Norberto A. Guzman

Laser-induced fluorescence and fluorescence microscopy for capillary electrophoresis zone detection☆

A procedure to improve on-column fluorescence detection for capillary zone electrophoresis is reported. A fluorescence detector was built using an epillumination fluorescence microscope and an argon-ion air-cooled laser. The 488-nm line was isolated with a band pass filter to eliminate the ultraviolet line. A dichroic mirror which reflected wavelengths under 510 nm and a 0.75 numerical aperture (NA), 0.3 mm working distance objective of the microscope condensed the laser beam on a fused-silica capillary and a cross-shaped fluorescence spot was observed. The emitted light was collected with the same objective, filtered with a 520-nm high pass filter, a spatial filter and a notch filter, and focused on a photodetector. The photodetector was either a gallium-arsenide or a multialkali photomultiplier tube. The signal generated was fed to a current-to-voltage converter and registered on a strip chart recorder. The detector was tested with fluorescein-derivatized amino acids. For the fluorescein thiocarbamyl (FTC)-amino acids the limit of concentration detection varied according to the amino acid. Taking FTC-arginine as the best example, the limit of concentration detection (LOCD) was 3.75·10−2M. Assuming that 1 nl was injeted, the limit of mass detection (LOMD) was 3.75·10−3 amol or about 2250 molecules. This represents an improvement of about three orders of magnitude with respect to previous laser induced fluorescence on-column detection. The potential and advantages when using an epillumination fluorescence microscope with laser induced fluorescence as a detection system for capillary electrophoresis are discussed.

Immunoaffinity capillary electrophoresis as a powerful strategy for the quantification of low-abundance biomarkers, drugs, and metabolites in biological matrices.

In the last few years, there has been a greater appreciation by the scientific community of how separation science has contributed to the advancement of biomedical research. Despite past contributions in facilitating several biomedical breakthroughs, separation sciences still urgently need the development of improved methods for the separation and detection of biological and chemical substances. In particular, the challenging task of quantifying small molecules and biomolecules, found in low abundance in complex matrices (e.g., serum), is a particular area in need of new high‐efficiency techniques. The tandem or on‐line coupling of highly selective antibody capture agents with the high‐resolving power of CE is being recognized as a powerful analytical tool for the enrichment and quantification of ultra‐low abundance analytes in complex matrices. This development will have a significant impact on the identification and characterization of many putative biomarkers and on biomedical research in general. Immunoaffinity CE (IACE) technology is rapidly emerging as the most promising method for the analysis of low‐abundance biomarkers; its power comes from a three‐step procedure: (i) bioselective adsorption and (ii) subsequent recovery of compounds from an immobilized affinity ligand followed by (iii) separation of the enriched compounds. This technology is highly suited to automation and can be engineered to as a multiplex instrument capable of routinely performing hundreds of assays per day. Furthermore, a significant enhancement in sensitivity can be achieved for the purified and enriched affinity targeted analytes. Thus, a compound that exists in a complex biological matrix at a concentration far below its LOD is easily brought to well within its range of quantification. The present review summarizes several applications of IACE, as well as a chronological description of the improvements made in the fabrication of the analyte concentrator–microreactor device leading to the development of a multidimensional biomarker analyzer.

Biomedical Applications of On-Line Preconcentration-Capillary Electrophoresis Using an Analyte Concentrator: Investigation of Design Options

Abstract A method to perform on-line sample preconcentration of serum immunoglobulin E by affinity capture is described. Purified anti-IgE antibodies were covalently bound to an analyte concentrator-reaction chamber or cartridge. The immunoglobulins (IgE) were bound to and eluted from the cartridge by the optimum dissociating buffer system, and the eluent(s) were then subjected to capillary electrophoresis. The first design used was a 5 mm solid-phase cartridge fabricated by assembling a bundle of multiple microcapillaries in which a monoclonal antibody directed against IgE was covalently bound to the surface of every microcapillary. The whole assembly was connected, through sleeve connectors, to the capillary column for affinity capillary electrophoresis. The second design used consisted of an analyte concentrator-reaction chamber that was fabricated from a solid rod of glass. Several small diameter passages or through holes containing a similar surface area was tested for the same experiments and perfor...

Detection and quantification of capillary electrophoresis zones by fluorescence microscopy

Abstract A fluorescence detector for capillary electrophoresis was built using an epillumination fluorescence microscope equipped with a 50-W mercury lamp and a photodiode. The performance of the detector was tested with riboflavin and fluorescamine-derivatized amino acids and amphetamine. We compared fluorite and conventional glass objectives and found that, by using a 25-μm I.D. capillary and a fluorite objective, an improvement in fluorescence detection by a factor of 56 can be achieved. A detection level of 500 amol for riboflavin was reached (using on-column detection). This sensitivity was at least comparable to that reported for other capillary electrophoresis systems using on-column non-coherent light fluorescence detectors. The potential and advantages of using a fluorescence microscope for capillary electrophoresis applications are discussed.

Immunoaffinity capillary electrophoresis: a new versatile tool for determining protein biomarkers in inflammatory processes.

Many diseases caused by inflammatory processes can progress to a chronic state causing deterioration in the quality of life and a poor prognosis for long‐term survival. To address inflammatory diseases effectively, early detection and novel therapeutics are required. However, this can be challenging, in part because of the lack of early predictive biomarkers and the limited availability of adequate technologies capable of the identification/characterization of key predictive biomarkers present in biological materials, especially those found at picomolar concentrations and below. This review highlights the need for state‐of‐the art methodologies, with high‐sensitivity and high‐throughput capabilities, for determination of multiple biomarkers. Although many new biomarkers have been discovered recently, existing technology has failed to successfully bring this advancement to the patients bedside. We present an overview of the various advances available today to extend the discovery of predictive biomarkers of inflammatory diseases; in particular, we review the technology of immunoaffinity capillary electrophoresis (IACE), which combines the use of antibodies as highly selective capture agents with the high resolving power of capillary electrophoresis. This two‐dimensional hybrid technology permits the quantification and characterization of several protein biomarkers simultaneously, including subtle structural changes such as variants, isoforms, peptide fragments, and post‐translational modifications. Furthermore, the results are rapid, sensitive, can be performed at a relatively low cost, without the introduction of false positive or false negative data. The IACE instrumentation can have relevance to medical, pharmaceutical, environmental, military, cultural heritage (authenticity of art work), forensic science, industrial and research fields, and in particular as a point‐of‐care biomarker analyzer in translational medicine.

Collinear laser-induced fluorescence detector for capillary electrophoresis: Analysis of glutamic acid in brain dialysates

Abstract Experiments with capillary electrophoresis using a laser-induced fluorescence detector with a collinear optical arrangement demonstrated several important points. First, increasing the numerical aperture of the microscope objective that is used simultaneously for focusing the excitation laser light as well as collection of emitted fluorescence enhances the signal used for the measurement of the emitted fluorescence and at the same time decreases the noise of interfering light. Second, detection of fluorescein-labelled amphetamine was performed at high-picomolar (10−10M) levels. Third, the signal-to-noise ratio of 280 found at the above-mentioned picomolar concentrations indicates that the measurement of low-picomolar concentrations (10−12M) of this compound in biological samples should be possible. Fourth, narrow -bore capillaries (5–10 μm internal diameter) were used to detect the neurotransmitters glutamic acid and aspartic acid as their naphthalene-2,3-dicarboxaldehyde derivatives in brain dialysates obtained from a freely moving rat. A mathematical model was developed to explain the relationship between numerical aperture, working distance, magnification of the lens, noise due to laser scattering and signal due to fluorescence. The model correctly predicted the observed values of photomultiplier tube current due to both laser scattering and fluorescence. The potential of the application of capillary electrophoresis with laser-induced fluorescence detection in the neurosciences is discussed.

Bidirectional microdialysis in vivo shows differential dopaminergic potency of cocaine, procaine and lidocaine in the nucleus accumbens using capillary electrophoresis for calibration of drug outward diffusion

Cocaine and two other local anesthetics were applied directly into the nucleus accumbens for 20 min by diffusion from a 4 mm microdialysis probe in freely moving rats. Cocaine (7.3 mM) increased the extracellular concentration of dopamine (DA). Equimolar procaine did also, but was not as potent as cocaine. Equimolar lidocaine had no effect. The concentration of these drugs outside the probe as measured by capillary electrophoresis in vitro was about 28% of that inside the probe, i.e. 72% remained inside. However, an in vivo test showed that about 53% cocaine and procaine, and 37% lidocaine remained in the perfusion fluid after passing through a probe inserted in the brain. This suggests that in vivo about 68 nmol cocaine diffused into the nucleus accumbens (NAC) during the 20 min. Five conclusions are drawn: (1) this confirms our earlier finding that local injection of cocaine increases extracellular DA, but in this case the cocaine was infused via the probe without disturbing the animal; (2) the action of cocaine on dopamine terminals in the accumbens is independent of local anesthesia; (3) procaine may enhance mood by a cocaine-like effect; (4) capillary electrophoresis has potential for measuring cocaine levels in small samples and (5) in vitro calibrations are of limited value to evaluate in vivo performance of microdialysis probes.

Capillary Electrophoresis as A Diagnostic Tool: Determination of Biological Constituents Present in Urine of Normal and Pathological Individuals

Abstract A quantitative ultraviolet detection method for the determination of urinary metabolites is described using capillary zone electrophoresis. The determination of these metabolites is simple, fast. reproducible and utilizes very small amounts of sample. This method is linear between 1.0 × 10−4 and 1.0 × 10−2 M for creatinine and between 1.0 × 10−1 and 1.0 M for urea. The ultraviolet method shows detection limits for creatinine in the picogram (femtomol) range, and for urea in the nanogram (picomol) range.

Capillary electrophoresis for the analytical separation and semi-preparative collection of monoclonal antibodies

Abstract A preparation of a highly purified monoclonal antibody was subjected to capillary electrophoretic determination. Through the use of multiple parallel capillaries it was possible to increase both the detector response and sample load in direct proportion to the number of capillaries. While the amount of antibody solution assessed by ultraviolet detection was in the range of tenths of μg ml −1 , values in the range of tenths of ng ml −1 were obtained using fluorescence detection. The results are discussed in terms of their practical values for biological samples, targeted for human consumption as pharmaceutical drugs.

An emerging micro-scale immuno-analytical diagnostic tool to see the unseen. Holding promise for precision medicine and P4 medicine

Over the years, analytical chemistry and immunology have contributed significantly to the field of clinical diagnosis by introducing quantitative techniques that can detect crucial and distinct chemical, biochemical and cellular biomarkers present in biosamples. Currently, quantitative two-dimensional hybrid immuno-analytical separation technologies are emerging as powerful tools for the sequential isolation, separation and detection of protein panels, including those with subtle structural changes such as variants, isoforms, peptide fragments, and post-translational modifications. One such technique to perform this challenging task is immunoaffinity capillary electrophoresis (IACE), which combines the use of antibodies and/or other affinity ligands as highly selective capture agents with the superior resolving power of capillary electrophoresis. Since affinity ligands can be polyreactive, i.e., binding and capturing more than one molecule, they may generate false positive results when tested under mono-dimensional procedures; one such application is enzyme-linked immunosorbent assay (ELISA). IACE, on the other hand, is a two-dimensional technique that captures (isolation and enrichment), releases, separates and detects (quantification, identification and characterization) a single or a panel of analytes from a sample, when coupled to one or more detectors simultaneously, without the presence of false positive or false negative data. This disruptive technique, capable of preconcentrate on-line results in enhanced sensitivity even in the analysis of complex matrices, may change the traditional system of testing biomarkers to obtain more accurate diagnosis of diseases, ideally before symptoms of a specific disease manifest. In this manuscript, we will present examples of the determination of biomarkers by IACE and the design of a miniaturized multi-dimensional IACE apparatus capable of improved sensitivity, specificity and throughput, with the potential of being used as a point-of-care instrument and holding promise for precision medicine and P4 medicine.