On-chip type cation-exchange chromatography with ferrocene-labeled anti-hemoglobin antibody and electrochemical detector for determination of hemoglobin A1c level

An on-chip type cation-exchange chromatography system with electrochemical detection of HbA_1c, which is one of the most important diabetes marker protein, was developed using ferrocene-conjugated anti-human hemoglobin (Hb) monoclonal antibody (FcAb). The FcAb was used as an electrochemical probe for the detection of each Hb. The system contains syringe pump, on-chip type cation-exchange column consisted of PDMS and cation-exchange resin beads, and a three-electrode flow-cell system. The separation conditions of HbA_1c in blood calibrator samples from other Hbs, e.g. HbA₀, HbA_1a or HbA_1b, were optimized using the on-chip type system. The electrochemical oxidation current from FcAb reacting with each Hb was measured at 350 mV (vs. Ag/AgCl). Hbs including HbA_1a and HbA_1b, HbA_1c and HbA₀ fractions were eluted in this order. A linear relationship between HbA_1c levels and electrochemical oxidation currents was obtained in the range from 4.0% to 12.6% HbA_1c. All procedure including antigen-antibody reaction was completed in 15 min. Furthermore, a good correlation was obtained between KO500 method (HPLC) and our proposed method. These results indicate that the on-chip type system with electrochemical detection can be applied to a novel POCT device for rapid and precise detection of HbA_1c.     

Electrogenerated chemiluminescence and electrochemical bi-functional sensors for H2O2 based on CdS nanocrystals/hemoglobin multilayers

A novel bi-functional sensor, based on CdS nanocrystals (NCs) and hemoglobin (Hb) multilayer films, designated as {Hb/CdS}_n, modified glassy carbon electrode (GCE) by layer-by-layer (LbL) assembly, has been presented. The electrogenerated chemiluminescence (ECL) and electrochemical properties of {Hb/CdS}_n have been investigated in detail. Hb in the multilayer films can enhance the stability of electrogenerated species of CdS NCs, and CdS NCs can also promote the direct electron transfer between Hb and GCE. As a consequence experimentally, the multilayer films modified GCE is suitable to be used as a bi-functional sensor, ECL sensor and electrochemical sensor, to determine H₂O₂ in obviously different concentration. In high concentration of H₂O₂, this sensor as an ECL sensor shows a linear response from 15 μM up to 18 mM. In the lower concentration of H₂O₂, it as an amperometric one shows two linear ranges of amperometric responses to the concentration of H₂O₂ ranging from 6.0 to 31.0 μM and from 6.0 μM down to 40 nM with a detection limit of 20 nM, based on the high stability of ECL by {Hb/CdS}_n and the excellent electrocatalytical ability of Hb to H₂O₂. Thus, {CdS/Hb}_n modified electrodes would have a great merit to expand the application of biosensors to life science and environmental science.     

A new modified conducting carbon composite electrode as sensor for ascorbate and biosensor for glucose

A new carbon-based conducting composite has been developed as electrochemical sensor and biosensor for the amperometric detection of ascorbate and glucose. Electrocatalytic oxidation of ascorbate has been done successfully at unmodified cellulose acetate-graphite composite electrodes, the sensor being highly sensitive, selective and with a low detection limit at 0.0 V vs. SCE and was successfully applied for ascorbate determination in commercial fruit juice samples. An interference free glucose biosensor has also been developed, based on the immobilisation of glucose oxidase by cross-linking with glutaraldehyde on poly (neutral red) modified composite electrodes. The biosensor exhibits a higher sensitivity of 31.5 ± 1.7 µA cm^− 2 mM^− 1 than other carbon-composite-based glucose biosensors, a detection limit of 20.3 µM and a very short response time.     

Label-Free Electrochemical Sensor Based on Manganese Doped Titanium Dioxide Nanoparticles for Myoglobin Detection: Biomarker for Acute Myocardial Infarction

A label free electrochemical sensor based on pure titanium oxide and manganese (Mn)-doped titanium oxide (TiO₂) nanoparticles are fabricated and characterized for the sensitive detection of myoglobin (Mb) levels to analyze the cardiovascular infarction. Pristine and Mn-doped TiO₂ nanoparticles were synthesized via the sol-gel method and characterized in order to understand their structure, morphologies, composition and optical properties. The structural properties revealed that the pure- and doped-TiO₂ nanoparticles possess different TiO₂ planes. FTIR studies confirm the formation of metal oxide nanoparticles by exhibiting a well-defined peak in the range of 600–650 cm⁻¹. The values of the optical band gap, estimated from UV-Vis spectroscopy, are decreased for the Mn-doped TiO₂ nanoparticles. UV-Vis spectra in the presence of myoglobin (Mb) indicated interaction between the TiO₂ nanoparticles and myoglobin. The SPE electrodes were then fabricated by printing powder film over the working electrode and tested for label-free electrochemical detection of myoglobin (Mb) in the concentration range of 0–15 nM Mb. The fabricated electrochemical sensor exhibited a high sensitivity of 100.40 μA-cm^−2/nM with a lowest detection limit of 0.013 nM (0.22 ng/mL) and a response time of ≤10 ms for sample S3. An interference study with cyt-c and Human Serum Albumin (HSA) of the sensors show the selective response towards Mb in 1:1 mixture.     

Glycated hemoglobin biosensing integration formed on Au nanoparticle-dotted tubular TiO2 nanoarray

Excessive glucose present in the blood of diabetic patients binds with the hemoglobin of red blood cells resulting in the formation of glycated hemoglobin (HbA_1c). Measurement of HbA_1c levels may help in identifying the efficacy of the ongoing treatment and hence provide a better control over the disease. In the present study, we have synthesized a sensitive and stable scaffold, which consists of Au nanoparticles (GNPs)-dotted tubular TiO₂, for the construction of an electrochemical HbA_1c biosensor. 12-phosphotungstic acid has been used as a reducer after depositing well-dispersed GNPs on TiO₂ nanotubes (TiO₂ NTs) and an electron mediator by accelerating the electron transfer between the conductor and protein. The fabricated electrode was characterized using scanning electron microscopy (SEM), cyclic voltammetry (CV), Fourier transform infrared spectroscopy (FTIR) and electrochemical impedance spectroscopic analysis (EIS). Biosensor exhibited low detection limit (0.5 μM), fast response time (3 s) and wide linearity (from 0.5 to 2000 μM). The working electrode was used 100 times over 4 months, when stored at 4 °C. The HbA1c biosensor was then effectively used to measure the % of HbA_1c in the blood of apparently healthy persons and diabetic patients.     

Synthesis of Ferrocene Based Naphthoquinones and its Application as Novel Non‐enzymatic Hydrogen Peroxide

At present, a highly sensitive hydrogen peroxide (H₂O₂) sensor is fabricated by ferrocene based naphthaquinone derivatives as 2,3‐Diferrocenyl‐1,4‐naphthoquinone and 2‐bromo‐3‐ferrocenyl‐1,4‐naphthoquinone. These ferrocene based naphthaquinone derivatives are characterized by H‐NMR and C‐NMR. The electrochemical properties of these ferrocene based naphthaquinone are investigated by cyclic voltammetry (CV), differential pulse voltammetry (DPV) and electrochemical impedance spectroscopy (EIS) on modified glassy carbon electrode (GCE). The modified electrode with ferrocene based naphthaquinone derivatives exhibits an improved voltammetric response to the H₂O₂ redox reaction. 2‐bromo‐3‐ferrocenyl‐1,4‐naphthoquinone show excellent non‐enzymatic sensing ability towards H₂O₂ response with a detection limitation of 2.7 μmol/L a wide detection range from 10 μM to 400 μM in H₂O₂ detection. The sensor also exhibits short response time (1 s) and good sensitivity of 71.4 μA mM^?1 cm^?2 and stability. Furthermore, the DPV method exhibited very high sensitivity (18999 μA mM^?1 cm^?2) and low detection limit (0.66 μM) compared to the CA method. Ferrocene based naphthaquinone derivative based sensors have a lower cost and high stability. Thus, this novel non‐enzyme sensor has potential application in H₂O₂ detection.     

Development of a peroxide biosensor made of a thiolated-viologen and hemoglobin-modified gold electrode

A gold electrode modified with thiolated-viologen is used to design a biosensor in corporate with hemoglobin (Hb). A highly stable self-assembled monolayer (SAM) of thiol-based viologen is immobilized onto the gold electrode. Hb is then immobilized onto the viologen-modified electrode. The modified electrode is very stable. By incorporating with SAMs of viologen and Hb, viologen can act as an electron transfer mediator for Hb to the gold electrode. The potential of Hb was found to be about − 135 mV versus Ag/AgCl for ferro and ferri active centers. The electrochemistry of Hb provides an opportunity to manufacture a third generation of biosensors. Experimental conditions influencing the biosensor performances such as pH, and potential are optimized and assessed. This sensor offered an excellent electrochemical response for H₂O₂ concentration below the μmol level with high sensitivity and selectivity with a short time response.     

Electrochemical Sensing of Pb2+ and Cd2+ Ions with the Use of Electrode Modified with Carbon-Covered Halloysite and Carbon Nanotubes

A novel voltammetric method for the sensitive and selective determination of cadmium and lead ions using screen-printed carbon electrodes (SPCEs) modified with carbon-deposited natural halloysite (C_Hal) and multi-walled carbon nanotubes (MWCNTs) was developed. The electrochemical properties of the proposed sensor were investigated by electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV), while the morphology and structure were established by scanning electron microscopy (SEM) and X-ray powder diffraction (XRD). A two-factorial central composite design (CCD) was employed to select the composition of the nanocomposite modifying the electrode surface. The optimal measuring parameters of differential pulse anodic stripping voltammetry (DPASV) used for quantitative analysis were established with the Nelder–Mead simplex method. In the analytical investigation of Cd(II) and Pb(II) ions by DPASV, the MWCNTs/C_Hal/Nafion/SPCE exhibited a linear response in the concentration range of 0.1–10.0 µmol L⁻¹ (for both ions) with a detection limit of 0.0051 and 0.0106 µmol L⁻¹ for Pb(II) and Cd(II), respectively. The proposed sensor was successfully applied for the determination of metal ions in different natural water and honey samples with recovery values of 96.4–101.6%.     

Portable Wireless Intelligent Electrochemical Sensor for the Ultrasensitive Detection of Rutin Using Functionalized Black Phosphorene Nanocomposite

To build a portable and sensitive method for monitoring the concentration of the flavonoid rutin, a new electrochemical sensing procedure was established. By using nitrogen-doped carbonized polymer dots (N-CPDs) anchoring few-layer black phosphorene (N-CPDs@FLBP) 0D-2D heterostructure and gold nanoparticles (AuNPs) as the modifiers, a carbon ionic liquid electrode and a screen-printed electrode (SPE) were used as the substrate electrodes to construct a conventional electrochemical sensor and a portable wireless intelligent electrochemical sensor, respectively. The electrochemical behavior of rutin on the fabricated electrochemical sensors was explored in detail, with the analytical performances investigated. Due to the electroactive groups of rutin, and the specific π-π stacking and cation–π interaction between the nanocomposite with rutin, the electrochemical responses of rutin were greatly enhanced on the AuNPs/N-CPDs@FLBP-modified electrodes. Under the optimal conditions, ultra-sensitive detection of rutin could be realized on AuNPs/N-CPDs@FLBP/SPE with the detection range of 1.0 nmol L⁻¹ to 220.0 μmol L⁻¹ and the detection limit of 0.33 nmol L⁻¹ (S/N = 3). Finally, two kinds of sensors were applied to test the real samples with satisfactory results.     

An enzymatic method for the rapid measurement of the hemoglobin A1c by a flow-injection system comprised of an electrochemical detector with a specific enzyme-reactor and a spectrophotometer

A flow-injection analytical (FIA) system, comprised of an electrochemical detector with a fructosyl-peptide oxidase (FPOX-CET) reactor and a flow-type spectrophotometer, was proposed for the simultaneous measurement of glycohemoglobin and total hemoglobin in blood cell. The blood cell samples were hemolyzed with a surfactant and then treated with protease. In the first stage of operation, total hemoglobin in digested sample was determined spectrophotometrically. In the second stage, fructosyl valyl histidine (FVH) released from glycohemoglobin by the selective proteolysis was determined specifically using the electrochemical detector with the FPOX-CET reactor. The FIA system could be automatically processed at an analytical speed of 40 samples per hour. The proposed assay method could determine selectively only the glycated N-terminal residue of β-chain in glycohemoglobin and total hemoglobin in blood cell. The enzymatic hemoglobin A_1c (HbA_1c) value calculated by the concentration ratio of the FVH to total hemoglobin, was closely correlated with the HbA_1c values certified by the Japan Diabetic Society (JDS) and the International Federation of Clinical Chemistry (IFCC).     

Zirconium Molybdate Nanocomposites’ Sensing Platform for the Sensitive and Selective Electrochemical Detection of Adefovir

Adefovir (ADV) is an anti-retroviral drug, which can be used to treat acquired immune deficiency syndrome (AIDS) and chronic hepatitis B (CHB), so its quantitative analysis is of great significance. In this work, zirconium molybdate (ZrMo₂O₈) was synthesized by a wet chemical method, and a composite with multi-walled carbon nanotubes (MWCNTs) was made. ZrMo₂O₈-MWCNTs composite was dropped onto the surface of a glassy carbon electrode (GCE) to prepare ZrMo₂O₈-MWCNTs/GCE, and ZrMo₂O₈-MWCNTs/GCE was used in the electrochemical detection of ADV for the first time. The preparation method is fast and simple. The materials were characterized by X-ray powder diffraction (XRD), scanning electron microscopy (SEM), energy dispersive spectroscopy (EDS) and cyclic voltammetry (CV). It was electrochemically analysed by differential pulse voltammetry (DPV). Compared with single-material modified electrodes, ZrMo₂O₈-MWCNTs/GCE showed a vastly improved electrochemical response to ADV. Moreover, the sensor complements the study of the electrochemical detection of ADV. Under optimal conditions, the proposed electrochemical method showed a wide linear range (from 1 to 100 μM) and a low detection limit (0.253 μM). It was successfully tested in serum and urine. In addition, the sensor has the advantages of a simple preparation, fast response, good reproducibility and repeatability. It may be helpful in the potential applications of other substances with similar structures.     

Reduction of cytochrome c at a lipid bilayer modified electrode

The reduction of horse heart cytochrome c has been investigated at a platinum electrode modified with a lipid bilayer membrane (BLM) which immobilized vinyl ferrocene as an electron mediator. The current—voltage curves show that the direct electrochemistry of cytochrome c at the metal electrode occurs quite efficiently. An adsorption equilibrium constant for cytochrome at the BLM surface, as well as an electron transfer rate constant between the protein and the modified electrode have been estimated from these results. The values of both parameters are much higher than those reported with other types of electrode modifications, indicating that a lipid bilayer-modified platinum electrode system using vinyl ferrocene as a mediator provides substantial improvements in electrochemical activity of cytochrome c at metal electrodes. The potential for modifying and utilizing this new class of “biomembrane-like” electrode surface for metalloprotein electrochemistry is briefly discussed.     

Sacrificial template synthesis of ultrathin polyaniline nanosheets and their application in highly sensitive electrochemical dopamine detection

Unique functional nanomaterials as electroactive media for efficiently electrochemical biosensing have always been an ever-increasing topic in biotechnology and environmental fields. In this study, we report a simple sacrificial template-guided polymerization strategy to fabricate ultrathin two-dimensional (2D) polyaniline (PANI) nanosheets for electrochemical detection of dopamine (DA). By using vanadium pentoxide nanosheets as both sacrificial templates and oxidants, the resulting PANI nanosheets show an ultrathin thickness of ca. 4 nm with a favorable electrical conductivity of ca. 10 S cm^?1. Furthermore, PANI nanosheets have been modified on a glass carbon electrode for highly sensitive DA detection. The proposed DA sensor delivers a linear range of 0.5–300 μM with a low detection limit of 0.118 μM (S/N = 3). In addition, the as-fabricated electrochemical sensor exhibits an outstanding selectivity, stability, reproducibility, and repeatability, enabling its feasible application for DA detection in real samples. Therefore, the ultrathin PANI nanosheets reported here are good candidates as electrodes for the sensitive and selective DA detection.     

Electrochemical characteristics of a platinum electrode modified with a matrix of polyvinyl butyral and colloidal Ag containing immobilized horseradish peroxidase

A new hydrogen peroxide biosensor was constructed, which consisted of a platinum electrode modified by a matrix of polyvinyl butyral (PVB) and nanometer-sized Ag colloid containing immobilized horseradish peroxidase (HRP), and using Co(bpy)₃³⁺ as mediator in the hydrogen peroxide solution. The electrochemical characteristics of the biosensor were studied by cyclic voltammetry and chronoamperometry. The modified process was characterized by electrochemical impedance spectroscopy and cyclic voltammetry. The HRP immobilized on colloidal Ag was stable and retained its biological activity. The sensor displays excellent electrocatalytic response to the reduction of H₂O₂. Analytical parameters such as pH and temperature were also studied. Linear calibration for H₂O₂ was obtained in the range of 1×10^–5 to 1×10^–2 M under optimized conditions. The sensor was highly sensitive to H₂O₂, with a detection limit of 2×10^–6 M, and the sensor achieved 95% of steady-state current within 10 s. The sensor exhibited high sensitivity, selectivity and stability.     

A multi-channel sensor based on 8-hydroxyquinoline ferrocenoate for probing Hg(II) ion

A new sensing molecule 8-hydroxyquinoline ferrocenoate (Fc-Q) which combines ferrocene and 8-hydroxyquinoline moieties was synthesized and applied as a multi-channel sensor for the detection of Hg²⁺ ion. Fc-Q can coordinate with Hg²⁺ to give colorimetric, fluorescent and electrochemical responses. Upon complexation with Hg²⁺ ion, the characteristic absorption peak is red-shifted (Δλ = 45 nm), the fluorescent intensity is quenched at 303 nm, and the oxidation peak is cathodic shifted (ΔE_1/2 = −149 mV). Quantitatively analyzed Hg²⁺ ions at the range of ppb level could be achieved by electrochemical response. For the practical application of sensing Hg²⁺ in real world water, Fc-Q modified screen-printed carbon electrodes were obtained for facile, sensitive, and on-site analysis of Hg²⁺.     

Immobilization of hemoglobin on SBA-15 applied to the electrocatalytic reduction of H<Subscript>2</Subscript>O<Subscript>2</Subscript>

The direct electron transfer between hemoglobin (Hb) and an electrode was realized by first immobilizing the protein onto SBA-15.The results of the immobilization showed that the adsorption was pH-dependent with a maximum adsorption near the isoelectric point of the protein, and SBA-15 with a larger pore diameter showed greater adsorption capacity for Hb. UV–vis spectroscopy and nitrogen adsorption analysis indicated that Hb was adsorbed within the channel of SBA-15 and no significant denaturation occurred to the protein. The Hb/SBA-15 composite obtained was used for the fabrication of a Hb biosensor to detect hydrogen peroxide. A pair of well-defined redox peaks at −0.337 and −0.370 V on the Hb/SBA-15 composite modified glassy carbon electrode was observed, and the electrode reactions showed a surface-controlled process with a single proton transfer at a scan rate range from 20 to 1,000 mV/s. The sensor showed a fast amperometric response, a low detection limit (2.3 × 10⁻⁹ M) and good stability for the detection of H₂O₂. The electrochemical results indicated that the immobilized Hb still retained its biological activity.     

Computational Modelling and Sustainable Synthesis of a Highly Selective Electrochemical MIP-Based Sensor for Citalopram Detection

A novel molecularly imprinted polymer (MIP) has been developed based on a simple and sustainable strategy for the selective determination of citalopram (CTL) using screen-printed carbon electrodes (SPCEs). The MIP layer was prepared by electrochemical in situ polymerization of the 3-amino-4 hydroxybenzoic acid (AHBA) functional monomer and CTL as a template molecule. To simulate the polymerization mixture and predict the most suitable ratio between the template and functional monomer, computational studies, namely molecular dynamics (MD) simulations, were carried out. During the experimental preparation process, essential parameters controlling the performance of the MIP sensor, including CTL:AHBA concentration, number of polymerization cycles, and square wave voltammetry (SWV) frequency were investigated and optimized. The electrochemical characteristics of the prepared MIP sensor were evaluated by both cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) techniques. Based on the optimal conditions, a linear electrochemical response of the sensor was obtained by SWV measurements from 0.1 to 1.25 µmol L⁻¹ with a limit of detection (LOD) of 0.162 µmol L⁻¹ (S/N = 3). Moreover, the MIP sensor revealed excellent CTL selectivity against very close analogues, as well as high imprinting factor of 22. Its applicability in spiked river water samples demonstrated its potential for adequate monitoring of CTL. This sensor offers a facile strategy to achieve portability while expressing a willingness to care for the environment.     

Electrochemical sensor for determination of aflatoxin B1 based on multiwalled carbon nanotubes-supported Au/Pt bimetallic nanoparticles

A sensitive and selective imprinted electrochemical sensor for the determination of aflatoxin B₁ (AFB₁) was constructed on a glassy carbon electrode by stepwise modification of functional multiwalled carbon nanotubes (MCNTs), Au/Pt bimetallic nanoparticles (Au/PtNPs), and a thin imprinted film. The fabrication of a homogeneous porous poly o-phenylenediamine (POPD)-grafted Au/Pt bimetallic multiwalled carbon nanotubes nanocomposite film was conducted by controllable electrodepositing technology. The sensitivity of the sensor was improved greatly because of the nanocomposite functional layer; the proposed sensor exhibited excellent selectivity toward AFB₁ owing to the porous molecular imprinted polymer (MIP) film. The surface morphologies of the modified electrodes were characterized using a scanning electron microscope. The performance of the imprinted sensor was investigated by cyclic voltammetry, differential pulse voltammetry, and electrochemical impedance spectroscopy in detail. A linear relationship between the sensor response signal and the logarithm of AFB₁ concentrations ranging from 1?×?10^?10 to 1?×?10^?5 mol L^?1 was obtained with a detection limit of 0.03 nmol L^?1. It was applied to detect AFB₁ in hogwash oil successfully.     

Determination of hemoglobin at a novel NH2/ITO ion implantation modified electrode

A novel electrode was prepared by implanting NH₂ ⁺ into an ITO film (NH₂/ITO). Gold nanoparticles were deposited on the surface of NH₂/ITO electrode. The NH₂/ITO and Au/NH₂/ITO electrodes were used to determine hemoglobin (Hb) immobilized on the electrodes surfaces. The relationship of the reductive peak current value of Hb among different electrodes was: Hb/ITO:Hb/Au/ITO:Hb/NH₂/ITO:Hb/Au/NH₂/ITO=1:1.5:2:4. The linkage between the –NH₂ implanted into ITO film and the –COOH of Hb was recognized to be the reason for the increase of active Hb coverage on NH₂/ITO electrode compared with the ITO electrode. Increase of active Hb coverage on Au/NH₂/ITO compared with Au/ITO was attributed to the different amount of gold nanoparticles deposited. The determination of Hb at an Au/NH₂/ITO electrode was optimized. Calibration curve was obtained over the range of 1.0 × 10⁻⁸ – 1.0 × 10⁻⁶ mol · L⁻¹ with a detection limit of 1.0 × 10⁻⁸ mol · L⁻¹. Results showed that the novel NH₂/ITO and Au/NH₂/ITO electrodes exhibited good stability, reproducibility besides better electrochemical performance. Correspondence: Jing Bo Hu, Department of Chemistry, Beijing Normal University, Beijing 100875, China     

Environmental effects of chitosan as an immobilization medium for electrochemically active small molecules

Abstract

A number of challenges arise when using ferrocene as a component of electrochemical biosensors, including solubility in aqueous solutions. Therefore, entrapment of the biotin-ferrocene molecules within the chitosan film provides a route for immobilization on an electroactive surface such as an electrode while making the system water compatible. The use of the chitosan-ferrocene bioconjugate thin film on the electrode surface produces a signal that can be monitored in aqueous media. Herein, we discuss a series of modified ferrocene molecules that contain various linkers that provide non-covalent entanglement points to the chitosan medium. The electrochemical analysis and electron microscopy results show marked differences in the ferrocene loaded chitosan polymers when the termini of the ferrocene-linker vary between –SH and –NH₂. The –SH modified systems showed increased reversible and robust electrochemical signals relative to the –NH₂ congeners. Further studies showed that non-covalent impregnation strategy used is robust to degradation and less than 1% of the ferrocene molecules were leached over time. These results indicate that there are specific considerations needed when using chitosan-ferrocene systems as components in sensor arrays in future studies.