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Recently, the amount of carnitine in saliva has been considered a biomarker for pancreatic cancer. Traditional analytical instrument such as liquid chromatography and mass-spectroscopy (MS) has been employed for carnitine detection. Only FET based biosensor was reported for carnitine, but the limit of detection was far above the required concentration. Here, we describe a highly sensitive and selective enzyme biosensor that we have developed for carnitine by using an enzyme-modified electrode combined with cathodic stripping voltammetry. The biosensor exhibits high sensitivity because of the preconcentration on the gold electrode of CoA, which was generated by the enzymatic reaction between carnitine and acetyl coenzyme A (acetyl-CoA) with carnitine acetyltransferase. Although both acetyl-CoA and CoA can adsorb on the gold electrode, the potential difference of reductive desorption detects CoA selectively. Under optimized conditions, the biosensor exhibited a linear range from 0.025 to 25 μM with a detection limit (S/N = 3) of 0.025 μM for carnitine determination. Moreover, the recovery experiments determination results show a stable range from 96.6 to 97.2 %. This developed carnitine biosensor has a potential in clinical detection due to the excellent performance.

A thick matrix-free monomolecular layer is highly suitable for detecting small target molecules, because recognition occurs close to the metal surface when the surface plasmon resonance method and electrochemical measurement are used. The detection limit for lectins (carbohydrate recognition proteins) on the monolayer is 100 times better than when using a thick polymer matrix. A thin ligand layer can also be useful for detecting much larger protein interactions, such as antigen–antibody interactions. Specifically, an antibody attached to a monolayer surface performs well as regards to secondary antibody modification once the target molecule has been captured, because there is no steric hindrance with the monolayer. As a result, secondary modification occurs effectively. Using a thin layer, we can realize a 10-fold enhancement with secondary modification.

We report the electrochemical determination of mite allergen in real house dust by using a thin layer electrochemical flow cell and magnetic beads. Der p1, which is an allergen from Dermatophagoides pteronyssinus, was immunochemically sandwiched between two dispersed monoclonal antibodies; one was modified on the surface of magnetic beads and the other was modified with alkaline phosphatase. After washing the beads, a small volume of p-aminophenol phosphate (p-APP) was added to produce p-aminophenol (p-AP). And then the p-AP concentration was measured electrochemically with a homemade electrochemical cell. The Der p1 assay was completed within 30 min and a low detection limit of 0.3 ng/mL was achieved. This is because the diffusion distance of Der p1 and the detection antibody was reduced to 22.3 μm by using dispersed magnetic beads. Only 10 min was required to complete the entire immunoreaction, and 54% of the Der p1 was confirmed to have immunoreacted in only 1 min of mixing. Furthermore, the p-APP volume could be reduced using the thin-layer electrochemical flow cell. This is advantageous in terms of concentrating p-AP, and provides a high signal-to-noise ratio measurement in a short time. We achieved a high correlation (r=0.967, p

Herein, we report on the fabrication of an extended-gated organic field-effect transistor (OFET)-based immunosensor and its application in the detection of human chromogranin A (hCgA). The fabricated OFET device possesses an extended-gate electrode immobilized with an anti-CgA antibody. The titration results of hCgA showed that the electrical changes in the OFET characteristics corresponded to the glycoprotein recognition ability of the monoclonal antibody (anti-CgA). The observed sensitivity (detection limit: 0.11 µg/mL) and selectivity indicate that the OFET-based immunosensor can be potentially applied to the rapid detection of the glycoprotein concentration without any labeling.

Cytosine methylation in DNA was determined by an enzyme linked immunosorbent assay (ELISA) with electrochemiluminescence (ECL) detection and employed for the DNA methylation assay of a long and real genomic sample for the first time. The developed method employed an antimethyl cytosine antibody labeled with acetylcholinesterase, which was added to recognize single methylated cytosine in a DNA oligomer. The acetylcholinesterase converted acetylthiocholine (substrate) to thiocholine (product), which was accumulated on a gold electrode surface via gold-thiol binding. This surface accumulated preconcentration made it possible to observe bright and distinctive ECL by applying a potential to the gold electrode in the presence of a tris(2,2-bipyridyl)ruthenium complex luminophore when the analyte DNA contained a methylation region. Methyl-cytosine was measured quantitatively in the 1-100 pmol range, which exhibits sufficiently high sensitivity to achieve real DNA measurements without amplification by a polymerase chain reaction (PCR). The proposed ECL method also exhibited high selectivity for methyl-cytosine against nonmethylated cytosine, guanine, thymine, and adenine nucleotides. Finally, original and methylated DNA samples were clearly distinguished with our method using a real DNA bacteriophage sample (48,502 base pairs).

An on-chip enzyme-linked immunosorbent assay combined with an electrochemical detection method (EC-ELISA) was employed to detect a leptin, one of the most important adipose derived hormones, using gold electrodes modified with a tri(ethylene glycol) terminated short alkanethiol (TEGCnSH, Cn = (CH(2))n, n = 2, 4, 6, and 8) monolayer. These TEGCnSH monolayers on gold electrodes can suppress non-specific protein adsorption without affecting the electrochemical activity required for detecting p-aminophenol (PAP), which is an alkaline phosphatase (ALP) product. We measured leptin with a highly sensitive detection range (100 pg mL(-1) to 10 ng mL(-1) level) and with the desired detection limit (13.6 pg mL(-1)) by using electrochemical detection. For detecting leptin, the EC-ELISA method using TEGC4SH modified gold electrode with a poly(dimethylsiloxane) based microchannel was superior to the conventional ELISA method. With the EC-ELISA method, we were able to measure leptin with a satisfactory detection range and a pg level detection limit within 30 min, which is a much lower detection level than that obtained with conventional plate based ELISA.

Ferrocene-attached polymyxin B (PMB-Fc) was prepared by the reaction of polymyxin B with ferrocenoyl chloride in a toluene/pyridine mixture. An electrochemical detection of lipopolysaccharide (LPS) was carried out using a combination of PMB-Fc and an enzyme-modified electrode constructed from a glassy carbon electrode modified with a bovine serum albumin membrane containing glucose oxidase. The ferrocene units of the PMB-Fc molecules were oxidized on the electrode, and then reduced to the original neutral form by a glucose oxidase-catalyzed reaction in the presence of D-glucose. The consumption/regeneration cycle for PMB-Fc resulted in a chemically amplified current response. The current response for PMB-Fc decreased in association with its complexation with LPS, and the magnitude of this current decrease caused by LPS was also amplified by the recycling process. The enzyme-modified electrode exhibited a rapid response of 5 min for LPS with the detection limit as low as 50 ng ml(-1). Further, the addition of D-solbitol or poly(vinyl alcohol) of high concentration over 1 mg ml(-1) substantially induced no response, and three kinds of LPS from different strains exhibited similar magnitudes of current response for the same concentrations; these results suggest the advantages of this detection system for practical applications. Ferrocene-attached colistin, an analogue of PMB-Fc, was also effective for the LPS detection using the glucose oxidase-modified electrode.

We have developed a miniaturized electrochemical surface plasmon resonance biosensor for measuring two biomolecules that have very different molecular sizes, one is transferrin (MW = 75 kDa) as a disease marker protein, the other is creatinine (MW = 113) as a calibration marker for the accurate measurement of human urinary samples. The sensor has a PDMS based microchannel that is 2 mm wide and 20 μm deep. Two gold films were integrated in the microchannel; one was modified with anti-transferrin antibody for immuno-reaction, and the other was modified with osmium-poly-vinylpyridine wired horseradish peroxidase (Os-gel-HRP). We further immobilized a tri-enzyme layer of creatininase, creatinase and sarcosine oxidase in order to measure creatinine by converting it to hydrogen peroxide in the upstream channel. We measured the transferrin concentration from the refractive index change involved in an immuno-complex formation, and we were simultaneously able to measure creatinine by employing the refractive index change in the Os-gel-HRP caused by oxidation with the hydrogen peroxide produced from creatinine by the tri-enzyme. The effects of ascorbic acid and uric acid in urine samples were sufficiently eliminated by adding ascorbate oxidase and uricase to the urine samples during sampling. We were able to measure two analyte concentrations within 15 min by one simple injection of 50 μL of diluted human urine into our sensor. The detectable transferrin and creatinine ranges were 20 ng/mL to 10 μg/mL, and 10 μM to 10 mM, respectively, which are sufficient levels for clinical tests. Finally, we compared the results obtained using our sensor with those obtained with a conventional immunoassay and the Jaffe method. We obtained a similar trend that can reduce the fluctuation in the urinary transferrin concentration from three different samples by calibrating the creatinine concentration.

A novel electrochemical enzyme immunoassay system with a detection limit of 2 pM (10 ng L −1 ) was developed for the determination of insulin, an important hormone for clinical diagnosis of diabetes. An insulin-containing sample solution was added to a solution containing a certain concentration of glucose oxidase (GO x )-labeled anti-insulin antibody to undergo an immunological reaction, and unbound GO x –antibody conjugate was trapped on the insulin-immobilized glass plate. Then hydrogen peroxide generated by the enzymatic reaction with the trapped conjugate was coulometrically determined as follows. The hydrogen peroxide generated oxidized [Os(bpy) 2 Cl] + to [Os(bpy) 2 Cl] 2+ in a horseradish peroxidase (HRP)-containing polymer layer on an electrode under the open-circuit condition, and then the [Os(bpy) 2 Cl] 2+ accumulated was reduced by applying a negative potential. The accumulation brought about a large current response and a low detection limit for hydrogen peroxide (0.5 nM): the detection limit was lowered by more two orders of magnitude than that obtained for conventional amperometric method using the same polymer-coated electrode. The enhanced sensitivity for hydrogen peroxide enabled the determination of a trace concentration of insulin with the immunoassay using the antibody modified with a hydrogen peroxide-producing enzyme. The assay time was ca. 30 min. The results suggested the possible application of the proposed method to an on-site diabetic sensing system.

Quantitative analyses of olive oil for lipophilic antioxidants, such as α-tocopherol and phenolics, by simple electrochemical measurements were conducted in a bicontinuous microemulsion (BME), which was bicontinuously composed of saline and toluene microphases with a surfactant system. Lipophilic antioxidants in oils were directly monitored in BME solutions using a lipophilic, fluorinated nanocarbon-film electrode (F-ECR). The combination of a well-balanced BME and extremely biased electrodes, such as strongly hydrophilic indium/tin oxide and strongly lipophilic (hydrophobic) F-ECR, allowed individual monitoring of hydrophilic and lipophilic antioxidants in the same BME solution without any required extraction. Furthermore, values for the charge Q, integrated from observed currents, showed good linear relationships with the results of conventional assays for antioxidant activity, namely, total phenolics and oxygen radical absorbance capacity assays, even with practical food samples. This proposed methodology provided a very simple, rapid, easily serviceable, and highly reproducible analysis that possesses great potential for applications to a wide range of chemical mixtures, in terms of analyte and media, beyond food oils.

B-type natriuretic peptide (BNP), a peptide hormone, was determined by electrochemical enzyme immunoassay through the following procedure. First, a certain concentration of sample BNP was added to the solution containing anti-BNP antibody modified with acetylcholinesterase (AChE) to undergo the immunological reaction. Then BNP-modified gold particles were added into the BNP/AChE-labeled anti-BNP antibody solution so that the unreacted AChE-labeled anti-BNP antibody was removed from the assay solution. Finally the AChE activity in the solution was measured, based on the chemisorption/electrochemical reductive desorption process of a thiol compound on a silver electrode. The chemisorption (accumulation) of thiocholine, which was produced through the enzymatic reaction, resulted in the enhanced sensitivity for the determination of AChE, and accordingly, for that of BNP. BNP could be determined in the concentration range from 20 to 200 ppt (pg mL−1), which indicated that the proposed enzyme immunoassay procedure was high enough for the measurement of the peptide hormone in blood samples.

We developed a microfluidic device for the continuous monitoring of catecholamine levels and studied the performance of two components of the device, namely an enzyme-modified pre-reactor and an interdigitated array (IDA) electrode. The enzyme-modified pre-reactor and IDA electrode were integrated on a chip. The integrated pre-reactor with a 3D structure, consisted of rectangular-shaped micropillars immobilized with ascorbate oxidase, and was used for the enzymatic removal of the electroactive interferent, l -ascorbic acid (AA). It exhibited elimination efficiencies of 99.7% and 98% for AA concentrations of 10 and 100 μM, respectively. We used a carbon-based IDA electrode (2 μm line and space) fabricated in a microflow channel for the highly sensitive detection of CAs. Redox cycling at the IDA electrode and an increase in the mass transport to the electrode contributed to the highly sensitive detection of dopamine (DA) and adrenaline (AD). The detection limits of the device for DA and AD standard solution in the absence of AA were 108 and 440 pM, respectively.

Catecholamine, a well-known neurotransmitter, is released to restore heart function after the occurrence of cardiac ischemia. Endogenous ascorbic acid interferes considerably with the monitoring of neurotransmitters such as catecholamine and glutamate. In this work we have successfully developed a nanoliter volume monolithic enzyme reactor and integrated it with a carbon film electrode for monitoring dopamine in order to scavenge such electroactive interferents as ascorbic acid. A monolithic silica support prepared by the sol-gel process has a large through-pore structure and a nanoporous surface. The low back pressure resulting from the large though-pore structure makes it possible to immobilize the enzyme and introduce fluid into the flow monitoring system by using a microsyringe pump. Ascorbate oxidase (AOx) was immobilized on the nanoporous surface of a monolithic matrix by physical adsorption and used for converting the ascorbic acid into its electrochemically inert form, dehydroascorbic acid, while monitoring dopamine. We showed that more than 99.8% of 100 μM of ascorbic acid could be converted to its oxidized form in the monolithic enzyme reactor. We succeeded in monitoring dopamine at a concentration of less than 100 nM in the presence of 50 μM of ascorbic acid. Therefore, this work demonstrated that a monolithic silica support offers excellent potential in regards to realizing a highly selective enzymatic reactor for biosensors.

Qualitative and quantitative analyses of hydrophilic and lipophilic antioxidants, such as polyphenols, by simple electrochemical measurements were conducted in a bicontinuous microemulsion (BME), in which water and oil phases coexisted bicontinuously on a microscopic scale. Hydrophilic and lipophilic antioxidants were individually monitored in the same BME solution using a hydrophilic indium tin oxide (ITO) electrode and a lipophilic fluorinated nanocarbon film electrode (F-ECR), respectively. The combination of well-balanced BME and extremely biased electrodes, such as ITO and F-ECR, in terms of hydrophilic-lipophilic balance allowed us to achieve individual monitoring of hydrophilic and lipophilic antioxidants in the same BME solution without extraction. Furthermore, the antioxidant activities of functional liquid foods, such as coffee and olive oil, were also evaluated by means of electrochemical measurements in BME solutions containing analytes in concentrations of several percent. The technique we propose provides a very simple, rapid, easily serviceable, and highly reproducible analysis and can be extended to a wide range of analytes and media.

A microfluidic device integrated with a micro-pre-reactor and glucose- and lactate-sensing microelectrodes in a dual channel was fabricated for in vivo monitoring using a micromachining technique. The device consists of two glass plates bonded together with UV-curable resin. The microelectrodes were made of carbon film, and a thin layer flow channel and a flow separator between the glucose and lactate electrodes were fabricated from 23-μm-thick positive photoresist. One carbon electrode was modified with a bilayer of glucose oxidase (GOD)/osmium-poly(vinylpyridine) wired horseradish peroxidase (Os-gel-HRP) and the other was modified with a bilayer of lactate oxidase (LOX)/Os-gel-HRP. The other glass plate has two rectangular flow channels created by chopper cutting with a dicing saw to enable a microdialysis (MD) probe and an outlet tube to be fitted. The cathodic currents of both the glucose and lactate electrodes started to increase rapidly after a microdialysis probe was dipped in glucose or lactate solution, and quickly reached a steady state value. We observed no chemical crosstalk between the glucose and lactate electrodes under our measurement conditions despite the short distance between them. By combining the device with a microdialysis probe, we obtained a linear relationship between the cathodic currents and glucose and lactate concentrations of 5 μM to 5 mM, which covers a concentration range sufficient for measuring brain glucose and lactate. The effect of l -ascorbic acid ( l -AA), which is known to cause major interference during in vivo measurement, was eliminated when ascorbate oxidase was incorporated in the device upstream of the thin-layer flow channel. We successfully observed a glucose concentration decrease and lactate concentration increase in a rat brain when we perfused the striatum with veratridine solution.

A novel biosensor for immunoglobulin G (IgG) detection based on an extended-gate type organic field effect transistor (OFET) has been developed that possesses an anti-IgG antibody on its extended-gate electrode and can be operated below 3 V. The titration results from the target IgG in the presence of a bovine serum albumin interferent, clearly exhibiting a negative shift in the OFET transfer curve with increasing IgG concentration. This is presumed to be due an interaction between target IgG and the immobilized anti-IgG antibody on the extended-gate electrode. As a result, a linear range from 0 to 10 µg/mL was achieved with a relatively low detection limit of 0.62 µg/mL (=4 nM). We believe that these results open up opportunities for applying extended-gate-type OFETs to immunosensing.

We fabricated a microfluidic device for the optical detection of airborne benzene, toluene, ethylbenzene and xylenes (BTEX). The device consists of concentration and detection cells formed of 3 cm x 1 cm Pyrex plates. The concentration cell is composed of an adsorbent to concentrate the BTEX gases and a thin-film heater todesorb the concentrated gases from the adsorbent thermally. The collected gases are introduced into the detection cell, which is connected to optical fibers, to measure their absorption spectra. We optimized the device's operating conditions by studying the thermal characteristics of the concentration cell and the time profile of the gas concentration flowing in the detection cell. We used the device under optimized operating conditions to detect toluene gas as a typical example BTEX. The gas concentration amplification rate was approximately 2 orders of magnitude, and we successfully measured parts-per-million levels of toluene gas with this device.

We used a micromachining technique to fabricate a very reliable amperometric glutamate sensor consisting of dual channel thin layer flow cells for the highly selective measurement of glutamate released from cultured nerve cells. Our microfabricated sensor has two carbon film electrodes separated by a separator in the thin layer channel flow cells. We modified one carbon electrode with a bilayer of Os-gel-HRP/BSA film containing GluOx, and the other with a bilayer of Os-gel-HRP/BSA film without GluOx. Therefore, any difference between the currents at the dual electrodes should be due to the oxidation of glutamate by GluOx. This dual mode measurement technique can eliminate current changes caused by the change in the double layer capacitance when KCl solution is added to stimulate the cells or by baseline fluctuations resulting from pumping noise. As a result, we were able to monitor the transient peak caused by the glutamate release from cultured rat cortex cells with high selectivity by stimulating the cells with 100 mM of KCl solution. The specificity of the GluOx for glutamate was improved when the sensor was pre-treated with 6-diazo-5-oxo- l -norleucine (DON), which is known to be a glutaminase inhibitor. The signal we obtained at the DON-treated sensor when we measured the extracellular solution was lower than that at an untreated sensor. This result shows that the current caused by extracellular glutamine was eliminated by the DON treatment. Moreover, the extracellular glutamate concentration measured with the DON pre-treated sensor was consistent with that obtained by HPLC.

Histamine release from rat basophilic leukemia (RBL-2H3) cells was continuously measured using a histamine oxidase (HO)-immobilized on-line electrochemical sensor. The sensor has a carbon-based electrode modified with a bilayer of osmium-polyvinylpyrridine-based gel containing horseradish peroxidase (HRP) and BSA containing HO. The high specificity of the enzyme made the sensor highly selective with regards to diamines such as putrescine and cadaverine. We obtained a low detection limit of 23 nM owing to the low operation potential of the sensor (below 0 V vs. Ag/AgCl) and the high activity of the enzyme after immobilization. Moreover, the sensor had a high conversion efficiency. The sensor response was unaffected by 400 min of continuous histamine measurement and by more than 2 weeks of storage in a refrigerator. We combined the histamine sensor with a microdialysis (MD) sampling probe and used it to measure the histamine concentration of RBL-2H3 cells stimulated with dinitrophenylated bovine serum albumin (DNP-BSA) after incubation with IgE. We also fabricated a small volume histamine sensor by the microfabrication technique and were able to measure the histamine release from a small mast cell colony in real time with the sensor by drawing the extracellular solution continuously through a sampling capillary located near the cells.

We have developed a highly sensitive and selective on-line biosensor for detecting the reduced form of nicotinamide adenine dinucleotide (NADH) produced by the enzymatic reactions of dehydrogenases with various substrates such as glutamate. The sensor consists of a glassy carbon electrode modified with an osmium-polyvinylpyridine-based bottom layer containing horseradish peroxidase, and a bovine serum albumin (BSA)–gluteraldehyde (Glut) top layer containing NADH oxidase (NOX) or glutamate dehydrogenase (GluDH) and NOX. We assembled the modified electrode in a thin-layer radial flow cell and sample solution was continuously introduced into the cell with a syringe pump. We optimized the sensitivity of the NADH sensor by adjusting the glutaraldehyde amount in the immobilized layer, the applied potential and the pH of buffer solution. We examined the flow-rate effect on the current response and the conversion efficiency of NADH at the modified electrode. As a result, we achieved a sensitivity of 48.8 nA cm−2 μM−1, a detectable concentration range of 25 nM∼10 μM and a detection limit of 20 nM (S/N=3) for the NADH sensor. The interference from ascorbic acid and other electroactive interferents can be greatly reduced since the sensor can be operated below 0 mV versus Ag/AgCl. The NADH sensor is relatively stable since it retains 70% of its original response after 1 month if stored at 2–8°C in a dry state after use. Furthermore, we fabricated a glutamate sensor by coimmobilizing GluDH and NOX in the BSA–Glut top layer. The detectable glutamate concentration range is from 0.1 to 10 μM and the detection limit is 0.1 μM (S/N=3). Our glutamate dehydrogenase-based sensor offers good selectivity as regards other amino acids.

A highly sensitive amperometric detector for the liquid chromatographic analysis of carbohydrates has been developed based on the optimization of the Ni content in a Ni–Ti alloy electrode. An optimum nickel content of 30 at. % was obtained from a signal-to-noise analysis in a flow injection experiment on glucose in 0.1 M NaOH. An optimum working potential of 0.5 V vs. Ag/AgCl was obtained from a hydrodynamic voltammogram. The glucose detection limit in the flow injection analysis was 11 fmol. The electrode was applied to an LC detector with a microbore column and the detection limit was 50 fmol.

We have developed an on-line electrochemical sensor for the continuous measurement of acetylcholine (ACh). The sensor comprises a small volume prereactor containing immobilized choline oxidase (ChOx) and catalase (or horseradish peroxidase, HRP), a small volume enzymatic reactor containing immobilized acetylcholine esterase (AChE) and ChOx and a glassy carbon electrode modified with Os-polyvinylpyrridine -HRP. The sensor can measure ACh selectively even when the choline (Ch) concentration is more than 1000 times that of ACh, when th ChOx and catalase are immobilized in the prereactor. The sensor shows a wide linear relationship from 5 nM to 1 μM and a detection limit of 5 nM (S/N = 2), which is comparable to the ACh detection limit achieved by liquid chromatography/electrochemistry. It also shows high selectivity against other neurotransmitters and interferents. The properties of the sensor combined with a microdialysis (MD) probe were also studied by measuring a solution containing a much higher concentration of Ch than ACh.

Tetrahydrobiopterin and seven of its analogues (neopterin, xanthopterin, biopterin, pterin, tetrahydropterin, 6-methyltetrahydropterin, and 6-methylpterin) were separated on a 1 × 150 mm C18 microbore column. These analytes were detected by dual-electrode amperometry and UV absorption. Both a conventional glassy carbon electrode and an interdigitated array microelectrode were used. Low fmol amounts of xanthopterin, tetrahydropterin, tetrahydrobiopterin and 6-methyltetrahydropterin could be determined by electrochemical detection in the oxidative mode, but pmol amounts of the other analogues were determined by electrochemical detection in the reductive mode and with UV detection. Catecholamines and their metabolites do not interfere with the determination of tetrahydrobiopterin and its analogues in biological samples. The developed method was explored for the determination of tetrahydrobiopterin in samples of human urine and rat tissue (brain, liver, and kidney).

A small volume L-glutamate online sensor was developed in order to monitor changes in the local concentration of L-glutamate released from cultured nerve cells. Syringe pump in the suction mode is used to sample extracellular fluid continuously from a glass micro-capillary and the concentration of L-glutamate can be determined by using a glassy carbon (GC) electrode modified with an Os-polyvinylpyridine mediator bottom film containing horseradish peroxidase and a bovine serum albumin top layer containing L-glutamate oxidase. The overall efficiency of L-glutamate detection with a sensor is 71% under optimum conditions due to an efficient enzymatic reaction at the modified electrode in the thin layer radial flow cell. As a result, we achieved a detection limit of 7–15 nM and a linear range of 50 nM to 10 μM. In an in vitro experiment, the extracellular fluid near a particular nerve cell can be sampled with this micro-pipet and continuously introduced into the modified GC electrode in the radial flow cell via suction provided by a syringe pump. The nerve cells are stimulated by KCl in a glass capillary and the L-glutamate concentration change can be monitored by changing the distance between the sampling pipet and the nerve cells.

To determine the basal acetylcholine level in the dialysate of rat frontal cortex, a horseradish peroxidase-osmium redox polymer-modified glassy carbon electrode (HRP-GCE) was employed instead of the conventional platinum electrode used in high-performance liquid chromatography-electrochemical detection (HPLC-ED). In initial experiments, an oxidizable unknown compound interfered with the detection of basal acetylcholine release on HPLC-HRP-GCE. An immobilized peroxidase-choline oxidase precolumn (pre-reactor) was included in the HPLC system, to eliminate the interference from the unknown compound. This combination could detect less than 10 fmol of standard acetylcholine and basal acetylcholine levels in the dialysate from a conventional concentric design microdialysis probe, without the use of cholinesterase inhibitor, and may facilitate physiological investigation of cholinergic neuronal activity in the central nervous system.

A method for the highly sensitive determination of acetylcholine (ACh) and choline (Ch) was developed using platinum (Pt) black microarray electrodes as detectors in a microbore liquid chromatography (LC) with a post column enzyme reactor. The electrodes were prepared by plating a gold (Au) film electrode with sub-μm Pt-black particles. Since hydrogen peroxide generated by the enzymatic reaction of ACh and Ch was oxidized only at the Pt-black microarray electrode, each Pt-black particle (typically 0.1–0.2 μm in size) operated as an ultramicroelectrode. A high signal-to-noise ratio was achieved because of the high current density at the Pt-black microarray electrodes and because the Au film has a much lower baseline noise than the Pt. Detection limits of 5.7 (ACh) and 6.0 (Ch) fmol were obtained, with a wide linear range. The ACh and Ch signals with an Au film electrode modified with Pt-black particles retained more than 70% of their initial value after 5 days with continuous potential application. This is better stability than for a bare platinum electrode which retained only 40% of its initial response under comparable conditions.

A hybrid functional biomolecular interface designed at a molecular size level is very effective at capturing an analyte with high sensitivity even if the interaction is very weak, as when detecting proteins with carbohydrate. We designed and processed a protein (lectin) recognition molecular interface taking the following points into consideration: (1) the height (molecular length) difference between the capturing and spacer molecules; (2) the ratio of capturing molecules in the recognition interface. When the height difference between the maltoside part (Concanavalin A (Con A) recognition group) and the OH group terminated spacer molecules exceeded ((CH(2))(6)), the association rate constant (k(a)) became larger (k(a)(1/Ms): ∼2.6 times) and the dissociation constant (K(D)) became much smaller (K(D)(M): 1.0 × 10(-6): ∼0.17 times) compared with the similar heights (lengths) of both molecular interfaces. With regard to maltoside density, a 100% maltoside monolayer was unsuitable for detecting Con A. We constructed a nanostructured recognition site with a maltoside part of 10%, which was the most suitable ratio for Con A detection. The binding interaction between Con A and the maltoside group was changed from monovalent binding to bivalent binding when the maltoside part was diluted in the recognition interface. From electrochemical measurements, even though there was a small amount of maltoside component on the suitable recognition monolayer, quality similar to that of 100% maltoside was observed.

A small-volume voltammetric detection of 4-aminophenol (PAP) has been developed using an interdigitated array (IDA) microelectrode cell in order to apply the IDA to electrochemical enzyme immunoassay. The signal of PAP at the IDA was steady state, and its magnitude was amplified compared with that of the usual single electrode due to redox cycling of PAP between the two finger sets of the IDA. A linear relationship between PAP concentration and cathodic limiting current was obtained from 1 to 1000 microM, reproducibly. The minimum sample volume in the measurement was reduced to 800 nL. High sample throughput of less than 1-min detection time per sample was achieved on 2-10-microL PAP samples. This IDA cell was applied to the electrochemical enzyme immunoassay of mouse IgG. Alkaline phosphatase was used as the enzyme label. The mouse IgG concentration was evaluated by detecting the concentration of PAP, which is the product of enzymatic reaction of the substrate, 4-aminophenyl phosphate (PAPP). Anti-mouse IgG was covalently immobilized on the glass surface of the small-volume immunowells by carbodiimide coupling. The assay range was 10-1000 ng/mL using 10-microL sample and 20-microL substrate solutions.

The highly sensitive detection of catecholamine has been achieved by using an interdigitated array (IDA) microelectrode as an electrochemical detector in a microbore high-performance liquid chromatography (HPLC) system. The current responses for catecholamine are measured for IDA electrodes with different gaps between their adjacent band electrodes. An IDA electrode with a narrow gap produces a high current density and reduces the influence of the auxiliary electrode position on the current response. The current response measured for an IDA electrode incorporated in the microbore HPLC system is about four times greater than that measured for a combination of the same IDA electrode and an ordinary sized column due to current enhancement by redox cycling in the slower stream.

This paper reports the development and characterization of a highly sensitive enzyme linked immunosorbent assay realized by the electrogenerated chemiluminescence (ECL) detection of a thiol monolayer formed by an enzyme labeled antibody. We used two monoclonal anti tumor necrosis factor-alpha (TNF-alpha) antibodies for a sandwich immunoassay. One was a capture antibody, and the other was a detection antibody labeled with an enzyme via an avidin-biotin interaction. Acetylcholinesterase was used as the labeling enzyme to convert acetylthiocholine to thiocholine. Then the thiocholine was collected on a gold electrode surface by gold-thiol binding. A bright and distinctive emission was observed at 1150 mV (vs Ag-AgCl) on the gold electrode with a thiocholine monolayer as a coreactant in the presence of tris(2,2'-bipyridyl)ruthenium complex. This method can greatly enhance the immunoassay signal since a large number of coreactant molecules can be generated by the enzymatic reaction, which is advantageous compared with a previously reported ECL based immunoassay that directly labels the detection antibody with a coreactant or luminophore. In addition, a surface accumulated coreactant is superior to the previously reported coreactant system in a bulk solution, because ECL emission occurs only very close to an electrode surface. As a result, high sensitivity and a low detection limit of 0.2 pM (3.4 pg/mL) TNF-alpha were achieved with excellent reproducibility by optimizing the conditions for the immuno-reaction, thiocholine accumulation, and ECL generation.

This paper reports a miniaturized immunosensor designed to determine a trace level cardiac marker, B-type natriuretic peptide (BNP), using a microfluidic device combined with a portable surface plasmon resonance (SPR) sensor system. Sample BNP solution was introduced into the microchannel after an immunoreaction with acetylcholine esterase-labeled antibody (conjugate), and only unbound conjugate was trapped on the BNP-immobilized surface in the flow channel. Then, the thiol compound generated by the enzymatic reaction with the trapped conjugate was accumulated on a gold thin film located downstream in the microchannel to monitor the real-time SPR angle shift. We achieved a detectable concentration range of 5 pg/mL-100 ng/mL by monitoring the SPR angle shift, which covers the required detection range for the BNP concentrations found in blood. This success resulted from the use of a T-shaped microfluidic device structure, which prevents the sample solution from flowing over the gold film used for SPR detection. We were able to measure trace levels of BNP peptide (15 fg) within 30 min since the procedure with our immunosensor is simpler than a multistep immunoassay through the simultaneous use of a labeled enzymatic reaction and the real-time monitoring of enzymatic product accumulation in the microfluidic device. We employed the procedure to detect serum BNP by using spiked samples in human serum and achieved satisfactory recovery for heat-treated samples to denature the esterase in the serum before the immunoreaction.

A novel electrochemical technique for lipopolysaccharide (LPS) detection has been developed using a combination of ferrocenylboronic acid derivatives and an enzyme-modified electrode. The enzyme-modified electrode was constructed from a gold electrode modified with a bovine serum albumin membrane containing diaphorase. Ferrocenylboronic acid derivatives are oxidized on the electrode, and then regenerated by a diaphorase-catalyzed reaction in the presence of NADH. The consumption/regeneration cycle for ferrocenylboronic acid derivatives resulted in a chemically amplified current response. The current response for ferrocenylboronic acid derivatives decreased in association with its complexation with glycosyl units of LPS, and this current decrease caused by LPS was also amplified by the recycling process. On the other hand, the addition of a monosaccharide such as D-mannose or D-galactose induced no response at the same LPS concentration. The enzyme membrane immobilized on the electrode plays an important role in selectivity as well as chemical amplification. In addition, the enzyme-modified electrode exhibited a rapid response of 5 min for LPS, which is much faster than the currently used method. The detection limit of LPS from Escherichia coli O127:B8 was as low as 50 ng ml-1.

A novel electrochemical enzyme immunoassay system with a 10 ng L(-1) level detection limit was developed for the determination of B-type natriuretic peptide (BNP), an important marker for the diagnosis of heart failure. Sample BNP was added to a solution containing a certain concentration of acetylcholinesterase(AChE)-labeled anti-BNP antibody to undergo an immunological reaction. After the immunological reaction, we proposed two assay schemes. One involves measuring the amount of antibody-enzyme conjugate that reacted with two BNP molecules (reacted conjugate). The other involves measuring the amount of antibody-enzyme conjugate with at least one free binding site (unreacted conjugate). Then the amount of reacted or unreacted conjugate was determined by measuring the AChE activity after the recovery of each conjugate from the immunological reaction mixture. To determine the trace level of the recovered antibody-enzyme conjugate, the AChE activity was determined with high sensitivity on the basis of the chemisorption/electrochemical desorption process of thiocholine, which was produced through the enzymatic reaction, on a silver surface. The thiocholine chemisorption (i.e., accumulation) on the silver electrode surface resulted in a sensitivity for the electrochemical determination of the AChE activity that was 2 orders of magnitude greater than that obtained when using direct measurement without accumulation. The procedure for determining the AChE activity of unreacted conjugate after its recovery on a BNP-modified disk was applied to the determination of BNP in serum samples. This procedure involves the removal of the immunological reaction mixture before the enzymatic reaction process, which allows the AChE activity to be measured without any interference from endogenous pseudocholinesterase, which exists with high activity in serum. With both procedures, the BNP could be measured within an hour. The detection limits were 20 and 40 ng L(-1) using the reacted and unreacted conjugate measuring procedures, respectively.

We have developed a microfluidic device consisting of a gold film working electrode modified with lactate oxidase and Os-poly(vinylpyridine) mediator containing horseradish peroxide, and reference and counter electrodes in a microflow detection channel separated by a microdialysis membrane from another microflow channel used for sample injection. The dialysis membrane is cellulose with a molecular weight cut off of 10 kDa. We achieved control over a wide recovery rate range of 3-94% because the device is capable of controlling both flow rates in the dual thin-layer channels. We were able to measure the lactate concentration in blood samples within a few minutes without any pretreatment because biomolecules are simultaneously separated by molecular weight and detected in the device. We achieved quantitative and reproducible measurements of the lactate concentration in blood samples, and obtained a relative standard deviation of 1.5% (n = 8). With our device, the lactate concentration in dog whole blood was measured with high stability without any pretreatment.

We fabricated a micro-fluidic device for the highly selective detection of the histamine released from rat basophilic leukemia (RBL) 2H3 cells. The device has two thin layer flow channels, each with one working electrode. One electrode was modified with Os-polyvinylpyridine based mediator containing horseradish peroxidase (Os-gel-HRP) and histamine oxidase (HAOx), the other was modified with Os-gel-HRP without any HAOx. We employed the device for differential measurement by using the HAOx modified electrode for detection and the unmodified electrode as a reference. The detection limit was greatly improved from 190 to 25 nM since the baseline noise level was suppressed. We used differential measurement to observe the histamine released from RBL-2H3 cells when stimulated with dinitrophenylated bovine serum albumin (DNP-BSA) as an antigen. We injected 5 microM of histamine solution into our device and it remained stable for more than 8 h.

This paper proposes a very simple procedure for preparing a biocompatible sensor based on a protein (bovine serum albumin, BSA), enzyme and vinylferrocene (VF) composite membrane modified electrode. The membrane was prepared simply by first casting vinylferrocene and then coating it with BSA and glucose oxidase immobilised with glutaraldehyde. The sensor response was independent of dissolved oxygen concentration from 3 to 10 ppm and showed good stability for serum sample measurement, unlike the commonly used BSA/enzyme modified electrode. The sensor response was almost unchanged over the measurement time (>10 h) whereas the responses of a BSA and glucose oxidase modified platinum electrode and an osmium-polyvinylpyrridine wired horseradish peroxidase modified electrode ( Ohara et al., 1993 ) fell to 68% of their initial value in a serum sample containing 10 mM glucose.

An IDA carbon microelectrode for HPLC was fabricated by phototithographic techniques from the carbon film of pyrolyzed 3,4,9,10-perylenetetracarboxytic dianhydride on thermally oxidized silicon wafers. The IDA electrode consisted of 300 pairs, the finger widths and gaps were 5 μm, and each finger was 2 mm long. The electrode in a small-volume thin-layer cell incorporated in a microbore HPLC system achieved a low detection limit for dopamine due to current enhancement by redox cycling and low background noise at the carbon IDA microelectrode. The detection limit of 0.5 fmol of dopamine can be realized because of the low noise level and high current density

A surface plasmon resonance (SPR) based biosensor was developed for monitoring 2,4-dichlorophenol, a known dioxin precursor, using an indirect competitive immunoassay. The SPR sensor was fabricated by immobilizing a gold-thin layer on the surface of an SPR sensor chip with an anti-(2,4-dichlorophenol) antibody using a gold binding polypeptide (GBP) and protein G. The SPR response based on the antigen-antibody reaction in a flow system was measured by injecting a 2,4-dichlorophenol sample solution into the flow system in which the SPR sensor was located. In a direct immunoassay system using the modified sensor chip, no significant SPR angle shift less than 0.001 degrees was observed when a 25 ppm of 2,4-dichlorophenol solution was injected. In order to improve the sensitivity of the SPR sensor, an indirect competitive immunoassay method was used in conjunction with the SPR sensor system using 2,4-dichlorophenol conjugated with bovine serum albumin (BSA). In the competitive assay, a 350 ppm 2,4-dichlorophenol-BSA conjugate solution containing 2,4-dichlorophenol at various concentrations (10-250 ppb) were injected into the SPR sensor system. The sensitivity of this indirect immunoassay was found to be extremely sensitive, compared to the direct one, and a detection limit of 20 ppb was estimated. Verification that the use of GBP for immobilizing the antibody on the sensor chip enhanced the sensitivity to 2,4-dichlorophenol was obtained by comparing the procedure with another modification, in which BSA was used instead of GBP for immobilizing the antibody on the sensor chip. The affinity constant of 2,4-dichlorophenol and its conjugate to the antibody were estimated form the SPR response.

To detect low levels of histamine, we developed a histamine microsensor using recombinant histamine oxidase. Histamine oxidase with a histidine tag was readily purified using a histidine affinity column. The enzyme showed higher catalytic activity on histamine than diamines (e.g., putrescine and cadaverine) or N(tau)-methylhistamine. The sensor had three carbon film electrodes modified with osmium-polyvinylpyridine-based gel containing horseradish peroxidase, histamine oxidase, and Ag. When a standard solution of histamine was aspirated at a flow rate of 2 microl/min, the detected current was proportional to the histamine concentration and the lower detection limit was 11.3 nM. When rat basophilic leukemia cells (1 x 10(6)) were stimulated by various concentrations of antigen (2, 20, and 200 ng/ml), the histamine concentrations were 0.32, 2.7, and 1.3 microM, respectively, and 20 ng/ml of antigen was found to be the optimal concentration for the antigen-antibody reaction. In contrast, when thapsigargin, an inhibitor of Ca-ATPase in the endoplasmic reticulum, was added (50, 100, and 500 nM), the detected current increased with thapsigargin concentrations and the measured histamine concentrations were 28 nM, 1.3 microM, and 2.7 microM, respectively. These results indicate that the microsensor is useful for the analysis of histamine release from mast cells.

In this Letter, we describe an accurate antibody detection method using a fabricated extended-gate type organic field-effect-transistor (OFET), which can be operated at below 3 V. The protein-sensing portion of the designed device is the gate electrode functionalized with streptavidin. Streptavidin possesses high molecular recognition ability for biotin, which specifically allows for the detection of biotinylated proteins. Here, we attempted to detect biotinylated immunoglobulin G (IgG) and observed a shift of threshold voltage of the OFET upon the addition of the antibody in an aqueous solution with a competing bovine serum albumin interferent. The detection limit for the biotinylated IgG was 8 nM, which indicates the potential utility of the designed device in healthcare applications.

A chip-type thin-layer radial flow cell was developed as an amperometric detector for capillary electrophoresis. We fabricated a carbon film-based interdigitated ring-shaped array (IDRA) microelectrode with a 2 microm bandwidth and an almost 1 microm gap on a glass plate and used it as a working electrode. A fused-silica capillary was arranged above the IDRA electrode using a guide hole drilled through the acryl plate that formed the flow cell lid. A flow channel for use in connecting the outlet capillary was also fabricated in the acryl plate. We characterized the analytical performance of the IDRA electrode in the microchip flow cell in terms of linear concentration range, sensitivity and concentration detection limit. We achieved a collection efficiency and catechol redox cycle at the IDRA microelectrode of 65% and 1.71, respectively, and thus a high sensitivity and low detection limit of 392.9 pA/microM and 15 nM for dopamine hydrochloride. We examined the reproducibility of the detector and found that the run-to-run and detector-to-detector relative standard deviations were both less than 10%.

Ring and split-disk plastic film carbon electrodes (PFCEs) were fabricated for use in thin-layer radial flow cells which were coupled to a microdialysis sampling system. PFCEs, were initially coated with osmium poly(vinylpyridine) redox polymer horseradish peroxidase (Os-gel-HRP). Then a second coat of oxidase enzyme was applied to produce enzyme bilayer (oxidase/Os-gel-HRP) PFCEs which were subsequently over-coated with cellulose acetate for use in the determination of glucose or lactate at 0 mV (vs Ag/AgCl). Split-disk electrode geometry enabled different oxidase enzymes to be immobilized on each half of a split-disk, Os-gel-HRP-coated, PFCE to facilitate the electrochemically independent yet continuous on-line determination of these two analytes from a single dialysate. In continuous-flow experiments, cellulose acetate overcoated oxidase/Os-gel-HRP cast-coated PFCEs were quick to stabilize background current and displayed linear and sensitive responses to substrates. The effect of ascorbic acid was minimal and cross talk between partner split-disk electrodes was demonstrated to be acceptable for in vivo applications. The utility of this analytical system is demonstrated by the quantitative on-line continuous assay of changes in dialysate striatal extracellular glucose and lactate from a conscious rat during (a) local stimulation of neurons by perfusion with the depolarizing agent, Veratridine, and (b) physical restraint.

An on-line acetylcholine (ACh) sensor was developed in order to determine extracellular ACh concentrations without interference from choline (Ch). The sensor is composed of a small-volume enzymatic prereactor (22-microL inner volume) in which choline oxidase and catalase are immobilized in series. Carbon electrodes were modified with an acetylcholine esterase (AChE), choline oxidase (ChOx), and osmium poly(vinylpyridine)-based redox polymer containing horseradish peroxidase (Os-gel-HRP). The sensor sensitivity was 43.7 nA/microM (+/- 0.15, n = 3) for ACh under optimized conditions. Almost no response was seen when 100 microM Ch was continuously injected. The detection limit for ACh with the sensor was comparable to that obtained using liquid chromatography with electrochemical detection combined with an enzymatic reactor. The electrical stimulation of cultured rat hippocampal tissue resulted in an extracellular ACh increase of 20 nM (+/- 11 nM, n = 3). This increase was observed continuously with our online sensor combined with a microcapillary sampling probe located very close to the tissue. The continuous measurement of ACh and Ch using a split disk carbon film dual electrode in which one electrode surface was modified with ChOx/Os-gel-HRP and the other with AChE-ChOx/Os-gel-HRP bilayer film was also demonstrated to improve the response time by eliminating the prereactor.

A sensitive, enzymatic glucose electrode was coupled with the microdialysis sampling technique to enable the continuous, on-line measurement of dialysate glucose. The glucose sensitive electrode was fabricated by immobilizing glucose oxidase onto the surface of an osmium-polyvinylpyrridine horse radish peroxidase gel (Os-gel-HRP) which had been cast coated onto a glassy carbon electrode. This 'bilayer' electrode generated a reductive current to glucose at a potential of 0 mV thereby minimizing faradic oxidative interferences. The system utilized the continuous mixing of two fluids immediately prior to the 'bilayer' electrode. One fluid was the dialysate. The other was an oxygenated, low pH phosphate buffer which minimized oxidative interference, buffered the electrode from variations of pH and maximized enzyme efficiency. In practical terms, the 'bilayer' electrode was simple to manufacture, quick to reach stable basal currents (less than 60 min), sensitive (2.5 microM glucose could be detected in the dialysate) and durable (usable for up to 3 days). In vivo experiments, used the smallest commercially available microdialysis probes to demonstrate that on-line, continuous measurements of EC striatal glucose in the dialysate were receptive to pharmacological (local perfusion with veratridine (50 microM), systemic hyperglycemia (1.5 ml of 0.55 M glucose intraperitoneal (i.p.)) and anesthesia (Nembutal 40 mg/kg i.p.)) and behavioral (restraint) manipulations. This technique allows for greater temporal resolution than conventional HPLC procedures whilst requiring significantly less technical outlay or analytical expertise. The high sensitivity of the analytical technique could facilitate the study of EC glucose levels in very localized regions of the brain if coupled to microdialysis probes of small dimensions.

We report the first on-line electrochemical sensor for the continuous measurement of gamma-aminobutyric acid (GABA), which is a well-known inhibitory neurotransmitter in the nervous system. The sensor is composed of a glutamate oxidase (GluOx) and catalase immobilized small-volume enzymatic reactor and a glassy carbon (GC) electrode modified with a top layer film consisting of gabase and GluOx coimmobilized bovine serum albumin and an Ospoly(vinylpyrridine) bottom layer film containing horseradish peroxidase. The response of the sensor depends on the alpha-ketoglutarate concentration and is almost saturated when its concentration is 100 times higher than GABA. The sensor exhibits a sensitivity of 1.56 nA/microM for GABA under optimized conditions and shows almost no response when 10 microM glutamate is continuously injected. A detection limit of 0.1 microM is obtained with a linear range of 0.1-10 microM. GABA can be measured in the absence of alpha-ketoglutarate when there is L-glutamate in the sample solution, which is a typical condition for the extracellular measurement of cultured nerve cells.

We developed an on-line sensor with a micro-machining technique for the real-time measurement of the excitatory neurotransmitter, L-glutamate, released from cultured rat nerve cells. The sensor was made by bonding two glass plates, together. One plate has a rectangular flow channel and the other has a carbon film based electrode deposited on it by chemical vapor deposition and pyrolysis. The electrode was modified with glutamate oxidase and mediator. The sensor was operated at a slow flow rate of 0.1 μl – 4 μl/min by drawing the solution near the target nerve cell with a syringe pump.