Voltammetric Detection of a Specific DNA Sequence of Avian Influenza Virus H5N1 Using HS‐ssDNA Probe Deposited onto Gold Electrode

The working principle of a genosensor is based on the mechanism of ion‐channel mimetic sensors. The analytical signals generated upon hybridization processes were recorded by a redox active marker [Fe(CN)₆]^3?/4? present in the sample solution using voltammetric techniques. The developed genosensor was suitable for determination of 20‐mer complementary oligonucleotide sequence, and also of the PCR products containing the complementary 20‐mer sequence in various positions, with detection limits in the 10 pM range. The noncomplementary 20‐mer oligonucleotide sequence as well as the PCR product without complementary region generated very weak response. The good discrimination of the position of the complementary part in the PCR products was observed.     

Indigo Carmine as New Label in PNA Biosensor for Detection of Short Sequence of p53 Tumor Suppressor Gene

A new electrochemical PNA hybridization biosensor for detection of a 15‐mer sequence unique to p53 using indigo carmine (IC) as an electrochemical detector is described in this work. This genosensor is based on the hybridization of target oligonucleotide with its complementary probe immobilized on the gold electrode by self‐assembled monolayer formation. Because this label is electroactive in acidic medium, the interaction between IC and short sequence of p53 is studied by differential pulse voltammety (DPV) in 0.1 M H₂SO₄. The results of electrochemical impedance spectroscopy and cyclic voltammetry in the solution of [Fe(CN)₆]^3?/4? shows no breakage in PNA‐DNA duplex. A decrease in the voltammetric peak currents of IC is observed upon hybridization of the probe with the target DNA. The influence of probe concentration on effective discrimination against non‐complementary oligonucleotides is investigated and a concentration of 10^?7 M is selected. The diagnostic performance of the PNA sensor is described and the detection limit is found to be 4.31×10^?12 M.     

Tethered thiazole orange intercalating dye for development of fibre-optic nucleic acid biosensors

Single-stranded DNA (ssDNA) oligonucleotide in solution, or that is immobilized onto a surface to create a biosensor, can be used as a selective probe to bind to a complementary single-stranded sequence. Fluorescence enhancement of thiazole orange (TO) occurs when the dye intercalates into double-stranded DNA (dsDNA). TO dye has been covalently attached to probe oligonucleotides (homopolymer and mixed base 10mer and 20mer) through the 5′ terminal phosphate group using polyethylene glycol linker. The tethered TO dye was able to intercalate when dsDNA formed in solution, and also at fused silica surfaces using immobilized ssDNA. The results indicated the potential for development of a self-contained biosensor where the fluorescent label was available as part of the immobilized oligonucleotide probe chemistry. The approach was shown to be able to operate in a reversible manner for multiple cycles of detection of targeted DNA sequences.     

Rapid and sensitive DNA target detection using enzyme amplified electrochemical detection based on microchip

A rapid and sensitive DNA targets detection using enzyme amplified electrochemical detection (ED) based on microchip was described. We employed a biotin‐modified DNA, which reacted with avidin‐conjugated horseradish peroxidase (avidin–HRP) to obtain the HRP‐labeled DNA probe and hybridized with its complementary target. After hybridization, the mixture containing dsDNA‐HRP, excess ssDNA‐HRP, and remaining avidin–HRP was separated by MCE. The separations were performed at a separation voltage of +1.6 kV and were completed in less than 100 s. The HRP was used as catalytic labels to catalyze H₂O₂/o‐aminophenol reaction. Target DNA could be detected by the HRP‐catalyzed reduction with ED. With this protocol, the limits of quantification for the hybridization assay of 21‐ and 39‐mer DNA fragments were of 8×10^?12 M and 1.2×10^?11 M, respectively. The proposed method has been applied satisfactorily in the analysis of Escherichia coli genomic DNA. We selected the detection of PCR amplifications from the gene of E. coli to test the real applicability of our method. By using an asymmetric PCR protocol, we obtained ssDNA targets of 148 bp that could be directly hybridized by the single‐stranded probe and detected with ED.     

Impedance‐Based DNA Biosensor Employing Molecular Beacon DNA as Probe and Thionine as Charge Neutralizer

In this paper, we present an impedance‐based DNA biosensor using thionine intercalation to amplify DNA hybridization signal. Beacon single‐stranded DNA (ssDNA) probe and mercaptoacetic acid were self‐assembled onto a Au electrode by forming Au? S bonds. These beacon ssDNAs were hybridized with the complementary sequences around the loop structure. Then thionine was intercalated into the double‐stranded DNA (dsDNA) immobilized on the Au electrode surface. Due to the neutralization of the negative charges of dsDNA by the intercalated thionine, the electronic transfer resistance (R_et) of the DNA modified Au electrode was significantly diminished. Herein, the decreased value of R_et resulted from the thionine intercalating into dsDNA was employed as the hybridization signal. SDS was used to reduce the unspecific adsorption between ssDNA and thionine. Several experimental conditions, including the surface coverage of ssDNA probe on Au electrode, the hybridization temperature and time were all optimized. Moreover, the hybridization reactions of the unstructured linear ssDNA probe and the structured beacon ssDNA probe with their complementary sequences were compared in this work. The sensitivity of the presented DNA biosensor highlighted that the intercalation of thionine into dsDNA was an efficient approach to amplify the hybridization signal using impedance detection technique. Additionally, in this DNA biosensing protocol, beacon ssDNA has a good ability to distinguish target DNA sequences. This results in a higher specificity than using traditional unstructured DNA probe.     

Electrochemical DNA Biosensor Based on Partially Reduced Graphene Oxide Modified Carbon Ionic Liquid Electrode for the Detection of Transgenic Soybean A2704‐12 Gene Sequence

In this work a partially reduced graphene oxide (p‐RGO) modified carbon ionic liquid electrode (CILE) was prepared as the platform to fabricate an electrochemical DNA sensor, which was used for the sensitive detection of target ssDNA sequence related to transgenic soybean A2704‐12 sequence. The CILE was fabricated by using 1‐butylpyridinium hexafluorophosphate as the binder and then p‐RGO was deposited on the surface of CILE by controlling the electroreduction conditions. NH₂ modified ssDNA probe sequences were immobilized on the electrode surface via covalent bonds between the unreduced oxygen groups on the p‐RGO surface and the amine group at the 5′‐end of ssDNA, which was denoted as ssDNA/p‐RGO/CILE and further used to hybridize with the target ssDNA sequence. Methylene blue (MB) was used as electrochemical indicator to monitor the DNA hybridization. The reduction peak current of MB after hybridization was proportional to the concentration of target A2704‐12 ssDNA sequences in the range from 1.0×10^?12 to 1.0×10^?6 mol/L with a detection limit of 2.9×10^?13 mol/L (3σ). The electrochemical DNA biosensor was further used for the detection of PCR products of transgenic soybean with satisfactory results.     

Nano‐Gold Modified Genosensor for Direct Detection of DNA Hybridization

In this paper, nano‐gold modified carbon paste electrode (NGMCPE) was employed to develop an electrochemical DNA hybridization biosensor. The proposed sensor was made up by immobilization of 15‐mer single stranded oligonucleotide probe for detection of target DNA. Hybridization detection relies on the alternation in guanine oxidation signal following hybridization of the probe with complementary genomic DNA. The guanine oxidation was monitored using differential pulse voltammetry (DPV). Different factors such as activation potential, activation time and probe immobilization conditions were optimized. The selectivity of the sensor was investigated by non‐complementary oligonucleotides. Diagnostic performance of the biosensor was described and the detection limit was found 1.9 × 10^?13 M at the NGMCPE surface. All of the investigations were performed in both CPE and NGMCPE and finally their results were compared.     

Low‐Background,Highly Sensitive DNA Biosensor by Using an Electrically Neutral Cobalt(II) Complex as the Redox Hybridization Indicator

An electrically neutral cobalt complex, [Co(GA)₂(phen)] (GA=glycollic acid, phen=1,10‐phenathroline), was synthesized and its interactions with double‐stranded DNA (dsDNA) were studied by using electrochemical methods on a glassy carbon electrode (GCE). We found that [Co(GA)₂(phen)] could intercalate into the DNA duplex through the planar phen ligand with a high binding constant of 6.2(±0.2)×10⁵ M ^?1. Surface studies showed that the cobalt complex could electrochemically accumulate within the modified dsDNA layer, rather than within the single‐stranded DNA (ssDNA) layer. Based on this feature, the complex was applied as a redox‐active hybridization indicator to detect 18‐base oligonucleotides from the CaMV35S promoter gene. This biosensor presented a very low background signal during hybridization detection and could realize the detection over a wide kinetic range from 1.0×10^?14 M to 1.0×10^?8 M , with a low detection limit of 2.0 fM towards the target sequences. The hybridization selectivity experiments further revealed that the complementary sequence, the one‐base‐mismatched sequence, and the non‐complementary sequence could be well‐distinguished by the cobalt‐complex‐based biosensor.     

Rolling‐Circle Amplification Detection of Thrombin Using Surface‐Enhanced Raman Spectroscopy with Core‐Shell Nanoparticle Probe

An ultrasensitive surface‐enhanced Raman spectroscopy (SERS) sensor based on rolling‐circle amplification (RCA)‐increased “hot‐spot” was developed for the detection of thrombin. The sensor contains a SERS gold nanoparticle@Raman label@SiO₂ core‐shell nanoparticle probe in which the Raman reporter molecules are sandwiched between a gold nanoparticle core and a thin silica shell by a layer‐by‐layer method. Thrombin aptamer sequences were immobilized onto the magnetic beads (MBs) through hybridization with their complementary strand. In the presence of thrombin, the aptamer sequence was released; this allowed the remaining single‐stranded DNA (ssDNA) to act as primer and initiate in situ RCA reaction to produce long ssDNAs. Then, a large number of SERS probes were attached on the long ssDNA templates, causing thousands of SERS probes to be involved in each biomolecular recognition event. This SERS method achieved the detection of thrombin in the range from 1.0×10^?12 to 1.0×10^?8 M and a detection limit of 4.2×10^?13 M , and showed good performance in real serum samples.     

Developing a Nano‐Biosensor for DNA Hybridization Using a New Electroactive Label

Development of electrochemical DNA hybridization biosensors based on carbon paste electrode (CPE) and gold nanoparticle modified carbon paste electrode (NGMCPE) as transducers and ethyl green (EG) as a new electroactive label is described. Electrochemical impedance spectroscopy and cyclic voltammetry techniques were applied for the investigation and comparison of bare CPE and NGMCPE surfaces. Our voltammetric and spectroscopic studies showed gold nanoparticles are enable to facilitate electron transfer between the accumulated label on DNA probe modified electrode and electrode surface and enhance the electrical signals and lead to an improved detection limit. The immobilization of a 15‐mer single strand oligonucleotide probe on the working electrodes and hybridization event between the probe and its complementary sequence as a target were investigated by differential pulse voltammetry (DPV) responses of the EG accumulated on the electrodes. The effects of some experimental variables on the performance of the biosensors were investigated and optimum conditions were suggested. The selectivity of the biosensors was studied using some non‐complementary oligonucleotides. Finally the detection limits were calculated as 1.35×10^?10 mol/L and 5.16×10^?11 mol/L on the CPE and NEGCPE, respectively. In addition, the biosensors exhibited a good selectivity, reproducibility and stability for the determination of DNA sequences.     

Detection of Aeromonas hydrophila DNA oligonucleotide sequence using a biosensor design based on Ceria nanoparticles decorated reduced graphene oxide and Fast Fourier transform square wave voltammetry

A new strategy was introduced for ssDNA immobilization on a modified glassy carbon electrode. The electrode surface was modified using polyaniline and chemically reduced graphene oxide decorated cerium oxide nanoparticles (CeO₂NPs-RGO). A single-stranded DNA (ssDNA) probe was immobilized on the modified electrode surface. Fast Fourier transform square wave voltammetry (FFT-SWV) was applied as detection technique and [Ru(bpy)₃]^2+/3+ redox signal was used as electrochemical marker. The hybridization of ssDNA with its complementary target caused a dramatic decrease in [Ru(bpy)₃]^2+/3+ FFT-SW signal. The proposed electrochemical biosensor was able to detect Aeromonas hydrophila DNA oligonucleotide sequence encoding aerolysin protein. Under optimal conditions, the biosensor showed excellent selectivity toward complementary sequence in comparison with noncomplementary and two-base mismatch sequences. The dynamic linear range of this electrochemical DNA biosensor for detecting 20-mer oligonucleotide sequence of A. hydrophila was from 1 × 10⁻¹⁵ to 1 × 10⁻⁸ mol L⁻¹. The proposed biosensor was successfully applied for the detection of DNA extracted from A. hydrophila in fish pond water up to 0.01 μg mL⁻¹ with RSD of 5%. Besides, molecular docking was applied to consider the [Ru(bpy)₃]^2+/3+ interaction with ssDNA before and after hybridization.     

Nanostructured zirconium oxide based genosensor for Escherichia coli detection

A deoxyribonucleic acid (DNA) biosensor has been fabricated via immobilization of 17 base terminal single stranded DNA (ssDNA) identified from the 16s rRNA coding region of Escherichia coli onto sol–gel derived nanostructured zirconium oxide (NanoZrO₂) film. An oligonucleotide probe with a terminal 5′-phosphate group has been attached to the surface of the electrode via affinity of NanoZrO₂ for phosphate. The results of hybridization studies carried out with the complementary, non-complementary and genomic DNA reveal that ssDNA/NanoZrO₂/ITO bioelectrode has a high selectivity and sensitivity towards hybridization detection with limits of 10^?6–10⁶ pM of complementary DNA.     

A low-cost surface plasmon resonance instrument based on detection of resonance excitation wavelength

A laboratory-made surface plasmon resonance (SPR) instrument based on the detection of resonance excitation wavelength has been successfully fabricated. The performance and workability of the SPR instrument was demonstrated as a DNA biosensor. Biotinylated single-stranded oligonucleotides (ssDNA) were chemically immobilized on a gold-film surface of the SPR instrument as a DNA probe for the detection of its fully complementary, half-complementary and non-complementary ssDNA. The immobilization of the ssDNA probe was done by avidin-biotin linkage. The ssDNA used were 12-mer oligonucleotides. The sensing mechanism was based on the shift in resonance wavelength of an excitation light beam as the target ssDNA hybridized with the ssDNA on the gold-film surface. The linear dynamic ranges of the DNA biosensor for fully complementary and half-complementary ssDNA are 0.04-1.2 pM and 0.08-1.1 pM, respectively. The DNA biosensor showed higher sensitivity to fully complementary ssDNA than to half-complementary ssDNA. But no shift of resonance wavelength to the non-complementary ssDNA was observed.     

Lable‐Free Electrochemical DNA Sensor Based on Gold Nanoparticles/Poly(neutral red) Modified Electrode

We present a new strategy for the label‐free electrochemical detection of DNA hybridization based on gold nanoparticles (AuNPs)/poly(neutral red) (PNR) modified electrode. Probe oligonucledotides with thiol groups at the 5‐end were covalently linked onto the surface of AuNPs/PNR modified electrode via S‐Au binding. The hybridization event was monitored by using differential pulse voltammetry (DPV) upon hybridization generates electrochemical changes at the PNR‐solution interface. A significant decrease in the peak current was observed upon hybridization of probe with complementary target ssDNA, whereas no obvious change was observed with noncomplementary target ssDNA. And the DNA sensor also showed a high selectivity for detecting one‐mismatched and three‐mismatched target ssDNA and a high sensitivity for detecting complementary target ssDNA, the detection limit is 4.2×10^?12 M for complementary target ssDNA. In addition, the DNA biosensor showed an excellent reproducibility and stability under the DNA‐hybridization conditions.     

rmpM Genosensor for Detection of Human Brain Bacterial Meningitis in Cerebrospinal Fluid

Human brain bacterial meningitis is a life-threatening disease caused mainly by Neisseria meningitidis, lead to damage of the outer membrane covering (meninges) of brain or even death. The usual methods of diagnosis are either time-consuming or have some limitations. The specific rmpM (reduction-modifiable protein M) virulent gene based genosensor is more sensitive, specific, and can detect N. meningitidis directly from the patient cerebrospinal fluid in 30 min including 1-min response time. 5′-Thiol-labeled single-stranded DNA (ssDNA) probe was immobilized onto screen-printed gold electrode (SPGE) and hybridized with denatured (95 °C) single-stranded genomic DNA (ssG-DNA) for 10 min at 25 °C. The electrochemical response was measured by cyclic voltammetry, differential pulse voltammetry (DPV) and electrochemical impedance using redox indicators. The sensitivity of the genosensor was 9.5087?(μA/cm²)/ng with DPV and limit of detection was 3 ng/6 μL ssG-DNA. The immobilization of the ssDNA probe and hybridization with ssG-DNA from N. meningitidis was characterized by atomic force microscopy and Fourier transform infrared spectroscopy. The rmpM genosensor was stable for 6 months at 4 °C with 10 % loss in initial DPV current. The advantage of rmpM genosensor is to detect bacterial meningitis simultaneously in multiple patients using SPGE array during an outbreak of the disease.     

Colloid Au-enhanced DNA immobilization for the electrochemical detection of sequence-specific DNA

We use colloidal Au to enhance the DNA immobilization amount on a gold electrode and ultimately lower the detection limit of our electrochemical DNA biosensor. Self-assembly of approximately 16-nm diameter colloidal Au onto a cysteamine modified gold electrode resulted in an easier attachment of an oligonucleotide with a mercaptohexyl group at the 5′-phosphate end, and therefore an increased capacity for nucleic acid detection. Quantitative results showed that the surface densities of oligonucleotides on the Au colloid modified gold electrode were approximately (1–4)×10¹⁴ molecules cm⁻². Hybridization was induced by exposure of the ssDNA-containing gold electrode to ferrocenecarboxaldehyde labeled complementary ssDNA in solution. The detection limit is 5×10⁻¹⁰ mol l⁻¹ of complementary ssDNA, which is much lower than our previous electrochemical DNA biosensors. The Au nanoparticle films on the Au electrode provide a novel means for ssDNA immobilization and sequence-specific DNA detection.     

DNA Hybridization at Magnetic Nanoparticles with Electrochemical Stripping Detection

A simple and practical method for electrochemical DNA hybridization assay has been developed to take advantage of magnetic nanoparticles for ssDNA immobilization and zinc sulfide nanoparticle as oligonucleotide label. Magnetic nanoparticles were prepared by coprecipitation of Fe²⁺ and Fe³⁺ with NH₄OH, and then amino silane was coated onto the surface of magnetite nanoparticles. The magnetic nanoparticles have the advantages of easy preparation, easy surface modification and low cost. The target ssDNA with the phosphate group at the 5′ end was then covalently immobilized to the amino group of magnetite nanoparticles by forming a phosphoramidate bond in the presence of 1‐ethyl‐3‐(3‐dimeth‐ylaminopropyl)carbodiimide (EDAC). The zinc sulfide (ZnS) nanoparticle‐labeled oligonucleotides probe was used to identify the target ssDNA immobilized on the magnetic nanoparticles based on a specific hybridization reaction. The hybridization events were assessed by the dissolution of the zinc sulfide nanoparticles anchored on the hybrids and the indirect determination of the dissolved zinc ions by anodic stripping voltammetry (ASV) at a mercury film glassy carbon electrode (GCE). The proposed method couples the high sensitivity of anodic stripping analysis for zinc ions with effective magnetic separation for eliminating nonspecific adsorption effects and offers great promise for DNA hybridization analysis.     

Direct selective detection of genomic DNA from coliform using a fiber optic biosensor

Fiber optic biosensors operated in a total internal reflection format were prepared based on covalent immobilization of 25mer lacZ single-stranded nucleic acid probe. Genomic DNA from Escherichia coli was extracted and then sheared by sonication to prepare fragments of approximately 300mer length. Other targets included a 25mer fully complementary lacZ sequence, 100mer polymerase chain reaction (PCR) products containing the lacZ sequence at various locations, and non-complementary DNA including genomic samples from salmon sperm. Non-selective adsorption of non-complementary oligonucleotides (ncDNA) was found to occur at a significantly faster rate than hybridization of complementary oligomers (cDNA) in all cases. The presence of ncDNA oligonucleotides did not inhibit selective interactions between immobilized DNA and cDNA in solution. The presence of high concentrations of non-complementary genomic DNA had little effect on extent or speed of hybridization of complementary oligonucleotides. Detection of genomic fragments containing the lacZ sequence was possible in as little as 20 s by observation of the steady-state fluorescence intensity increase or by time-dependent rate of fluorescence intensity changes.     

Modeling of DNA hybridization detection using methylene blue as an electroactive label

Mathematical modeling of methylene blue (MB) signal in ssDNA and dsDNA on pencil graphite electrode is described. A DNA biosensor was developed based on MB signal. The probe and target DNAs were 20 mer oligonucleotides corresponding to consensus sequence of HPV major capsid protein L1 gene. Hybrids of various complementary and non-complementary oligonucleotides with the probe were considered as dsDNA with different hybridization degrees. Modeling was developed by incorporation of only the stable forms of dsDNA hybrids. Effect of hybridization degree on current signal in various forms was studied. A factor named AHP (Average Hybridization Percentage) for verifying the hybridization events was defined. Results showed that there is a significant mathematical relation between the calculated AHP and MB signals.     

Duplex‐forming Oligonucleotide of Triazole‐linked RNA

An oligonucleotide of triazole‐linked RNA (^TLRNA) was synthesized by performing consecutive copper‐catalyzed azide‐alkyne cycloaddition reactions for elongation. The reaction conditions that had been optimized for the synthesis of 3‐mer ^TLRNA were found to be inappropriate for longer oligonucleotides, and the conditions were reoptimized for the solid‐phase synthesis of an 11‐mer ^TLRNA oligonucleotide. Duplex formation of the 11‐mer ^TLRNA oligonucleotide was examined with the complementary oligonucleotide of natural RNA to reveal the effects of the 2′‐OH groups on the duplex stability.