Analysis of Aß (1-40) and Aß (1-42) monomer and fibrils by capillary electrophoresis.

A method based on capillary electrophoresis (CE) with UV absorbance detection is presented to characterize synthetic amyloid beta (Aß) peptide preparations at different aggregation states. Aggregation of Aß (1-40) and Aß (1-42) is closely linked to Alzheimer's disease (AD), and studying how Aß peptides self-assemble to form aggregates is the focus of intense research. Developing methods capable of identifying, characterizing and quantifying a wide range of Aß species from monomers to fully formed fibrils is critical for AD research and is a major analytical challenge. Monomer and fibril samples of Aß (1-40) and Aß (1-42) were prepared and characterized for this study. The monomer-equivalent concentration for each sample was determined by HPLC-UV, and aggregate formation was confirmed and characterized by transmission electron microscopy. The same samples were studied using CE with UV absorbance detection. Analysis by mass spectrometry of collected CE fractions was used to confirm the presence of Aß for some CE-UV peaks. The CE-UV method reported here clearly indicates that monomeric and aggregated Aß were electrophoretically separated, and substantial differences in the electrophoretic profiles between samples of Aß (1-40) and Aß (1-42) were observed. This CE-UV method can differentiate between Aß monomer, oligomeric intermediates, and mature fibrils.

Analysis of monomeric Abeta (1-40) peptide by capillary electrophoresis.

A method was developed to characterize and quantify preparations of monomeric beta-amyloid (Abeta) peptide using capillary electrophoresis (CE) with UV absorbance detection. The detection limit for Abeta monomer using this method was 0.5 microM (19 pg). The self-assembly of Abeta to form amyloid fibrils is closely linked to Alzheimer's disease and is the subject of intense investigations. Consistent preparation of Abeta monomer samples at known concentrations and free of aggregates is a significant challenge for researchers studying the mechanism of Abeta fibril formation and searching for small molecules that inhibit Abeta fibril formation. Samples of Abeta monomer are known to sometimes contain pre-existing aggregates that can affect the kinetics and structure of amyloid fibrils. The CE method presented here showed that some of the monomeric Abeta samples prepared for this study contained a species producing a second peak (in addition to the major monomer peak). The aggregation was monitored using a thioflavin T fluorescence assay, and the resulting fibrils were characterized by transmission electron microscopy. Monomer samples containing the additional peak based on CE analysis were shown to aggregate more rapidly than monomer samples that were free of this putative Abeta aggregate peak.

Negative ion-atmospheric pressure photoionization: electron capture, dissociative electron capture, proton transfer, and anion attachment.

To better guide the development of liquid chromatography/electron capture-atmospheric pressure photoionization-mass spectrometry (LC/EC-APPI-MS) in analysis of low polarity compounds, the ionization mechanism of 19 compounds was studied using dopant assisted negative ion-APPI. Four ionization mechanisms, i.e., EC, dissociative EC, proton transfer, and anion attachment, were identified as being responsible for the ionization of the studied compounds. The mechanisms were found to sometimes compete with each other, resulting in multiple ionization products from the same molecule. However, dissociative EC and proton transfer could also combine to generate the same [M - H](-) ions. Experimental evidence suggests that O(2)(-*), which was directly observed in the APPI source, plays a key role in the formation of [M - H](-) ions by way of proton transfer. Introduction of anions more basic than O(2)(-*), i.e., C(6)H(5)CH(2)(-), into the APPI source, via addition of di-tert-butyl peroxide in the solvent and/or dopant, i.e., toluene, enhanced the deprotonation ability of negative ion-APPI. Although the use of halogenated solvents could hinder efficient EC, dissociative EC, and proton transfer of negative ion-APPI due to their EC ability, the subsequently generated halide anions promoted halide attachment to compounds that otherwise could not be efficiently ionized. With the four available ionization mechanisms, it becomes obvious that negative ion-APPI is capable of ionizing a wider range of compounds than negative ion chemical ionization (NICI), negative ion-atmospheric pressure chemical ionization (negative ion-APCI) or negative ion-electrospray ionization (negative ion-ESI).

Multiplexed, waveguide approach to magnetically assisted transport evanescent field fluoroassays.

This paper, expanding upon the recently developed magnetically assisted transport evanescent field fluoroassays (MATEFFs), takes advantage of several innovations in order to successfully integrate a microfluidic platform and planar waveguide technology for exploitation of multiplexing advantages. In the current adaptation of MATEFFs, a multiple internal reflection element (waveguide) is created using a simple microscope slide and PDMS microfluidic architecture, allowing simultaneous detection of multiple samples. Furthermore, the magnetic beads are manipulated using a passive pumping technique and a simple external permanent magnet, thereby circumventing the need for electromagnetic fabrication or complicated architectures and equipment. Initial testing, optimization, and calibration were performed using a model sandwich immunoassay system for the detection of rabbit IgG, with which we demonstrate a linear dynamic range of 3 orders of magnitude and physiologically relevant detection limits of nanograms per milliliter. Further work employed a sandwich immunoassay for the detection of interleukin-4, a cytokine that promotes proliferation and differentiation of B cells, to demonstrate technique reproducibility with RSD values of 5% and reported LOD of 10 ng/mL. The use of harvesting magnetic beads resulted in assays with mass-sensing behavior. Using IgG as a model cross-reactant with the interleukin-4 system, we additionally illustrate technique selectivity and multiplexing capability. A DNA hybridization assay is carried out using magnetic bead-immobilized single-stranded DNA with hybridization detected via ethidium bromide intercalation, further establishing technique versatility.

Electron capture atmospheric pressure photoionization mass spectrometry: analysis of fullerenes, perfluorinated compounds, and pentafluorobenzyl derivatives.

An electron capture (EC) ionization mechanism has been found to be highly efficient in negative-ion atmospheric pressure photoionization (APPI) for the analysis of compounds with positive electron affinity (EA). Using negative-ion APPI, we first report the sensitive detection of natural electrophores with limited polarity, such as fullerenes and perfluorinated compounds, by mass spectrometry (MS). Using direct infusion on a quadrupole time-of-flight (QTOF) mass spectrometer, the limits of detection (LODs) for C(60) and perfluoromethylcyclohexane were determined to be 0.15 pg (0.2 fmol) and 1 femtoliter (fL) ( approximately 1.5 pg or 4.3 fmol), respectively. As the EA of the analyte increases, the detection sensitivity is enhanced. Making use of the accurate mass measurement capability of the QTOF mass spectrometer, we were able to investigate the elemental composition of the ions in each spectrum and attribute the observed high sensitivity to an EC-initiated ionization process. The proposed EC ionization mechanism is further supported by the observation of a dissociative EC reaction of pentafluorobenzyl (PFB)-derivatized phenols. The analysis of phenols by EC-APPI of their PFB derivatives resulted in very high sensitivity, with the lowest reported LOD of approximately 0.17 pg (0.5 fmol) being for 2,4-dinitrophenol. For future LC/EC-APPI-MS applications, the effect of additives and solvents on sensitivity was also tested and reported.

Separation and detection of individual submicron particles by capillary electrophoresis with laser-light-scattering detection.

Separation and detection of individual submicron polystyrene spheres using capillary electrophoresis with laser-light-scattering detection has been demonstrated. Electrophoretically separated particles were passed through a focused laser beam and light scattered from individual particles was collected at 90 degrees. Each diameter of polystyrene spheres injected (from 110 to 992 nm) resulted in the observation of a well-defined migration window containing multiple peaks, each arising from the light scattered by an individual particle. The migration time window for individual particles of a particular size corresponded well to the migration time of a peak from a population of particles of the same size detected using a UV absorbance detector. The electrophoretic mobility and scattered light intensity were determined for each particle detected. The average scattered light intensity for each particle size was consistent with Mie scattering theory. Particles as small as 110 nm in diameter were detected individually using this method, but particles with a diameter of 57 nm could not be individually detected. The number of single particle scattering events was counted and compared to the theoretical number of particles injected electrokinetically, and the detection efficiency determined ranged from 38 to 57% for polystyrene spheres of different sizes. The laser-light-scattering detection method was directly compared to laser-induced fluorescence detection using fluorescent polystyrene microspheres. The number of particles detected individually by each method was in agreement.

Magnetically-assisted transport evanescent field fluoroimmunoassay.

The immunoassay, based on specific recognition of an antigen by its antibody, has garnered widespread use in clinical analysis as well as application in such areas as food industry and environmental monitoring. Fluoroimmunoassays (FIAs) are especially attractive due to the inherent sensitivity of fluorescence spectroscopy and the availability of a wide range of commercial antibodies and fluorescent labels. In current form, however, FIAs can be cumbersome, multistep procedures and often lack versatility when there is interest in measuring many different target antigens. This report is proof of a concept paper introducing a new FIA approach, Magnetically-Assisted Transport Evanescent Field Fluoroimmunoassays (MATEFFs), which seeks to preserve the advantages of current approaches to FIAs while attempting to address some of the drawbacks. MATEFFs utilize magnetic microspheres as solid supports for the fluoroimmunoassay with direct detection of bound analyte within the sample mixture effected by selectively driving the functionalized beads to a prism surface using an external magnet. An evanescent wave is generated by total internal reflection of a laser beam at the optical interface between the prism and sample and serves to excite the fluorescent species magnetically delivered into the localized field. This technique eliminates wash steps without compromising sensitivity, all the while minimizing interference from fluorescing species present in the sample matrix. Preliminary optimization studies assessing the impact of background interfering agents, incident angle, magnetic field direction, laser power density via focusing, and bead concentration on MATEFFs performance characteristics are discussed herein along with a detailed description of the experimental platform. Utilizing a model sandwich assay system with biotinylated anti-IgG as the capture antibody, rabbit IgG as the antigen, and anti-IgG-R-phycoerythrin as the reporter antibody, we demonstrate a linear dynamic range of 3 orders of magnitude, physiologically relevant detection limits of low nanograms per milliliter, and RSD values of less than 5%.