Biotinylated Detection Probe Research Articles

A Rapid and Sensitive Aptamer‐Based Electrochemical Biosensor for Direct Detection of Escherichia Coli O111

AbstractA sensitive and specific electrochemical biosensor based on target‐induced aptamer displacement was developed for direct detection of Escherichia coli O111. The aptamer for Escherichia coli O111 was immobilized on a gold electrode by hybridization with the capture probe anchored on the electrode surface through Au‐thiol binding. In the presence of Escherichia coli O111, the aptamer was dissociated from the capture probe‐aptamer duplex due to the stronger interaction between the aptamer and the Escherichia coli O111. The consequent single‐strand capture probe could be hybridized with biotinylated detection probe and tagged with streptavidin‐alkaline phosphatase, producing sensitive enzyme‐catalyzed electrochemical response to Escherichia coli O111. The designed biosensor showed weak electrochemical signal to Salmonella typhimurium, Staphylococcus aureus and common non‐pathogenic Escherichia coli, indicating high specificity for Escherichia coli O111. Under the optimal conditions, the proposed strategy could directly detect Escherichia coli O111 with the detection limit of 112 CFU mL−1 in phosphate buffer saline and 305 CFU mL−1 in milk within 3.5 h, demonstrated the sensitive and accurate quantification of target pathogenic bacteria. The designed biosensor could become a powerful tool for pathogenic microorganisms screening in clinical diagnostics, food safety, biothreat detection and environmental monitoring.

Gold nanoparticles/water-soluble carbon nanotubes/aromatic diamine polymer composite films for highly sensitive detection of cellobiose dehydrogenase gene

An electrochemical sensor based on gold nanoparticles (GNPs)/multiwalled carbon nanotubes (MWCNTs)/poly (1,5-naphthalenediamine) films modified glassy carbon electrode (GCE) was fabricated. The effectiveness of the sensor was confirmed by sensitive detection of cellobiose dehydrogenase (CDH) gene which was extracted from Phanerochaete chrysosporium using polymerase chain reaction (PCR). The monomer of 1,5-naphthalenediamine was electropolymerized on the GCE surface with abundant free amino groups which enhanced the stability of MWCNTs modified electrode. Congo red (CR)-functionalized MWCNTs possess excellent conductivity as well as high solubility in water which enabled to form the uniform and stable network nanostructures easily and created a large number of binding sites for electrodeposition of GNPs. The continuous GNPs together with MWCNTs greatly increased the surface area, conductivity and electrocatalytic activity. This electrode structure significantly improved the sensitivity of sensor and enhanced the DNA immobilization and hybridization. The thiol modified capture probes were immobilized onto the composite films-modified GCE by a direct formation of thiol–Au bond and horseradish peroxidase–streptavidin (HRP–SA) conjugates were labeled to the biotinylated detection probes through biotin–streptavidin bond. Scanning electron microscopy (SEM), electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) were used to investigate the film assembly and DNA hybridization processes. The amperometric current response to HRP-catalyzed reaction was linearly related to the common logarithm of the target nucleic acid concentration in the range of 1.0 × 10 −15–1.0 × 10 −10 M, with the detection limit of 1.2 × 10 −16 M. In addition, the electrochemical biosensor exhibited high sensitivity, selectivity, stability and reproducibility.

Antibody-free peptide substrate screening of serine/threonine kinase (protein kinase A) with a biotinylated detection probe

Being different from anti-phosphotyrosine antibodies, anti-phosphoserine- or anti-phosphothreonine-specific antibodies with high affinity for the detection of serine/threonine kinase substrates are not readily available. Therefore, chemical modification methods were developed for the detection of phosphoserine or threonine in the screening of protein kinase substrates based on β-elimination and Michael addition. We have developed a biotin-based detection probe for identification of the phosphorylated serine or threonine residue. A biotin derivative induced a color reaction using alkaline phosphate-conjugated streptavidin that amplified the signal. It was effective for the detection and separation of the target peptide on the resin. The detection probe was successfully used in identifying PKA substrates from peptide libraries on resin beads. The peptide library was prepared as a ladder-type, such that the active peptides on the colored resin beads were readily sequenced with the truncated peptide fragments by MALDI-TOF/MS analysis after releasing the peptides from the resin bead through photolysis.

An ultrasensitive DNA biosensor based on enzyme-catalyzed deposition of cupric hexacyanoferrate nanoparticles

An ultrasensitive electrochemical biosensor is demonstrated for the detection of a synthetic single-stranded DNA (ss-DNA) with a detection limit down to femtomolar levels. The synthetic ss-DNA with sequence associated with the influenza A virus RNA was used as our model target DNA. The detection scheme was based on a sandwich structure involving a capture probe, the target DNA and a detection probe. An avidin-labeled glucose oxidase (GOx) was attached to a biotinylated detection probe via biotin–avidin interaction. Subsequently, the DNA hybridization event was transduced and amplified by an in-situ enzyme-catalyzed deposition of cupric hexacyanoferrate (CuHCF) nanoparticles in the presence of glucose, cupric ions and ferricyanide. The peak current in differential pulse voltammetry of the deposited CuHCF nanoparticles directly correlated to the concentration of the target DNA. DNA was therefore quantified in the concentration range from 1.0 fM to 10 pM with a detection limit of 1.0 fM and a relative standard derivation of ≤11% at 100 fM. Excellent mismatch discrimination was demonstrated in the proposed biosensor.

Sensitive detection of lip genes by electrochemical DNA sensor and its application in polymerase chain reaction amplicons from Phanerochaete chrysosporium

An electrochemical DNA sensor based on the sandwich hybridization recognition of target sequence of lignin peroxidase ( lip) genes on a gold electrode was developed. A monolayer of thiolated capture probe was formed on a gold electrode through self-assembling. Following hybridizations with target nucleic acid and biotinylated detection probe, streptavidin–horseradish peroxidase (HRP) conjugate was applied to the electrode. The DNA conformation and surface coverage on electrode were characterized by impedance spectroscopy and square wave voltammetry. The experimental variables were optimized to maximize the hybridization efficiency, detection sensitivity and speed up the assay time. The amperometric current response to HRP-catalyzed reaction was linearly related to the natural logarithm of the target nucleic acid concentration in the range from 0.6 to 30 nM, with the correlation coefficient of 0.9722. The detection limit was 0.03 nM. Synthesized oligonucleotide as well as Phanerochaete chrysosporium lip gene fragments amplified using polymerase chain reaction and digested by restriction endonucleases were tested. The DNA sensor exhibited good precision, stability, sensitivity, and selectivity, and discriminated satisfactorily against mismatched nucleic acid samples of similar lengths.

Electrochemical immunosensor with aptamer-based enzymatic amplification

An electrochemical immunosensor is reported by using aptamer-based enzymatic amplification with immunoglobulin E (IgE) as the model analyte. In this method, the IgE antibody is covalently immobilized as the capture probe on the gold electrode via a self-assembled monolayer of cysteamine. After the target is captured, the biotinylated anti-IgE aptamer is used as the detection probe. The specific interaction of streptavidin-conjugated alkaline phosphatase to the surface-bound biotinylated detection probe mediates a catalytic reaction of ascorbic acid 2-phosphate substrate to produce a reducing agent ascorbic acid. Then silver ions in the solution can be reduced, leading to the deposition of metallic silver on the electrode surface. The amount of deposited silver, which is determined by the amount of IgE target bound on the electrode surface, can be quantified using the stripping voltammetry. The results obtained demonstrated that the electrochemical immunosensor possesses high specificity and a wide dynamic range with a low detection limit that possibly arises from the combination of the highly specific aptamer and the highly sensitive stripping determination of enzymatically deposited silver.

Electrochemical detection of point mutation based on surface ligation reaction and biometallization

A highly sensitive electrochemical method for point mutation detection based on surface enzymatic ligation reaction and biometallization is demonstrated. In this method the surface-immobilized allele-specific probe, complementary to the mutant target, undergoes allele-specific ligation with the 5′-phosphorylated ligation probe in the presence of the mutant oligonucleotide target and E. coli DNA ligase. If there is an allele mismatch, no ligation takes place. After thermal treatment at 90°C, the formed duplex melts apart, which merely allows the ligation product to remain on the electrode surface. Then, biotinylated detection probes hybridize with the ligation product. With the binding of streptavidin-alkaline phosphatase (SA-ALP) to the biotinylated probes, a non-reductive substrate of alkaline phosphatase, ascorbic acid 2-phosphate (AA-P), can be converted into ascorbic acid (AA) at the electrode surface. Silver ions in solution are then reduced by AA, resulting in the deposition of silver metal onto the electrode surface. Linear sweep voltammetry (LSV) is used to detect the amount of deposited silver. The proposed approach has been successfully implemented for the identification of single base mutation in codon 12 of K-ras oncogene target with a detection limit of 80fM, demonstrating that this method provides a highly specific, sensitive and cost-efficient approach for point mutation detection.

Enzyme-amplified electrochemical detection of DNA using electrocatalysis of ferrocenyl-tethered dendrimer.

We have developed a sandwich-type enzyme-linked DNA sensor as a new electrochemical method to detect DNA hybridization. A partially ferrocenyl-tethered poly(amidoamine) dendrimer (Fc-D) was used as an electrocatalyst to enhance the electronic signals of DNA detection as well as a building block to immobilize capture probes. Fc-D was immobilized on a carboxylic acid-terminated self-assembled monolayer (SAM) by covalent coupling of unreacted amine in Fc-D to the acid. Thiolated capture probe was attached to the remaining amine groups of Fc-D on the SAM via a bifunctional linker. The target DNA was hybridized with the capture probe, and an extension in the DNA of the target was then hybridized with a biotinylated detection probe. Avidin-conjugated alkaline phosphatase was bound to the detection probe and allowed to generate the electroactive label, p-aminophenol, from p-aminophenyl phosphate enzymatically. p-Aminophenol diffuses into the Fc-D layer and is then electrocatalytically oxidized by the electronic mediation of the immobilized Fc-D, which leads to a great enhancement in signal. Consequently, the amount of hybridized target can be estimated using the intensity of electrocatalytic current. This DNA sensor exhibits a detection limit of 20 fmol. Our method was also successfully applied to the sequence-selective discrimination between perfectly matched and single-base mismatched target oligonucleotides.

RT-PCR-ELOSA tests on pooled sample units for the detection of virus Y in potato tubers

A highly sensitive RT-PCR protocol able to detect potato virus Y (PVY) in pooled sample units (tubers) was developed. PVY-specific primers selected in the coat protein gene were found to amplify a 359 bp fragment from diluted crude extract of infected tubers. For the detection of the amplification products, a colorimetric detection procedure in microtiter plates was established. The amplicons are hybridized between a covalently linked capture probe and a specific biotinylated detection probe ELOSA tests. This detection method detects at least 50 pg of virus per reaction for the four cultivars tested. The RT-PCR-ELOSA assay was adapted to pooled units in order to increase the sample size while reducing the number of tests.

Development of routine RT‐PCR tests for certification of fruit tree multiplication material*

Current developments in certification procedures for propagating material require the availability of rapid, sensitive, reliable and user‐friendly detection protocols applicable for routine testing. Our research concerns the possible use of reverse transcriptase‐polymerase chain reaction (RT‐PCR) for the detection of the pathogens listed for virus‐tested pome and stone‐fruit propagating material in Belgium. Although RT‐PCR satisfies the need for rapidity and sensitivity, the usual protocols relying on the use of purified nucleic acid preparations as template and ethidium bromide‐stained agarose gels for detection are not appropriate for routine use. We therefore first optimized the parameters and cycling conditions of the RT‐PCR reactions to allow direct use of crude extracts of either leaf or bark material as a template. Sandwich hybridization between a covalently linked capture probe and a biotinylated detection probe was then used for the detection of the specific amplicons (Lambdatech S.A. kits in development). These assays have the sensitivity and specificity of the RT‐PCR, enhanced by sandwich hybridization with specific probes, and ease of sample preparation and detection of the amplicons. They make it possible to analyse a great number of samples and are thus well adapted for routine quality‐control testing of propagating material.

DNA probe hybridisation in microwells using a new bioluminescent system for the detection of PCR-amplified HIV-1 proviral DNA

A new bioluminescent detection system combined with a sandwich DNA hybridisation reaction in microwells has been developed for the detection of human immunodeficiency virus type 1 (HIV-1) provirus DNA amplified by the polymerase chain reaction (PCR). First, a fragment of the HIV-1 gag gene was amplified. The amplified DNA fragments were denatured and hybridised to a capture probe immobilised in microwells and to a biotinylated detection probe. A streptavidin-pyruvate kinase conjugate could then react on the biotinylated probe and the kinase activity detected by means of the luciferin-luciferase system, with production of a bioluminescent signal. This sandwich assay followed by a bioluminescent reaction detected as little as 7 amol of target DNA. The bioluminescent assay detected 5 HIV copies generated after one round of PCR, even if no band was seen on an agarose gel. The assay was applied to the detection of HIV-proviral DNA in peripheral blood mononuclear cells after one round of PCR and allowed to clearly identify a positive sample as compared to nested PCR.

Novel application of streptavidin-hapten derivatives as protein-tracer conjugate in competitive-type immunoassays involving biotinylated detection probes

To investigate the use of streptavidin-hapten derivatives as potential protein-tracer conjugates for competitive-type immunoassays, we labeled streptavidin with cortisol and compared biotin-binding activity of the conjugates with that of unlabeled streptavidin. In this model system, streptavidin labeled with one to approximately 17 cortisol molecules retained its capability to cross-link a biotinylated protein on microtiter wells to a biotin-based general detection reagent developed for time-resolved fluorometry. Compared with unlabeled streptavidin, there was no reduction in the binding activity of the conjugate carrying as many as 2.6 cortisol molecules per molecule of streptavidin. Conjugation ratios greater than 4.4 showed a slight decrease in binding activity, presumably because of the aggregate formation evident at these labeling ratios. As expected, the conjugates were also capable of linking a solid-phase-bound anti-cortisol monoclonal antibody to the biotinylated detection reagent. The fluorescence signal generated increased almost linearly with increasing conjugation ratios from about three to nine cortisol molecules per molecule of streptavidin. At greater ratios, the assay response plateaued. The calibration curves obtained were typical for competitive-type immunoassays when the conjugates were incorporated in a cortisol assay based on a second-antibody immobilization approach.