disease developing at a young age, essential for a better understanding of disease heterogeneity.

Sulfur source-mediated in situ growth of highly conductive nanocomposites for sensitive detection of miRNA-21

The accurate detection of miRNA-21, one of the emerging biomarkers for breast cancer, is extremely important for facilitating early diagnosis. In this paper, we present a novel nanocomposite material Co9S8@WS2/DWCNT was prepared through a one-pot hydrothermal method. WS2 (Tungsten disulfide) semiconductor nanosheets were used as negative carriers to provide binding sites. TAA (Thioacetamide) was used as a sulfur source and cross-linking agent to modify ZIF-67 (Co-based zeolite imidazole framework) into Co9S8 with excellent electrical conductivity and to grow it in situ on the WS2 surface. DWCNT (Double-walled carbon nanotubes) was then stacked with WS2 by π-π conjugation. To further enhance the electrical conductivity and biocompatibility of the composite nanomaterials, it was doped by AgNPs (Silver nanoparticles) to obtain Co9S8@WS2/DWCNT-AgNPs nanocomposites with excellent electrochemical properties. The capture probe cDNA was immobilized on the electrode through Ag-S bonding to enable sensitive detection of miRNA-21. With a concentration response range of 10−18-10−11 M and a detection limit as low as 1.54 aM, the detection of miRNA-21 in human serum samples has been successfully achieved applying to this biosensing platform.

An Ultrasensitive Voltammetric Genosensor for the Detection of Bacteria Vibrio cholerae in Vegetable and Environmental Water Samples.

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).