Frank A. Gomez

Use of a partial-filling technique in affinity capillary electrophoresis for determining binding constants of ligands to receptors

This work evaluates the concept of a partial-filling technique in affinity capillary electrophoresis (ACE) using two model systems: vancomycin from Streptomyces orientalis and carbonic anhydrase B (CAB, EC 4.2.1.1). In this technique the capillary is first partially-filled with ligand followed by a sample of receptor and non-interacting standard and electrophoresed. Analysis of the change in the mobility ratio, M, of the receptor, relative to the non-interacting standard, as a function of the concentration of the ligand, yields a value for the binding constant. These values agree well with those estimated using other binding and ACE techniques. Data demonstrating the quantitative potential of this method is presented.

Use of mobility ratios to estimate binding constants of ligands to proteins in affinity capillary electrophoresis

This work evaluates the use of mobility ratios (M) to estimate binding constants of proteins to ligands using affinity capillary electrophoresis (ACE). This concept is demonstrated using two model systems: vancomycin (Van) from Streptomyces orientalis and carbonic anhydrase B (CAB, EC 4.2.1.1). A plot of change in M (deltaM) over the concentration of ligand [L] versus deltaM yields a more useful representation of the Scatchard plot in capillary electrophoresis (CE) than traditional plots of the change in mobility delta mu over [L] versus delta mu in a wide set of circumstances, especially when comparing electropherograms obtained in the presence of substantial variations in electroosmotic flow. Altering the voltage and/or capillary length of the CE system produced only small variations in M, but much larger changes in the more standard measures of migration used by the mu form of analysis. The use of M in the Scatchard analysis offers a new approach to estimating binding constants of ligands to proteins using ACE.

Point of care testing: The impact of nanotechnology

Point-of-care (POC) diagnostic devices are integral in the health care system and particularly for the diagnosis and monitoring of diseases. POC testing has a variety of advantages including the ability to provide rapid and accurate results, ease of use, low cost, and little need for specialized equipment. One of the goals of POC testing is the development of a chip-based, miniaturized, portable, and self-containing system that allows for the assay of different analytes in complex samples. To achieve these goals, many researchers have focused on paper-based and printed electrode technologies as the material for fabricating POC diagnostic systems. These technologies are affordable, sensitive, user-friendly, rapid, and scalable for manufacturing. Moreover, the combination such devices with nanomaterials provide a path for the development of highly sensitive and selective biosensors for future generation POC tools. This review article discusses present technologies in on-site or at home POC diagnostic assays implemented in paper-based microfluidic and screen printing devices over the past decade as well as in the near future. In addition, recent advances in the application of nanomaterials such as gold nanoparticles, carbon nanotubes (CNTs), magnetic nanoparticles, and graphene in POC devices will be reviewed. The factors that limit POC testing to become real world products and future directions are also identified.

Recent advances in affinity capillary electrophoresis (2007).

Papers detailing the use of affinity capillary electrophoresis (ACE) in examining binding parameters between biological species are summarized in this review. The works cited in this review were published during 2007 and were drawn from the major chemical and biochemical journals and especially the major analytical chemistry literature.

Multiple-plug binding assays using affinity capillary electrophoresis electrophoresis

This work evaluates the concept of a multiple-plug binding assay to estimate binding constants of proteins to ligands using affinity capillary electrophoresis (ACE). This concept is demonstrated using two model systems: carbonic anhydrase B (CAB, EC 4.2.1.1) and vancomycin from Streptomyces orientalis. Multiple plugs of protein, and non-interacting neutral and protein standards, are injected and analysis of the electrophoretic mobilities of the individual protein plugs, relative to the non-interacting neutral standard, as a function of the concentration of ligand yields values for their binding constants to the protein. These values agree well with those estimated using other assay and ACE techniques. This technique offers a new and expeditions approach to estimating binding constants of ligands to proteins.

Using Capillary Electrophoresis To Follow the Acetylation of the Amino Groups of Insulin and To Estimate Their Basicities

Analytical Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Addition/Correction Using Capillary Electrophoresis To Follow the Acetylation of the Amino Groups of Insulin and To Estimate Their Basicities Jinming Gao, Milan Mrksich, Frank A. Gomez, and George M. Whitesides Anal. Chem., 1996, 68 (13), 2287-2287• DOI: 10.1021/ac9614001 • Publication Date (Web): 01 July 1996 Downloaded from http://pubs.acs.org on May 14, 2009

Determination of the binding of β-cyclodextrin derivatives to adamantane carboxylic acids using capillary electrophoresis

SummaryBinding constants between adamantane carboxylic acids and β-cyclodextrin derivatives were determined by capillary electrophoresis (CE) using indirect detection. In this procedure, a mixture of adamantane derivative and non-interacting anionic standards, is injected and analysis of the electrophoretic mobility, relative to the noninteracting anionic standards, as a function of the concentration of cyclodextrin, yields values for their binding constants to the adamantane derivative. The usefulness of the method for quantification of binding constants is demonstrated.

Multiple-step ligand injection affinity capillary electrophoresis for determining binding constants of ligands to receptors.

This work demonstrates the use of multiple-step ligand injection affinity capillary electrophoresis (ACE) using two model systems: vancomycin from Streptomyces orientalis and carbonic anhydrase B (CAB, EC 4.2.1.1). In this technique a sample plug of receptor and non-interacting standards is injected by pressure and electrophoresed in a buffer containing a given concentration of ligand. The sequence is repeated for all concentrations of ligand generating a single electropherogram containing a series of individual sample plugs superimposed on environments of buffer containing increasing concentrations of ligand. Analysis of the change in the relative migration time ratio, RMTR, relative to the non-interacting standards, as a function of the concentration of the ligand, yields a value for the binding constant. A competitive assay using the technique is also demonstrated using neutral ligands for CAB. These values agree well with those estimated using other binding and ACE techniques. Data demonstrating the quantitative potential of this method are presented.

On-column derivatization and analysis of amino acids, peptides, and alkylamines by anhydrides using capillary electrophoresis

This work demonstrates the use of an in‐capillary procedure for derivatization of amino acids, peptides, and alkylamines by anhydrides using capillary electrophoresis (CE). Migrating in an uncoated fused‐silica capillary, plugs of substrate and anhydride are injected separately and electrophoresed. Differential transport velocities permit the separate zones to penetrate each other under an applied field, thereby facilitating reaction. In initial experiments the extent of reaction between tryptophan and acetic anhydride was examined and product amounts quantitated by CE. In separate experiments a series of amino acids and peptides were injected into the capillary and reacted with phthalic anhydride on‐column to yield the phthalic derivatized species. Finally, on‐column derivatization of alkylamines with phthalic anhydride was investigated and electrophoretic mobility related to molecular weight of the derivatized amines. These procedures illustrate the use of the capillary as a microreactor in the facile synthesis of derivatized molecules and ease of quantitation of reaction products under conditions of electrophoresis.

Analysis and stability study of temozolomide using capillary electrophoresis

The applicability of micellar electrokinetic capillary chromatography (MEKC) for the analysis of temozolomide (TMZ) and its degradants, 3-methyl-(triazen-1-yl)imidazole-4-carboxamide (MTIC) and 5-amino-imidazole-4-carboxamide (AIC) has been studied. Using short-end injection, the analysis of TMZ and its degradants could be performed within 1.2 min. The obtained precision of migration times was better than 1.6 RSD%, and the limit of quantitation (LOQ) was 0.31-0.93 microg/mL. The therapeutic concentration of TMZ in blood samples can be determined after direct sample injection and conventional on-capillary UV detection. The proposed MEKC method was applied to study the stability of TMZ in water and serum at different pH values. It was established that the half-life of the TMZ in vitro serum at room temperature was 33 min, close to the half-life (28 min) obtained in water at pH 7.9.