Non-invasive electrochemical hand-held biosensor as diagnostic indicator of dental diseases

A new hand-held amperometric biosensor for assay of salivary peroxidase in human secretions is described. The biosensor comprises a flow through amperometric detector coupled with a micropipette, and electronics (an amplifier, peak detector, microprocessor, and display). A micropipette, amplifier, peak detector, and microprocessor have been built in-house. All operations, such as sample injection, electrochemical detection and data processing are accomplished using only one action, by pressing the plunger of the micropipette. Because the use of a peristaltic pump is completely eliminated, the consumption of both samples and reagents is significantly reduced. The biosensor has been tested as a diagnostic indicator of dental diseases. It is shown that there is a functional relationship between the total level of salivary peroxidase and the clinical status of the periodontal disease. By using commercial available kits with peroxidase-labeled conjugates, the biosensor has an excellent potential for detection of different hormones, drugs, viruses, antibodies, and bacteria.     

Organic-inorganic matrix for electrochemical immunoassay: Detection of human IgG based on ZnO/chitosan composite

A new strategy to construct amperometric immunosensor for human IgG assay based on ZnO/chitosan composite as sensing platform has been described. This material, which combined the advantages of inorganic species, ZnO and organic polymer, chitosan, can maintain biological activity well. A sequential sandwich immunoassay format was performed on the ZnO/chitosan composite supported by glass carbon electrode (GCE) using goat-anti-human IgG antibody (IgG Ab) and human IgG as a model system. Amperometry was used to determine the amount of horse-radish peroxidase (HRP) fixed on the sensor surface, which was related to the content of the desired human IgG. Assay conditions that were optimized included the amount of labeled antibody, the incubation time and temperature, the pH of the substrate solution, etc. Using hydroquinone as a mediator, amperometric detection at −150 mV (versus SCE) resulted in a detection range 2.5-500 ng mL⁻¹, with a detection limit of 1.2 ng mL⁻¹. The simple manipulations of the construction of ZnO/chitosan composite, as well as low-cost and broad linear range, are the main features of the proposed immunosensing method.     

Catalytic oxidation of thiols at preheated glassy carbon electrode modified with abrasive immobilization of multiwall carbon nanotubes: applications to amperometric detection of thiocytosine, l-cysteine and glutathione

The performance of preheated glassy carbon electrode modified with carbon nanotubes is described. First glassy carbon electrode is heated for 5 min at 50 °C, then abrasive immobilization of multiwall carbon nanotubes on a preheated glassy carbon electrode was achieved by gentle rubbing of electrode surface on a filter paper supporting carbon nanotubes. Carbon nanotubes (CNTs)-modified glassy carbon electrodes exhibit strong and stable electrocatalytic response toward thiols oxidation in wide pH range. These properties permit an important decrease in over voltage for the oxidation of thiocytosine, glutathione and l-cysteine, as well as a dramatic increase in the peak currents in comparison with bare glassy carbon electrode. Furthermore, the thiols amperometric response of the coated electrodes is extremely stable, with more than 95% of the initial activity after 30 min stirring of 0.1 mM thiols. The electrocatalytic behavior is further exploited as a sensitive detection scheme for thiols detection by hydrodynamic amperometry. The substantial decrease in the overvoltage of the thiols oxidation associated with a stable amperometric response and antifouling properties of nanotubes films allow the development of highly sensitive thiols sensor without using any redox mediator. Such ability of carbon nanotubes to promote the thiols electron transfer reaction, short response time (5 s) and long-term stability, low detection limit, extended linear concentration range, high sensitivity suggest great promise for thiols amperometric sensors and detector for chromatographic analysis of thiol derivatives.     

Affinity biosensors based on disposable screen-printed electrodes modified with DNA

Simple and sensitive DNA sensors have been developed on a base on graphite screen-printed electrodes modified with DNA and enzymes. Cholinesterase and peroxidase immobilized by treatment with glutaraldehyde were used for the detection of human DNA antibodies of systemic lupus erythematosus and bronchial asthma patients. The amperometric signal was measured at +680 mV versus Ag/AgCl for DNA-cholinesterase sensor and −150 mV for DNA-peroxidase sensor 5 min after the injection of acethylthiocholine and hydroquinone, respectively. The addition of serum samples results in the sharp decrease of the signal due to the formation of DNA-antibody adducts followed by the suppression of the access of substrate to the enzyme active site. Sulfonamide medicines suppress the DNA-antibody interaction due to the competitive binding along DNA minor grooves. DNA sensor labeled with peroxidase showed the linear calibration range of 5×10⁻⁹ to 7×10⁻⁵ mol l⁻¹ of sulfamethoxazole and of 5×10⁻⁸ to 1×10⁻⁴ mol l⁻¹ of sulfathiazole.     

Amperometric immunosensor for detection of antibodies of Salmonella typhi in patient serum

An amperometric sensor for detection of antibodies to Salmonella typhi in the serum of patients was developed. This involved usage of screen-printed electrodes and recombinant flagellin fusion protein. An indirect enzyme-linked immunosorbant assay was used for detection of antibodies to S. typhi in the patient serum. The screen-printed electrodes were made using polystyrene and graphite. These electrodes were tested for their ability to detect 1-naphthol, which is the product formed due to the hydrolysis of the substrate 1-naphthyl phosphate by the enzyme alkaline phosphatase. These electrodes were coated with recombinant flagellin fusion protein made by recombinant DNA technology and blocked with bovine serum albumin (BSA). Further they were incubated with patient serum and goat anti-human alkaline phosphatase conjugate. The immunosensing was performed by using amperometric method. Pooled human serum samples from apparently healthy individuals were used as control. Both the pooled healthy human serum samples and patient sera were subjected to Widal agglutination test and amperometric method. A 100% correlation was found between the Widal test and amperometric method. The time taken for the detection by electrochemical method is 1 h and 15 min, while the time taken by Widal test is 18 h.     

A novel amperometric sensor for the detection of difenidol hydrochloride based on the modification of Ru(bpy)3 on a glassy carbon electrode

An amperometric sensor for the detection of difenidol, a tertiary amine-containing analyte, was proposed. Ruthenium(II) tris(bipyridine)/multi-walled carbon nanotubes/Nafion composite film was suggested to modify the glassy carbon electrode. The modified electrode was shown to be an excellent amperometric sensor for the detection of difenidol hydrochloride. The linear range is from 1.0 × 10⁻⁶ to 3.3 × 10⁻⁵ M with a correlation coefficient of 0.998. The limit of detection was 5 × 10⁻⁷ M, which was obtained through experimental determination based on a signal-to-noise ratio of three. The sensor was employed to the determination of the active ingredients in the tablets containing difenidol hydrochloride.     

A label-free amperometric immunosenor based on multi-layer assembly of polymerized o-phenylenediamine and gold nanoparticles for determination of Japanese B encephalitis vaccine

A label-free amperometric immunosensor for fast and sensitive assay of Japanese B encephalitis vaccine is presented. Antiserum of Japanese B encephalitis were immobilized on bilayer nano-Au/o-phenylenediamine polymer film with deposited Prussian blue as an electronic mediator on the Pt electrode. The electrochemical behavior of the biosensor was studied with Fe^2+/3+ as probe on the Pt surface using cyclic voltammetry technique. The variation of amperometric response to the concentration of Japanese B encephalitis vaccine, the target antigen, was evaluated by cyclic voltammetry in PBS. The immunosensor showed a specific response to Japanese B encephalitis vaccine in the range 1.1 × 10⁻⁸ to 1.9 × 10⁻⁶ lg pfu/ml (pfu/ml is plaque forming unit and lg is common logarithm) with a detection limit of 6 × 10⁻⁹ lg pfu/ml. The correlation coefficient is 0.9955. The incubation time, incubation temperature, pH, reproducibility and stability of the immunosensor were also studied. The present work supplied a promising test method for biological products.     

An end-channel amperometric detector for microchip capillary electrophoresis

An end-channel amperometric detector with a guide tube for working electrode was designed and integrated on a home-made glass microchip. The guide tube was directly patterned and fabricated at the end of the detection reservoir, which made the fixation and alignment of working electrode relatively easy. The fabrication was carried out in a two-step etching process. A 30 μm carbon fiber microdisk electrode and Pt cathode were also integrated onto the amperometric detector. The characteristics and primary performance of the home-made microchip capillary electrophoresis (MCCE) were investigated with neurotransmitters. The baseline separation of dopamine (DA), catechol (CA) and epinephrine (EP) was achieved within 80 s. Separation parameters such as injection time, buffer components, pH of the buffer were studied. Relative standard deviations of not more than 6.0% were obtained for both peak currents and migration times. Under the selected separation conditions, the response for DA was linear from 5 to 200 μM and from 20 to 800 μM for CA. The limits of detection of DA and CA were 0.51 and 2.9 μM, respectively (S/N=3).     

Electrode modified with a composite film of ZnO nanorods and Ag nanoparticles as a sensor for hydrogen peroxide

A conducting fluorine-doped tin oxide (FTO) electrode, first modified with zinc oxide nanorods (ZnONRs) and subsequently attached with photosynthesized silver nanoparticles (AgNPs), designated as AgNPs/ZnONRs/FTO electrode, was used as an amperometric sensor for the determination of hydrogen peroxide. The first layer (ZnONRs) was obtained by chemical bath deposition (CBD), and was utilized simultaneously as the catalyst for the photoreduction of Ag ions under UV irradiation and as the matrix for the immobilization of AgNPs. The aspect ratio of ZnONRs to be deposited was optimized by controlling the number of their CBDs to render enough surface area for Ag deposition, and the amount of AgNPs to be attached was controlled by adjusting the UV-irradiation time. The immobilized AgNPs showed excellent electrocatalytic response to the reduction of hydrogen peroxide. The resultant amperometric sensor showed 10-fold enhanced sensitivity for the detection of H₂O₂, compared to that without AgNPs, i.e., only with a layer of ZnONRs. Amperometric determination of H₂O₂ at −0.55 V gave a limit of detection of 0.9 μM (S/N = 3) and a sensitivity of 152.1 mA M⁻¹ cm⁻² up to 0.983 mM, with a response time (steady-state, t₉₅) of 30-40 s. The selectivity of the sensor was investigated against ascorbic acid (AA) and uric acid (UA). Energy dispersive X-ray (EDX) analysis, transmission electron microscopic (TEM) image, X-ray diffraction (XRD) patterns, cyclic voltammetry (CV), and scanning electron microscopic (SEM) images were utilized to characterize the modified electrode. Sensing properties of the modified electrode were studied both by CV and amperometric analysis.     

Cobalt phthalocyanine as a biomimetic catalyst in the amperometric quantification of dipyrone using FIA

A biomimetic sensor for the determination of dipyrone was prepared by modifying carbon paste with cobalt phthalocyanine (CoPc), and used as an amperometric detector in a flow injection analysis (FIA) system. The results of cyclic voltammetry experiments suggested that CoPc behaved as a biomimetic catalyst in the electrocatalytic oxidation of dipyrone, which involved the transfer of one electron. The optimized FIA procedure employed a flow rate of 1.5 mL min⁻¹, a 75 μL sample loop, a 0.1 mol L⁻¹ phosphate buffer carrier solution at pH 7.0 and amperometric detection at a potential of 0.3 V vs. Ag/AgCl. Under these conditions, the proposed method showed a linear response for dipyrone concentrations in the range 5.0 × 10⁻⁶-6.3 × 10⁻³ mol L⁻¹. Selectivity and interference studies were carried out in order to validate the system for use with pharmaceutical and environmental samples. In addition to being environmentally friendly, the proposed method is a sensitive and selective analytical tool for the determination of dipyrone.     

Development of a disposable amperometric immunosensor for the detection of ecstasy and its analogues using screen-printed electrodes

An amperometric immunosensor for the specific and simple detection of 3,4-methylenedioxyamphetamine (MDA) and its analogues, 3,4-methylenedioxymethamphetamine (MDMA) and 3,4-methylenedioxyethylamphetamine (MDEA) in saliva and urine was developed. A direct competitive assay in which free analyte and horseradish peroxidase labelled species were simultaneously added to an immobilised polyclonal antibody was employed. Both MDA and MDMA could be labelled with the enzyme and the use of an MDMA-HRP tracer greatly enhanced the sensitivity of the assay. Amperometric detection was performed at +100 mV versus Ag/AgCl, using tetramethylbenzidine (TMB)/H₂O₂ as substrate. The antibody, raised specifically against the methylenedioxy moiety of an MDA-BSA immunogen allowed highly specific detection of these analogues with negligible cross-reactivity towards any other amphetamine related compounds. Total assay time was 45 min and the standard curve using MDA could be evaluated within the range 0.61-400 ng ml⁻¹ with corresponding limit of detection (LOD) of 0.36 and 0.042 ng ml⁻¹ for saliva and urine, respectively. The cross-reactivity pattern of the analytes was determined and showed the order of sensitivity increased with increased alkyl chain length (MDA < MDMA < MDEA). The overall performance of the sensor, working range, precision and sensitivity demonstrate its usefulness for rapid and direct measurement of methylenedioxy analogues of ecstasy in saliva and urine. The sensor has better specificity than any previous method for ecstasy, with greater sensitivity than ELISA methods, is less expensive/assay with an “easier to use” format than previous methods. The detection works in saliva or urine, eliminating requirement of blood sampling, with improved precision.     

Flow electrochemical biosensors based on enzymatic porous reactor and tubular detector of silver solid amalgam

A flow amperometric enzymatic biosensor for the determination of glucose was constructed. The biosensor consists of a flow reactor based on porous silver solid amalgam (AgSA) and a flow tubular detector based on compact AgSA. The preparation of the sensor and the determination of glucose occurred in three steps. First, a self-assembled monolayer of 11-mercaptoundecanoic acid (MUA) was formed at the porous surface of the reactor. Second, enzyme glucose oxidase (GOx) was covalently immobilized at MUA-layer using N-ethyl-N′-(3-dimethylaminopropyl) carboimide and N-hydroxysuccinimide chemistry. Finally, a decrease of oxygen concentration (directly proportional to the concentration of glucose) during enzymatic reaction was amperometrically measured on the tubular detector under flow injection conditions. The following parameters of glucose determination were optimized with respect to amperometric response: composition of the mobile phase, its concentration, the potential of detection and the flow rate. The calibration curve of glucose was linear in the concentration range of 0.02–0.80 mmol L⁻¹ with detection limit of 0.01 mmol L⁻¹. The content of glucose in the sample of honey was determined as 35.5 ± 1.0 mass % (number of the repeated measurements n = 7; standard deviation SD = 1.2%; relative standard deviation RSD = 3.2%) which corresponds well with the declared values. The tested biosensor proved good long-term stability (77% of the current response of glucose was retained after 35 days).     

Ultra-sensitive hydrazine chemical sensor based on high-aspect-ratio ZnO nanowires

High-aspect-ratio ZnO nanowires based ultra-sensitive hydrazine amperometric sensor has been fabricated which showed a high and reproducible sensitivity of 12.76 μA cm⁻² nM⁻¹, detection limit, based on S/N ratio, 84.7 nM, response time less than 5 s, linear range from 500 to 1200 nM and correlation coefficient of R = 0.9989. This is the first report in which such a very high-sensitivity and low detection limit has been achieved for the hydrazine sensors by using ZnO nanostructures modified electrodes. Therefore, this work opens a way to utilize simply grown ZnO nanostructures as an efficient electron mediator to fabricate efficient hydrazine sensors.     

A chemiluminescence sensor for the determination of hydrogen peroxide

A chemiluminescence one-shot sensor for hydrogen peroxide is described. It is prepared by immobilization of cobalt chloride and sodium lauryl sulphate in hydroxyethyl cellulose matrix cast on a microscope cover glass. Luminol, sodium phosphate and the sample are mixed before use and applied on the membrane by a micropipette. The calibration graph is linear in the range 20-1600 μg/L, and the detection limit of the method (3σ) is 9 μg/L. A relative standard deviation of 4.5% was obtained for 100 μg/L H₂O₂ (n = 11). The sensor has been applied successfully to the determination of hydrogen peroxide in rainwater.     

Fast and direct determination of butylated hydroxyanisole in biodiesel by batch injection analysis with amperometric detection

We report here, for the first time, application of batch injection analysis (BIA) with amperometric detection for determination of the phenolic antioxidant butylated hydroxyanisole (BHA) in biodiesel. A sample plug was directly injected onto a boron-doped diamond electrode immersed in 50% v/v hydroethanolic solution with 0.1 mol L⁻¹ HClO₄ using an electronic micropipette. Importantly, the only preparation step required for biodiesel analysis is dilution in the same hydroethanolic electrolyte solution. Our proposed method has several advantages for routine biodiesel analysis, including: a low relative standard deviation between injections (0.29%, n = 20), high analytical frequency (120 h⁻¹), adequate recovery values (93-101%) for spiked samples, satisfactory accuracy (based on comparative determinations by high-performance liquid-chromatography), and a low detection limit (100 ng of BHA per g of biodiesel). Finally, our method can be adapted for the determination of other antioxidants in biodiesel samples.     

Modification of carbon ceramic electrode prepared with sol-gel technique by a thin film of chlorogenic acid: application to amperometric detection of NADH

The carbon ceramic electrode prepared with sol-gel technique is modified by a thin film of chlorogenic acid (CGA). By immersing the carbon ceramic electrode in aqueous solution of chlorogenic acid at less than 2 s a thin film of chlorogenic acid adsorbed strongly and irreversibly on the surface of electrode. The cyclic voltammetry of the resulting modified CCE prepared at optimum conditions shows a well-defined stable reversible redox couple due to hydroquinone/quinone system in both acidic and basic solutions. The modified electrode showed excellent electrocatalytic activity toward NADH oxidation and it also showed a high analytical performance for amperometric detection of NADH. The catalytic rate constant of the modified carbon ceramic electrode for the oxidation of NADH is determined by cyclic voltammetry measurement. Under the optimised conditions the calibration curve is linear in the concentration range 1-120 μm. The detection limit (S/N = 3) and sensitivity are 0.2 μM and 25 nA μM⁻¹.The results of six successive measurement-regeneration cycles show relative standard deviations of 2.5% for electrolyte solution containing 1 mM NADH, indicating that the electrode renewal gives a good reproducible and antifouling surface. The advantages of this amperometric detector are: high sensitivity, excellent catalytic activity, short response time t < 2 s, remarkable long-term stability, simplicity of preparation at short time and good reproducibility.     

Cobalt hexacyanoferrate modified multi-walled carbon nanotubes/graphite composite electrode as electrochemical sensor on microfluidic chip

Nanomaterial-based electrochemical sensor has received significant interest. In this work, cobalt hexacyanoferrate modified multi-walled carbon nanotubes/graphite composite electrode was electrochemically prepared and exploited as an amperometric detector for microchip electrophoresis. The prepared sensor displayed rapid and sensitive response towards hydrazine and isoniazid oxidation, which was attributed to synergetic electrocatalytic effect of cobalt hexacyanoferrate and multi-walled carbon nanotubes. The sensitivity enhancement with nearly two orders of magnitude was gained, compared with the bare carbon paste electrode, with the detection limit of 0.91 μM (S/N = 3) for hydrazine. Acceptable repeatability of the microanalysis system was verified by consecutive eleven injections of hydrazine without chip and electrode treatments, the RSDs for peak current and migration time were 3.4% and 2.1%, respectively. Meanwhile, well-shaped electrophoretic peaks were observed, mainly due to fast electron transfer of electroactive species on the modified electrode. The developed microchip-electrochemistry setup was successfully applied to the determination of hydrazine and isoniazid in river water and pharmaceutical preparation, respectively. Several merits of the novel electrochemical sensor coupled with microfluidic platform, such as comparative stability, easy fabrication and high sensitivity, hold great potential for hydrazine compounds assay in the lab-on-a-chip system.     

A novel non-enzymatic glucose sensor based on Cu nanoparticle modified graphene sheets electrode

A novel, stable and sensitive non-enzymatic glucose sensor was developed by potentiostatically electrodepositing metallic Cu nanoparticles on graphene sheets. The electrochemical performance of the Cu-graphene sheets electrode for detection of glucose was investigated by cyclic voltammetry and chronamperometry. The Cu-graphene sheets electrode displayed a synergistic effect of copper nanoparticles and graphene sheets towards the oxidation of glucose in alkaline solution, showing higher oxidation current and negative shift in peak potential. At detection potential of 500 mV, the Cu-graphene electrode sensor presented a wide linear range up to 4.5 mM glucose with a detection limit of 0.5 μM (signal/noise = 3). In addition, the sensor responds very quickly (<2 s) with addition of glucose. Furthermore, the Cu-graphene sheets electrode exhibits high stability and selectivity to glucose, and the poisoning by chloride ion as well as interference from the oxidation of common interfering species (ascorbic, dopamine, uric acid and carbohydrate) are effectively avoided. The Cu-graphene sheets electrode allows highly selective and sensitive, stable and fast amperometric sensing of glucose, which is promising for the development of non-enzymatic glucose sensor.     

Amperometric morphine sensing using a molecularly imprinted polymer-modified electrode

This study incorporates morphine into a molecularly imprinted polymer (MIP) for the amperometric detection of morphine. The polymer, poly(3,4-ethylenedioxythiophene), PEDOT, is an electroactive film that catalyzes morphine oxidation and lowers the oxidization potential on an indium tin oxide (ITO) electrode. The MIP-PEDOT modified electrode is prepared by electropolymerizing PEDOT onto an ITO electrode in a 0.1 M LiClO₄ solution with template addition (morphine). After template molecule extraction, the oxidizing current of the MIP-PEDOT modified electrode is measured in a 0.1 M KCl solution (pH = 5.3) at 0.75 V (versus Ag/AgCl/sat’d KCl) with the morphine concentration varying in the 0.1-5 mM range. A linear range, displaying the relationship between steady-state currents and morphine concentrations, from 0.1 to 1 mM, is obtained. The proposed amperometric sensor could be used for morphine detection with a sensitivity of 91.86 μA/cm² per mM. A detection limit of 0.2 mM at a signal-to-noise ratio of 3 is achieved. Moreover, the proposed method can discriminate between morphine and its analogs, such as codeine.