Eva M. Abad-Villar

High-voltage contactless conductivity-detection for lab-on-chip devices using external electrodes on the holder

The detection of ionic species in a polymeric planar electrophoresis device by contactless conductivity measurement is described. To our knowledge this is the first report of such measurements carried out with external electrodes which are part of the holder rather than the separation chip itself. The approach allows the use of bare devices as used for optical measurements, which greatly simplifies the method. The use of a sine wave of 100 kHz of a high amplitude of 500 V for cell excitation assures high sensitivity which is demonstrated with electropherograms for alkali and heavy metal ions as well as inorganic anions and carboxylates at concentrations between 10 and 50 µM. The determination of underivatized amino acids was also possible by using a buffer in the alkaline region where these species are present in anionic form. Detection limits were found to be in the order of 1–5 µM for the inorganic ions and between about 5 and 50 µM for the organic species.

Flow injection electrochemical enzyme immunoassay based on the use of gold bands

Abstract A flow system for the development of immunoassays, which employs an immunoreactor and an electrochemical detector based on gold bands, was designed. Gold is sputtered over Kapton (a polyimide) to form the solid support in which either the immunological interaction or the electronic transference takes place. IgG labelled with alkaline phosphatase (AP) is physically adsorbed on the gold surface of the reactor. The employment of two eight-way valves allows an independent treatment of reactor and detector. The enzymatic reaction (hydrolysis of naphthylphosphate (NP) by AP) and the immunological interaction (between IgG–AP and anti-AP) are studied. They are followed by the amount of naphthol produced. As anti-AP inhibits the enzymatic activity of AP, a decrease in the signal is observed. The oxidation signals of naphthol coming either from solutions or from the enzymatic hydrolysis of NP in the same or different reactors are highly reproducible.

Gold bands as a suitable surface for enzyme immunoassays

Gold bands sputtered over a polymeric material, Kapton, are employed for the development of enzyme immunoassays. The immunological interaction takes place between human IgM and alkaline phosphatase (AP) conjugated anti-IgM. The model analyte (IgM) could be determined following a non-competitive design in the range of 0.05-5 ppm, with a limit of detection of 50 ppb. After the interaction, gold bands are sequentially inserted in a flow system and the extension of the reaction is followed through the enzymatic hydrolysis of naphthylphosphate, AP substrate. The product, naphthol, is oxidised to naphtoquinone in the gold band of the flow cell that constitutes the detector. Parameters affecting the interaction are studied and calibration curves are performed. The reproducibility between different bands (RSD=4%, n=5) and possibilities of regeneration are also detailed.

Voltammetric and flow amperometric methods for the determination of melatonin in pharmaceuticals

Melatonin can be sensitively detected in pharmaceuticals by two different and simple electrochemical methods: cyclic voltammetry (CV) and amperometric detection in a flow injection analysis system (FIA-ED). An adequate pre-treatment of the carbon paste electrode in the first case and the employ of a high flow rate in the second one were the key for obtaining a very good reproducibility (R.S.D. values of 1.5 (n=10) and 1.3% (n=20), respectively). Low limits of detection were achieved and with the coupling of a flow system a linear dynamic range of three orders of magnitude (from 10(-8) to 10(-5) M) was obtained. Both methods were applied to the determination of melatonin in pharmaceuticals. In order to best validate these methodologies a fluorescent procedure was developed to contrast the results. As no interferences from the matrix were found the employ of a separation technique is not necessary. In this way the procedure is fastened and simplified. Moreover, the low price, ease of handling, possibility of automation and high sample throughput are important advantages that convert the flow methodology in an attractive alternative for quality control of pharmaceuticals.

Simultaneous and sequential enzyme immunoassays on gold bands with flow electrochemical detection

Abstract The feasibility of carrying out enzyme (AP) immunoassays on thin gold bands is demonstrated here for the determination of an analyte (IgG) model, following two different formats: simultaneous and sequential. The detection, that also employs a gold band as the working electrode of the potentionstatic system, is amperometrically performed in a flow system. After the reaction between IgG and IgG–AP for binding anti-IgG places, the enzymatic hydrolysis of naphthylphosphate occurs. The naphtol generated in the immunoreactor is conducted to the detector where it is oxidised to naphtoquinone providing highly reproducible analytical signals. Comparison of both formats of immunoassays was made in terms of analysis time and sensitivity. Since the gold bands where the immunoreactions took place have to be inserted in the flow system for the detection, a novel single flow cell that enabled their rapid insertion was specially designed.

Corneal Penetration of Polyhexamethylene Biguanide and Chlorhexidine Digluconate

Objective: Cationic antiseptics, such as polyhexamethylene biguanide (PHMB) and chlorhexidine digluconate (CHG), are widely used for the topical treatment of Acanthamoeba keratitis. The aim of this study was to investigate the corneal penetration of PHMB and CHG topically administered as eye drops and to study the effect of PHMB and CHG on the epithelial barrier function. Methods: The penetration was evaluated in vitro in rabbit corneas clamped in artificial perfusion chambers. Two different preparations of PHMB 0.02% (Cosmocil and Lavasept) and CHG 0.02% eye drops were administered twice each hour for up to 8 hours to the rabbit corneas with and without epithelium. The amount of drug penetrating into the anterior chamber was measured using capillary electrophoresis with contactless conductivity detection. The integrity of the epithelial barrier function was evaluated by adding fluorescein to the PHMB or CHG eye drops. The fluorescence of the anterior chamber perfusate was measured continuously throughout the experiment. Corneas treated with fluorescein alone (in either NaCl 0.9% or benzalkoniumchloride (BAC) eye drops) served as controls. Results: Neither PHMB nor CHG were detectable at any time in the anterior chamber perfusate of either the corneas with or without epithelium. PHMB and CHG treatment resulted in a minimal increase of fluorescein penetration as compared to the controls treated with 0.9% NaCl/0.05% fluorescein eye drops indicating a slight disruption of the epithelial barrier function caused by the biguanides. In contrast fluorescein penetration was much further enhanced when BAC 0.01% control eye drops were administered. Conclusion: This study showed that neither PHMB nor CHG readily penetrated through the cornea to the anterior chamber, which may explain why treatment of Acanthamoeba keratitis requires many months of sustained topical drug administration. PHMB and CHG had little effect on the epithelial barrier function compared to BAC, which is widely used as a preservative in eye drops.