Drug response assay system in a microchip using human hepatoma cells.

A microchip-based cell response assay system to an anticancer agent was developed. The hepatoma cell line HepG2 was used to assess the effects of an anticancer agent, doxorubicin. The required cell number was reduced by two orders, and the observation of the time course of cell response became possible. The system clearly showed that treatment with higher doses of the drug or longer exposure times gave more effects to cells. The possibilities of novel drug response studies or toxicity assay system were demonstrated.     

Disposable integrated bismuth citrate-modified screen-printed immunosensor for ultrasensitive quantum dot-based electrochemical assay of C-reactive protein in human serum

A novel immunosensor based on graphite screen-printed electrodes (SPEs) modified with bismuth citrate was developed for the voltammetric determination of C-reactive protein (CRP) in human serum using quantum dots (QDs) labels. The sandwich-type immunoassay involved physisorption of CRP capture antibody on the surface of the sensor, sequential immunoreactions with CRP and biotinylated CRP reporter antibody and finally reaction with streptavidin-conjugated PbS QDs. The quantification of the target protein was performed with acidic dissolution of the PbS QDs and anodic stripping voltammetric detection of the Pb(II) released. Detection was performed at bismuth nanodomains formed on the sensor surface during the electrolytic preconcentration step, as bismuth citrate was reduced to metallic bismuth simultaneously with the deposition of Pb on the surface of the immunosensor. Under optimal conditions, the response was linear over the range 0.2–100 ng mL⁻¹ CRP and the limit of detection was 0.05 ng mL⁻¹ CRP. Since the modified SPE serves as both the biorecognition element and the QDs reader, the analytical procedure is simplified, the drawbacks of existing electroplated immunosensors are minimized while the proposed disposable sensing platform provides convenient, low-cost and ultrasensitive detection of proteins and wider scope for mass-production.     

Micro-flow injection system for the urinary protein assay

A urinary protein assay has been investigated, employing a micro-flow injection analysis (μFIA) combined with an adsorptive separation of protein from analyte. The adsorptive separation part of protein in the artificial urine with ceramic hydroxyapatite is integrated on the μFIA chip, since the interference of other components coexisting in urine occurs in the conventional FIA system. The typical FI peak can be obtained following the adsorption–elution process of the protein prior to the detection, and the protein concentration in artificial urine can be quantitatively determined.     

Application of microchip CGE for the analysis of PEG-modified recombinant human granulocyte-colony stimulating factors

The purpose of this study was to evaluate the microchip CGE (MCGE) for the analysis of PEG-modified granulocyte-colony stimulating factor (PEG-G-CSF) prepared with PEG-aldehydes. The unmodified and PEG-modified G-CSFs were analyzed by Protein 80 and 230 Labchips on the Agilent 2100 Bioanalyzer. The MCGE allowed size-based separation and quantitation of PEG-G-CSF. The Protein 80 Labchip was useful for PEG-5K-G-CSF, while the Protein 230 Labchip was more suitable for PEG-20K-G-CSF. The MCGE was also used to monitor a search for optimal PEG-modification (PEGylation) conditions to produce mono-PEG-G-CSF. This study demonstrates the usefulness of MCGE for monitoring and optimizing the PEGylation of G-CSF with the advantages of speed, minimal sample consumption, and automatic quantitation.     

A new analytical procedure for assay of lysozyme in human tear and saliva with immobilized reagents in flow injection chemiluminescence system.

A novel analytical procedure based on chemiluminescence (CL) detection was described for the determination of lysozyme at ng ml(-1) level by using controlled-reagent-release technology in a flow injection system. The analytical reagents involved in the CL reaction, including luminol and periodate, were both immobilized on the anion-exchange resins in the flow injection system. Through water injection, luminol and periodate were eluted from the anion-exchange column to generate the chemiluminescence, which was inhibited in the presence of lysozyme. By measuring the decrease of CL intensity, one could analyze the lysozyme quantitatively. The decrement of CL emission was linear over the logarithm of lysozyme concentration in the range of 30-1000 ng ml(-1). A typical analytical procedure, including sampling and washing, could be performed in 0.5 min at a flow rate of 2.0 ml min(-1), giving a throughput of 120 h(-1), with a relative standard deviation of less than 3.0%. The proposed method was applied successfully to the determination of lysozyme in human tear and saliva samples, and the recovery was from 92.0% to 105.7%.     

Culture and leukocyte adhesion assay of human arterial endothelial cells in a glass microchip.

Cells are frequently exploited as processing components for integrated chemical systems, such as biochemical reactors and bioassay systems. By culturing vascular endothelial cells (ECs) in integrated chemical devices, vascular models have also been fabricated. Here, we utilized a thermally fused-glass microchip which is chemically and physically stable and favorable for optical detections, and cultured human arterial ECs (HAECs) in it. HAECs reached confluence within 4 days. Survival and tolerance for high shear stress (25 dyn/cm2) of the HAECs were confirmed. Furthermore, HAECs responded to inflammatory cytokine, tumor necrosis facor-alpha (TNF-alpha) and attached to more leukocyte cell line, HL-60 cells than unstimulated HAECs. Our developed device can be applied as a human arterial model, and we propose it as a new method for vascular studies.     

Development and validation of an HPLC-UV detection assay for the determination of rufinamide in human plasma and saliva

The development of a simple and rapid high-performance liquid chromatography (HPLC) method for the determination of the new antiepileptic drug rufinamide (RFN) in human plasma and saliva is reported. Samples (250 μl) are alkalinized with ammonium hydroxide (pH 9.25) and extracted with dichloromethane using metoclopramide as internal standard. Separation is achieved with a Spherisorb silica column (250 × 4.6 mm i.d., 5 μm) at 30 °C using as mobile phase a solution of methanol/dichloromethane/n-hexane 10/25/65 (vol/vol/vol) mixed with 6 ml ammonium hydroxide. The instrument used was a Shimadzu LC-10Av chromatograph and flow rate was 1.5 ml min^-1, with a LaChrom L-7400 UV detector set at 230 nm. Calibration curves are linear [r ² = 0.998 ± 0.002 for plasma (n = 10) and r ² = 0.999 ± 0.001 for saliva (n = 9)] over the range of 0.25–20.0 μg ml^-1, with a limit of quantification at 0.25 μg ml^-1. Precision and accuracy are within current acceptability standards. The assay is suitable for pharmacokinetic studies in humans and for therapeutic drug monitoring.     

A CE‐based assay for human protein kinase CK2 activity measurement and inhibitor screening

A new assay for protein kinase CK2 activity determination based on the quantification of a phosphorylated substrate was developed. The common CK2 substrate peptide RRRDDDSDDD, conjugated with the fluorophore 5‐[(2‐aminoethyl)amino]naphthalene‐1‐sulfonic acid at the C‐terminus served as the analyte. By means of CZE using 2 mol/L acetic acid as electrolyte and UV detection at 214 nm, the non‐phosphorylated and the phosphorylated peptide variants could be resolved within 6 min from a complex assay mixture. By this means, activity of human CK2 could be monitored by a kinetic, as well as an endpoint, method. Inhibition of human recombinant CK2 holoenzyme by 6‐methyl‐1,3,8‐trihydroxyanthraquinone and 4,5,6,7‐tetrabromobenzotriazole resulted in IC₅₀ values of 1.33 and 0.27 μM, respectively, which were similar to those obtained with the standard radiometric assay. These results suggest that the CE/UV strategy described here is a straightforward assay for CK2 inhibitor testing.     

Immunosensor for electrodetection of the C-reactive protein in serum

Epidemiological studies have demonstrated an association between the risk of cardiovascular events and increasing C-reactive protein (CRP) concentration. This paper reports the development of an immunosensor for the assessment of the cardiovascular process using anti-C-reactive protein antibody immobilized onto a gold-printed screen electrode. Positive and negative human sera were successfully evaluated using electrochemical impedance spectroscopy (EIS), differential pulse voltammetry (DPV), and atomic force microscopy (AFM). EIS results show that, after the incubation with positive serum for myocardial infarction, the resistance increased about two times in relation to the negative serum. A linear range from 6.25 to 50 μg mL^?1 and detection limit of 0.78 μg mL^?1 using DPV were obtained. The immunosensor developed for the CRP detection using gold electrode revealed efficacy and a potential use for the diagnosis and monitoring of the progression of cardiovascular diseases.     

Multiple enzyme linked immunosorbent assay system on a capillary-assembled microchip integrating valving and immuno-reaction functions

Gastrodin is a bioactive constituent of rhizome in Gastrodia elata Blume (Orchidaceae) The aim of this study is to develop a rapid and sensitive liquid chromatographic method coupled to microdialysis sampling system to measure the unbound of gastrodin in rat blood, brain and bile. Microdialysis probes were simultaneously inserted into the jugular vein, brain striatum and bile duct of each anesthetized rat for sampling after the administration of gastrodin (100 or 300 mg kg⁻¹) through the femoral vein. Separation of unbound gastrodin from various biological fluids was applied to an RP-select B column (250 mm × 4.6 mm i.d., 5 μm). The mobile phase consisted of acetonitrile–50 mM potassium dihydrogen phosphate buffer–triethylamine (5:95:0.1, v/v/v, adjusted to pH 2.5 with orthophosphoric acid) with a flow rate of 1 mL min⁻¹. The UV detector wavelength was set at 221 nm. Fifteen minutes after the administration, the gastrodin reached the peak concentration in brain and bile. In addition, the results indicate that gastrodin penetrates the blood-brain barrier (BBB) and goes through hepatobiliary excretion.     

Nonradioactive telomerase activity assay by microchip electrophoresis: privileges to the classical gel electrophoresis assay

The present study accents on the privileges of microchip-based electrophoresis to the conventional gel electrophoresis in separation of telomerase repeat amplification protocol/polymerase chain reaction (PCR) ladder products obtained in telomerase-catalyzed reaction in cancer cells. We try to clarify the interpretation of the results obtained by both electrophoretic procedures and to avoid misinterpretation as a result of PCR-dependent artefacts.     

Zeolite nanoparticle modified microchip reactor for efficient protein digestion

An enzymatic microreactor has been fabricated based on the poly(methyl methacrylate) (PMMA) microchchip surface-modified with zeolite nanoparticles. By introducing the silanol functional groups, the surface of PMMA microchannel has been successfully modified with silicalite-1 nanoparticle for the first time due to its large external surface area and high dispersibility in solutions. Trypsin can be stably immobilized in the microchannel to form a bioreactor using silica sol-gel matrix. The immobilization of enzyme can be realized with a stable gel network through a silicon-oxygen-silicon bridge via tethering to those silanol groups, which has been investigated by scanning electron microscopy and microchip capillary electrophoresis with laser-induced fluorescence detection. The maximum proteolytic rate constant of the immobilized trypsin is measured to be about 6.6 mM s(-1). Using matrix assisted laser desorption and ionization time-of-flight mass spectrometry, the proposed microreactor provides an efficient digestion of cytochrome c and bovine serum albumin at a fast flow rate of 4.0 microL min(-1), which affords a very short reaction time of less than 5 s.     

A three-dimensionally adjustable amperometric detector for microchip electrophoretic measurement of nitroaromatic pollutants

A microchip capillary electrophoresis (CE)–amperometric detection (AD) system has been fabricated by integrating a two-dimensionally adjustable CE microchip and an amperometric detection cell containing a one-dimensionally adjustable disc detection electrode in a Plexiglas holder. It facilitates the precise three-dimensional alignment between the channel outlet and the detection electrode without a complicated three-dimensional manipulator. The performance of this unique system was demonstrated by separating four nitroaromatic pollutants (nitrobenzene, 2,4-dinitrotoluene, 2,4,6-trinitrotoluene, and p-nitrobenzene). Factors influencing their separation and detection processes were examined and optimised. The four analytes have been well-separated within 120 s in a 75 cm long separation channel at a separation voltage of +2000 V using an electrophoretic separation medium containing 15 mM borax and 15 mM sodium dodecyl sulfate (pH 9.2). Highly linear response is obtained for the four analytes over the range of 0–5 ppm with the detection limits ranging from 12 to 52 ppb. The present system demonstrated long-term stability and reproducibility with relative standard deviations of less than 5% for the peak current (n = 9). The new approach for the microchannel–electrode alignment should find a wide range of applications in other microfluidic analysis systems.     

High-throughput nitric oxide assay in biological fluids using microchip capillary electrophoresis

In order to develop a high-throughput screening method for the nitrogen monoxide metabolites, nitrite and nitrate, in biological fluids, we have investigated the simultaneous determination of these metabolites using microchip capillary electrophoresis (MCE). In this study, the control of applied voltage to obtain higher sensitivity by increasing the sample injection volume was investigated. Also, the improvement of reproducibility by correcting the injection volume using the internal standard was investigated. By increasing the sample volume, the limits of detection achieved for nitrite and nitrate were 24 and 12 microM, respectively. Because we used a 10-fold diluted sample when detecting nitrite and nitrate in human serum, it was necessary to increase the sensitivity by a factor of 10-50. The run-to-run and day-to-day relative standard deviations achieved were improved to less than 10% by using an internal standard to correct the injection volume. Moreover, we obtained successful separation of nitrite and nitrate in spiked human serum within 6.5 s under optimum analytical conditions. As a result, although it is necessary to obtain greater sensitivity, it was concluded that determination of the amount of NO metabolites in biological fluids using MCE is possible.     

Method development for proteome stabilization in human saliva

Human saliva is a biological fluid with emerging early detection and diagnostic potentials. However, the salivary proteome suffers from rapid degradation and thus compromises its translational and clinical utilities. Therefore, easy, reliable and practical methods are urgently required for the storage of human saliva samples. In this study, saliva samples from healthy subjects were collected and stored at room temperature (RT) and 4 °C for different lengths of time with and without specific protein stabilization treatments. SDS-PAGE was run to compare the protein profiling between samples. Reference proteins, β-actin and interleukin-1 β (IL1β), were chosen to evaluate salivary protein stability. Immunoassay was used for the detection of these target proteins. All data was compared with the positive control that had been kept at −80 °C. The results show that the salivary proteome that has been stored at 4 °C with added protease inhibitors was stable for approximately two weeks without significant degradation. By adding ethanol to the samples, the salivary proteome was stabilized at RT. After optimization, a simple, robust and convenient method is developed for the stabilization of proteins in human saliva that does not affect the downstream translational and clinical applications. The salivary proteome could be stabilized without significant degradation by adding ethanol at RT for about two weeks. This optimized method could greatly accelerate the clinical usage of saliva for future diagnosis.     

Sensitive, label-free protein assay using 1-ethyl-3-methylimidazolium tetrafluoroborate-supported microchip electrophoresis with laser-induced fluorescence detection

Based on the dimer-monomer equilibrium movement of the fluorescent dye Pyronin Y (PY), a rapid, simple, highly sensitive, label-free method for protein detection was developed by microchip electrophoresis with LIF detection. PY formed a nonfluorescent dimer induced by the premicellar aggregation of an anionic surfactant, SDS, however, the fluorescence intensity of the system increased dramatically when proteins such as BSA, bovine hemoglobin, cytochrome c, and trypsin were added to the solution due to the transition of dimer to fluorescent monomer. Furthermore, 1-ethyl-3-methylimidazolium tetrafluoroborate (EMImBF4) instead of PBS was applied as running buffers in microchip electrophoresis. Due to the excellent properties of EMImBF4, not only nonspecific protein adsorption was more efficiently suppressed, but also approximately ten-fold higher fluorescence intensity enhancement was obtained than that using PBS. Under the optimal conditions, detection limits for BSA, bovine hemoglobin, cytochrome c, and trypsin were 1.00x10(-6), 2x10(-6), 7x10(-7), and 5x10(-7) mg/mL, respectively. Thus, without covalent modification of the protein, a protein assay method with high sensitivity was achieved on microchips.     

Automated direct assay system for the measurement of sex steroid hormones in serum using high-performance liquid chromatography

An automated direct assay system using high-performance liquid chromatography was developed for the simultaneous measurement of estradiol, estrone, progesterone, 17 alpha-hydroxyprogesterone, 20 alpha-hydroxyprogesterone, testosterone and androstenedione in biological fluids. A comparison between the values measured by this method and by radioimmunoassay revealed good correlation for estradiol (r = 0.938, p less than 0.001) and progesterone (r = 0.903, p less than 0.001). Estradiol and estrone could be analysed above the level of 250 pg/ml, and progesterone, 17 alpha-hydroxyprogesterone, 20 alpha-hydroxyprogesterone, testosterone and androstenedione could be analysed above the level of 5.0-7.5 ng/ml. The method was applied to the clinical appraisal of placental function and maturation of ovarian follicles.     

A disposable biosensor for the determination of alpha-amylase in human saliva

A disposable tri-enzymatic biosensor is presented for the determination of α-amylase in human saliva. It is based on the quantity of maltose generated by hydrolysis of maltopentose in the presence of salivary α-amylase. The biosensor is fabricated by co-immobilization of the enzymes α-glucosidase, glucose oxidase, and mutarotase on screen-printed electrodes modified with Prussian Blue. The assay can be performed with a “drop” of sample, this allowing for ease and simplicity. A linear relationship is found for the range from 5 to 250 units per mL, with an LOD of 5 units per mL. The biosensor is stable for at least one month and over this time retains 80% of its original activity. The system was then evaluated for matrix effects of human saliva and compared to a spectrometric method using a commercially available kit.     

Range of separation of potential tool for bioseparation, microchip electrophoresis system, for DNA polymorphisms on the human Y-chromosome.

For the requirement of a high, fast and sufficient technology to suit the needs of 21st century biotechnology, the separation range of a microchip electrophoresis system was studied. Two DNA fragments on the human Y-chromosome, SY594 (82 bp) and 12f2 (88 bp), were successfully separated with a reproducibility of 1.9% and an accuracy of 2.8%. Then, a mixture of 10 DNA markers ranging from 61 bp to 189 bp was successfully separated with high resolution. All of these results demonstrate the superiority of microchip electrophoresis as a tool for 21st century bioseparation.