Streptavidin-alkaline Phosphatase Research Articles

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.

Nanobody-based magnetic chemiluminescence immunoassay for one-pot detection of ochratoxin A.

Ochratoxin A (OTA) contamination seriously threatens food safety and human health and requires sensitive and rapid tools for monitoring. In this study, a convenient enzyme-linked immunosorbent assay based on Avi-labeled nanobody Nb-2G/streptavidin-alkaline phosphatase and magnetic beads (MBS-ELISA) was established for the sensitive detection of OTA, which could be used for one-pot detection without immobilization. After optimization, the 50% inhibitory concentration (IC50) and the lowest limit of detection value of the MBS-ELISA was 1.17ng/mL and 0.07ng/mL and the linear range was 248.8 pg/mL-5.28ng/mL, respectively, which accords with state criteria for food safety. The developed one-step MBS-ELISA was almost 20-times more sensitive than the classic BA-ELISA and could generate results within 15min, which was significantly less than the classic BA-ELISA at approximately 3h. The MBS-ELISA indicated good recovery (86.4-114.3%) in spiked sorghum, buckwheat, and mung bean. Thus, MBS-ELISA represents a very promising strategy for the simple, rapid, and accurate detection of OTA and other toxic and hazardous contaminants.

Genosensing on a 3D-printed nanocarbon electrode

In this paper we present the characterization of 3D-printed nanocarbon electrodes (3DnCes) and their application in electrochemical enzyme-linked detection of DNA hybridization. The approach takes advantage of a facile procedure based on adsorption of target DNA on the electrode surface followed by hybridization with a biotinylated probe and binding of streptavidin–alkaline phosphatase conjugate. The alkaline phosphatase converts 1-naphthyl phosphate in the background electrolyte into electrochemically oxidizable 1-naphthol, which is subsequently detected using linear sweep voltammetry. The preparation, characterization, and analytical performance of the 3DnCes are reported. The results show the applicability of such 3DnCes in detection of target DNA hybridization specifically with the complementary biotinylated probe, and indicate the potential of 3D printed electrodes for use in various bioanalytical approaches.

SARS-CoV-2 viral RNA detection using the novel CoVradar device associated with the CoVreader smartphone app

The COVID-19 pandemic has highlighted the need for innovative approaches to its diagnosis. Here we present CoVradar, a novel and simple colorimetric method that combines nucleic acid analysis with dynamic chemical labeling (DCL) technology and the Spin-Tube device to detect SARS-CoV-2 RNA in saliva samples. The assay includes a fragmentation step to increase the number of RNA templates for analysis, using abasic peptide nucleic acid probes (DGL probes) immobilized to nylon membranes in a specific dot pattern to capture RNA fragments. Duplexes are formed by labeling complementary RNA fragments with biotinylated SMART bases, which act as templates for DCL. Signals are generated by recognizing biotin with streptavidin alkaline phosphatase and incubating with a chromogenic substrate to produce a blue precipitate. CoVradar results are analysed by CoVreader, a smartphone-based image processing system that can display and interpret the blotch pattern. CoVradar and CoVreader provide a unique molecular assay capable of detecting SARS-CoV-2 viral RNA without the need for extraction, preamplification, or pre-labeling steps, offering advantages in terms of time (∼3 h/test), cost (∼€1/test manufacturing cost) and simplicity (does not require large equipment). This solution is also promising for developing assays for other infectious diseases.

Multilocus genetic analysis of two-breed chicken hybrids

Abstract. Background. Due to the fact that now many new forms of poultry are created by crossing existing breeds and populations, a comprehensive genetic analysis of hybrid chickens as a ground for further breeding work is of particular importance. Aim. To study the features of the population and genetic organization of the genomes of inter-breed chicken hybrids. Materials and methodology. The experimental work was based on the use of an oligonucleotide probe (GTG)5, which was labeled with digoxygenin. The probe was hybridized with genomic DNA on a nylon filter, and then labeled DNA fragments were visualized using a streptavidin-alkaline phosphatase conjugate chemistry. The number and distribution of DNA fragments was highly specific for each individual. These parameters under study include similarity coefficient (BS), genetic distances between groups (D) and average heterozygosity levels (H). Scientific novelty. For the first time, marker DNA fragments characterizing individual groups of birds have been identified; these fragments can be used in the certification of populations. The novelty of the work also lies in the determination of the main genetic characteristics in new groups of hybrid chickens, which will be used to consolidate the desired breeding traits. Results. Based on the data obtained, it can be concluded that there are relatively small genetic differences between various hybrid forms, which is the result of using the same source breeds. Brahma × Sussex Light and Uzbek Game × Amrock hybrids were relatively distant from each other (D = 0.070). Sussex Light× Amrock hybrids were the most genetically diverse according to the criterion of average heterozygosity (P = 0.66).

Plasmodium relictum MSP-1 capture antigen-based ELISA for detection of avian malaria antibodies in African penguins (Spheniscus demersus).

Avian malaria, caused by Plasmodium spp. and transmitted by mosquitos, is a leading cause of mortality of captive penguins. Antimalarial drugs are currently used to control infections in penguins. However, the effectiveness of treatment reduces significantly by the time the clinical signs appear, while early and unnecessary treatment interferes with development of protective immunity. Therefore, for suppressing parasitemia without affecting the development of immunity in captive penguins, antimalaria drugs need to be administered at the right time, which requires reliable diagnostic tools that can determine the levels of circulating antimalaria antibodies. In the present study, we have developed an enzyme-linked immunosorbent assay (ELISA) diagnostic assay based on the merozoite surface protein 1 (MSP-1) of P. relictum isolate SGS1 to specifically detect and relatively quantify antimalaria antibodies in penguins. We expressed and purified a truncated P. relictum isolate SGS1 MSP-1 and optimized its biotinylation and subsequent conjugation to streptavidin alkaline phosphatase for signal generation in ELISA. We tested the assay by analyzing sera obtained from penguins at the Baltimore Zoo, from Spring through Fall, and found that levels of detectable antibodies against MSP-1 varied seasonally for individual penguins, consistent with the expected seasonal variations in avian malaria prevalence. Corroboratively, we analyzed the sensitivity of the assay by titrating positive sera and found that the signal intensity generated was serum concentration-dependent, thus validating the ability of the assay to detect and relatively quantify the levels of antimalaria antibodies in penguin sera.

Fluorescent and colorimetric detection of Norfloxacin with a bifunctional ligand and enzymatic signal amplification system

We report a dual signal read-out enzyme-linked immunosorbent assay (ELISA) strategy for the sensitive detection of Norfloxacin (Nor) using a bifunctional ligand and enzymatic signal amplification system. In the proposed method, the immune response of an anti-Nor antibody against Nor-Biotin bifunctional ligands triggers the immobilization of streptavidin–alkaline phosphatase and the alkaline-phosphatase could continuous triggers the hydrolysis of L-ascorbic acid-phosphoric acid to produce abundant L-ascorbic acid (AA). Colorimetric signal is generated from the aggregation of azide and alkyne-based colloidal gold generated by a click-chemistry reaction with Cu+ catalysis (reduced by the AA), which changes its color from red to blue. A fluorescence signal is generated by the opening of the spirolactam ring caused by unreacted Cu2+ binding to the Cu2+ probe. Our method was successfully illustrated by detecting Nor in the range of3.18 × 10−2 to 6.88 × 103 pg/mL.The efficient detection of Nor was further confirmed using Nor added to commercially available milk, with a recovery in the range of 98.65%–104.01%. This work provides a promising method to detect Nor and other veterinary drug residues to guarantee food safety.

I2/I−-mediated fluorescence quenching of an Ag+-doped gold nanocluster-based immunoassay for sensitive detection of Escherichia coli O157:H7 in milk

Escherichia coli O157:H7 is a type of hazardous bacteria in the field of food safety. A sensitive and effective method is urgently needed to detect it, avoiding enormous harm for the human health. In this study, we synthesized stable Ag+-doped gold nanoclusters (Ag-AuNC) with a fluorescence intensity 4.8 times stronger than that of AuNC. It was further demonstrated that Ag0 existing in the AuNC core and a fraction of Ag+ anchored on the AuNC shell eliminated the surface defects and improved the luminescent properties of AuNC. A combination of I2 and I- was used to quench fluorescence-enhanced Ag-AuNC, which was first applied in ELISA for detecting E. coli O157:H7 to improve the sensitivity. In the presence of E. coli O157:H7, the biotinylated anti-E. coli O157:H7 mAb and streptavidin-alkaline phosphatase would be immobilized and catalyze l-ascorbic acid 2-phosphate sesquimagnesium salt hydrate to produce ascorbic acid. After addition of KIO3, I2/I- were generated. The I2 could trigger oxidative etching of Ag-AuNC and I- could combine with Ag+ to decrease the Ag+ concentration of Ag-AuNC, which resulted in fluorescence quenching of Ag-AuNC. Under optimal conditions, the linear range of I2/I--mediated fluorescence quenching of Ag-AuNC-based immunoassay for detecting E. coli O157:H7 was 3.3 × 103 to 106 cfu/mL, with a detection limit of 9.2 × 102 cfu/mL, 10.7-fold lower than that of the traditional ELISA. The proposed immunoassay exhibits excellent sensitivity, specificity, recovery, and accuracy, which is useful for quantitative detection of E. coli O157:H7 in food safety.

Construction of a dephosphorylation-mediated chemiluminescent biosensor for multiplexed detection of DNA glycosylases in cancer cells.

DNA glycosylases are engaged in the base excision repair process and play a vital role in maintaining genomic integrity. It remains a challenge for multiplexed detection of DNA glycosylases in cancer cells. Herein, we demonstrate the construction of a dephosphorylation-mediated chemiluminescent biosensor for multiplexed detection of human alkyladenine DNA glycosylase (hAAG) and uracil DNA glycosylase (UDG) in cancer cells. In this biosensor, the generation of chemiluminescence signals relies on the dephosphorylation of 3-(2'-spiroadamantyl)-4-methoxy-4-(3''-phosphoryloxyphenyl)-1,2-dioxetane (AMPPD) catalyzed by alkaline phosphatase (ALP). We design a bifunctional double-stranded DNA (dsDNA) substrate, a biotin-labelled poly-(T) probe, and two capture probes for the hAAG and UDG assay. This assay involves four steps including (1) the cleavage of the bifunctional dsDNA substrate induced by DNA glycosylases, (2) the recognition of the 3'-OH terminus of the primer by TdT and the subsequent TdT-mediated polymerization reaction, (3) the construction of the AuNPs-dsDNA-ALP nanostructures, and (4) the streptavidin-alkaline phosphatase (SA-ALP)-initiated dephosphorylation of AMPPD for the generation of an enhanced chemiluminescence signal. By taking advantage of the unique features of TdT-mediated polymerization and the intrinsic superiority of the ALP-AMPPD-based chemiluminescence system, this biosensor exhibits good specificity and high sensitivity with a detection limit of 1.53 × 10-6 U mL-1 for hAAG and 1.77 × 10-6 U mL-1 for UDG, and it can even quantify multiple DNA glycosylases at the single-cell level. Moreover, this biosensor can be applied for the measurement of kinetic parameters and the screening of DNA glycosylase inhibitors, holding great potential in DNA damage-related biomedical research and disease diagnostics.

Rapid Collection and Aptamer-Based Sensitive Electrochemical Detection of Soybean Rust Fungi Airborne Urediniospores

Plants are the central source of food for humans around the world. Unfortunately, plants can be negatively affected by diverse kinds of diseases that are responsible for major economic losses worldwide. Thus, monitoring plant health and early detection of pathogens are essential to reduce disease spread and facilitate effective management practices. Various detection approaches are currently practiced. These methods mainly include visual inspection and laboratory tests. Nonetheless, these methods are labor-intensive, time-consuming, expensive, and inefficient in the early stages of infection. Thus, it is extremely important to detect diseases at the early stages of the epidemic. Here, we would like to present a fast, sensitive, and reliable electrochemical sensing platform for the detection of airborne soybean rust spores. The suspected airborne soybean rust spores are first collected and trapped inside a carbon 3D electrode matrix by high-capacity air-sampling means. Then, a specific biotinylated aptamer, suitable to target specific sites of soybean rust spores is applied. This aptamer agent binds to the surface of the collected spores on the electrode. Finally, spore-bound aptamer units are incubated with a streptavidin–alkaline phosphatase agent leading to the enzymatic formation of p-nitrophenol, which is characterized by its unique electrochemical properties. Our method allows for the rapid (ca. 2 min), selective, and sensitive collection and detection of soybean rust spores (down to the limit of 100–200 collected spores per cm2 of electrode area). This method could be further optimized for its sensitivity and applied to the future multiplex early detection of various airborne plant diseases.

A whole area scanning-enabled direct-counting strategy for studying blocking efficiency in mitigating protein-solid surface binding.

The study of protein-solid surface binding as well as blocking efficiency of blocking agents plays an important role in the development of high-performance immunoassays. Although conventional colorimetric based assays are widely employed to monitor protein non-specific binding on the surface of microplate wells and evaluate the performance of blocking agents, there is still a great need to develop new methods to achieve the same goal from a new perspective. In this study, an innovative whole area scanning (WAS)-enabled direct-counting strategy was developed and validated through studying the blocking efficiency of different blocking agents on the non-specific binding of streptavidin-alkaline phosphatase conjugate (Strep-ALP, a model protein) to the surface of 96-well microplates. After non-specific binding of Strep-ALP in wells with or without blocking agents' treatment and loading of ELF™ 97 phosphate (ELFP), ALP in Strep-ALP conjugates converts ELFP to water-insoluble ELF™ 97 alcohol (ELFA), which precipitates locally, self-assembles into large needle structures, and glows green fluorescence upon excitation. After quenching the reaction, WAS of the whole wells allows us to directly count the number of individual fluorescent precipitates, which can be used to calculate and compare the blocking efficiency of three commonly used blocking agents (BSA, casein, and dry milk) based on mitigating the non-specific binding of Strep-ALP. WAS-enabled counting of individual needle-type precipitates opens a new avenue to investigate protein-solid surface binding as well as the efficiency of blocking agents with high sensitivity.

Fast enzyme-linked electrochemical sensing of DNA hybridization at pencil graphite electrodes. Application to detect gene deletion in a human cell culture

In this paper we present a rapid electrochemical enzyme-linked DNA hybridization assay using disposable pencil graphite electrodes (PeGE) to detect target DNA (tDNA) sequences in DNA fragments amplified by polymerase chain reaction. The procedure consists of several short (1–2 min) incubation steps, including adsorption of the tDNA at unpretreated PeGE from denaturing medium, surface blocking with milk proteins, hybridization with a biotinylated oligonucleotide probe and binding of streptavidin-alkaline phosphatase conjugate to the biotin tags. Then the PeGE is transferred into background electrolyte solution containing 1-naphthyl phosphate, which is enzymatically dephosphorylated to give electrochemically oxidizable indicator 1-naphthol. The assay, which can be performed within 7–8 min, offers a perfect discrimination between specific and nonspecific DNA amplicons and easy detection of about ~40 femtomoles of tDNA in large excesses of non-complementary DNA. An application on the detection of p53 gene deletion in a human cell culture, featuring a real biological model, is presented. The designed setup has a potential to be applied as one of simple, fast, robust ultralow cost “do-it-yourself “instruments recently introduced as diagnostic tools for third world countries.

An electrochemical sandwich-type aptasensor for determination of lipocalin-2 based on graphene oxide/polymer composite and gold nanoparticles

In this work, we reported an electrochemical aptasensor based on the poly-3-amino-1,2,4-triazole-5-thiol/graphene oxide composite (P(ATT)-GO) and gold nanoparticles (AuNPs) modified graphite screen-printed electrode (GSPE) (GSPE/P(ATT)-GO/AuNPs) for determination of lipocalin-2 (LCN2) (neutrophil gelatinase-associated lipocalin). A sandwich based strategy was utilized to enhance the electrochemical signal. First, a thiol tethered DNA aptamer was immobilized onto the composite electrode. Then, the LCN2 solution was incubated with the aptamer modified GSPE/P(ATT)-GO/AuNPs. Secondary aptamer (Apt2) peculiar to the LCN2 and labeled with biotin was interacted with the LCN2. A streptavidin-alkaline phosphatase conjugate was then applied to the surface. The determination of LCN2 was performed by using the electroactive property of α-naphthol which is acquired the product from the interaction between alkaline phosphatase and α-naphthyl phosphate. The constructed electrode was characterized by scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The aptamer modified GSPE/P(ATT)-GO/AuNPs showed the superior electrocatalytic performance towards the voltammetric determination of LCN2 with a wide linear range (1.0–1000.0 ng/mL) and a low limit of detection (LOD) (0.3 ng/mL). The proposed aptasensor revealed the excellent sensitivity, anti-interference ability and reproducibility which approved that the GSPE/P (ATT)-GO/AuNPs is a promising composite for the sensitive detection of LCN2. The fabricated aptasensor was applied for the determination of LCN2 in fetal bovine serum samples using the standard addition method and the recovery values were in the range of 99.2% and 103.22%.

Cord-Based Microfluidic Chips as A Platform for ELISA and Glucose Assays.

This paper describes the development and application of microfluidic cord-based analytical devices (µCADs) in two enzyme-linked immunosorbent assays (ELISAs) and glucose assay. In this study, biotinylated goat anti-mouse immunoglobulin (IgG) antibody, rabbit IgG antibody, and glucose are quantitatively detected. In the ELISA systems, the antibody is spotted on the cord at the detection site and a series of washes, followed by streptavidin-alkaline phosphatase (Strep-ALP) or alkaline phosphatase (ALP)-conjugated secondary antibody and colorimetric substrate, completing the experiment. The devices are subsequently scanned and analyzed yielding a correlation between inverse yellow or inverse blue intensity and antibody concentration. For the first ELISA, a linear range of detection was observed at lower concentrations (2.50 × 10−4–1.75 × 10−3 mg/mL) of Strep-ALP with saturation of the enzyme achieved at higher concentrations (>2.50 × 10−4). For the second ELISA, the L50 was demonstrated to be 167.6 fmol/zone. The glucose assay consisted of spotting increasing concentrations of glucose on the analysis sites and transporting, via capillary action, a solution containing glucose oxidase (GOx), horseradish peroxidase (HRP), and potassium iodide (KI) to the detection sites realizing a yellow-brown color indicating oxidation of iodide to iodine. The device was then dried, scanned, and analyzed to show the correlation between yellow inverse intensity and glucose. Glucose in artificial urine showed good correlation using the devices.

Electrochemical aptasensing strategy for kanamycin detection based on target-triggered single-strand DNA adsorption on MoS2 nanosheets and enzymatic signal amplification

A selective and sensitive electrochemical aptasensing strategy was developed for kanamycin detection based on target-triggered single-strand DNA adsorption on MoS2 nanosheets and enzymatic signal amplification. Firstly, a Y-shape double-stranded DNA (dsDNA) was designed containing probe DNA, assist DNA (labeled with biotin at its 3′-terminal) and aptamer DNA. Then, in the presence of kanamycin, aptamer DNA can be released from the Y-shape dsDNA structure due to its interaction with kanamycin to form stable conjugate, which will destroy the Y-shape dsDNA structure and lead to the presence of two-fragment of single-stranded DNA (ssDNA) on the remained half-hybrid DNA structure (containing probe DNA and assist DNA). Based on the strong interaction between ssDNA and MoS2 nanosheets, the half-hybrid DNA structure can be captured on MoS2 nanosheets modified electrode surface, which triggers the subsequent immobilization of streptavidin-alkaline phosphatase (SA-ALP) compound. Under the catalysis of ALP, p-nitrophenol phosphate (PNPP) is hydrolyzed to produce p-nitrophenol (PNP). Based on the linear relationship between the electrochemical oxidation signal of PNP and kanamycin concentration, kanamycin can be detected with the detection limit of 0.029 nM (S/N = 3). The aptamer-MoS2 nanosheets based electrochemical biosensor shows good selectivity, reproducibility for kanamycin detection.

Electrochemical enzyme-linked oligonucleotide array for aflatoxin B1 detection

In this work, an electrochemical enzyme-linked oligonucleotide array to achieve simple and rapid multidetection of aflatoxin B1 (AFB1) is presented. The assay is based on a competitive format and disposable screen-printed cells (SPCs). Firstly, the electrodeposition of poly(aniline-anthranilic acid) copolymer (PANI-PAA) on graphite screen-printed working electrodes was performed by means of cyclic voltammetry (CV). Aflatoxin B1 conjugated with bovine serum albumin (AFB1-BSA) was then immobilized by covalent binding on PANI-PAA copolymer. After performing the affinity reaction between AFB1 and the biotinylated DNA-aptamer (apt-BIO), the solution was dropped on the modified SPCs and the competition was carried out. The biotinylated complexes formed onto the sensor surface were coupled with a streptavidin-alkaline phosphatase conjugate. 1-naphthyl phosphate was used as enzymatic substrate; the electroactive product was detected by differential pulse voltammetry (DPV). The response of the enzyme-linked oligonucleotide assay was signal-off, according to the competitive format. A dose-response curve was obtained between 0.1 ng mL−1 and 10 ng mL−1 and a limit of detection of 0.086 ng mL−1 was achieved. Finally, preliminary experiments in maize flour samples spiked with AFB1 were also performed.

Development of an Electrochemical Biosensor for Rapid and Effective Detection of Pathogenic Escherichia coli in Licorice Extract

An aptamer-based electrochemical biosensor was successfully developed and applied in the rapid detection of pathogenic Escherichia coli (E. coli) in licorice extract. The thiolated capture probes were firstly immobilized on a gold electrode, and then the biotinylated aptamer probes for E. coli were introduced by hybridization with the capture probes. Due to the stronger interaction between the aptamer and the E. coli, a part of the biotinylated aptamers will dissociate from the capture probes in the presence of E. coli. The residual biotinylated aptamer probes can quantitatively bind with streptavidin-alkaline phosphatase. Subsequently, α-naphthyl phosphate substrate was catalytically hydrolyzed to generate electrochemical response, which could be recorded by a differential pulse voltammetry. The dependence of the peak current on the logarithm of E. coli concentration in the range from 5.0 × 102 colony forming units (CFU)/mL to 5.0 × 107 CFU/mL exhibited a linear trend with a detection limit of 80 CFU/mL. The relative standard deviation of 5 successive scans was 5.3%, 4.5% and 1.1% for 5.0 × 102, 5.0 × 105 and 5.0 × 107 CFU/mL E. coli, respectively. In the detection of the licorice extract samples, the results obtained from the proposed strategy and traditional culture counting method were close to each other, but the time consumption was only ~1/30 compared with the traditional method. These results demonstrate that the designed biosensor can be potentially utilized for rapid microbial examination in traditional Chinese medicine and relevant fields.

Electrochemical Detection of SNP in Human Mitochondrial DNA Using Cyclic Primer Extension with Biotinylated Nucletides and Enzymatic Labeling at Disposable Pencil Graphite Electrodes

AbstractA novel method of SNP typing in human mitochondrial DNA utilizing enzymatic labeling and electrochemical detection at disposable pencil graphite electrodes is described. The procedure is based on amplification of DNA stretches by cyclic primer extension (PEx) of SNP‐specific diagnostic primers in a mixture of biotinylated and natural nucleotides. The diagnostic primers are designed to recognize, by its 3'‐terminal nucleotide, the SNP‐site in target template. Under optimized conditions of the PEx reaction, efficient polymerase synthesis of biotin‐labeled strands takes place only in the case of full complementarity between the diagnostic primer and the target SNP site. There is also benefit from introducing many biotin molecules per extended DNA strand, resulting in another level of signal amplification. After adsorption of biotinylated PEx products at the electrode surface, streptavidin‐alkaline phosphatase conjugate was bound to the biotin tags, 1‐naphthol was enzymatically produced and electrochemically detected. Several critical steps and parameters of the assay, including termination of 3’‐OH ends of residual amplification primers, temperature for annealing of diagnostic primers, relative amount of biotinylated deoxynucleoside triphosphate in the PEx mixture and number of PEx cycles were optimized in this study to attain best SNP resolution, and reduction of time needed for the analysis.

DNA-Based Sensor for the Detection of an Organophosphorus Pesticide: Profenofos.

In this work, we propose an electrochemical DNA aptasensor for the detection of profenofos, an organophosphorus pesticide, based on a competitive format and disposable graphite screen-printed electrodes (GSPEs). A thiol-tethered DNA capture probe, which results to be complementary to the chosen aptamer sequence, was immobilised on gold nanoparticles/polyaniline composite film-modified electrodes (AuNPs/PANI/GSPE). Different profenofos solutions containing a fixed amount of the biotinylated DNA aptamer were dropped onto the realized aptasensors. The hybridisation reaction was measured using a streptavidin-alkaline phosphatase enzyme conjugate, which catalyses the hydrolysis of 1-naphthyl -phosphate. The 1-naphtol enzymatic product was detected by means of differential pulse voltammetry (DPV). The aptasensor showed itself to work as a signal off sensor, according to the competitive format used. A dose response curve was obtained between 0.10 μM and 10 μM with a detection limit of 0.27 μM.

A novel electrochemical DNA biosensor for Ebola virus detection

The aim of this study was to fabricate a novel electrochemical-based DNA-sensing device for Ebola virus DNA diagnostic by an enzyme-amplified detection, which improves the sensitivity and selectivity of the sensor. A thiolated DNA capture probe sequence was immobilized on the screen printed electrode surface and hybridized with biotinylated target strand DNA for the fabrication of Ebola DNA-sensing devices. Prior to the electrochemical detection of the enzymatic product by differential pulse voltammetry (DPV) method, the biotinylated hybrid was labeled with a streptavidin-alkaline phosphatase conjugate on the surface of the working electrode. All the experiment steps were optimized using electrochemical impedance spectroscopy (EIS) and the optimum condition for biosensor fabrication was achieved. A detection limit of 4.7 nM complementary oligonucleotides was obtained using the fabricated biosensor and the standard deviation of the blank solution. The last step, the selectivity and reproducibility of fabricated electrochemical DNA biosensor was obtained.