Analysis of structural specificity in antibody—antigen reactions by capillary electrophoresis with laser-induced fluorescence detection

A rapid and simple procedure for screening antibodies for binding to an antigen is proposed. A fluorescent hapten —dye conjugate was prepared by labeling the amino moiety of the hapten with a commercially available reactive cyanine dye, Cy5 (excitation maximum: 650 nm, emission maximum: 670 nm). A fixed amount of the Cy5—hapten was titrated with serial dilution of the antibody. Each of the titration mixture was analyzed by capillary electrophoresis (25 cm × 20 μm column) monitored by laser-induced fluorescence (laser: 10 mW helium-neon, 632.8 nm). Free and antibody-bound Cy5-hapten were analyzed simultaneously on the electropherogram. Competitive immunoassay of hapten was demonstrated with low-end sensitivity of 5 · 10⁻⁸ M, about 10× more sensitive than the present drug screening methods. Using morphine as an example, the screening of various antibodies (from different vendors) and cross-reactivity of morphine analogues using the present procedure will be discussed.     

毛细管电泳-间接激光光热干涉检测方法研究

本文提出毛细管电泳-间接激光光热干涉检测新方法。采用吸收系数大、吸收波长与泵浦激光(He-Ne)波长匹配性能较好的亚甲蓝溶液为背景电解质溶液,加入乙醇减少了毛细管壁对亚甲蓝的吸附作用。并将间接光热干涉检测法用于氨基酸毛细管电泳分离检测,对赖氨酸检测限达5×10^-6mol/L(S/N=2).     

Ultraviolet laser-induced fluorescence detection strategies in capillary electrophoresis: determination of naphthalene sulphonates in river water

Various UV-laser-induced fluorescence detection strategies for capillary electrophoresis (CE) are compared, i.e. two UV-laser systems (a pulsed laser providing up to 25 mW of tunable emission, applied at 280, 290 and 325 nm, and a continuous wave (cw) laser providing up to 100 mW of 257 nm emission) and different methods to collect the fluorescence emission signal and to reduce the background. Attention is focused on the determination of amino- and hydroxy-substituted naphthalene sulphonates (NS) in river water; these analytes exhibit native fluorescence upon UV excitation. Optimum results were obtained by applying only a minor portion of the available (average) laser powers, viz. 0.7 mW at 280 nm for the pulsed laser, and 5 mW for the cw laser. For emission collection, the most favourable results were obtained with a mirror-based microscope objective, which facilitates efficient spatial filtering and does not produce impurity fluorescence upon UV-laser irradiation. For standard solutions, the cw laser gave around 20-fold better detection limits (10⁻⁹–10⁻¹⁰ M) than the pulsed laser. For river water, excitation of interferences (presumably humic acids, which exhibit native fluorescence) could be much better suppressed if the pulsed laser was used with selective excitation at 280 nm. Therefore, for real-sample analysis the latter combination is to be preferred. The set-up was used for the identification and quantification (at the 1–35 μg l⁻¹ level) of NS in a river Elbe sample.     

Semi-quantitative trace analysis of nuclear fast red by surface enhanced resonance Raman scattering

The dye nuclear fast red has been detected and determined semi-quantitatively by means of surface enhanced resonance Raman scattering (SERRS) and surface enhanced Raman scattering (SERS), using laser exciting wavelengths of 514.5 and 632.8 nm, respectively, by employing a citrate-reduced silver colloid. A good linear correlation is observed for the dependence of the intensities of the SERRS bands at 989 cm⁻¹ (R=0.9897) and 1278 cm⁻¹ (R=0.9872) on dye concentration over the range 10⁻⁹ to 10⁻⁷ M, when using an exciting wavelength of 514.5 nm. At dye concentrations above 10⁻⁷ M, the concentration dependence of the SERRS signals is non-linear. This is almost certainly due to the coverage of the colloidal silver particles being in excess of a full monolayer of the dye. A linear correlation is also observed for the dependence of the intensities of the SERS bands at 989 cm⁻¹ (R=0.9739) and 1278 cm⁻¹ (R=0.9838) on the dye concentration over the range 10⁻⁸ to 10⁻⁶ M when using an exciting wavelength of 632.8 nm. Strong fluorescence prevented collection of resonance Raman scattering (RRS) spectra from powdered samples or aqueous solutions of the dye using an exciting wavelength of 514.5 nm, but weak bands were observed in the spectra obtained from both powdered and aqueous samples of the dye using an exciting wavelength of 632.8 nm. A study of the pH dependence of SERRS/SERS and UV–VIS absorption spectra revealed the presence of different ionisation states of the dye. The limits of detection for nuclear fast red by SERRS (514.5 nm), SERS (632.8 nm) and visible spectroscopy (535 nm) are 9, 89 and 1000 ng ml⁻¹, respectively.     

Biological effects of helium-neon (HeNe) laser irradiation on acrosome reaction in bull sperm cells

The purpose of this study was to determine the effect of He---Ne laser irradiation (632.8 nm, 10 mW) on the induction of acrosome reaction and mortality in bull sperm cells in comparison with two important capacitation agents; calcium and heparin. Frozen-thawed bull sperm cells were washed in percoll gradient and suspended at a concentration of 1 × 10⁶ ml⁻¹ in sp-TALP medium, capacitated in the presence of 2 mM CaCl₂, 10 μg ml⁻¹ heparin, or irradiated at fluences from 2 to 16 J cm⁻², and incubated for 0, 30, 60 and 90 minutes. At the end of the incubation period, the percentage of sperm that were acrosome-reacted and dead was determined. The results obtained indicated that laser irradiation at all fluences produced a significant increase (p < 0.001) in the percentage of sperm cells that were acrosome reacted, and a significant decrease (p < 0.001) in the percentage of dead sperm at 90 minutes of incubation in comparison to other capacitation agents and the control group. The percentage of sperm cells with acrosome reaction was increased with increasing fluences of laser irradiation and time of incubation. It is conclude that the application of He---Ne laser irradiation at fluences from 2 to 16 J cm⁻² induced the acrosome reaction and decreased the sperm mortality percentage in vitro of bull sperm cells.     

Comparison of the effects of visible femtosecond laser pulses and continuous wave laser radiation of low average intensity on the clonogenicity of Escherichia coli.

To evaluate the contribution of local pulsed heating of light-absorbing microregions to biochemical activity, irradiation of Escherichia coli was carried out using femtosecond laser pulses (λ = 620 nm, τ_p=3 × 10⁻¹³ s, f_p = 0.5 Hz, E_p = 1.1 × 10⁻³J cm⁻², I_av = 5.5 × 10⁻⁴ W cm⁻², I_p = 10⁹ W cm⁻²) and continuous wave (CW) laser radiation (λ = 632.8 nm, I = 1.3 W cm⁻²). The irradiation dose required to produce a similar biological effect (a 160%–190% increase in the clonogenic activity of the irradiated cells compared with the non-irradiated controls) is a factor of about 10³ lower for pulsed radiation than for CW radiation (3.3 × 10⁻¹ and 7.8 × 10² J cm⁻² respectively). The minimum size of the microregions transiently heated on irradiation with femtosecond laser pulses is estimated to be about 10 Å, which corresponds to the size of the chromophores of hypothetical primary photoacceptors—respiratory chain components.     

Sulfur speciation by capillary electrophoresis with indirect spectrophotometric detection: In search of a suitable carrier electrolyte to maximize sensitivity

Various probes have been evaluated as alternative ions to chromate, which is most frequently used in the analysis of small inorganic anions by capillary electrophoresis with indirect UV detection. Sulfur species (S₂O₃²⁻, SO₄²⁻, S₄O₆²⁻, S(−II)) have been determined. The optimization of the method was particularly focused on S(−II) since this species rapidly yields S₂O₃²⁻ and SO₄²⁻ in the presence of oxidizing agents. Therefore, it could not be analysed by capillary electrophoresis with chromate as the background electrolyte. The alternative probe ions all contain aromatic rings (benzene or naphthalene) to provide the intrinsic background absorbance for indirect detection. They are all fully ionized at the pH chosen for this application (TRIS buffer, pH=8) and the range of their mobilities is large enough to suit the analytes mobilities. They have no oxidizing properties. Transfer ratios have been determined experimentally and compared to calculated values derived from the Kohlrausch regulation function. All experimental values were lower than expected from the calculations, which proves the limitations of the Kohlrausch theory concerning the configuration (electrolyte/analyte) of this study. However, maximizing (z_A/z_E)·ε_E (with z_A and z_E being the charges of the analyte and the probe, respectively, and ε_E the molar absorptivity of the probe) and keeping the mobility of the probe close to those of the analytes will give a good hint for the choice of the most suitable UV-absorbing probe. Pyromellitate and naphthalenetrisulfonate, the mobilities of which are close to that of S(−II), give the best sensitivity for this species, with good resolution and sensitivity for all other species.     

Capillary electrophoresis for measuring major and trace anions in thermal water and condensed-steam samples from hydrothermal springs and fumaroles

A new application of capillary electrophoresis for measuring major and trace anions in thermal water and condensed-steam samples is presented. Ten fluid samples were collected from hydrothermal springs and fumaroles located in a volcanic zone of Deception Island, Antarctica. Anion separation was achieved in less than 6 min using indirect UV detection at 254 nm with a negative power supply (−15 kV). The electrolyte consisted of 4.7 mM sodium chromate, 4.0 mM electroosmotic flow modifier (OFM) hydroxide, 10 mM 2-(N-cyclohexylamino)ethanesulfonic acid and 0.1 mM calcium gluconate (pH 9.1). Major anions (Cl⁻, SO₄², PO₄H²⁻, and CO₃H⁻) were measured using hydrostatic injection (10 cm for 30 s) at 25°C. Trace amounts of anions (F⁻, Br⁻, and NO₃⁻) were better determined by electromigration injection (4 kV, 10 s) at 15°C. Good reproducibility of the migration times (<0.72% RSD), a satisfactory linear response and accuracy as well as acceptable detection limits were successfully obtained.     

A collinear light-emitting diode-induced fluorescence detector for capillary electrophoresis

A novel fluorescence detector based on collinear scheme using a brightness light-emitting diode emitting at 470 nm as excitation source is described. The detector is assembled by all-solid-state optical-electronic components and coupled with capillary electrophoresis using on-column detection mode. Fluorescein isothiocyanate (FITC) and FITC-labeled amino acids and small molecule peptide as test analyte were used to evaluate the detector. The concentration limit of detection for FITC-labeled phenylalanine was 10 nM at a signal-to-noise ratio (S/N) of 3. The system exhibited good linear responses in the range of 1 × 10⁻⁷ to 2 × 10⁻⁵ M (R² = 0.999).     

Biomodulative effects induced by 805 nm laser light irradiation of normal and tumor cells

The influence of light emitted from a diode laser centred at λ = 805 nm was investigated on murine skeletal myotubes (C2), normal urothelial cells (HCV29), human squamous carcinoma cells of the gingival muscosa (ZMK) and urothelial carcinoma cells (J82) in a computer-controlled irradiation chamber. Cells were treated with varying fluences between 0 and 20 J cm⁻². The response was tested by analysis of the mitotic index using single cell counting after Orcein staining and proliferation index based on BrdU incorporation during DNA synthesis. While the mitotic index of C2, HCV29 and J82 cells increased at a fluence of 4 J cm⁻², irradiation with fluences of 20 J cm⁻² resulted in a slight decrease. ZMK tumor cells showed a decrease of the mitotic index with both fluences. No significant differences could be determined when using irradiances between 10 mW cm⁻² and 150 mW cm⁻². The BrdU test after irradiation showed no significant effects compared to the controls in each cell line.     

End-column electrochemical detection for inorganic and organic species in high-voltage capillary electrophoresis

The electronics and construction for an end-column ultramicroelectrode (3–10 μm) detection system that permits the use of medium-sized capillaries (25 μm I.D.) without appreciable effects from the potential field at the end of the capillary. Normal peak-to-peak noise over 10 s was 0.01–0.1 pA. The background noise observed for a 200 × μm carbon-fiber electrode placed either 180 μm within a 25-μm capillary or at a point 500 μm away from the capillary was essentially the same. A study of detector response as a function of the position of the electrode has shown that accurate location of the electrode is important for sensitive and reproducible detection. These studies also showed that differences between the density of the electrolyte existing the capillary and the electrolyte in the detection cell could cause anomalous electrode response depending on the location of the electrode relative to the end of the capillary. Application of a carbon fiber or an Hg film electrode gave detection limits (twice the peak-to-peak noise over 10 s) of 2 · 10⁻⁸ mol/l for Pb²⁺, 1 · 10^{− 5} mol/l for NO₂⁻ and 5 · 10⁻¹⁰ mol/l for catechol.     

Immobilization enzyme fluorescence capillary analysis for determination of lactic acid

A new method for the determination of lactic acid based on the immobilization enzyme fluorescence capillary analysis (IE-FCA) was proposed. Lactic dehydrogenase (LDH) was immobilized on inner surface of a capillary with glutaraldehyde, and an immobilized enzyme lactate capillary bioreactor (IE-LCBR) was formed for the determination of lactic acid. After nicotinamide adenine dinucleotide (NAD⁺) is mixed with lactic acid solution, it was sucked into the IE-LCBR and was detected at λ_ex 353 nm/λ_em 466 nm. Optimized conditions are as follows: the temperature is 38 °C; the reaction time is 15 min; the concentrations of Tris buffer (pH 8.8) and NAD⁺ are 0.1 mol L⁻¹ and 4 mmol L⁻¹, respectively; the concentration of LDH used for immobilization is 15 kU L⁻¹. The concentration of lactic acid is directly proportional to the fluorescence intensity measured from 0.50 to 2.0 mmol L⁻¹; and the analytical recovery of added lactic acid was 99–105%. The minimum detection limit of the method is 0.40 mmol L⁻¹ and sensitivity of the IE-CBR is 4.6 F mmol⁻¹ L⁻¹ lactate. Its relative standard deviation (R.S.D.) is ≤2.0%. This IE-FCA method was employed for determination of lactate in milk drink.     

Simultaneous detection and quantitation of sodium, potassium calcium and magnesium in ocular lenses by high- performance capillary electrophoresis with indirect photometric detection

A high-performance capillary electrophoresis (HPCE) method which can be used to quantitatively determine Na⁺, K⁺, Ca²⁺ and Mg²⁺ simultaneously in ocular lenses has been developed. The proteins in the lens aqueous homogenates were precipitated by 10% trichloroacetic acid. The precipitated proteins were removed after a brief centrifugation, and the supernatant containing the cations was washed with ether and directly used for HPCE analysis. A 50 μm × 75 cm fused-silica capillary was used for separation and the detection wavelength was set at 214 nm. A 20-mM imidazole at pH 6.0 containing 0.1% hydroxypropyl methyl cellulose was used as background electrolyte. Sample solution was injected at 15 kV for 10 s, and the electrophoresis was carried out at 15 kV. All the cations can be separated and quantified from the peak areas within 9 min. The values obtained by this method were comparable with commonly used flame atomic absorption and flame atomic emission spectroscopy. It is demonstrated that this HPCE method can be used to quantify all the cation levels simultaneously within a short time even in a small single rat or mice lens.     

Capillary electrophoresis with electrochemiluminescence detection of procyclidine in human urine pretreated by ion-exchange cartridge

This paper describe a Ru(bpy)₃²⁺ based electrochemiluminescence (ECL) method to detect procyclidine in human urine following separation by capillary electrophoresis (CE). An ECL detection cell was designed for post-column addition of Ru(bpy)₃²⁺. Parameters affecting separation and detection were optimized, leading to a detection limit of 1×10⁻⁹ mol/l in an on-capillary stacking mode. For application in urine, a cartridge packed with slightly acidic cation-exchange resin was used to eliminate the matrix effects of urine and improve the detection sensitivity. Extraction recovery was nearly 90%.     

Far-UV laser flash photolysis in solution. A study of the chemistry of 1,1-dimethylsilene in hydrocarbon solvents

The far-UV (193 nm) laser flash photolysis of nitrogen-saturated isooctane solutions of 1,1-dimethylsiletane allows the direct detection of 1,1-dimethylsilene as a transient species, which (at low laser intensities) decays with pseudo-first-order kinetics (τ 10 μs) and exhibits a UV absorption spectrum with λ_max 255 nm. Characteristic rapid quenching is observed for the silene with methanol (k_McOH = (4.9 ± 0.2) × 10⁹ M⁻¹ s⁻¹), tert-butanol (k_BuOH = (1.8 ± 0.1) × 10⁹ M⁻¹ s⁻¹) and oxygen (k_O2 = (2.0 ± 0.5) × 10⁸ M⁻¹ s⁻¹). The Arrhenius activation parameters for the reaction with methanol have been determined to be E_a = −2.6 ± 0.6 kcal mol⁻¹ and log A = 7.7 ± 0.3.     

Capillary electrochromatographic separation of metal ion species with on-line detection by inductively coupled plasma mass spectrometry

A bonded phase capillary column containing macrocyclic polyamine, [28]ane-N₆O₂ functional groups was used for the electrophoretic separation of arsenic, chromium and selenium species. A simple device interfacing this capillary electrochromatography (CEC) systems to an inductively coupled plasma mass spectrometer (ICPMS) is described. The dimension of the capillary column was 160 cm×100 μm i.d. To accommodate this electrophoretic separation, an auxiliary capillary was used with nitric acid (0.05 M) as makeup liquid. With the electrokinetic method at –20 kV, 20 s and a nebulizer gas flow rate of 1 l min⁻¹, the sample injected was analyzed with an applied potential of −20 kV. The background electrolyte buffer for the separation of CrO₄²⁻–Cr³⁺ was phosphate (20 mM, pH 6.5). That for HAsO₄²⁻–Ph₄As⁺ was pyromellitate (20 mM, pH 6.0) and for SeO₄²⁻–SeO₃²⁻ was acetate (20 mM, pH 6.0). The role of the buffer’s anion was also discussed. The separation efficiency of the bonded phase was compared with the bare fused silica. Concentration detection limits for these metal ions were in the low ppb range. In addition, the matrix effect of the established system with the bonded phase was found smaller than that with the bare fused silica.     

Forward-scattering degenerate four-wave mixing as a potential laser-based absorption detection method in liquid separation systems: Coupling to conventional-size liquid chromatography

An explorative study on the compatibility of liquid separation systems, such as (micro) liquid chromatography (LC) and capillary electrophoresis (CE), and forward-scattering degenerate four-wave mixing (F-D4WM) as a detection method is presented. F-D4WM is a laser-based technique showing some analogy with holographic spectroscopy: a signal on a theoretical dark background is observed as a result of light absorption by an analyte. Parameters considered are solvent composition focussing on acetonitrile, methanol and water; mobile phases in LC and CE), detector cell construction, and influences of laser beam powers. A specially designed detector cell has been developed to meet the Brewster condition, both at the air-quartz and the quartz-liquid boundaries. For practical reasons, the tested cell has an optical pathlength of 1 mm; reduction to 100 μm is required to apply the cell in microseparations. The F-D4WM technique has been involved for detection in a conventional-size, reversed-phase LC separation of 1- and 2-aminoanthraquinones. The detection limit obtained (for the 1 mm cell) is 2 × 10⁻⁵ absorbance units. The experiments indicate that further reduction of background deserves explicit attention.     

Separation and indirect detection by capillary zone electrophoresis of ppb (w/w) levels of aluminum ions in solutions of multiple cations

Solutions of multiple cations in aqueous solutions at concentrations as low as 200 ppb were analyzed by capillary zone electrophoresis. Aluminum ions were cleanly separated from Li⁺, K⁺, Ca²⁺, Cr³⁺, Zn²⁺, CU²⁺, and other ions less than 6 min after injection of the solution on a 50 cm × 50 μm I.D. uncoated fused-silica capillary column at 15 kV. Indirect detection at 204 nm was carried out using a pH 2.8 background electrolyte containing 5.2 mM ephedrine as a UV-absorbing co-ion and 4.7 mM -hydroxyisobutyric acid as a completing counter ion. Mobilities for Al³⁺ and 14 other complexed cations were determined for this electrolyte.     

Photodegradation of a textile dye catalyzed by sol–gel derived nanocrystalline TiO2 via ultrasonic irradiation

Nanocrstalline pure anatase titania were prepared by sol–gel process at room temperature followed by ultrasonication (Ti–US). The photocatalytic activity of Ti–US has been evaluated by the degradation of textile dye, Methylene Blue in presence and absence of common inorganic salts (NO₃⁻, C₂O₄²⁻, SO₄²⁻, citrate). It was observed that, in presence of anions, the degradation of the dye increases significantly. The influence of the presence of H₂O₂ on the degradation rate was studied. The dependence of photodegradation of the dye rates on various parameters such as dye concentration, photocatalyst concentration and pH were also investigated. The photodegradation rate follows first order kinetics. H₂O₂ and UV light have a negligible effect in absence of Ti–US catalyst. The relative photonic efficiency of the system is reported using phenol as a standard organic compound.     

A new immune resonance scattering spectral assay for trace fibrinogen with gold nanoparticle label

An on-line stacking method based on moving reaction boundary (MRB) was developed for the sensitive determination of barbital and phenobarbital in human urine via capillary electrophoresis (CE). The optimized conditions for the method are: 60 mmol L⁻¹ pH 11.0 Gly–NaOH as the background electrolyte, 10 mmol L⁻¹ pH 5.5 Gly–HCl as sample buffer, secobarbital as the internal standard (IS), 12.5 kV, 1.4 psi 10 s sample injection, 75 μm ID 60.2 cm total length (50 cm effective length) capillary and 214 nm detect wavelength. Under the optimized conditions, the method can well stack and separate barbital and phenobarbital in urine samples and result in 20.5-fold and 22.6-fold improvement in concentration sensitivity for barbital and phenobarbital, respectively. Furthermore, the method holds: (1) good linear calibration functions for the two target compounds (correlation coefficients r > 0.999), (2) low limits of detection (0.27 μg mL⁻¹ for barbital and 0.26 μg mL⁻¹ for phenobarbital), (3) low limits of quantification (0.92 μg mL⁻¹ for barbital and 0.87 μg mL⁻¹ for phenobarbital), (4) good precision (R.S.D. of intra-day and inter-day less than 5.38% for barbital and 1.67% for phenobarbital, respectively) and (5) high recoveries at three concentration levels (90.27–106.36% for barbital and 93.05–113.60% for phenobarbital in urine). The method is simple, sensitive and efficient, and can fit to the need of clinical and forensic toxicology.