Sensitive label-free electrochemical analysis of human IgE using an aptasensor with cDNA amplification.

In this study, we developed an ultrasensitive label-free aptamer-based electrochemical biosensor, featuring a highly specific anti-human immunoglobulin E (IgE) aptamer as a capture probe, for human IgE detection. Construction of the aptasensor began with the electrodeposition of gold nanoparticles (AuNPs) onto a graphite-based screen-printed electrode (SPE). After immobilizing the thiol-capped anti-human IgE aptamer onto the AuNPs through self-assembly, we treated the electrode with mercaptohexanol (MCH) to ensure that the remaining unoccupied surfaces of the AuNPs would not undergo nonspecific binding. We employed a designed complementary DNA featuring a guanine-rich section in its sequence (cDNA G1) as a detection probe to bind with the unbound anti-human IgE aptamer. We measured the redox current of methylene blue (MB) to determine the concentration of human IgE in the sample. When the aptamer captured human IgE, the binding of cDNA G1 to the aptamer was inhibited. Using cDNA G1 in the assay greatly amplified the redox signal of MB bound to the detection probe. Accordingly, this approach allowed the linear range (coefficient of determination: 0.996) for the analysis of human IgE to extend from 1 to 100,000pM; the limit of detection was 0.16pM. The fabricated aptasensor exhibited good selectivity toward human IgE even when human IgG, thrombin, and human serum albumin were present at 100-fold concentrations. This method should be readily applicable to the detection of other analytes, merely by replacing the anti-human IgE aptamer/cDNA G1 pair with a suitable anti-target molecule aptamer and cDNA.

1-Butyl-3-methylimidazolium-based ionic liquids and an anionic surfactant: excellent background electrolyte modifiers for the analysis of benzodiazepines through capillary electrophoresis.

In this study, we found that adding 1-butyl-3-methylimidazolium-based ionic liquids (ILs) and sodium dodecyl sulfate (SDS) as modifiers in the background electrolyte (BGE) for capillary electrophoresis enhanced the separation of benzodiazepines. In particular, 1-butyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide ([BMIM][NTf2]) was the best IL additive for the separation system because its anionic moiety interacted favorably with the benzodiazepines. We added SDS because of its known effect on the separation of hydrophobic analytes. We optimized the separation conditions in terms of the concentrations of the IL, SDS, and organic solvent, the pH, and the BGE's ionic strength. The optimal BGE, containing 170 mM [BMIM][NTf2] and 10 mM SDS, provided baseline separation, high efficiency, and satisfactory peak shapes for the benzodiazepines. The separation mechanism was based on heteroassociation between the anionic moiety of the IL and the benzodiazepines, with SDS improving the resolution of the separation. The limits of detection for the seven analytes ranged from 2.74 to 4.42 microg/mL. We subjected a urine sample to off-line solid phase extraction (SPE) prior to the analysis of its benzodiazepine content. Our experimental results reveal that the combination of [BMIM][NTf2] and SDS provides adequate separation efficiency for its application to CE analyses of benzodiazepines after SPE concentration.

Separation of open-cage fullerenes using nonaqueous capillary electrophoresis.

In this study, nonaqueous capillary electrophoresis (NACE) was used to separate three open-cage fullerenes. Trifluoroacetic acid (TFA) was used as the nonaqueous background electrolyte to change the analytes' mobilities. The selectivity and separation efficiency were critically affected by the nature of the buffer system, the choice of organic solvent, and the concentrations of TFA and sodium acetate (NaOAc) in the background electrolyte. The optimized separation occurred using 200 mM TFA/20mM NaOAc in MeOH/acetonitrile (10:90, v/v), providing highly efficient baseline separation of the open-cage fullerenes within 5 min. The migration time repeatability for the three analytes was less than 1% (relative standard deviation). Thus, NACE is a rapid, useful alternative to high-performance liquid chromatography for the separation of open-cage fullerenes.

Using the cationic surfactants N-cetyl-N-methylpyrrolidinium bromide and 1-cetyl-3-methylimidazolium bromide for sweeping-micellar electrokinetic chromatography.

This paper describes a sweeping-micellar electrokinetic chromatography (sweeping-MEKC) technique for the determination of seven benzodiazepines, using, as sweeping carriers, the ionic liquid-type cationic surfactants 1-cetyl-3-methylimidazolium bromide (C(16)MIMBr) and N-cetyl-N-methylpyrrolidinium bromide (C(16)MPYB). These surfactants resemble the commonly employed cationic surfactant cetyltrimethylammonium bromide (CTAB), but they provide different separation efficiencies. We optimized the separation and sweeping conditions, including the pH, the concentrations of organic modifier and surfactant, and the sample injection volume. Adding C(16)MIMBr or C(16)MPYB to the background electrolyte enhanced the separation efficiency and detection sensitivity during the sweeping-MEKC analyses of the benzodiazepines. C(16)MIMBr enhanced the sensitivity for each benzodiazepine 31-59-fold; C(16)MPYB, 86-165-fold. In the presence of C(16)MPYB, the limits of detection for the seven analytes ranged from 4.68 to 9.75 ng/mL. We adopted the sweeping-MEKC conditions optimized for C(16)MPYB to satisfactorily analyze a human urine sample spiked with the seven benzodiazepines. To minimize the matrix effects, we subjected this urine sample to off-line solid phase extraction (SPE) prior to analysis. The recoveries of the analytes after SPE were satisfactory (ca. 77.0-88.3%). Our experimental results reveal that the cationic surfactant C(16)MPYB exhibits superior sweeping power relative to those of C(16)MIMBr and CTAB and that it can be applied in sweeping-MEKC analyses for the on-line concentrating and analyzing of benzodiazepines present in real samples at nanogram-per-milliliter concentrations.

Using sweeping micellar electrokinetic chromatography to analyze Delta9-tetrahydrocannabinol and its major metabolites.

We have applied sweeping micellar electrokinetic chromatography (sweeping-MEKC) to the simultaneous determination of Delta(9)-tetrahydrocannabinol (THC) and its major metabolites, 11-hydroxy-Delta(9)-tetrahydrocannabinol (THC-OH) and 11-nor-9-carboxy-Delta(9)-tetrahydrocannabinol (THC-COOH). We monitored the effects of several of the sweeping-MEKC parameters, including the proportion of organic modifier, the concentration of sodium dodecyl sulfate (SDS), the pH, and the sample injection volume, to optimize the separation process. The optimal buffer for the analysis of the three analytes was 25 mM citric acid/disodium hydrogenphosphate (pH 2.6) containing 40% methanol and 75 mM SDS. Under the optimized separation parameters, the enrichment factors for THC, THC-COOH, and THC-OH when using sweeping-MEKC (relative to MEKC) were 77, 139, and 200, respectively. The limits of detection (LODs) for the three compounds in standard solutions ranged from 3.87 to 15.2 ng/mL. We combined the sweeping-MEKC method with solid-phase extraction to successfully detect THC, THC-COOH, and THC-OH in human urine with acceptable repeatability. The LODs of these analytes in urine samples ranged from 17.2 to 23.3 ng/mL. Therefore, this sweeping-MEKC method is useful for determining, with high sensitivity, the amounts of THC and its metabolites in the urine of suspected THC users.

Determination of cocaine and its metabolites using cation-selective exhaustive injection and sweeping-MEKC.

We have employed a high-sensitivity on-line preconcentration method, cation-selective exhaustive injection (CSEI) and sweeping MEKC, for the analysis of cocaine, benzoylecgonine, norcocaine, and cocaethylene. We monitored the effects of several of the CSEI-sweeping-MEKC parameters - including the pH, the concentrations of SDS and organic modifier, the injection length of the high-conductivity buffer, and the injection time of the sample - to optimize the separation process. The optimal BGE was 100 mM phosphoric acid (pH 1.8) containing 75 mM SDS with 10% 2-propanol and 10% tetrahydrofuran as the organic modifier. In addition, electrokinetic injection of the sample at 15 kV for 900 s provided both high separation efficiency and enhanced sweeping sensitivity. The sensitivity enhancements for cocaine, norcocaine, and cocaethylene ranged from 2.06 x 10(4) to 3.96 x 10(4); for benzoylecgonine it was 1.75 x 10(3); the coefficients of determination exceeded 0.9958. The LODs, based on an S/N ratio of 3:1, of sweeping-MEKC ranged from 33.5 to 52.8 ng/mL; in contrast, when using CSEI-sweeping-MEKC the sensitivity increased to range from 29.7 to 236 pg/mL. Under the optimal conditions, we analyzed cocaine in a human urine sample prepared using off-line SPE to minimize the influence of the matrix. The recovery of the SPE efficiency was satisfactory (ca. 74.9-87.6%). Our experimental results suggest that, under the optimal conditions, the CSEI-sweeping-MEKC method can be used to determine cocaine and its metabolites with high sensitivity in human urine.

Using cation-selective exhaustive injection and sweeping micellar electrokinetic chromatography to determine selective serotonin reuptake inhibitors.

We have employed a rapid and highly efficient on-line preconcentration method, cation-selective exhaustive injection and sweeping micellar electrokinetic chromatography (CSEI-sweeping-MEKC), for the analysis of selective serotonin reuptake inhibitors (SSRIs) of antidepressant drugs. We monitored the effects of several of the CSEI-sweeping-MEKC parameters - including the pH, the concentrations of high-conductivity buffer (HCB), sodium dodecyl sulfate (SDS), and organic modifier, the injection length of the HCB, and the injection time of the sample - to optimize the separation process. The optimal background electrolyte was 50 mM citric acid/disodium hydrogenphosphate buffer (pH 2.2) containing 100 mM SDS and 22% isopropyl alcohol. The sensitivity enhancements of the SSRIs sertraline, fluoxetine, paroxetine, fluvoxamine, and citalopram ranged from 5.7 x 10(4) to 1.2 x 10(5); the coefficients of determination exceeded 0.9938 and the relative standard deviations of the peak heights were less than 3.2%; the detection limits ranged from 0.056 to 0.22 ng/mL. We employed the optimal conditions to analyze these five SSRIs in a plasma sample prepared using solid-phase extraction (SPE) to minimize the influence of the matrix. Although the limits of detection of the SSRIs in human plasma were higher than those in pure water, this present technique is more sensitive than other, more-conventional methods. The recovery of the SPE extraction efficiency was satisfactory (up to 89%). Our findings suggest that, under the optimal conditions, the CSEI-sweeping-MEKC method can be used successfully to determine these five SSRIs in human plasma.

Chaotropic salts: novel modifiers for the capillary electrophoretic analysis of benzodiazepines.

This paper describes a CE method for analyzing benzodiazepines using the chaotropic salts lithium trifluoromethanesulfonate (LiOTf), lithium hexafluorophosphate (LiPF(6)), and lithium bis(trifluoromethanesulfonyl)imide (LiNTf(2)) as modifiers in the running buffer. Although adequate resolution of seven benzodiazepine analytes occurred under the influence of each of the chaotropic anions, the separation efficiency was highest when bis(trifluoromethanesulfonyl)imide (Tf(2)N(-)) was the modifier. We applied affinity CE in conjunction with linear analysis to determine the association constants for the formation of complexes between the Tf(2)N(-) anion and the benzodiazepines. According to the estimated Gibbs free energies, the interactions between this chaotropic anion and the benzodiazepines were either ion-dipole or ion-induced dipole interactions. Adding chaotropic salts as modifiers into CE buffers is a simple and reproducible technique for separating benzodiazepines.

In-channel simplified decoupler with renewable electrochemical detection for microchip capillary electrophoresis.

Electrochemical (EC) detection is comparable to fluorescence detection in that it is simple to perform, economical, and highly sensitive. In this study, we used replica molding to fabricate a PDMS microchip for microchip capillary electrophoresis (CE). A decoupler electrode and a working electrode were implanted into the PDMS chip during the molding process to prevent leakage into the electrode channel. The working electrode could be renewed readily through its slight withdrawal (ca. 3 mm) from the PDMS; its detection ability was highly reproducible in the microchip CE-EC system. The relative standard deviation (R.S.D.) of the detecting current for the renewed working electrode was 1.2% (n=5). The calibration curves were linear for both dopamine and catechol analytes over the concentration range 10-1000 microM, with coefficients of determination (R(2)) of 0.999 and 0.976, respectively. The number of theoretical plates (N/m) for these analytes was greater than 133,000.

On-line preconcentration and determination of ketamine and norketamine by micellar electrokinetic chromatography. Complementary method to gas chromatography/mass spectrometry.

We have investigated a rapid, simple, and highly efficient on-line preconcentration method using in micellar electrokinetic chromatography (MEKC) for the analysis of abused drugs. Ketamine is an anesthetic that has been abused as a hallucinogen. We applied the sample sweeping technique first to ketamine and its major metabolite, norketamine, and separated the analytes with MEKC. Several of the sweeping MEKC parameters to effect successful separations, such as the concentration of sodium dodecyl sulfate (SDS), the injection time, and the applied voltage were optimized. The improvements in the number of theoretical plates under the different separation conditions are presented clearly in a three-dimensional representation. The limits of detection were 2.8, 3.4, and 3.3 ng/mL for ketamine, norketamine, and ketamine-D(4), respectively. The enrichment factor for each compound was within the range of 540-800. Experimental results are in agreement with those of analysis conducted by gas chromatography/mass spectroscopy (GC/MS). Therefore, we believe that sweeping, combined with MEKC, represents a suitable complementary method to GC/MS for use in clinical and forensic analyses of ketamine and norketamine.