Biotinylated Detection Probe Research Articles

Duplex-specific nuclease-based electrochemical biosensor for the detection of microRNAs by conversion of homogeneous assay into surface-tethered electrochemical analysis

We proposed a simple and sensitive strategy for the detection of microRNAs (miRNAs) by converting homogeneous assay into surface-tethered electrochemical analysis. Specifically, the biotinylated detection probes (biotin-DNA-biotin) can trigger the in-situ assembly of tetrameric streptavidin (SA) proteins on an electrode surface via the SA-biotin interactions. The (SA-biotin-DNA-biotin)n assemblies electrically insulated the electrode interface, thereby blocking the electron transfer of [Fe(CN)6]3-/4-. When the probe was hybridized with the target miRNA, it would be cleaved into small fragments (denoted as biotin-DNA) by duplex-specific nuclease (DSN). The released target miRNA can enter into the next hybridization-enzymolysis cycle, thus leading to the generation of considerable amounts of biotin-DNA fragments. The released biotin-DNA competed with the detection probe to bind SA, thus limiting the in-situ formation of (SA-biotin-DNA-biotin)n assemblies. The surface-tethered electrochemical analysis by the dual signal amplification of DSN and (SA-biotin-DNA-biotin)n assemblies has been used for the determination of miRNAs in cell lysate with a satisfactory result. The method showed a detection limit down to 10 aM. The “one-step” immobilization-free strategy can be used to design novel biosensors for the detection of other biomarkers.

Femtomolar direct voltammetric determination of circulating miRNAs in sera of cancer patients using an enzymeless biosensor

A disposable enzyme−free biosensing platform for the sensitive and selective voltammetric determination of miRNAs is reported. The bioplatform implies a sandwich−type hybridization configuration involving the use of two synthetic DNA probes that hybridize contiguously with the target miRNA−21. A thiolated capture probe was immobilized through thiol chemistry on disposable carbon electrodes modified with a hybrid nanomaterial composed of reduced graphene oxide (rGO) and gold nanoparticles (AuNPs). A biotinylated detection probe was conjugated with ferrocene-capped AuNPs modified with streptavidin (Fc−AuNPs−Strep) which were used as labeling nanocarriers. The extent of the hybridization event was followed by differential pulse voltammetric measurement of the Fc oxidation peak. Under the optimized conditions, the developed biosensor provides attractive characteristics for the determination of the synthetic target miRNA, with a linear range between 10 fM and 2 pM and a limit of detection (LOD) of 5 fM, fully discrimination towards a highly homologous miRNA (with just one mismatched base) and a storage stability of at least two months. The biosensor was able to determine accurately the target miRNA directly in scarcely diluted serum from breast cancer (BC) patients with no need for a previous total RNA (RNAt) extraction and in a very small amount of RNAt extracted from breast adenocarcinoma cells without the need for amplification or reverse transcription to complementary DNA.

Target-induced aptamer displacement on gold nanoparticles and rolling circle amplification for ultrasensitive live Salmonella typhimurium electrochemical biosensing

Salmonella typhimurium (S. typhimurium) infection disease is harm to public through food have become a life-threatening problem stresses an urgent need for development of rapid, robust and cost-effective alternative technology to help control the spread of S. typhimurium. Herein, a simple and facile aptasensor has been developed for ultrasensitive determination of live S. typhimurium by combining target-induced aptamer displacement on gold nanoparticles (AuNPs) deposited electrode with rolling circle amplification (RCA). The heterogeneous recognition and specific binding of target S. typhimurium with aptamer, releasing the primer binding part, leading to anchoring multiple circular templates on the aptasensor surface. Then, the RCA reaction was initiated to produce massive long DNA molecules with multiple tandem-repeat sequences. These RCA products could hybridize with the biotinylated detection probes for enzyme-amplified electrochemical readout on the surface of the aptasensor. The designed electrochemical aptasensor showed very high sensitivity for S. typhimurium down to 16 CFU mL−1 with a wide linear detection range of 20 to 2 × 108 CFU mL−1, and exhibited remarkable signal amplification performance, acceptable reproducibility and low matrix effect. This proposed strategy presented a simple and facile platform towards ultrasensitive and handy bacterial detection, which would become an effective tool for microorganism detection in food safety, clinical diagnostics and even biological threats monitoring.

Application of DNA-based electrochemical biosensor in rapid detection of Escherichia coli exist in licorice decoction

A new method for detection of Escherichia coli exist in licorice decoction was developed by using DNA-based electrochemical biosensor. The thiolated capture probe was immobilized on a gold electrode at first. Then the aptamer for Escherichia coli was combined with the capture probe by hybridization. Due to the stronger interaction between the aptamer and the E. coli, the aptamer can dissociate from the capture probe in the presence of E. coli in licorice decoction. The biotinylated detection probe was hybridized with the single-strand capture probe. As a result, the electrochemical response to Escherichia coli can be measured by using differential pulse voltammetric in the presence of α-naphthyl phosphate. The plot of peak current vs. the logarithm of concentration in the range from 2.7×10² to 2.7×10⁸ CFU·mL⁻¹ displayed a linear relationship with a detection limit of 50 CFU·mL⁻¹. The relative standard deviation of 3 successive scans was 2.5%，2.1%，4.6% for 2×10²，2×10⁴，2×106：⁶ CFU·mL⁻¹ E. coli, respectively. The proposed procedure showed better specificity to E. coli in comparison to Pseudomonas aeruginosa, Staphylococcus aureus and Bacillus subtilis. In the detection of the real extractum glycyrrhizae, the results between the proposed strategy and the GB assay showed high degree of agreement, demonstrating the designed biosensor could be utilized as a powerful tool for microbial examination for traditional Chinese medicine.

Spectrophotometric and Electrochemical Determination of MicroRNA-155 Using Sandwich Hybridization Magnetic Beads

ABSTRACTHere a new colorimetric assay and electrochemical biosensor for microRNA detection based on magnetic bead separation and enzyme amplification are reported. MicroRNA-155 was selected as a model because of its involvement in breast and lung cancer. A novel platform was developed for label-free microRNA detection based on a sandwich assembly using streptavidin magnetic beads coupled with a biotinylated capture probe that hybridized only a portion of the microRNA, the remainder hybridized with biotinylated detection probe linked to streptavidin-alkaline phosphatase. After appropriate reaction with the substrate, the enzymatic product was evaluated with colorimetric and electrochemical detection. The electrochemical method showed high sensitivity, allowing microRNA detection at 29 pmol L−1, which correspond to 1.4 fmole in 50 μL with a linear range from 81 pmol L−1 to 1.2 nmol L−1. This simple and efficient biosensor may have application in clinical molecular diagnostics and biomedical research.

Characterization of paraben substituted cyclotriphosphazenes, and a DNA interaction study with a real-time electrochemical profiling based biosensor

This paper describes an amperometric method for studying DNA-drug candidate interactions. It uses an automatted electrochemical biosensor (MiSens®) based on real-time electrochemical profiling and gold nanoparticles. A biochip was prepared from a 10 x 20 mm silicon dioxide wafer. The biochip surface is modified with a self-assembled monolayer and integrated into the microfluidic system. All the steps of the DNA-drug interaction assay have been performed during fluid flow. Biotinylated surface DNA has been captured on a NeutrAvidin -modified biochip surface. Hybridization of the complementary target sequence and biotinylated detection probe to the surface DNA strand was studied with and without the addition of newly synthesised drug candidates. NeutrAvidin and enzyme modified gold nanoparticles were then injected to bind to the biochip surface. The real-time reading of the amperometric response during the substrate injection results in the biosensor signal. The DNA interaction analysis was exemplarily applied to test the activity of paraben-substituted cyclotriphosphazenes as potential anticancer agents. Two of the synthesised compounds were identified that are capable of inducing DNA damage by 27 and 34%, respectively.

Ultrasensitive electrochemical biosensing strategy for microRNA-21 detection based on homogeneous target-initiated transcription amplification

In this work, we have developed a simple and rapid biosensing method for highly sensitive detection of microRNA-21 (miRNA-21) by integrating homogeneous target-initiated transcription amplification (HTITA) with interfacial sensing process. MiRNA-21, as a target, recognizes and binds with the corresponding site of the multifunction straight probe, which would trigger extension reaction to form T7 promoters in the presence of Klenow fragment (KF exo−) and dNTPs. The remaining sites of multifunction straight probe serve as templates for transcription amplification based on T7 RNA polymerase (T7 pol.). As a result, a great amount of single-stranded RNA products are obtained, which hybridize with the biotinylated detection probes in the solution, forming transcription products. Finally, the resulted transcription products hybridize with the capture probes immobilized on the surface of electrode, producing electrochemical signal read-out. The developed method achieved a detection limit as low as 0.6fM with a dynamic response range from 1fM to 10nM and could discriminate single-nucleotide differences in microRNA (miRNA) sequences. In addition, the established electrochemical strategy was applied to directly detect miRNA-21 in total RNA extracted from HepG2 cells. Thus, this simple and sensitive electrochemical biosensing method might be a potential alternative tool for the quantitative analysis of miRNAs in physiological fluids, biomedical research and point-of-care testing (POCT) in the future.

Ultrasensitive electrochemical biosensor for specific detection of DNA based on molecular beacon mediated circular strand displacement polymerization and hyperbranched rolling circle amplification

Using a cascade signal amplification strategy, an ultrasensitive electrochemical biosensor for specific detection of DNA based on molecular beacon (MB) mediated circular strand displacement polymerization (CSDP) and hyperbranched rolling circle amplification (HRCA) was proposed. The hybridization of MB probe to target DNA resulted in a conformational change of the MB and triggered the CSDP in the presence of bio-primer and Klenow fragment (KF exo−), leading to multiple biotin-tagged DNA duplex. Furthermore, the HRCA was implemented to product amounts of double-stranded DNA (ds-DNA) fragments using phi29 DNA polymerase via biotin-streptavidin interaction. After the product of HRCA binded numerous biotinylated detection probes, an ultrasensitive electrochemical readout by further employing the streptavidin-alkaline phosphatase. The proposed biosensor exhibited excellent detection sensitivity and specificity with a log-linear response to target DNA from 0.01 fM to 10 pM as low as 8.9 aM. The proposed method allowed DNA detection with simplicity, rapidness, low cost and high specificity, which might have the potential for application in clinical molecular diagnostics and environmental monitoring.

Species-specific optical genosensors for the detection of mycotoxigenic Fusarium fungi in food samples

Plant-pathogenic Fusarium species, Fusarium verticillioides and Fusarium proliferatum, are the major producers of fumonisins which are one of the most common mycotoxins found in maize. Herein, we report the development of specific and sensitive genosensors for detecting these two closely related Fusarium species in food samples. The sensors are based on species-specific capture and detection probes, which bind to the intergenic spacer region of rDNA (IGS). Oligonucleotide functionalized magnetic microbeads are used to capture the target DNA which is then detected using biotinylated detection probes and a streptavidin-coupled label. The developed genosensors had detection limits of 1.8 pM and 3.0 pM for F. proliferatum and F. verticillioides, respectively, using synthetic DNA targets. Furthermore, the biosensors were used to analyze natural fungal contamination of commercial maize samples. After amplification of the genomic DNA the sensors detected the presence of the fungi, in accordance with previous results obtained with PCR. No cross-reactivity between F. verticillioides and F. proliferatum, or other fungi species tested, was observed. The developed biosensors can provide a valuable tool to evaluate the potential for mycotoxin contamination in conditions where detection of mycotoxins directly is challenging.

Electrochemical Aptasensor for Rapid and Sensitive Determination of Salmonella Based on Target-Induced Strand Displacement and Gold Nanoparticle Amplification

ABSTRACTA simple, rapid, and sensitive electrochemical aptasensor based on target-induced strand displacement and gold nanoparticle amplification was developed for the determination of Salmonella. The aptamer for Salmonella was captured on the sensing interface by hybridizing with a capture probe. In the presence of Salmonella, the aptamer dissociated from the capture probe as the stronger interaction between the aptamer and the Salmonella. The single-strand capture probe was then hybridized with the biotinylated detection probe assembled on gold nanoparticles and catalyzed by streptavidin–alkaline phosphatase, providing a sensitive electrochemical response. The gold nanoparticles significantly amplified the detection probe signal and increased the sensitivity. The linear dynamic range was from 2 × 101 to 2 × 106 CFU mL−1 with a detection limit of 20 CFU mL−1. This strategy was utilized for the determination of Salmonella in milk, demonstrating potential application in clinical diagnostics, food safety, and environmental monitoring.

A facile and pragmatic electrochemical biosensing strategy for ultrasensitive detection of DNA in real sample based on defective T junction induced transcription amplification.

A novel and pragmatic electrochemical sensing strategy was developed for ultrasensitive and specific detection of nucleic acids by combining with defective T junction induced transcription amplification (DTITA). The homogeneous recognition and specific binding of target DNA with a pair of designed probes formed a defective T junction, further triggered primer extension reaction and in vitro transcription amplification to produce numerous single-stranded RNA. These RNA products of DTITA could hybridized with the biotinylated detection probes and immobilized capture probes for enzyme-amplified electrochemical detection on the surface of the biosensor. The proposed isothermal DTITA strategy displayed remarkable signal amplification performance and reproducibility. The electrochemical DNA biosensor showed very high sensitivity for target DNA with a low detection limit of 0.4 fM (240 molecules of the synthetic DNA), and can directly detect target pathogenic gene of Group B Streptococci (GBS) from as low as 400 copies of genomic DNA. Moreover, the established biosensor was successfully verified for directly identifying GBS in clinical samples. This proposed strategy presented a simple and pragmatic platform toward ultrasensitive and handy nucleic acids detection, and would become a potential tool for general application in point-of-care setting.

An electrochemical biosensor for highly sensitive determination of microRNA based on enzymatic and molecular beacon mediated strand displacement amplification

Abstract In this study, a simple electrochemical biosensor has been established for highly sensitive and specific detection of target microRNA using molecular beacon mediated strand displacement amplification and enzymatic amplification. In the presence of target microRNA, the specific hybridization of microRNA with corresponding domain opens the hairpin structure of a molecular beacon (MB), which triggers strand displacement amplification (SDA) to release nicking DNA triggers. The part of DNA triggers binds to the capture probes immobilized on the gold electrode. Another part of DNA triggers hybridizes with the biotinylated detection probes. The electrochemical signal is obtained by using streptavidin linked to alkaline phosphatase (ST-AP) toward the synthetic enzyme substrate α-naphthyl phosphate (α-NP). Under the optimal conditions, the established biosensor shows high sensitivity and selectivity in a dynamic response range from 50 pM to 10 nM with a detection limit as low as 40 pM (S/N = 3), and good specificity and acceptable reproducibility are achieved for the miRNA-222 detection. This simple and highly efficient biosensor can provide a sensing platform for clinical molecular diagnostics and biomedical research.

A novel chemiluminescence immunoassay for highly sensitive and specific detection of protein using rolling circle amplification and the multiplex binding system

A simple, sensitive and specific chemiluminescence immunoassay (CLIA) strategy has been developed for protein detection by integrating rolling circle amplification (RCA), multiplex binding of the biotin-streptavidin (B-SA) system and enzymatic amplification. The cascade signal amplification methodology is initiated by specifically recognition of target protein based on sandwich immunoassay, which can combine with circular DNA for triggering RCA via biotin-streptavidin. Upon RCA, thousands of repeated sequences are generated for hybridizing with biotinylated detection probes. Then the streptavidin-horseradish peroxidases (ST-HRPs) are bound to biotinylated detection probes, which subsequently catalyze the oxidation of luminol by H2O2 and yield an enhanced chemiluminescence (CL) signal. Taking human prolactin (PRL) as a model, under optimal conditions, the CL intensity was proportional to the logarithm value over six orders from 10fgmL−1 to 10ngmL−1 with a detection limit of 0.16fgmL−1. The established approach was successfully applied for the detection of human PRL in serum samples. Thus, it might be a potential tool for protein detection in clinic biomedical application.

Microfluidics and nanoparticles based amperometric biosensor for the detection of cyanobacteria (Planktothrix agardhii NIVA-CYA 116) DNA.

Some of the cyanobacteria produce protease inhibitor oligopeptides such as cyanopeptolins and cause drinking water contamination; hence, their detection has great importance to monitor the well-being of water sources that is used for human consumption. In the current study, a fast and sensitive nucleic acid biosensor assay has been described where cyanopeptolin coding region of one of the cyanobacteria (Planktothrix agardhii NIVA-CYA 116) genome has been used as target for monitoring of the fresh water resources. A biochip that has two sets of Au electrode arrays, each consist of shared reference/counter electrodes and 3 working electrodes has been used for the assay. The biochip has been integrated to a microfluidics system and all steps of the assay have been performed during the reagent flow to achieve fast and sensitive DNA detection. On-line hybridization of the target on to the capture probe immobilized surface resulted in a very short assay duration with respect to the conventional static assays. The binding of the avidin and enzyme modified Au nanoparticles to the biotinylated detection probe and the subsequent injection of the substrate enabled a real-time amperometric measurement with a detection limit of 6×10(-12) M target DNA (calibration curve r(2)=0.98). The developed assay enables fast and sensitive detection of cyanopeptolin producing cyanobacteria from freshwater samples and hence shows a promising technology for toxic microorganism detection from environmental samples.

Electrochemical aptasensor for highly sensitive determination of cocaine using a supramolecular aptamer and rolling circle amplification

We report on a novel strategy for the electrochemical detection of cocaine. It is based on the use of a supramolecular aptamer, rolling circle amplification (RCA), and multiplex binding of a biotin-strepavidin system. The aptamer fragments were assembled to a supramolecular aptamer which, in the presence of cocaine, conjugates to streptavidin for anchoring of biotinylated circular DNA. This initiates RCA and enables sensitive electrochemical-enzymatic readout. A significant signal amplification was obtained by using streptavidin linked to alkaline phosphatase that binds to the remaining biotinylated detection probes and catalyzes the hydrolysis of the synthetic enzyme substrate α-naphthylphosphate. This dual amplification strategy tremendously increases the detection limit of the aptasensor. Under optimal conditions and using differential pulse voltammetry, cocaine can be detected in the concentration range between 2 and 500 nM with a detection limit as low as 1.3 nM (at S/N = 3). The method is specific and acceptably reproducible. It was successfully applied to the detection of cocaine in (spiked) urine samples. The data were in good agreement with those obtained by the GC-MS reference method.

A novel electrochemical biosensor for ultrasensitive and specific detection of DNA based on molecular beacon mediated circular strand displacement and rolling circle amplification

A novel electrochemical biosensing strategy was developed for ultrasensitive and specific detection of target DNA using a cascade signal amplification based on molecular beacon (MB) mediated circular strand displacement (CSD), rolling circle amplification (RCA), biotin–strepavidin system, and enzymatic amplification. The target DNA hybridized with the loop portion of MB probe immobilized on the gold electrode and triggered the CSD, leading to multiple biotin-tagged DNA duplex. Furthermore, via biotin–streptavidin interaction, the RCA was implemented, producing long massive tandem-repeat DNA sequences for binding numerous biotinylated detection probes. This enabled an ultrasensitive electrochemical readout by further employing the streptavidin-alkaline phosphatase. The proposed biosensor showed very high sensitivity and selectivity with a dynamic response range from 1fM to 100pM. The proposed strategy could have the potential for applying in clinical molecular diagnostics and environmental monitoring.

Target-induced aptamer release strategy based on electrochemical detection of staphylococcal enterotoxin B using GNPs-ZrO2-Chits film

A novel electrochemical aptasensor was developed for ultrasensitive detection of staphylococcal enterotoxin B (SEB) by combining signal amplification and target-induced aptamer release strategy. A gold electrode was modified with a nanocomposite made of gold nanoparticles reduced in situ, zirconia nanoparticles, and chitosan. The SEB aptamer was hybridized by anchoring the capture probe on the modified gold electrode surface through AuS binding. In the presence of SEB, the capture probe-aptamer duplex was compelled to open, releasing the aptamer from the electrode. The resulting single-strand capture probe was hybridized with a biotinylated detection probe and labeled with streptavidin-horseradish peroxidase, producing an ultrasensitive enzyme-catalyzed electrochemical signal. Under optimal conditions, the amperometric responses were proportional to the SEB concentrations ranging from 2 to 512ngmL−1, with a detection limit of as low as 0.24ngmL−1 (S/N=3). The aptasensor exhibited good stability, outstanding reproducibility, and high selectivity. The as-prepared aptasensor was used to analyze SEB in human serum specimens, and validated through enzyme-linked immunosorbent assay method. Analytical results suggest that the developed assay is a promising alternative approach for detecting SEB in clinical diagnosis.

Sensitive detection of enteropathogenic E. coli using a bfpA gene-based electrochemical sensor

We have developed a sensitive assay for enteropathogenic E. coli (EPEC) by integrating DNA extraction, specific polymerase chain reaction (PCR) and DNA detection using an electrode modified with the bundle-forming pilus (bfpA) structural gene. The PCR amplified products are captured on the electrode and hybridized with biotinylated detection probes to form a sandwich hybrid containing two biotinylated detection probes. The sandwich hybridization structure significantly combined the numerous streptavidin alkaline phosphatase on the electrode by biotin-streptavidin connectors. Electrochemical readout is based on dual signal amplification by both the sandwich hybridization structure and the enzyme. The electrode can satisfactorily discriminate complementary and mismatched oligonucleotides. Under optimal conditions, synthetic target DNA can be detected in the 1 pM to 10 nM concentration range, with a detection limit of 0.3 pM. EPEC can be quantified in the 10 to 107 CFU mL−1 levels within 3.5 h. The method also is believed to present a powerful platform for the screening of pathogenic microorganisms in clinical diagnostics, food safety and environmental monitoring.

Direct Detection of Bacterial Genomic DNA at Sub-Femtomolar Concentrations Using Single Molecule Arrays

We report a method for the sensitive measurement of genomic DNA based on the direct detection of single molecules of DNA in arrays of femtoliter wells. The method begins by generating short fragments of DNA from large, double-stranded molecules of genomic DNA using either restriction enzymes or sonication. Single-stranded fragments are then generated by melting the duplex, and these fragments are hybridized to complementary biotinylated detection probes and capture probes on paramagnetic beads. The resulting DNA complexes are then labeled with an enzyme (streptavidin-β-galactosidase), and single enzymes associated with these complexes on beads are detected in single molecule arrays (Simoa). DNA concentration is quantified by determining the average number of enzymes per bead via Poisson statistics (digital) or the average bead intensity (analog). The Simoa DNA assay was used to detect genomic DNA purified from S. aureus with an average limit of detection (LOD) of 0.07 fM, or 2100 DNA molecules per 50 μL sample. We used this assay to detect S. aureus spiked into (a) whole blood, with an average LOD of 1100 bacteria per 25 μL sample (0.074 fM), and (b) water from the Charles River, with an LOD of 1300 bacteria per 50 μL sample (0.042 fM). Bacteria were detected in river water without prior purification of DNA. The Simoa DNA assay, which directly detects target DNA molecules without molecular replication, is an attractive alternative to existing sensitive DNA detection technologies that rely on amplification using polymerases, such as the polymerase chain reaction (PCR).

A simple and sensitive electrochemical aptasensor for determination of Chloramphenicol in honey based on target-induced strand release

A novel electrochemical aptasensor for highly sensitive detection of Chloramphenicol (CAP) in honey was successfully developed based on target-induced strand release (TISR). CAP aptamer was immobilized on electrode and then hybridized with the complementary biotinylated detection probe to form aptamer/DNA duplex. In the presence of CAP, the TISR resulted in the dissociation of biotinylated detection probe from the electrode. Then the binding of streptavidin–alkaline phosphatase (ST–AP) to the remaining biotinylated detection probe led to enzyme-amplified electrochemical signal, which decreased with the increase of CAP concentration. Under optimal conditions, the electrochemical signal was linear with the logarithm of CAP concentrations in the range from 1nM to 1000nM with the detection limit of 0.29nM of CAP. In addition, the designed strategy allowed the direct analysis of real honey samples and the results showed a good agreement with conventional LC–MS/MS method.