A selective glucose sensor based on direct oxidation on a bimetal catalyst with a molecular imprinted polymer.

A selective nonenzymatic glucose sensor was developed based on the direct oxidation of glucose on hierarchical CuCo bimetal-coated with a glucose-imprinted polymer (GIP). Glucose was introduced into the GIP composed of Nafion and polyurethane along with aminophenyl boronic acid (APBA), which was formed on the bimetal electrode formed on a screen-printed electrode. The extraction of glucose from the GIP allowed for the selective permeation of glucose into the bimetal electrode surface for oxidation. The GIP-coated bimetal sensor probe was characterized using electrochemical and surface analytical methods. The GIP layer coated on the NaOH pre-treated bimetal electrode exhibited a dynamic range between 1.0µM and 25.0mM with a detection limit of 0.65±0.10µM in phosphate buffer solution (pH 7.4). The anodic responses of uric acid, acetaminophen, dopamine, ascorbic acid, L-cysteine, and other saccharides (monosaccharides: galactose, mannose, fructose, and xylose; disaccharides: sucrose, lactose, and maltose) were not detected using the GIP-coated bimetal sensor. The reliability of the sensor was evaluated by the determination of glucose in artificial and whole blood samples.

An amperometric nanobiosensor using a biocompatible conjugate for early detection of metastatic cancer cells in biological fluid.

Metastasis is the major cause of cancer-associated death in humans, and its early diagnosis will help clinicians to develop suitable therapeutic strategies which may save life of cancer patients. In this direction, we designed an amperometric biosensor using a biocompatible conjugate to diagnose cancer metastasis by detecting epithelial cell adhesion molecule expressing metastatic cancer cells (Ep-MCCs). The sensor probe is fabricated by immobilizing monoclonal capture antibody (CapAnti) on the gold nanoparticles (AuNPs)/conducting polymer composite layer. The detection relies on a sandwich-type approach using a bioconjugate composed of reporter antibody (RepAnti), nanostructured collagen (nCOL), AuNPs, and hydrazine (Hyd) which served as a nonenzymatic electrocatalyst for the reduction of H2O2. The binding of Ep-MCCs with the sensor probe was confirmed using electrochemical impedance spectroscopy, cyclic voltammetry, and chronoamperometry. A dynamic range for the Ep-MCCs detection is determined between 45 and 100,000 Ep-MCCs/mL with the detection limit of 28±3 Ep-MCCs/mL. The proposed immunosensor is successfully applied to detect Ep-MCCs in serum and mixed cell samples and interferences due to nontarget cells and molecules present in the real sample matrix are also examined. The early stage of Ep-MCCs was examined by fluorescence-activated cell sorting assay, which confirms that the developed biosensor has detected Ep-MCCs in its early stage.

Analysis of Phthalate Esters in Mammalian Cell Culture Using a Microfluidic Channel Coupled with an Electrochemical Sensor.

An analytical tool to monitor trace phthalate was developed using a microfluidic channel device coupled with a novel electrochemical biosensor. At first, the electrochemical sensor was constructed with biomimetic layers to reveal a large hydrogen over potential by controlling the surface charge and hydrophobicity through assembling with a lipid (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine) and a cationic molecule (toluidine blue O) bonded to a conductive polymer. The modified electrode possessing a highly negative polarization potential (approximately -1.8 V vs Ag/AgCl) can uptake sparingly soluble phthalate ester (PEs) compounds in aqueous media. Each sensor probe material was characterized employing SEM, AFM, XPS, QCM, TEM, UV-visible, and impedance spectroscopy. The microfluidic channel is used first to concentrate and separate trace amounts of phthalates, and then the sensor probe is installed at the end of channel. Experimental variables affecting the PEs analysis were assessed and optimized in terms of biomimetic layer composition and analytical conditions. The linear dynamic range and detection limits of the PEs were 0.15 nM-10.0 µM and ∼12.5 pM with relative standard deviations <5%. The proposed method was applied to evaluate the effect of endocrine disruptors on mammalian kidney cells, where the cell samples show in-taking percentages between 1.8 and 7.0% to the total PEs according to the incubation time.

Ultrasensitive detection of drug resistant cancer cells in biological matrixes using an amperometric nanobiosensor.

Multidrug resistance (MDR) is a key issue in the failure of cancer chemotherapy and its detection will be helpful to develop suitable therapeutic strategies for cancer patients and overcome the death rates. In this direction, we designed a new amperometric sensor (a medical device prototype) to detect drug resistant cancer cells by sensing "Permeability glycoprotein (P-gp)". The sensor probe is fabricated by immobilizing monoclonal P-gp antibody on the gold nanoparticles (AuNPs) conducting polymer composite. The detection relies on a sandwich-type approach using a bioconjugate, where the aminophenyl boronic acid (APBA) served as a recognition molecule which binds with the cell surface glycans and hydrazine (Hyd) served as an electrocatalyst for the reduction of H2O2 which are attached on multi-wall carbon nanotube (MWCNT) (APBA-MWCNT-Hyd). A linear range for the cancer cell detection is obtained between 50 and 100,000 cells/mL with the detection limit of 23±2 cells/mL. The proposed immunosensor is successfully applied to detect MDR cancer cells (MDRCC) in serum and mixed cell samples. Interferences by drug sensitive (SKBr-3 and HeLa), noncancerous cells (HEK-293 and OSE), and other chemical molecules present in the real sample matrix are examined. The sensitivity of the proposed immunosensor is excellent compared with the conventional reporter antibody based assay.

Implantable nonenzymatic glucose/O2 micro film fuel cells assembled with hierarchical AuZn electrodes.

Hierarchical AuZn dendrites revealed electrocatalytic properties towards the glucose oxidation and the four-electron O2 reduction. The micro fuel cell using AuZn electrodes generated a power density of 2.07 and 0.29 mW cm(-2) for glucose and human whole blood. The micro film fuel cell implanted into the rat brain produced ∼0.52 V continuously operating for more than 18 days.

Selective nonenzymatic bilirubin detection in blood samples using a Nafion/Mn-Cu sensor.

The specific detection of biological organics without the use of an enzyme is challenging, and it is crucial for analytical and clinical chemistry. We report specific nonenzymatic bilirubin detection through the catalytic oxidation of bilirubin molecule on the Nafion/Mn-Cu surface. The catalytic ability, true surface area, morphology, crystallinity, composition, and oxidation state of the sensor surface were assessed using voltammetry, coulometry, XPS, XRD, Brunauer-Emmett-Teller (BET), SEM, EDXS, and TOF-SIMS experiments. The results showed that the surface was composed of microporous Mn-Cu bimetallic crystal in flake shape with a large BET surface area (3.635 m(2)g(-1)), where the surface area and crystallinity mainly affected the sensor performance. Product analysis of the catalytic reaction on the sensor probe revealed a specific two-electron oxidation of dipyrromethane moiety to dipyrromethene in the bilirubin molecule. Experimental variables affecting the analysis of bilirubin were optimized in terms of probe composition, temperature, pH, and potential. At the optimized condition, the dynamic range was between 1.2 µM and 0.42 mM, which yielded the equation of ΔI (µA)=(1.03 ± 0.72)+(457.0 ± 4.03) [C] (mM) with 0.999 of correlation coefficient, and the detection limit was 25.0 ± 1.8 nM (n=5, k=3). The stability test, interference effects, and analysis of real clinical samples, human whole blood and certified serum samples were demonstrated to confirm the reliability of the proposed bilirubin sensor.

Cancer cell detection based on the interaction between an anticancer drug and cell membrane components.

Simple and general cancer cell detection methods are required in point-of-care diagnostics. Herein, the interaction between an anticancer drug, daunomycin, and cancer cell membrane components has been studied using an aptamer probe immobilized on a conducting polymer-gold nanoparticle composite film through electrochemical and fluorescence methods and applied to the quantitative detection of cancer cells. The developed method differentiates between cancerous and noncancerous cells effectively.

Investigation on the downregulation of dopamine by acetaminophen administration based on their simultaneous determination in urine.

A highly sensitive and selective method is developed for the simultaneous detection of dopamine (DA) and acetaminophen (AP) by reactive blue-4 (RB4) dye entrapped poly1,5-diaminonaphthalne (polyDAN) composite film layer. The polyDAN-RB4 composite is electrochemically developed at glassy carbon electrode. The polymeric film, characterized by XPS and SEM is able to catalyze the oxidation of DA and AP. Two well-defined oxidation peaks are observed in the differential pulse voltammogram (DPV). The experimental parameters affecting the analytical performance are optimized in terms of RB4 concentration, temperature, and pH. The dynamic range for DA and AP analysis is between 0.1-150 and 0.2-164µM with a detection limit of 0.061±0.002 and 0.083±0.003µM, respectively. The anionic form of the polyDAN-RB4 composite repels common metabolites present in serum and urine, and hence no interference is observed. The effect of AP on the DA concentrations in urine is also studied after the oral administration of a single as well as multiple doses. The DA concentrations have been found to decrease nearly 50±3% after prolonged AP administration.

A simple separation method with a microfluidic channel based on alternating current potential modulation.

A simple separation and detection system based on an electrochemical potential modulated microchannel (EPMM) device was developed for the first time. The application of alternating current (AC) potential to the microfluidic separation channel walls, which were composed of screen printed carbon electrodes, resulted in the oscillation and fluctuation of analytes and in the formation of a perfect flat flow front. These events resulted in an increase in the effective concentration and in the fine separation of samples. The performance of the EPMM device was examined through the analysis of endocrine disruptors (EDs) and heavy metal ions (HMIs) as model compounds. The analytical parameters that affected the separation and detection of EDs and HMIs were studied in terms of AC amplitude, AC frequency, flow rate, buffer concentration, pH, detection potential, and temperature. The separation efficiency was evaluated through measurements of the theoretical plate number (N), the retention time, and the half-peak width. Linear calibration plots for the detection of EDs and HMIs were obtained between 0.15 and 250.0 nM (detection limit 86.4 ± 2.9 pM) and between 0.01 and 10.0 nM (detection limit 9.5 ± 0.3 pM), respectively. The new device was successfully demonstrated with authentic and real samples.

In vivo detection of glutathione disulfide and oxidative stress monitoring using a biosensor.

A highly sensitive in vivo biosensor for glutathione disulfide (GSSG) is developed using covalently immobilized-glutathione reductase (GR) and -ß-nicotinamide adenine dinucleotide phosphate (NADPH) on gold nanoparticles deposited on poly[2,2':5',2″-terthiophene-3'-(p-benzoic acid)] (polyTTBA). The fabricated biosensor was characterized with SEM, TEM, XPS, and QCM. Analytical parameters affecting the biosensor performance were optimized in terms of applied potential, NADPH:GR ratio, temperature, and pH. A linear calibration plot is obtained using chronoamperometry in the dynamic range between 0.1 µM and 2.5 mM of GSSG, with a detection limit of 12.5 ± 0.5 nM. The developed biosensor is applied to detect GSSG in a real plasma sample. A microbiosensor was applied to detect the in vivo GSSG concentration to monitor the oxidative stress caused by diquat and t-butyl hydroperoxide. The results obtained are reliable, implying a promising approach for a GSSG biosensor in clinical diagnostics and oxidative stress monitoring.

In vitro monitoring of i-NOS concentrations with an immunosensor: the inhibitory effect of endocrine disruptors on i-NOS release.

The amperometric immunosensor has demonstrated the toxicity of endocrine disrupters (EDs) through monitoring the in vitro i-NOS concentration change, where the antibody of inducible nitric oxide synthase (i-NOS) was immobilized on the conducting polymer-gold nanoparticles composite. The performance of the sensor and the experimental parameters affecting the immunoreaction were optimized. Neuronal cells treated by EDs decreased in the in vitro i-NOS concentration. The effect of bisphenol A (BPA) on the i-NOS concentration released in the cells was investigated with different incubation times, and the interfering by nonspecific binding species present in a neuronal cell lysate was also examined. Of all the tested EDs, BPA showed the inhibitoriest effect and the minimum inhibitory concentration of BPA affecting the i-NOS concentration was 0.09 ± 0.005 µM. The result shows that monitoring of i-NOS in the neuronal cells treated by EDs will be a useful method to evaluate the toxic behavior of EDs.

Separation and simultaneous detection of anticancer drugs in a microfluidic device with an amperometric biosensor.

A simple and highly sensitive method for simultaneous detection of anticancer drugs is developed by integrating the preconcentration and separation steps in a microfluidic device with an amperometric biosensor. An amperometric detection with dsDNA and cardiolipin modified screen printed electrodes are used for the detection of anticancer drugs at the end of separation channel. The preconcentration capacity is enhanced thoroughly using field amplified sample stacking and field amplified sample injection techniques. The experimental parameters affecting the analytical performances, such as pH, temperature, buffer concentration, water plug length, and detection potential are optimized. A reproducible response is observed during multiple injections of samples with a RSD <5%. The calibration plots are linear with the correlation coefficient between 0.9913 and 0.9982 over the range of 2-60 pM. The detection limits of four drugs are determined to be between 1.2 (± 0.05) and 5.5 (± 0.3) fM. The applicability of the device to the direct analysis of anticancer drugs is successfully demonstrated in a real spiked urine sample. Device was also examined for interference effect of common chemicals present in real samples.

Detection of daunomycin using phosphatidylserine and aptamer co-immobilized on Au nanoparticles deposited conducting polymer.

A highly sensitive and selective sensor for daunomycin was developed using phosphatidylserine (PS) and aptamer as bioreceptors. The PS and aptamer were co-immobilized onto gold nanoparticles modified/functionalized [2,2':5',2″-terthiophene-3'-(p-benzoic acid)] (polyTTBA) conducting polymer. Direct electrochemistry of daunomycin was used to fabricate a label free sensor that monitors current at -0.61 V. The formation of each layer was confirmed with XPS, SEM, and QCM. Response of the sensor was compared with and without PS in terms of sensitivity and selectivity. Interaction between the sensor probe and daunomycin was determined with DPV. The experimental parameters affecting sensor performance were optimized in terms of concentration of immobilized aptamer, PS:aptamer ratio, temperature, pH, and reaction times. The dynamic range for daunomycin analysis ranged between 0.1 and 60.0 nM with a detection limit of 52.3 ± 2.1 pM. Sensor was also examined for interference effect of other drugs. The present sensor exhibited long term stability and successfully detected daunomycin in a real human urine spiked with daunomycin.

Ag(I)-cysteamine complex based electrochemical stripping immunoassay: ultrasensitive human IgG detection.

An ultrasensitive electrochemical immunosensor for a protein using a Ag (I)-cysteamine complex (Ag-Cys) as a label was fabricated. The low detection of a protein was based on the electrochemical stripping of Ag from the adsorbed Ag-Cys complex on the gold nanoparticles (AuNPs) conjugated human immunoglobulin G (anti-IgG) antibody (AuNPs-anti-IgG). The electrochemical immunosensor was fabricated by immobilizing anti-IgG antibody on a poly-5,2':5',2''-terthiophene-3'-carboxylic acid (polyTTCA) film grown on the glassy carbon electrode through the covalent bond formation between amine groups of anti-IgG and carboxylic acid groups of polyTTCA. The target protein, IgG was sandwiched between the anti-IgG antibody that covalently attached onto the polyTTCA layer and AuNPs-anti-IgG. Using square wave voltammetry, well defined Ag stripping voltammograms were obtained for the each target concentration. Various experimental parameters were optimized and interference effects from other proteins were checked out. The immunosensor exhibited a wide dynamic range with the detection limit of 0.4 ± 0.05 fg/mL. To evaluate the analytical reliability, the proposed immunosensor was applied to human IgG spiked serum samples and acceptable results were obtained indicating that the method can be readily extended to other bioaffinity assays of clinical or environmental significance.

A selective nitric oxide nanocomposite biosensor based on direct electron transfer of microperoxidase: removal of interferences by co-immobilized enzymes.

A highly selective nitric oxide (NO) biosensor was developed by immobilizing microperoxidase (MP) onto the MWCNT-poly-5,2':5',2″-terthiophene-3'-carboxylic acid (PTTCA) nanocomposite. Catalase (CAS) and superoxide dismutase (SOD) co-immobilized on the probe successfully protected the interferences of H(2)O(2) and O(2)(-) during NO detection. The nanocomposite layer showed the direct electron transfer processes of the immobilized CAS, SOD, and MP simultaneously at -0.11/+0.04, -0.33/-0.27, and -0.47/-0.40 V vs. Ag/AgCl. Au nanoparticles (AuNPs) were electrodeposited on a glassy carbon surface to enhance the sensitivity of the sensor probe. The layers of CAS/SOD/MP/MWCNT-PTTCA/AuNPs were characterized using SEM, XPS, and QCM. The CAS/SOD/MP/MWCNT-PTTCA/AuNPs probe showed an excellent performance in the electrocatalytic reduction of NO which was attributed to the heme group of MP. Experimental conditions affecting the sensitivity of the biosensor were optimized in terms of pH, temperature, and applied potential. The dynamic range of NO analysis was from 1.0 to 40 µM with the detection limit of 4.3 ± 0.2 nM. The reliability of biosensor was examined for the determination of NO released from the real samples of rat liver, stomach (AGS), and intestinal (HT-29) cancer cells.

Total analysis of endocrine disruptors in a microchip with gold nanoparticles.

The development of a simple, sensitive, and direct method for the total analysis of certain endocrine disruptors was performed by integrating preconcentration steps to a separation step on a microchip through the modification of the field-amplified sample stacking and field-amplified sample injection steps. To improve the preconcentration and separation performances, the preconcentration and separation buffers were modified with citrate-stabilized gold nanoparticles (AuNPs). For the detection of the separated samples, cellulose-dsDNA/AuNPs-modified carbon paste electrodes were used at the channel end. The experimental parameters affecting the analytical performances, such as the buffer concentration, water plug length, SDS concentration in the separation buffer, AuNPs concentration, preconcentration time, detection potential and electrode to channel distance, were examined. The detection limits of the test compounds were between 7.1 and 11.1 fM and that for 4-pentylphenol was 7.1 (±1.1) fM. Dynamic ranges were in the range from 0.15 to 600.0 pM. The experiments with real samples were performed to evaluate the reliability of the proposed method.

Triggering the redox reaction of cytochrome c on a biomimetic layer and elimination of interferences for NADH detection.

Biomimetic layers triggering the redox process of cytochrome c (cyt c) by beta-nicotinamide adenine dinucleotide (NADH) were fabricated and applied for the detection of NADH. A probe was constructed based on a conducting polymer (poly-5,2':5',2''-terthiophene-3'-carboxylic acid, poly-TTCA) formed on the Au nanoparticles, which were deposited on a screen-printed carbon electrode; the probe was modified with biomaterials including cyt c, lipids (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and cardiolipin), and ubiquinone, which are involved in electron transfer sequence in the cell membrane. To eliminate affection of foreign biological species, we assembled a lipid bilayer using the Langmuir-Blodgett technique by controlling the density of the outward lipid layer. The characteristics of the biomimetic layers were investigated by cyclic voltammetry, impedance spectrometry, quartz crystal microbalance, and X-ray photoelectron spectroscopy. The electron transfer process triggered by the presence of NADH was used to determine the NADH concentration. L-ascorbic acid, uric acid, and NADPH did not show any affection during the detection of NADH at the density controlled-lipid bilayer. Real sample analysis was successfully performed to evaluate the reliability of the proposed biomimetic probe.

Conjugated polymers and an iron complex as electrocatalytic materials for an enzyme-based biofuel cell.

Poly-5,2':5',2''-terthiophen-3'-carboxylic acid (polyTTCA) and poly-Fe(III)-[N,N'-bis[4-(5,2':5',2''-terthien-3'-yl)salicyliden]-1,2-ethanediamine] (polyFeTSED) were synthesized and electrochemically polymerized on an Au surface for use as mediators and catalysts for a biofuel cell. The atomic force microscopy (AFM) images of (a) the TTCA homopolymer (polyTTCA) and (b) the TTCA-FeTSED copolymer (poly(TTCA-FeTSED)) layers show a nanoparticle structure. The enzymes (glucose oxidase (GOx) or horseradish peroxidase (HRP)) were immobilized onto the conducting polymer layer through covalent bond formation, which allowed for direct electron transfer processes of the enzymes. A quartz crystal microbalance (QCM) revealed that the surface coverages of GOx and HRP were 4.21 x 10(-12)M and 9.65 x 10(-12)M, respectively. The anode with immobilized GOx and the cathode with immobilized HRP were used as model enzyme systems in biofuel cells for glucose and H(2)O(2) detection, respectively. The biofuel cell composed of the GOx/polyTTCA/Au and the HRP/poly(TTCA-FeTSED)/Au electrodes was assembled and examined for cell performance. The copolymer of polyFeTSED complex with polyTTCA revealed a catalytic activity for the electrochemical reduction of H(2)O(2) and resulted in approximately a sevenfold increase in the power density of the biofuel cell over that of polyTTCA itself. Moreover, the conjugated polymers extend the biofuel cell lifetime to 4 months through the stabilization of immobilized enzymes. The biofuel cell operated in a solution containing glucose and anode-produced H(2)O(2) generated an open-circuit voltage of approximately 366.0 mV, while the maximum electrical power density extracted from the cell was 5.12 microW cm(-2) at an external optimal load of 25.0 k Omega.

Immunosensors for detection of Annexin II and MUC5AC for early diagnosis of lung cancer.

Amperometric immunosensors were developed to diagnose lung cancer through the detection of Annexin II and MUC5AC. To fabricate the sensor probe, a conducting polymer (poly-terthiophene carboxylic acid; poly-TTCA) was electropolymerized onto a gold nanoparticle/glassy carbon electrode (AuNP/GCE) and a dendrimer (Den) was covalently bonded to the poly-TTCA through amide bond formation, where AuNPs were doped onto the dendrimer. To obtain the final sensor probe, an antibody (anti-Annexin II) and hydrazine (Hyd), which is a catalyst for the reduction of H(2)O(2) generated by glucose oxidase (GOx), were covalently attached onto the Den/AuNP-modified surface. Each surface was then characterized by SEM, impedance spectroscopy and XPS. The final sensor probe was examined before and after interaction with Annexin II and MUC5AC using impedance-spectroscopic, quartz crystal microbalance and amperometric methods. The performance of the immunosensor for the Annexin II was evaluated for the apical surface fluid labeled with GOx by the standard addition method. In this case, the detection limit of the proposed method was 0.051 ng/mL (k=3, n=5). The Annexin II concentration in the secretions collected from squamous metaplastic cells was determined to be 280+/-8.0 pg/mL (n=5).

Direct electrochemistry of laccase immobilized on au nanoparticles encapsulated-dendrimer bonded conducting polymer: application for a catechin sensor.

The direct electrochemistry of laccase was promoted by Au nanoparticle (AuNP)-encapsulated dendrimers (Den), which was applied for the detection of catechin. To increase the electrical properties, AuNPs were captured in the interiors of the dendrimer (Den-AuNPs) as opposed to attachment at the periphery of dendrimer. To prepare Den-AuNPs, the Au(III) ions were first coordinated in the interior of dendrimer with nitrogen ligands and then reduced to form AuNPs. The size of AuNPs encapsulated within the interior of the dendrimer was determined to be 1.7 +/- 0.4 nm. AuNPs-encapsulated dendrimers were then used to covalently immobilize laccase (PDATT/ Den(AuNPs)/laccase) through the formation of amide bonds between carboxylic acid groups of the dendrimer and the amine groups of laccase. Each layer of the PDATT/Den(AuNPs)/laccase probe was characterized using CV, EIS, QCM, XPS, SEM, and TEM. The PDATT/Den(AuNPs)/laccase probe displayed a well-defined direct electron-transfer (DET) process of laccase. The quasi-reversible redox peak of the Cu redox center of the laccase molecule was observed at -0.03/+0.13 V vs Ag/AgCl, and the electron-transfer rate constant was determined to be 1.28 s (-1). A catechin biosensor based on the electrocatalytic process by direct electrochemistry of laccase was developed. The linear range and the detection limit in the catechin analysis were determined to be 0.1-10 and 0.05 +/- 0.003 microM, respectively. Interference effects from various phenolic and polyphenolic compounds were also studied, and the general applicability of the biosensor was evaluated by selective analysis of real samples of catechin.