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Many bioanalytical methods rely on electrophoretic separation of structurally labile and surface active biomolecules such as proteins and peptides. Often poor separation efficiency is due to surfac ...

Detection of immune responses is important in the diagnosis of many diseases. For example, the detection of circulating autoantibodies against double-stranded DNA (dsDNA) is used in the diagnosis of Systemic Lupus Erythematosus (SLE). It is, however, difficult to reach satisfactory sensitivity, specificity, and accuracy with established assays. Also, existing methodologies for quantification of autoantibodies are challenging to transfer to a point-of-care setting. Here we present the use of flow-induced dispersion analysis (FIDA) for rapid (minutes) measurement of autoantibodies against dsDNA. The assay is based on Taylor dispersion analysis (TDA) and is fully automated with the use of standard capillary electrophoresis (CE) based equipment employing fluorescence detection. It is robust toward matrix effects as demonstrated by the direct analysis of samples composed of up to 85% plasma derived from human blood samples, and it allows for flexible exchange of the DNA sequences used to probe for the autoantibodies. Plasma samples from SLE positive patients were analyzed using the new FIDA methodology as well as by standard indirect immunofluorescence and solid-phase immunoassays. Interestingly, the patient antibodies bound DNA sequences with different affinities, suggesting pronounced heterogeneity among autoantibodies produced in SLE. The FIDA based methodology is a new approach for autoantibody detection and holds promise for being used for patient stratification and monitoring of disease activity.

Rapid and sensitive quantification of protein based biomarkers and drugs is a substantial challenge in diagnostics and biopharmaceutical drug development. Current technologies, such as ELISA, are characterized by being slow (hours), requiring relatively large amounts of sample and being subject to cumbersome and expensive assay development. In this work a new approach for quantification based on changes in diffusivity is presented. The apparent diffusivity of an indicator molecule interacting with the protein of interest is determined by Taylor Dispersion Analysis (TDA) in a hydrodynamic flow system. In the presence of the analyte the apparent diffusivity of the indicator changes due to complexation. This change in diffusivity is used to quantify the analyte. This approach, termed Flow Induced Dispersion Analysis (FIDA), is characterized by being fast (minutes), selective (quantification is possible in a blood plasma matrix), fully automated, and being subject to a simple assay development. FIDA is demonstrated for quantification of the protein Human Serum Albumin (HSA) in human plasma as well as for quantification of an antibody against HSA. The sensitivity of the FIDA assay depends on the indicator-analyte dissociation constant which in favourable cases is in the sub-nanomolar to picomolar range for antibody-antigen interactions.

Many bioanalytical methods rely on electrophoretic separation of structurally labile and surface active biomolecules such as proteins and peptides. Often poor separation efficiency is due to surface adsorption processes leading to protein denaturation and surface fouling in the separation channel. Flexible and reliable approaches for preventing unwanted protein adsorption in separation science are thus in high demand. We therefore present new coating approaches based on an automated in-capillary surface-initiated atom transfer radical polymerization process (covalent coating) as well as by electrostatically adsorbing a presynthesized polymer leading to functionalized molecular brushes. The electroosmotic flow was measured following each step of the covalent coating procedure providing a detailed characterization and quality control. Both approaches resulted in good fouling resistance against the four model proteins cytochrome c, myoglobin, ovalbumin, and human serum albumin in the pH range 3.4-8.4. Further, even samples containing 10% v/v plasma derived from human blood did not show signs of adsorbing to the coated capillaries. The covalent as well as the electrostatically adsorbed coating were both found to be stable and provided almost complete suppression of the electroosmotic flow in the pH range 3.4-8.4. The coating procedures may easily be integrated in fully automated capillary electrophoresis methodologies.

In this paper, we describe a capillary-based microfluidic device utilizing flow induced dispersion analysis (FIDA) for quantitative characterization of biomarkers. The microfluidic device is fabricated by micromilling technology and has incorporated buried optical fibers for light detection. The angle and distance between the fiber guiding the excitation light source and the fiber collecting fluorescent emission light were optimized to enhance signal-to-noise ratio (SNR) and limit of detection (LOD). The prototype achieves a LOD of 50 nM for the fluorescein indicator by using a low-cost Miniature Fiber Optic Spectrometer. The FIDA-based procedure employing fluorescein as the indicator and human serum albumin (HSA) as the analyte is carried out in the microfluidic device.

A temperature controlled (37 °C) metabolic reaction chamber with a volume of 1 mL was coupled directly to electrospray ionization mass spectrometry (ESI-MS) by the use of a 50 μm deep counter flow micro-chip electromembrane extraction (EME) system. The EME/ESI-MS system was used to study the in vitro metabolism of amitriptyline in real time. There was no need to stop the metabolisms by protein precipitation as in conventional metabolic studies, since the EME selectively extracted the drug and metabolites from the reaction solution comprised of rat liver microsomes in buffer. Compositional changes in the reaction chamber were continuously detected 9 seconds later in the MS. Most of this time delay was due to transport of the purified extract towards the ESI source. The EME step effectively removed the enzymatic material, buffer and salts from the reaction mixture, and prevented these species from being introduced into the ESI-MS system. The on-chip EME/ESI-MS system provided repeatability for the amitriptyline signal intensity within 3.1% relative standard deviation (RSD) (n = 6), gave a linear response for amitriptyline in the tested concentration range of 0.25 to 15 μM, and was found not to be prone to ion-suppression from major metabolites introduced simultaneously into the EME/ESI-MS system. The setup allowed the study of fast reactions kinetics. The half-life, t(1/2), for the metabolism of 10 μM amitriptyline was 1.4 minutes with a 12.6% RSD (n = 6).