Direct detection of renal function markers using microchip CE with pulsed electrochemical detection

Creatinine, creatine, and uric acid are three important compounds that are measured in a variety of clinical assays, most notably for renal function. Traditional clinical assays for these compounds have focused on the use of enzymes or chemical reactions. Electrophoretic microchips have the potential to integrate separation power of capillary electrophoresis with devices that are small, portable, and have the speed of conventional sensors. The development of a microchip CE system for the direct detection of creatinine, creatine, and uric acid is presented. The device uses pulsed amperometric detection (PAD) to detect the nitrogen-containing compounds as well as the easily oxidizable uric acid. Baseline separation of creatinine, creatine and uric acid was achieved using 30 mM borate buffer (pH = 9.4) in less than 200 s. Linear calibration curves were obtained with limits of detection of 80 microM, 250 microM and 270 microM for creatinine, creatine and uric acid respectively. An optimization of the separation conditions and a comparison of PAD with other amperometric detection modes is also shown. Finally, analysis of a real urine sample is presented with validation of creatinine concentrations using a clinical assay kit based on the Jaffé reaction.     

High-performance capillary zone electrophoretic assay for markers of diabetic nephropathy in plasma and urine

A new high-performance capillary zone electrophoretic assay for creatine (Cr), creatinine (Cn), urea (U) and uric acid (Ua), markers of human diabetic nephropathy, both in plasma and urine has been developed with UV detection at 200 nm. The plasma sample was deproteinized with trichloroacetic acid and centrifuged at 10 000 rpm for 10 min. The urine sample was diluted 20-fold with buffer before analysis. The optimum separation conditions for the markers was investigated with respect to the concentration of the buffer, the pH, the voltage and the capillary temperature. Baseline separation was achieved in 25 mmol/L phosphate buffer (pH 3.45) using a 21 cm x 75 microm I.D. fused-silica capillary at 40 degrees C with an electric field of 1190 V/cm. The calibration curves showed good linearity in the range 3.5-1000, 0.18-700, 500-5000 and 2-800 microM (r2 min > 0.998) for Cr, Cn, U and Ua, respectively. The proposed method also has a high reproducibility (peak area RSD max < 3%) and has been successfully applied to the determination of clinical samples.     

Highly sensitive flow detection of uric acid based on an intermediate regeneration of uricase

The principle of the signal amplification of a uric acid sensor based on dithiothreitol (DTT)-mediated intermediate regeneration of uricase was applied to a flow-injection system with an immobilized uricase reactor and a DTT-containing carrier. Highly sensitive detection for nM to microM order of uric acid was achieved when 10 mM TRIS-HCl buffer (pH 10.0) containing 20 mM DTT was used as a carrier at 0.6 ml min-1 and 37 degrees C. The sensitivity of the uric acid was much improved over a batch method using a uricase membrane-coupling electrode, and the detection limit (ca. peak current 8 nA) of uric acid was found to be down to 3 x 10(-10) M (amplification factor; more than 10,000). This chemically amplified flow-system is very useful for the direct assay of uric acid in highly diluted biological fluids (urine and serum) without complicated pretreatment of the samples, because this sensor has the potential to detect trace amounts (nM to microM) of uric acid in highly diluted body fluids in which the concentration of interfering constituents was decreased to negligible levels. Good correlation was observed between this system and conventional spectrophotometry. The immobilized uricase reactor could be re-used for at least 4 months of repeated analysis without loss of activity and was stable if stored at 4 degrees C in 10 mM TRIS-HCl buffer, pH 9.0.     

CE Determination of Creatinine and Uric Acid in Saliva and Urine During Exercise

A simple and reliable method based on capillary electrophoresis with electrochemical detection (CE–ED) was applied to study the effect of aerobic exercises on creatinine and uric acid concertration in saliva and urine. The pH value, the running buffer concentration, the SDS concentration, separation voltage, injection time and the potential applied to the working electrode were investigated to find the optimum conditions. The detection limits (S/N = 3) for creatinine and uric acid were 3.6 μmol L^?1 and 0.86 μmol L^?1, respectively. This method was successfully used in the rapid analysis of creatinine and uric acid in saliva samples. After aerobic exercises, creatinine concentration decreased, and uric acid concentration increased in saliva. In urine, the concentrations of creatinine and uric acid both increased after exercise.     

Direct measurements of xanthine in 2000-fold diluted xanthinuric urine with a nanoporous carbon fiber sensor

High selectivity and sensitivity is reported in the measurements of xanthine in urine by fast scan cyclic voltammetry (FSV) with a nanostructured carbon fiber sensor of 3.5 +/- 0.4 mum radius. Fabrication of the sensors for the measurements is described. Fabrication of the nanostructure at the carbon fiber sensor surface exposes surface pores. SEM images confirm the formation of the nanostructure. The results indicate that the nanostructure improves the sensitivity and limit of detection (LOD) in the measurements of xanthine and uric acid. The sensors allow rapid direct measurements of xanthine in 2000-fold diluted xanthinuric urine and of uric acid in 2000-fold diluted normal urine. The sensitivity and the LOD of xanthine is 0.40 +/- 0.02 nA microM(-1) (0.995) and 1 microM, respectively, and 0.99 +/- 0.01 nA microM(-1) (0.998) and 500 nM for uric acid. The concentration of xanthine in 2000-fold diluted xanthinuric urine is 1.6 +/- 0.2 muM from FSV and from HPLC. The concentration of xanthine and uric acid in urine can be determined by pre- or post-calibration of the sensor in buffer or by the method of standard addition.     

Fabrication and evaluation of a carbon-based dual-electrode detector for poly(dimethylsiloxane) electrophoresis chips

The first carbon-based dual-electrode detector for microchip capillary electrophoresis (CE) is described. The poly(dimethylsiloxane) (PDMS)-based microchip CE devices were constructed by reversibly sealing a PDMS layer containing separation and injection channels to another PDMS layer containing carbon fiber working electrodes. End-channel amperometric detection was employed and the performance of the chip was evaluated using catechol. The response was found to be linear between 1 and 600 microM with an experimentally determined limit of detection (LOD) of 500 nM and a sensitivity of 30 pA/microM. Collection efficiencies for catechol ranged from 36.0 to 43.7% at field strengths of 260-615 V/cm. The selectivity that can be gained with these devices is demonstrated by the first CE-based dual-electrode detection of a Cu(II) peptide complex. These devices illustrate the potential for a rugged and easily constructed microchip CE system with an integrated carbon-based detector of similar scale.     

Chromatographic Properties of Ethanol/Water Mobile Phases on Silica Based Monolithic C18

A simple and reliable method based on capillary electrophoresis with electrochemical detection (CE–ED) was applied to study the effect of aerobic exercises on creatinine and uric acid concertration in saliva and urine. The pH value, the running buffer concentration, the SDS concentration, separation voltage, injection time and the potential applied to the working electrode were investigated to find the optimum conditions. The detection limits (S/N = 3) for creatinine and uric acid were 3.6 μmol L⁻¹ and 0.86 μmol L⁻¹, respectively. This method was successfully used in the rapid analysis of creatinine and uric acid in saliva samples. After aerobic exercises, creatinine concentration decreased, and uric acid concentration increased in saliva. In urine, the concentrations of creatinine and uric acid both increased after exercise.

     

Nitrite and nitrate measurement in human urine by capillary electrophoresis.

Nitrite and nitrate have been widely used as markers for nitric oxide (NO) formation in vivo and represent the major NO oxidation products in biological fluids. In the present study, the use of capillary electrophoresis (CE) in the measurement of nitrite and nitrate in human urine is described. Urine samples were electrophoresed in an extended light path fused-silica capillary (104 cm; 75 microm ID) at an applied negative potential of 30 kV, and UV detection at 214 nm. Using electrokinetic sample injection (-6 kV x 20 s), we found that urine concentration, pH, sodium and chloride interfered with nitrite and nitrate detection. The detection of nitrite and nitrate was decreased when hydrodynamic sample injection was used (30 mbar x 60 s). However, basal levels of urinary nitrite (0.25 +/- 0.05 microM) and nitrate (591 +/- 115 microM) were detected and no interference by variations in urine concentration and pH was noted when hydrodynamic sample injection was used. Thus, hydrodynamic sample injection is convenient for the measurement of urinary nitrite and nitrate and avoids the effect of variations in urine matrices and pH on nitrite and nitrate detection.     

Evaluation of microchip capillary electrophoresis with external contactless conductivity detection for the determination of major inorganic ions and lithium in serum and urine samples

The determination of inorganic ions in clinical samples in less than 90 seconds was demonstrated for microchip capillary electrophoresis using capacitively coupled contactless conductivity detection (C(4)D). Bare electrophoresis chips were used in combination with external electrodes which were part of the chip holder. In order to achieve the required selectivity and sensitivity, an optimization of the electrode layout was carried out. Limits of detection (LOD) of 1 microM for K(+), 1.5 microM for Ca(2+), 3 microM for Na(+), 1.75 microM for Mg(2+) and 7.5 microM for Li(+) were achieved. The determination of inorganic cations (NH(4)(+), K(+), Na(+), Ca(2+), Mg(2+)) and anions (Cl(-), NO(3)(-), SO(4)(2-), phosphate) in blood serum and urine samples was possible in one common electrolyte solution containing 15 mM L-arginine, 10.75 mM maleic acid and 1.5 mM 18-crown-6 at pH 5.90 by simply switching the separation voltage from positive to negative polarity. Lithium, present at significant levels when used for therapeutic purposes, can also be determined in blood serum using a slightly modified background electrolyte solution.     

Separation of three water-soluble vitamins by poly(dimethylsiloxane) microchannel electrophoresis with electrochemical detection

A method for rapid separation and sensitive determination of three water-soluble vitamins, pyridoxine, ascorbic acid (VC), and p-aminobenzoic acid (PABA) has been developed by PDMS microchannel electrophoresis integrated with amperometric detection. After treatment of the microchip with oxygen plasma, the peak shapes of the three analytes were essentially improved. Pyridoxine, VC, and PABA were well separated within only 80 s in a running buffer of 20 mM borate solution (pH 8.5). Good linearity was obtained within the concentration range of 2-200 microM for the three water-soluble vitamins. The detection limits were 1.0 microM for pyridoxine and VC, and 1.5 microM for PABA. The proposed method has been successfully applied to real human urine sample, without solid phase extraction, with recoveries of 80-122% for the three water-soluble vitamins.     

A selective voltammetric method for uric acid detection at beta-cyclodextrin modified electrode incorporating carbon nanotubes

A beta-cyclodextrin-coated electrode incorporating carbon nanotubes was constructed and applied to the detection of uric acid in the presence of high concentration of ascorbic acid. The major obstacle of the overlapped oxidation potential of ascorbic acid was overcome owing to the distinct ability of the carbon nanotubes-modified electrode to yield a large anodic peak difference ca. 400 mV. The sensitive detection of uric acid has been further improved by the formation of a supramolecular complex between beta-cyclodextrin and uric acid. A linear calibration curve was obtained for 5 x 10(-7) to 5 x 10(-5) M in 0.2 M HAc-NaAc buffer (pH 4.5) with correlation coefficient of 0.998 and detection limit of 0.2 microM. The practical analytical application was illustrated by a selective measurement of uric acid in human urine without any preliminary treatment.     

Separation and simultaneous determination of uric acid and ascorbic acid on a dynamically modified poly(dimethylsiloxane) microchip.

Poly(dimethylsiloxane) microfluidic channels alternately modified by poly(diallyldimethylammonium chloride) and poly(sodium 4-styrenesulfonate) were successfully used to separate uric acid and ascorbic acid. Results show that uric acid and ascorbic acid can be well separated and detected simultaneously in modified microchips coupled with in-channel electrochemical detection. Under the optimal conditions, the linear ranges of uric acid and ascorbic acid were both from 25 to 600 microM, with the correlation coefficients of 0.997 and 0.996, respectively. The detection limits were 8 microM for uric acid and 5 microM for ascorbic acid. Factors influencing separation and detection, including buffer solution, detection potential and separation voltage, were investigated and optimized. In addition, the dependences of the current response on sensitivity and reproducibility were studied, and the stability of the device was also evaluated in detail. This method was successfully used to determine uric acid and ascorbic acid in human urine.     

Indirect laser-induced fluorescence detection of diuretics separated by capillary electrophoresis

Indirect LIF detection was applied to the detection of four acidic diuretics separated by CZE. Semiconductor laser was employed to provide the stable excitation of 473 nm. With an optimized electrophoretic buffer system which contained 5 mM of triethylamine, 0.1 microM of fluorescein, and 5% of n-butanol, fast separation of four diuretics (ethacrynic acid, chlorthalidone, bendroflumethiazide, and bumetanide) can be performed within 3 min with the detection limits of 0.2-2 microg/mL. The impacts of buffer components including the concentrations of the electrolytes, fluorescence probe, and the organic additives were demonstrated. The method was applied for the detection of diuretics in urine. As an alternative way for the fast analysis of diuretics, this indirect detection method provided the technical support for future microchip performances, in which diuretics may be detected in the microchip by the common LIF detector without derivatization.     

高效毛细管区带电泳测定人尿液中尿酸、肌酐和伪尿核苷的含量

报道了采用高效毛细管区带电泳技术直接将人尿液注入毛细管进行尿液中肌酐、尿酸及伪尿核苷含量测定的新方法。试验表明,以磷酸盐（ｐＨ６．１）作缓冲液,对人体尿液中肌酐、尿酸及伪尿核苷进行直接分析具有较高的灵敏度和较好的重复性。     

Incorporation of Disposable Screen‐Printed Electrodes for Use in Capillary Electrophoresis End‐Column Amperometric Detection System

The development and performance of an end‐column amperometric detection system integrated with disposable screen‐printed electrodes for capillary electrophoresis is presented. In this system, the electrode and capillary can be easily replaced and the capillary/electrode alignment procedure is straightforward. The use of easily replaceable screen‐printed electrodes offers a tremendous benefit for capillary electrophoresis applications requiring frequent replacement of the working electrode due to fouling. This simple and convenient system is very attractive for routine analyses by capillary electrophoresis with electrochemical detection. The separation and determination of uric acid in human urine is presented.     

Multicommutated Flow System with Amperometric Detection. Determination of Uric Acid in Urine

In this work an automated flow methodology based on a tubular amperometric detector coupled to a multicommutated flow system was developed and applied in the determination of uric acid in urine. The exploitation of the analytical potential of multicommutated flow systems allowed the implementation of an expeditious and easily controlled on‐line sample dilution, based on the zone sampling approach. The dilution capability exhibited by the developed methodology allowed a direct insertion of the samples in the flow system, without any pretreatment, assuring faster, simpler and less expensive analyses when compared to the enzymatic based methods with spectrophotometric detection commonly used in clinical analyses. The results obtained with the developed system in the determination of uric acid in urine were compared with those obtained by the enzymatic method used in clinical analysis laboratories, and no statistical difference between both methods (for a confidence level of 95%) was found. The proposed system showed good repeatability (RSD<3%, n=10) and a detection limit of 4×10^?7 mol L^?1.     

Determination of 8-hydroxy-2'-deoxyguanosine in untreated urine by capillary electrophoresis with UV detection

A capillary electrophoresis method with UV detection was developed for the determination of 8-hydroxy-2'-deoxyguanosine (8-OHdG) in untreated urine samples. The calibration graph for 8-OHdG in urine is linear in the concentration range 10-500 mg/l. and the detection limit is 5 mg/l (17 microM). 8-OHdG was determined in urine from oncological patients treated by radiation therapy. Its concentrations relative to creatinine were found to be in the range 10-47 microg 8-OHdG/l mg creatinine (4-19 micromol 8-OHdG/mmol creatinine). The overall time of the analysis of a urine sample was less than 15 min.     

Optimization of sample stacking for the simultaneous determination of nonsteroidal anti-inflammatory drugs with a wall-coated histidine capillary column

A wall-coated histidine capillary column was developed for the on-line preconcentration of nonsteroidal anti-inflammatory drugs (NSAIDs) in capillary electrochromatography (CEC). A wide variety of experimental parameters, such as the sample buffer, background electrolyte (BGE) composition, concentration, sample plug lengths, water plug, and the effect of organic modifiers were studied. The relationship between peak height and injection times for the NSAIDs by variation of sample and BGE buffer concentration was investigated. On addition of sodium chloride (0.3-0.6%) to the sample zone, the stacking efficiency was increased. With acetate buffer (100 mM, pH 5.0)/ethanol (20% v/v) as BGE and sample solution in acetate buffer (0.2 mM, pH 5.0)/ethanol (20% v/v)/NaCl (0.3% w/v), NSAIDs could be determined at low microM levels without sample matrix removal. The detection limit was 0.096 microM for indoprofen, 0.110 microM for ketoprofen, 0.012 microM for naproxen, 0.023 microM for ibuprofen, 0.110 microM for fenoprofen, 0.140 microM for flurbiprofen, and 0.120 microM for suprofen. The method could be successfully applied to the simultaneous determination of NSAIDs in urine. The recoveries were better than 82% for all the analytes. The present method enables simple manipulation with UV detection for the determination of NSAIDs at low concentration levels in complex matrix samples.     

A flow-based procedure with solenoid micro-pumps for the spectrophotometric determination of uric acid in urine

The determination of uric acid in urine shows clinical importance, once it can be related to human organism dysfunctions, such as gout. An analytical procedure employing a multicommuted flow system was developed for the determination of uric acid in urine samples. Cu(II) ions are reduced by uric acid to Cu(I) that can be quantified by spectrophotometry in the presence of 2,2′-biquinoline 4,4′-dicarboxylic acid (BCA). The analytical response was linear between 10 and 100 μmol L^− 1 uric acid with a detection limit of 3.0 μmol L^− 1 (99.7% confidence level). Coefficient of variation of 1.2% and sampling rate of 150 determinations per hour were achieved. Per determination, 32 μg of CuSO₄ and 200 μg of BCA were consumed, generating 2.0 mL of waste. Recoveries from 91 to 112% were estimated and the results for 7 urine samples agreed with those obtained by the commercially available enzymatic kit for determination of uric acid. The procedure required 100-fold dilution of urine samples, minimizing sample consumption and interfering effects. In order to avoid the manual dilution step, on-line sample dilution was achieved by a simple system reconfiguration attaining a sampling rate of 95 h^− 1.     

A fast and highly sensitive detection of cholesterol using polymer microfluidic devices and amperometric system

In this work, the rapid detection of cholesterol using poly(dimethylsiloxane) microchip capillary electrophoresis, based on the coupling of enzymatic assays and electrochemical detection, was developed. Direct amperometric detection for poly(dimethylsiloxane) (PDMS) microchip capillary electrophoresis was successfully applied to quantify cholesterol levels. Factors influencing the performance of the method (such as the concentration and pH value of buffer electrolyte, concentration of cholesterol oxidase enzyme (ChOx), effect of solvent on the cholesterol solubility, and interferences) were carefully investigated and optimized. The migration time of hydrogen peroxide, product of the reaction, was less than 100 s when using 40 mM phosphate buffer at pH 7.0 as the running buffer, a concentration of 0.68 U/mL of the ChOx, a separation voltage of +1.6 kV, an injection time of 20 s, and a detection potential of +0.5 V. PDMS microchip capillary electrophoresis showed linearity between 38.7 μg/dL (1 μM) and 270.6 mg/dL (7 mM) for the cholesterol standard; the detection limit was determined as 38.7 ng/dL (1 nM). To demonstrate the potential of this assay, the proposed method was applied to quantify cholesterol in bovine serum. The percentages of recoveries were assessed over the range of 98.9-101.8%. The sample throughput was found to be 60 samples per hour. Therefore, PDMS microchip capillary electrophoresis, based on the coupling of enzymatic assays and electrochemical detection, is very rapid, accurate and sensitive method for the determination of cholesterol levels.