Noninvasive picoliter volume thermometry based on backscatter interferometry

Using the on-chip refractive index (RI) detector based on backscatter interferometry, sensitive, small volume, noninvasive thermometry can be performed. The current optical configuration for the on-chip interferometric backscatter detector (OCIBD) is quite simple and consists of an unfocused laser, an unaltered chip with a hemispherical channel and a photodetector. Alignment is straightforward with the only requirement being that the beam fully fills the channel. The interaction of an unfocused laser beam with the uncoated etched channel with a curvature within the silica plate (chip) produces fringes whose positional changes scale with respect to the refractive index (RI), n, of the fluid in the channel. Due to the inherently high value of dn/dT for most fluids and the high sensitivity of OCIBD to RI changes, the measurement of small temperature variations in sub-nanoliter volumes is possible. Performing OCIBD with a 75 microm diameter laser beam on a silica chip that contains an etched channel with a 40 microm radius facilitates noninvasive thermometry on a N-(2-hydroxyethyl)piperazine-(2-ethanesulfonic acid) (HEPES) solution in a 188 x 10(-12) L probe volume with a temperature resolution of 9.9 x 10(-4) degrees C, at the 99% confidence level.     

A chip-scale universal detector for electrophoresis based on backscattering interferometry

An on-chip detector based on backscatter interferometry has been developed to perform sub-nanoliter volume refractive index measurements. The detection system consists of a simple, folded optical train based on the interaction of a laser beam and an etched channel in a silica (glass) plate. This etched channel is composed of two radii joined by a flat portion which define a curved surface in the shape of a half cylinder in a silica (glass) plate. The backscattered light from the channel takes on the form of a high contrast interference pattern that contains information related to the bulk properties of the fluid located within the probe volume. Positional changes of the interference pattern extrema (fringes) allow for the determination of refractive index changes at the 10(-6) level in a detection volume of 188 x 10(-12) L. Under capillary electrophoresis (CE) conditions, the injected mass detection limits for small molecules with little native absorption ranges from 530 fmol (0.18 ng) for sucrose to 720 fmol (0.43 nanograms) for raffinose. Fluorescein was also used to evaluate the technique for universal CE and under further optimized conditions can be quantified at the 150 microM level. Separation performance for the solutes tested ranged from about 2300 to 15,500 plates or 61,000 to 400,000 N m-1. The results presented here indicate there is potential for using the simple optical train of backscattering interferometry for on-chip universal solute analysis.     

Label-free molecular interaction determinations with nanoscale interferometry

Quantification of protein-protein and ligand-substrate interactions is central to understanding basic cellular function and for evaluating therapeutics. To mimic biological conditions, such studies are best executed without modifying the proteins or ligands (i.e., label-free). While tools for label-free assays exist, they have limitations making them difficult to fully integrate into microfluidic devices. Furthermore, it has been problematic to reduce detection volumes for on-channel universal analyte quantification without compromising sensitivity, as needed in label-free methods. Here we show how backscattering interferometry in rectangular channels (BIRC) facilitates label-free studies within picoliter volumes. The simple and unique optical train was based on rectangular microfluidic channels molded in poly(dimethylsiloxane) and low-power coherent radiation. Quantification of irreversible streptavidin-biotin binding and reversible protein A-human IgG Fc molecular interactions in a 225 pL detection volume was carried out label-free and noninvasively. Detection limits of 47 x 10(-15) mol of biotin reacted with surface-immobilized streptavidin were achieved. In the case of reversible interactions of protein A and the Fc fragment of human IgG, detection limits were determined to be 2 x 10(-15) mol of IgG Fc. These experiments demonstrate for the first time that (1) high-sensitivity universal solute quantification is possible using interferometry performed within micrometer-sized channels formed in inexpensive PDMS chips, (2) label-free reversible molecular interaction can be studied with femtomoles of solute, and (3) BIRC has the potential to quantify binding affinities in a high-throughput format.     

Noninvasive fluid flow measurements in microfluidic channels with backscatter interferometry

The ability to measure fluid velocity within picoliter volumes or on-chip noninvasively, is important toward fully realizing the potential of microfluidics and micrototal analysis systems, particularly in applications such as micro-high-performance liquid chromatography (HPLC) or in metering mixing where the flow rate must be quantified. Additionally, these measurements need to be performed directly on moving fluids in a noninvasive fashion. We presented here the proof of principle experiments showing nonintrusive fluid flow measurements can be accomplished on-chip using a pump and probe configuration with backscattering interferometry. The on-chip interferometric backscatter detector (OCIBD) is based on a fiber-coupled HeNe laser that illuminates a portion of an isotropically etched 40 microm radius channel and a position sensitive transducer to measure fringe pattern shifts. An infrared laser with a mechanical shutter is used to heat a section of a flowing volume and the resulting refractive index (RI) change is detected with the OCIBD downstream as a time-dependent RI perturbation. Fluid velocity is quantified as changes in the phase difference between the shutter signal and the OCIBD detected signal in the Fourier domain. The experiments are performed in the range of 3-6 microL/h with 3sigma detection limits determined to be 0.127 nL/s. Additionally, the RI response of the system is calibrated using temperature changes as well as glycerol solutions.     

Tris(2,2'-bipyridyl)ruthenium(II)-zirconia-Nafion composite films applied as solid-state electrochemiluminescence detector for capillary electrophoresis

The major goal of this work was to develop a new solid-state electrochemiluminescence (ECL) detector suitable for capillary electrophoresis (CE). The detector was fabricated by coating a sol-gel derived zirconia (ZrO(2))-Nafion composite film on a graphite electrode, then the zirconia-Nafion modified electrode was immersed in tris(2,2'-bipyridyl)ruthenium(II) (Ru(bpy)(3) (2+)) solution to immobilize this active chemiluminescence reagent. The voltammetric and ECL behaviors of the detector were investigated and optimized in tripropylamine solution. The ratio of 53% for zirconia in the zirconia-Nafion composite provided the highest luminescence intensity of immobilized Ru(bpy)(3) (2+). The ECL can maintain its stability very well in the phosphate solution in the period of 5-90 h when the solid-state ECL detector was immersed in the solution all the time. The optimum distance of capillary outlet to the solid-state ECL detector has been found to be ca. 50-80 microm for a 75 microm capillary. The effects of ionic strength and pH of ECL solution on peak height were investigated. The CE with solid-state ECL detector system was successfully used to detect tripropylamine, lidocaine, and proline. The detection limits (S/N = 3) were 5 x 10(-9) mol.L(-1) for tripropylamine, 1 x 10(-8) mol.L(-1) for lidocaine and 5 x 10(-6) mol.L(-1) for proline, and the linear ranges were from 1.0 x 10(-8) to 1.0 x 10(-5) mol.L(-1) for tripropylamine, 5.0 x 10(-7) mol.L(-1) to 1.0 x 10(-5) mol.L(-1) for lidocaine and 1.0 x 10(-5) to 1.0 x 10(-3) mol.L(-1) for proline, respectively.     

Quantification and evaluation of Joule heating in on-chip capillary electrophoresis

We present the use of a novel, picoliter volume interferometer to measure, for the first time, the extent of Joule heating in chip-scale capillary electrophoresis (CE). The simple optical configuration for the on-chip interferometric backscatter detector (OCIBD) consists of an unfocused laser, an unaltered silica chip with a half-cylinder channel and a photodetector. Using OCIBD for millidegree-level noninvasive thermometry, temperature changes associated with Joule heating (2.81 degrees C above ambient) in on-chip CE have been observed in 90 microm wide and 40 microm deep separation channels. The temporal response of Joule heating in isotropically etched channels was exponential, with it taking an excess of 2.7 s to reach equilibrium. Buffer viscosity changes have also been derived from empirical on-chip thermometry data, allowing for the determination of diffusion coefficients for solutes when separated in heated buffers. In addition, OCIBD has allowed the reduction in separation efficiency to be estimated in the absence of laminar flow and due to increased molecular diffusion and lower buffer viscosity. A 7% reduction in separation efficiency was determined for a high current drawing buffer such as Tris-boric acid under an applied field of just 400 V/cm. Results indicate that heating effects in on-chip CE have been underestimated and there is a need to readdress the theoretical model.     

胶束电动毛细管色谱安培检测中药马齿苋中多巴胺和去甲肾上腺素

建立了胶束电动毛细管色谱结合电化学安培检测同时分析中药马齿苋中多巴胺和去甲肾上腺素的方法。考察了缓冲液的浓度、pH值、十二烷基硫酸钠(SDS)浓度以及工作电极电势对分离检测的影响。在优化的条件下,多巴胺和去甲肾上腺素在1.0×10-6～5 0×10-4mol/L范围内有良好线性,浓度检测限(S/N=3)分别为8 7×10-7mol/L和4 2×10-7mol/L,质量检测限分别为1 45fmol和0 41fmol。该方法组分定性可靠,不需要衍生处理,选择性好。将该法应用于中药马齿苋样品的分析,获得了较好的结果。     

A sensitive and rapid immunoassay for quantification of CA125 in human sera by capillary electrophoresis with enhanced chemiluminescence detection

In this paper we have presented a sensitive and rapid immunoassay (IA) method by capillary electrophoresis with an enhanced chemiluminescence detection system (CE-CL) based on the catalytic effects of horseradish peroxidase (HRP) on the luminol-hydrogen peroxide reaction. The conditions for the CL reaction and electrophoresis were systematically investigated using HRP as a model sample. The linear range from 2.5 x 10(-11) to 1.0 x 10(-9) mol/L (R = 0.999), and the detection limit of 1.0 x 10(-12) mol/L (signal-to-noise ratio = 3) for HRP were achieved using para-iodophenol as CL enhancer. The relative standard deviations of the migration time and peak area for 5.0 x 10(-10) mol/L HRP (n = 7) were 0.26 and 4.8%, respectively, using a CE system with a home-built CL detector. Under the optimal condition, the HRP-labeled CA125 antibody (Ab) and the Ab-antigen complex were well separated within 4 min by CE using a high-pH buffer (pH 10.20). The assay was successfully used for quantification of CA125 in human sera from health controls and patients associated with ovarian cancer, and the recoveries of the standard addition experiments were 93-109%. Our primary results demonstrated that IA based on CE-CL detection is a powerful tool for clinical diagnosis combined with these commercial IA kits.     

亲和毛细管电泳法测定牛血清白蛋白和加替沙星的结合常数

利用亲和毛细管电泳法对牛血清白蛋白(BSA)与加替沙星(GT)间的结合反应及其相互作用做了初步探索,并应用淌度比(M)作为指标测定了两者的结合常数。以20 mmol/L pH 7.4的磷酸盐缓冲液作为运行缓冲液,分别以GT和BSA作为添加剂,另一组分为进样样品,内标为二甲基甲酰胺,于214 nm波长下检测。两种测定条件下得到的结合常数分别为4.4×104 L/mol和4.2×104 L/mol,与传统的荧光淬灭法测得的结果基本一致。该方法具有简单、高效的优点。     

Evaluation of band broadening in chemiluminescence detection coupled to pressurized capillary electrochromatography with an off-column coaxial flow interface

A system of off-column coaxial flow chemiluminescence (CL) detection coupled to pressurized CEC (pCEC) was described. The interface utilized a reactor that introduced postcolumn CL reagent into the capillary effluents in a sheathing flow profile. To compare and evaluate band broadening of analytes caused by the detector, the typical CL compounds luminol and N-(4-aminobutyl)-N-ethylisoluminol (ABEI) were separated and detected by pCEC or capillary HPLC (cHPLC) coupled to CL and UV detector, respectively. The results demonstrated that the band broadening caused by off-column detection interface was minimized due to the fast kinetic nature of the CL reaction. With the proposed pCEC-CL system, the detection limits of luminol and ABEI were 1.0x10(-8) and 8.0x10(-8) mol/L, respectively, which were approximately 100-fold more sensitive than those obtained with UV absorption. In addition, separation and detection of the ABEI-labeled L-lysine (L-Lys) and L-arginine (L-Arg) were accomplished by pCEC-CL method based on the principle of ABEI-potassium ferricyanide-alkaline medium CL reaction system. Under the optimum conditions, good results could be achieved compared with pCEC-UV.     

Capillary zone electrophoresis of amino acids on a hybrid poly(dimethylsiloxane)-glass chip

Poly(dimethylsiloxane) (PDMS)-PDMS and hybrid PDMS-glass devices have been characterized and compared in terms of current-voltage linearity, contact angle, electroosmotic velocity, electroosmotic mobility, and electrokinetic potential in dependence on the surface treatment. The hybrid PDMS-glass microfluidic devices have further been tested as on-chip capillary electrophoresis systems for the separation of fluorescently labeled amino acids. It has been demonstrated that different methods of surface pretreatment of the PDMS-glass devices result in significantly different separation performance, with plate numbers varying from 650 to 57 000 in dependence on the surface state and the nature of the amino acids. Electrophoretic separations of amino acids have been achieved within tens of seconds with detection limits of less than 2 microM (approximately 2 x 10(-16) to 2.5 x 10(-16) mol quantities at injection volumes of 110-120 pL). The detected amounts of fluorescein isothiocyante (FITC)-amino acids are at least ten times lower, since the amino acid:FITC ratio is 10:1 mol. The results demonstrate the perspective of such hybrid PDMS-glass microfluidic systems and the methods to modify their surfaces for on-chip separation methods for biomolecules.     

Capillary electrophoresis/electrochemical detection system with on-line deoxygenation

A capillary electrophoresis (CE)/electrochemical detection system with on-line deoxygenation was developed, consisting of a deoxygenation injector, a deoxygenation protector, and an electrochemical detection cell. When the system was utilized for 60 min, the steady-state current of oxygen detected could be dropped to 3% of the original value for the gold/mercury amalgam electrode and to 8% of the original value for the gold electrode, and the limit of detection could be decreased two orders of magnitude for the reducible analytes such as TI+ (from 3.1 x 10-5 mol/L to 8.0 x 10-7 mol/L) and metronidazole (from 3.8 x 10-5 mol/L to 4.0 x 10-7 mol/L).     

Rapid analysis of organic acids in plant extracts by capillary electrophoresis with indirect UV detection: directed metabolic analyses during metal stress

A fast, reliable capillary zone electrophoresis (CZE) method with indirect UV detection was optimized and validated to determine the main organic acids contained in plants. Citric, malic, succinic, oxalic, formic, fumaric, acetic acids, and phosphate were quantified. A rapid separation while keeping a good resolution was obtained by optimizing capillary length, separation voltage, electrolyte composition, and pH. Analyses were performed in a 30 cm uncoated fused-silica capillary (length to the detector window) in the co-electroosmotic mode with reversed electroosmotic flow and anodic detection using a -30 kV separation voltage. The pH 9.0 electrolyte contained 3 x 10(-4)mol/L tetradecyltrimethylammonium and 10(-2)mol/L trimellitate. Separation with baseline return was achieved in 100 s. Linearity, detection limits, repeatability, reproducibility, and recoveries were evaluated. Mean precision values of 0.2 and 3.4% for migration times and time-corrected peak areas, respectively, enabled accurate identification and quantification whether in standard solutions or in samples. Such performances were perfectly adapted to high-throughput routine determinations of organic acids in research or industry. Organic acids were assayed in different plant tissues and cells, including sycamore, arabidopsis, buttercup, and pea. Citrate and malate were the most abundant in all plants tested with concentrations reaching 18.9 and 22.3 micromol/g fresh matter, respectively. Cadmium effect on pea leaves metabolism was also assessed.     

Capillary gel electrophoresis for DNA sequencing. Laser-induced fluorescence detection with the sheath flow cuvette

Capillary polyacrylamide gel electrophoresis separation of dideoxycytidine chain-terminated DNA fragments is reported. A post-column laser-induced fluorescence detector based on the sheath flow cuvette was used to minimize background signals due to light scatter from the gel and capillary. A preliminary mass detection limit of 10(-20) mol of fluorescein-labeled DNA fragments was obtained. The system was used to analyze an actual DNA sequencing sample. Theoretical plate counts of 2 x 10(6) were produced. Gel stability limits the performance of the current system.     

Micellar electrokinetic chromatography of enkephalin-related peptides with laser-induced fluorescence detection

Eight enkephalin-related peptides were derivatized using fluorescein isothiocyanate (FITC), and the derivatization products were separated and detected by capillary electrophoresis with laser-induced fluorescence detection. The optimum molar ratio of FITC and peptide for derivatization was found to be 40:1, and 5 mmol/L sodium borate buffer (pH 9.2) was selected as derivatization media in order to get the high efficiency. Enkephalin-related peptides were completely separated in the pH range of 10.51-10.60 in a running buffer consisting of 100 mmol/L sodium borate and 60 mmol/L sodium dodecyl sulfate (SDS). The detection limit for these eight enkephalins ranged from 0.18 to 2.25 nmol/L, and the linear response range was 1.0 x 10(-6) to 1.0 x 10(-9) mol/L with correlation coefficients between 0.9947-0.9988. A separation efficiency as high as 380000 theoretical plates could be obtained for these analytes.     

毛细管胶束电动色谱法分析PTH氨基酸

建立了用毛细管胶束电动色谱法（MEKC）分离19种PTH氨基酸的方法,并探讨了电压、pH值、温度、胶束浓度对氨基酸迁移时间的影响。方法具有速度快、灵敏度高、样品用量少的优点。     

New technique for capillary electrophoresis directly coupled with end-column electrochemiluminescence detection

A new end-column electrochemiluminescence (ECL) detection technique coupling to capillary electrophoresis (CE) is characterized. A 300 microm diameter Pt working electrode was used to directly couple with a 75 microm inner diameter separation capillary without an electric field decoupler. The hydrodynamic cyclic voltammogram (CV) of Ru(bpy) 3 2+ showed that electrophoretic current did not affect the ECL reaction. The presence of high-voltage (HV) field only resulted in the shift of the ECL detection potential. The distance of capillary to electrode was an important parameter for optimizing detection performance as it determined the characteristics of mass transport toward the electrode and the actual concentration of Ru(bpy) 3 2+ in the detection region. The optimum distance of capillary to electrode was decided by the inner diameter of the capillary, too. For a 75 microm capillary, the working electrode should be placed away from the capillary outlet at a distance within the range of 220-260 microm. The effects of pH value of ECL solution and molecular structure of analytes on peak height and theoretical plate numbers were discussed. Using the 75 microm capillary, under the optimum conditions, the method provided a linear range for tripropylamine (TPA) between 1 x 10(-10) and 1 x 10(-5) mol/L with correlation coefficient of 0.998. The detection limit (signal-to-noise ratio S/N = 3) was 5.0 x 10(-11) mol/L. The relative standard deviation in peak height for eight consecutive injections was 5.6%. By this new technique lidocaine spiked in a urine sample was determined. The method exhibited the linear range for lidocaine from 5.0 x 10(-8) to 1.0 x 10(-5) mol/L with correlation efficient of 0.998. The limit of detection (S/N = 3) was 2.0 x 10(-8) mol/L.     

基于无标记的核酸适配体荧光生物传感器检测凝血酶

设计了一个简单、通用、基于核酸适配体无标记的高敏感、高专一检测凝血酶的荧光方法。以无标记凝血酶核酸适配体单链DNA为识别元素,Pico Green染料传导互补双链的荧光信号。Pico Green是一种不对称菁,当其单独存在时不产生荧光信号,而当其被吸附到互补的双链DNA上时,可产生很强的荧光信号,但被吸附到单链DNA上时,却无明显的信号改变。基于该性质,将其用于凝血酶的检测。该方法对凝血酶的响应线性范围为1.0×10~(-14)~1.0×10~(-7)mol/L,相关系数(r~2)为0.99,检出限为1.0×10~(-14)mol/L。1.0×10~(-8)mol/L两种干扰物质(牛血清蛋白和细胞色素C)的存在不影响凝血酶的检测,表明该方法对凝血酶具有非常好的专一性。该方法成功应用于对人血清样品的检测,其平均回收率为97%~102%。方法可简单、灵敏、特异性地检测凝血酶,有望用于医学临床诊断等领域。     

Quantitative determination of glutathione in single human erythrocytes by capillary zone electrophoresis with electrochemical detection

Glutathione (GSH) in single human erythrocytes is determined by capillary zone electrophoresis with end-column amperometric detection at a gold/mercury amalgam microelectrode. A capillary of 10 microm inner diameter is suitable for determination of GSH in an individual erythrocyte with a good signal-to-noise ratio. The limit of detection is 1 x 10(-7) mol/L or 26 amol and the linear dynamic range is 2 x 10(-7) to 2 X 10(-5) mol/L for the capillary. In this method, the calibration line is obtained with a capillary adsorbed before a certain amount of hemoglobin can be used for the quantification of GSH in the external standardization. The whole cell injection and the lack of necessity of a derivatization reaction lead to more accurate and precise results, which are closer to the macroscopic values of glutathione in human red blood cell (i.e., hemolysate) than those determined by indirect laser-induced fluorescence detection.     

毛细管电泳电化学法检测过氧化氢

以金微盘电极和离子液体修饰单壁碳纳米管糊微盘电极分别作为毛细管电泳电化学检测器,试验了两种电极对过氧化氢的响应情况,将金微盘电极与毛细管电泳联用,对过氧化氢进行了定性和定量检测.探讨了分离电压、缓冲溶液pH值和工作电位等条件对H2O2检测的影响.实验结果表明,峰电流与H2O2浓度在1.0×10-6～1.0×10-5mo...