Employing Label‐free Electrochemical Biosensor Based on 3D‐Reduced Graphene Oxide and Polyaniline Nanofibers for Ultrasensitive Detection of Breast Cancer BRCA1 Biomarker

A label‐free DNA biosensor based on three‐dimensional reduced graphene oxide (3D‐rGO) and polyaniline (PANI) nanofibers modified glassy carbon electrode (GCE) was successfully developed for supersensitive detection of breast cancer BRCA1. The results demonstrated that 3D‐rGO and PANI nanofibers had synergic effects for reducing the charge transfer resistance (R_ct), meaning a huge enhancement in electrochemical activity of 3D‐rGO‐PANI/GCE. Probe DNA could be immobilized on 3D‐rGO‐PANI/GCE for special and sensitive recognition of target DNA (1.0×10^?15–1.0×10^?7 M) with a theoretical LOD of 3.01×10^?16 M (3S/m). Furthermore, this proposed nano‐biosensor could directly detect BRCA1 in real blood samples.     

Reagentless Aptamer Based Impedance Biosensor for Monitoring Adenosine

A simple and highly sensitive electrochemical impedance spectroscopy (EIS) biosensor based on nano‐MnO₂ as a platform for the immobilization of the aptamer was developed for the determination of adenosine. In the measurement of adenosine, the change in interfacial electron transfer resistance (R_et) of the biosensor using a redox couple of [Fe(CN)₆]^3?/4? as the probe was monitored. The change of the electron transfer resistance (ΔR_et) of the biosensor was linear with the concentration of adenosine in the range from 1.0 nM to 100 nM. The fabricated sensor was shown to exhibit high sensitivity, desirable selectivity and good stability.     

An impedimetric biosensor based on poly(l-lysine)-decorated multiwall carbon nanotubes for the determination of diazinon in water and fruits

A new electrochemical biosensor is developed for the detection of diazinon. For this purpose, a glassy carbon electrode is modified with MWCNTs and poly-l-lysine to immobilize a double-strain DNA (ds-DNA) on the surface of the electrode. In the first step, the interaction of diazinon with ds-DNA is transduced by electrochemical impedance spectroscopy and UV–Vis spectroscopy to monitor the intercalation of diazinon with DNA helix. This interaction leads to reduced interfacial charge-transfer resistance (R_ct). The difference in the R_ct before and after the interaction is considered as a suitable signal for diazinon detection. The proposed biosensor has a low detection limit (0.3 nmol L⁻¹), a wide linear dynamic range (0.001‒100 µmol L⁻¹), and high selectivity for the determination of diazinon. Finally, the performance of the biosensor for detecting of diazinon is verified in real samples such as river water, agricultural wastewater, lettuce juice, and tomato juice.

     

Synthesis,Characterization, and DNA Interaction Studies of the Ruthenium(II) Complexes [Ru(bpy)2(ipbp)]2+ and [Ru(ipbp)(phen)2]2+ (ipbp=3‐(1H‐Imidazo[4,5‐f][1,10]phenanthrolin‐2‐yl)‐4H‐1‐benzopyran‐2‐one; bpy=2,2′‐Bipyridine; phen=1,10‐Phenanthroline)

A novel ligand 3‐(1H‐imidazo[4,5‐f][1,10]phenanthrolin‐2‐yl)‐4H‐1‐benzopyran‐4‐one (ipbp) and its ruthenium(II) complexes [Ru(bpy)₂(ipbp)]²⁺ ( 1 ) and [Ru(ipbp)(phen)₂]²⁺ ( 2 ) (bpy=2,2′‐bipyridine, phen=1,10‐phenanthroline) were synthesized and characterized by elemental analysis and mass, ¹H‐NMR, and electronic‐absorption spectroscopy. The electrochemical behavior of the complexes was studied by cyclic voltammetry. The DNA‐binding behavior of the complexes was investigated by spectroscopic methods and viscosity measurements. The results indicate that complexes 1 and 2 bind with calf‐thymus DNA in an intercalative mode. In addition, 1 and 2 promote cleavage of plasmid pBR 322 DNA from the supercoil form I to the open circular form II upon irradiation.     

Impedimetric DNA‐Based Biosensor for Silver Ions Detection with Hemin/G‐Quadruplex Nanowire as Enhancer

A DNA‐based biosensor was reported for detection of silver ions (Ag⁺) by electrochemical impedance spectroscopy (EIS) with [Fe(CN)₆]^4?/3? as redox probe and hybridization chain reaction (HCR) induced hemin/G‐quadruplex nanowire as enhanced label. In the present of target Ag⁺, Ag⁺ interacted with cytosine‐cytosine (C? C) mismatch to form the stable C? Ag⁺? C complex with the aim of immobilizing the primer DNA on electrode, which thus triggered the HCR to form inert hemin/G‐quadruplex nanowire with an amplified EIS signal. As a result, the DNA biosensor showed a high sensitivity with the concentration range spanning from 0.1 nM to 100 µM and a detection limit of 0.05 nM.     

Direct Detection of DNA and DNA‐Ligand Interaction by Impedance Spectroscopy

In this work we present an impedimetric detection system for DNA‐ligand interactions. The sensor system consists of thiol‐modified single‐stranded DNA chemisorbed to gold. Impedance measurements in the presence of the redox system ferri‐/ferrocyanide show an increase in charge transfer resistance (R_ct) after hybridisation of a complementary target. Different amounts of capture strands, used for gold electrode modification, result in surface coverages between 3 and 15 pmol/cm² ssDNA. The relative change in R_ct upon hybridisation increases with increasing amount of capture probe on the electrode from 1.5‐ to 4.5‐fold. Impedimetric detection of binding events of a metal‐intercalator ([Ru(phen)₃]²⁺) and a groove binder (spermine) to double‐stranded DNA is demonstrated. Binding of [Ru(phen)₃]²⁺ and spermine exhibits a decrease in charge transfer resistance. Here, the ligand’s interaction leads to electrostatic shielding of the negatively charged DNA backbone. The impedance changes have been evaluated in dependence on the concentration of both DNA binders. Furthermore, the association of a single‐stranded binding protein (SSBP) is found to cause an increase in charge transfer resistance only when incubated with single‐stranded DNA. The specific binding of an anti‐dsDNA antibody to the dsDNA‐modified electrode surface decreases in contrast the interfacial impedance.     

DNA Interactions of the Functionalized (Mixed Polypyridine)ruthenium(II) Complex Bis(2,2′‐bipyridine‐κN1,κN1′)(methyl dipyrido[3,2‐a : 2′,3′‐c]phenazine‐11‐carboxylate‐κN4,κN5)ruthenium(2+) ([Ru(bpy)2(dppz‐11‐CO2Me)]2+)

A novel polypyridine ligand, dipyrido[3,2‐a:2′,3′‐c]phenazine‐11‐carboxylic acid methyl ester (=dppz‐11‐CO₂Me), and its ruthenium(II) complex, [Ru(bpy)₂(dppz‐11‐CO₂Me)]²⁺ ( 1 ), were synthesized and characterized. The binding properties of this complex to calf‐thymus DNA (CT‐DNA) were investigated by different spectrophotometric methods and viscosity measurements. The results suggest that the complex binds to DNA in an intercalative mode and serves as a molecular ‘light switch’ for DNA. When irradiated at 365 nm, the complex 1 promoted the photocleavage of plasmid pBR‐322 DNA.     

Investigating Electrochemical Stability and Reliability of Gold Electrode‐electrolyte Systems to Develop Bioelectronic Nose Using Insect Olfactory Receptor

This article aims to demonstrate an electrochemically stable and reliable gold electrode‐electrolyte system to develop an insect odorant receptor (Drosophila melanogaster Or35a) based bioelectronic nose. Cyclic voltammograms (CVs) and electrochemical impedance spectroscopy (EIS) of bare gold electrodes, after modification with the self‐assembled monolayer (SAM) of 6‐mercaptohexanoic acid (MHA) and after immobilization with Or35a integrated into the lipid bilayers of liposomes were conducted in the presence of four different redox probes. Potassium ferri/ferrocyanide [Fe(CN)₆]^3?/[Fe (CN)₆]^4? and hydroquinone (H₂Q) redox probes revealed variable and irreversible signals at the time scale of our measurements, with atomic force microscopy (AFM) images and x‐ray photoelectron spectroscopy (XPS) results suggesting gold surface etching due to the presence of CN^? ions in case of [Fe(CN)₆]^3?/[Fe (CN)₆]^4?. Although the hexaammineruthenium complex showed stable electrochemical behaviour at all stages of biosensor development, changes in CV and EIS readings after each surface modifications were insignificant. PBS buffer as a non‐Faradaic medium, was found to provide reliable systems for electrochemical probing of modified gold electrodes with Or35a/liposomes in aqueous media. Using this system, we have shown that this novel biosensor can detect its known odorant E2‐hexenal selectively compared to methyl salicylate down to femtomolar concentration.     

Impedimetric Biosensor for Bovine Herpesvirus Type 1‐Antigen Detection

The bovine herpesvirus type 1 (BHV‐1) is a pathogen of great economic impact for livestock, which is related multi‐systemic infections that leads to mortality or morbidity of cattles. Thus, the search for cheap and practical methodologies that allow the selective detection of BHV‐1 antigen (BHV‐1 AG) is of utmost relevance. Therefore, an impedimetric label‐free immunosensor was herein, developed and its performance evaluated in biological samples enriched with BHV‐1 AG. Briefly, the biosensor construction was based on the immobilization of BHV‐1 antibody (BHV‐1 AB) and casein on the activated glassy carbon electrode surface. The BHV‐1 AB was isolated from egg yolk of immunized chickens, which is a less stressful protocol. The bio sensing principle was based on Electrochemical Impedance Spectroscopy by using Fe(CN)₆^4?/3? probe, which were also used to check variation of charge transfer resistance (?R_ct), when the electrode surface was increasingly blocked by immune complex. A linear relationship between ?R_ct and BHV‐1 AG concentration was verified in the range from 10 to 50 TCID₅₀ mL^?1, with LOQ of 2.00 TCID₅₀ mL^?1 and LOD of 0.66 TCID₅₀ mL^?1. Besides the suitable sensitivity, the immunosensor displayed accuracy, stability, and specificity to detect BHV‐1 AG in biological samples of serum, nasal secretions, semen and urine. Moreover, to the best of our knowledge this is the first immunosensor applied to BHV‐1 diagnostic.     

A Dendrimer Supported Electrochemical Immunosensor for the Detection of Alpha‐feto protein – a Cancer Biomarker

The electrochemical detection of alpha‐feto protein based on novel gold nanoparticles‐ poly(propylene imine) dendrimer platform is reported. The platform was prepared by co‐electrodeposition of gold nanoparticles and generation 3 poly (propylene imine) dendrimer on a glassy carbon electrode. Each modifying step was characterised by cyclic voltammetry and electrochemical impedance spectroscopy. The electrochemical measurements showed that the platform was stable, conducting and exhibited reversible electrochemistry. Results obtained from the electrochemical impedance spectroscopy interrogation in [Fe(CN)₆^3−/4−] redox probe showed a marked reduction in charge transfer resistance (R_ct) after each modification step. The immunosensor was prepared by immobilisation of a probe anti‐alpha feto protein (AFP) on the platform for 3 hrs at 35 °C followed by blocking the surface with bovine serum albumin to minimise non‐specific binding. The prepared immunosensor was used to detect AFP over a wide concentration range from 0.005 to 500 ng/mL and detection limits of 0.0022 and 0.00185 ng/mL were obtained for SWV and EIS measurements respectively. The immunosensor gave good stability over a period of fourteen days when stored at 4 °C.     

Carbon Nanotubes‐Based Amperometric Cholesterol Biosensor Fabricated Through Layer‐by‐Layer Technique

A carbon nanotubes‐based amperometric cholesterol biosensor has been fabricated through layer‐by‐layer (LBL) deposition of a cationic polyelectrolyte (PDDA, poly(diallyldimethylammonium chloride)) and cholesterol oxidase (ChOx) on multi‐walled carbon nanotubes (MWNTs)‐modified gold electrode, followed by electrochemical generation of a nonconducting poly(o‐phenylenediamine) (PPD) film as the protective coating. Electrochemical impedance measurements have shown that PDDA/ChOx multilayer film could be formed uniformly on MWNTs‐modified gold electrode. Due to the strong electrocatalytic properties of MWNTs toward H₂O₂ and the low permeability of PPD film for electroacitve species, such as ascorbic acid, uric acid and acetaminophen, the biosensor has shown high sensitivity and good anti‐interferent ability in the detection of cholesterol. The effect of the pH value of the detection solution on the response of the biosensor was also investigated. A linear range up to 6.0 mM has been observed for the biosensor with a detection limit of 0.2 mM. The apparent Michaelis‐Menten constant and the maximum response current density were calculated to be 7.17 mM and 7.32 μA cm^?2, respectively.     

A Peptide Nucleic Acid (PNA)‐DNA Ferrocenyl Intercalator for Electrochemical Sensing

A ferrocenyl intercalator was investigated to develop an electrochemical DNA biosensor employing a peptide nucleic acid (PNA) sequence as capture probe. After hybridization with single strand DNA sequence, a naphthalene diimide intercalator bearing ferrocene moieties (FND) was introduced to bind with the PNA‐DNA duplex and the electrochemical signal of the ferrocene molecules was used to monitor the DNA recognition. Electrochemical impedance spectroscopy was used to characterize the different modification steps. Differential pulse voltammetry was employed to evaluate the electrochemical signal of the FND intercalator related to its interaction with the complementary PNA‐DNA hybrid. The ferrocene oxidation peaks were utilised for the target DNA quantification. The developed biosensor demonstrated a good linear dependence of FND oxidation peak on DNA concentration in the range 1 fM to 100 nM of target DNA, with a low detection limit of 11.68 fM. Selectivity tests were also investigated with a non‐complementary DNA sequence, indicating that the FND intercalator exhibits a selective response to the target PNA‐DNA duplex.     

Disposable Amperometric Biosensor Based on Poly‐L‐lysine and Fe3O4 NPs‐chitosan Composite for the Detection of Tyramine in Cheese

An amperometric tyramine biosensor based on poly‐L‐lysine (PLL) and Fe₃O₄ nanoparticles (Fe₃O₄NP) modified screen printed carbon electrode (SPCE) was developed. PLL was formed on the SPCE by the electropolymerization of L‐lysine. Subsequently, Fe₃O₄NP suspension prepared in chitosan (CH) solution was casted onto the PLL/SPCE. Tyrosinase (Ty) enzyme was immobilized onto the modified Fe₃O₄?CH/PLL/SPCE and the electrode was coated with Nafion to fabricate the Ty/Fe₃O₄?CH/PLL/SPCE. Different techniques including scanning electron microscopy, chronoamperometry (i–t curve), cyclic voltammetry and electrochemical impedance spectroscopy were utilized to study the fabrication processes, electrochemical characteristics and performance parameters of the biosensor. The analytical performance of the tyramine biosensor was evaluated with respect to linear range, sensitivity, limit of detection, repeatability and reproducibility. The response of the biosensor to tyramine was linear between 4.9×10^?7–6.3×10^?5 M with a detection limit of 7.5×10^?8 M and sensitivity of 71.36 μA mM^?1 (595 μA mM^?1 cm^?2). The application of the developed biosensor for the determination of tyramine was successfully tested in cheese sample and mean analytical recovery of added tyramine in cheese extract was calculated as 101.2±2.1 %. The presented tyramine biosensor is a promising approach for tyramine analysis in real samples due to its high sensitivity, rapid response and easy fabrication.     

A Cole‐Cole Dielectric Relaxation Analysis of Albumin and γ‐Globulins for Protein Quantification by Electrical Impedance Spectroscopy

Optimal serum protein concentrations are vital for normal body functioning. Affordable while accurate protein quantification methods with minimum processing requirements are needed for diagnosis of related diseases. The standard automated chemistry analyzer is limited by high installation and maintenance costs. This study proposes the use of electrical impedimetric spectroscopy (EIS) as an alternative to current methods. Its practical applicability was tested using albumin and γ‐globulin or their miscellanea in three different media; water, serum and tissue‐mimicking phantoms at 25 °C. Impedance measurements were taken between frequency f=0.10 MHz to 300 MHz by an impedance analyzer. A Cole‐Cole analysis was used to elucidate the stepwise variations in the dielectric parameters of the protein medium so as to obtain empirical dielectric parameter‐protein concentration relationships and their correlation coefficients R². From the results, linear relationships between parameters and protein concentrations with high correlation coefficients over R²=0.90 were observed. Resistance to charge transfer R_ct and characteristic frequency f_c were significantly altered by changing protein concentrations as compared to bulk solution resistance R_s, relaxation time constant τ and shape factor α. The relationships developed would aid in monitoring changes in body fluid protein concentrations by EIS.     

Electrochemical Study of Schiff Bases by Means of Self‐Assembled Monolayers

In this paper, Schiffbases were investigated using cyclic voltammetry (CV) and impedance electrochemical spectroscopy (EIS) techniques by means of self‐assembled monolayers for the first time, where a 0.1 M KCl solution and the redox couple of Fe(CN)₆^3?/Fe(CN)₆^4?were used as the electrolyte and probing‐pin, respectively. The monolayers formed by the employed Schiff base were proved to be relatively stable, and its electrochemical response in the studied system with different pH values was also de scribed clearly with CV and EIS plots. The results show that the monolayer of Schiff bases could exist in the solution with pH value from 2 to 10. In the EIS measurement in the concentration range from 10^?5 M to 5× 10^?4 M, a nearly linear relation ship between the charge transfer resistance (R_ct) and the logarithm concentration of Cu²+was observed, suggesting that Cu²+ could be titrated with the EIS method quasi‐quantitatively. The phenomenon agreed with the former report very well. Using the self‐assembled monolayers to study Schiff bases with the electrochemical method is the major contribution of our work.     

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.     

Noncovalent Assembly of Picket‐Fence Porphyrins on Nitrogen‐Doped Carbon Nanotubes for Highly Efficient Catalysis and Biosensing

A water‐insoluble picket‐fence porphyrin was first assembled on nitrogen‐doped multiwalled carbon nanotubes (CN_x MWNTs) through Fe? N coordination for highly efficient catalysis and biosensing. Scanning electron micrographs, Raman spectra, X‐ray photoelectron spectra, UV/Vis absorption spectra, and electrochemical impedance spectra were employed to characterize this novel nanocomposite. By using electrochemical methods on the porphyrin at low potential in neutral aqueous solution, the presence of CN_x MWNTs led to the direct formation of a high‐valent iron(IV)–porphyrin unit, which produced excellent catalytic activity toward the oxidation of sulfite ions. By using sulfite ions, a widely used versatile additive and preservative in the food and beverage industries, as a model, a highly sensitive amperometric biosensor was proposed. The biosensor showed a linear range of four orders of magnitude from 8.0×10^?7 to 4.9×10^?3 mol L^?1 and a detection limit of 3.5×10^?7 mol L^?1 due to the highly efficient catalysis of the nanocomposite. The designed platform and method had good analytical performance and could be successfully applied in the determination of sulfite ions in beverages. The direct noncovalent assembly of porphyrin on CN_x MWNTs provided a facile way to design novel biofunctional materials for biosensing and photovoltaic devices.     

Design of Electrochemically Modified fMWCNT‐pencil Graphite Electrode Decorated with Cu and Ag Nanofilm and its Electrocatalytic Behavior Towards Imazethapyr

Herein, a facile procedure was developed for designing an electrochemical sensor based on pencil graphite electrode modified with electrochemically synthesized silver and copper nanoparticles (AgNP and CuNP) supported on functionalized multiwalled carbon nanotubes (f MWCNTs). The electrochemical and morphological characterization was carried out by cyclic voltammetry, Electrochemical Impedance Spectroscopy, Powder X‐ray diffraction, Field Emission Scanning Electron Microscopy, Transmission electron microscopy and Atomic Force Microscopy. The designed sensor exhibited electrocatalytic behavior towards the reduction of Imazethapyr. Results indicates the combination of AgNPs, CuNPs and f MWCNTs on PGE produced remarkable enhancement in electrocatalytic and sensing properties. Various electro‐kinetic parameters like R_ct, k_app, n, α, E⁰, k⁰, Γ, D and k have been evaluated by CV, impedance and Chronoamperometric studies. The electrochemical performance was improved by optimizing the effect of pH, scan rate, amount of f MWCNTs and deposition parameters of AgNP and CuNP. The sensor was efficaciously applied for determination of Imazethapyr and exhibited a linear correlation in the concentration range of 0.01–5.0 μg mL⁻¹ with low detection limits, 0.159 ng mL⁻¹ using AdSWV. The fabricated sensor exhibited good accuracy, acceptable stability and high efficacy for quantitative determination of Imazethapyr in real samples with notable recoveries ranging from 98 % to 100.2 %.     

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.     

An Electrochemical Impedance Immunosensor Based on Gold Nanoparticle‐Modified Electrodes for the Detection of HbA1c in Human Blood

A label‐free immunosensor for the detection of HbA1c was developed based on gold nanoparticle (AuNP)‐aryl diazonium salt modified glassy carbon (GC) electrode where transduction is achieved using electrochemical impedance spectroscopy (EIS). GC electrodes were first modified with 4‐aminophenyl (Ph‐NH₂) layers to which AuNPs were attached. Thereafter an oligo(ethylene glycol) (OEG‐COOH) species were covalently attached to the remaining free amine groups on the Ph‐NH₂ surface. The AuNP surfaces were further modified with Ph‐NH₂ followed by attachment of a glycosylated pentapeptide (GPP), an analogon to HbA1c. Exposure of this interface to anti‐HbA1c IgG resulted in a change in charge transfer resistance (R_ct) due to the anti‐HbA1c IgG selectively complexing to the surface bound GPP. To detect the amount of HbA1c, a competitive inhibition assay was employed where the surface bound GPP and HbA1c in solution compete for the anti‐HbA1c IgG antibodies. The higher the concentration of HbA1c, the less antibody binds to the sensing interface and the lower the change of R_ct. The response of the immunosensor is linear with the HbA1c% of total haemoglobin in the range of 0%–23.3%. This competitive inhibition assay can be used for the detection of HbA1c in human blood. The performance of the immunosensor for detection of HbA1c in human blood is comparable to the clinical laboratory method.