Here we report that nanopillar array structures have an intrinsic ability to suppress electroosmotic flow (EOF). Currently using glass chips for electrophoresis requires laborious surface coating to control EOF, which works as a counterflow to the electrophoresis mobility of negatively charged samples such as DNA and sodium dodecyl sulfate (SDS) denatured proteins. Due to the intrinsic ability of the nanopillar array to suppress the EOF, we carried out electrophoresis of SDS-protein complexes in nanopillar chips without adding any reagent to suppress protein adsorption and the EOF. We also show that the EOF profile inside a nanopillar region was deformed to an inverse parabolic flow. We used a combination of EOF measurements and fluorescence observations to compare EOF in microchannel, nanochannel, and nanopillar array chips. Our results of EOF measurements in micro- and nanochannel chips were in complete agreement with the conventional equation of the EOF mobility (μ(EOF-channel) = αC(i)(-0.5), where C(i) is the bulk concentration of the i-ions and α differs in micro- and nanochannels), whereas EOF in the nanopillar chips did not follow this equation. Therefore we developed a new modified form of the conventional EOF equation, μ(EOF-nanopillar) ≈ β[C(i) - (C(i)(2)/N(i))], where N(i) is the number of sites available to i-ions and β differs for each nanopillar chip because of different spacings or patterns, etc. The modified equation of the EOF mobility that we proposed here was in good agreement with our experimental results. In this equation, we showed that the charge density of the nanopillar region, that is, the total number of nanopillars inside the microchannel, affected the suppression of EOF, and the arrangement of nanopillars into a tilted or square array had no effect on it.

We report a CE-LIF method for the separation and detection of five synthetic amyloid-β peptides corresponding to an important family of CSF-biomarkers in the context of Alzheimer disease (AD). The presumed most relevant peptides (Aβ1-42, Aβ1-40, and Aβ1-38) that may support the differentiation between AD and healthy patients or other dementias were successfully detected in CSF by incorporating an immunoconcentration step prior to CE analysis of derivatized peptides. We labeled the Aβ peptides with a fluoroprobe dye before CE-LIF analysis. This reagent reacts with the amino groups of lysine residues and produced mostly ditagged Aβ peptides under the proposed experimental conditions. The labeling reaction displayed similar efficiency with each one of the five different synthetic Aβ peptides that were tested. The limit of detection of the CE-LIF method approached 280 attomoles of injected synthetic labeled Aβ peptides. We obtained excellent correlation between peak areas and peptide concentrations from 35 nM to 750 nM. For the detection of Aβ peptides in human CSF samples, we enriched the peptides by immunoprecipitation prior to the CE-LIF analysis. The comparison of the CE-LIF profiles obtained from CSF samples from 3 AD patients and 4 non-demented control subjects indicated noticeable differences, suggesting that this method, which relies on a multibiomarker approach, may have potential as a clinical diagnostic test for AD.

Online preconcentration of human serum albumin (HSA) and its immunocomplex with a monoclonal antibody by on-chip transient isotachophoresis is reported. An 800-fold signal enhancement was achieved following the preconcentration on standard cross-channel microchips made of poly (methyl methacrylate). Sample injection, preconcentration, and separation were done continuously and controlled solely by a sequential voltage switching program. The preconcentration was followed by on-chip nondenaturing gel electrophoresis in methylcellulose solution. The method was applied to microchip electrophoresis immunoassay of HSA. Baseline separation of HSA and its immunocomplex was achieved in 25 s in the first 1 cm of the microchannel. In a direct immunoassay, the minimum detectable concentration of fluorescent labeled HSA by laser-induced fluorescence detection was 7.5 pM.

The preferential aggregation of Aβ1-42 in amyloid plaques is one of the major neuropathological events in Alzheimer's disease. This is accompanied by a relative reduction of the concentration of Aβ1-42 in the cerebrospinal fluid (CSF) of patients developing the signs of Alzheimer's disease. Here, we describe a microchip gel electrophoresis method in polydimethylsiloxane (PDMS) chip that enables rapid profiling of major Aβ peptides in cerebrospinal fluid. To control the electroosmotic flow (EOF) in the PDMS channel and also to reduce the adsorption of the peptides to the surface of the channel, a new double coating using poly(dimethylacrylamide-co-allyl glycidyl ether) (PDMA-AGE) and methylcellulose-Tween-20 was developed. With this method, separation of five synthetic Aβ peptides (Aβ1-37, Aβ1-38, Aβ1-39, Aβ1-40, and Aβ1-42) was achieved, and relative abundance of Aβ1-42 to Aβ1-37 could be calculated in different standard mixtures. We applied our method for profiling of Aβ peptides in CSF samples from nonAlzheimer patients and patients with Alzheimer's disease. Aβ peptides in the CSF samples were captured and concentrated using a microfluidic system in which magnetic beads coated with anti-Aβ were self-organized into an affinity microcolumn under the a permanent magnetic field. Finally, we could detect two Aβ peptides (Aβ1-40 and Aβ1-42) in the CSF samples.

In this research, a simple on-line microchip gel electrophoresis with ITP was applied for the concentration and separation of BSA and its immunoassay complex with mAb in a single cross form PMMA microchip. We investigated the ITP concentration effect in PMMA MCE using combination of leading electrolytes, terminating electrolytes and other factors. We realized an ITP-based concentration and separation of BSA and its immunoassay complexes in standard cross-channel microchip gel electrophoresis, which exceeded 2000-fold concentration of BSA immunocomplex using Tris-H3PO4 as a leading electrolyte and Tris-gamma-amino butyric acid as a terminating electrolyte. In addition, we also realized concentration of BSA sample in water, which was more than 20 000-fold and was the result of the concentration effect from combining ITP and the sample stacking techniques.

Determination of amyloid b (Ab) isoforms and in particular the proportion of the Ab 1-42 isoform in cerebrospinal fluid (CSF) of patients suspected of Alzheimer's disease might help in early diagnosis and treatment of that illness. Due to the low concentration of Ab peptides in biological fluids, a preconcentration step prior to the detection step is often necessary. This study utilized on-chip immunoprecipitation, known as micro-immunoprecipitation (lIP). The technique uses an immunosorbent (IS) consisting of magnetic beads coated with specific anti-Ab antibodies organized into an affinity microcolumn by a magnetic field. Our goal was to thoroughly describe the critical steps in developing the IS, such as selecting the proper beads and anti-Ab antibodies, as well as optimizing the immobilization technique and lIP protocol. The latter includes selecting optimal elution conditions. Furthermore, we demonstrate the efficiency of anti-Ab IS for lIP and specific capture of 5 Ab peptides under optimized conditions using various subsequent analytical methods, including matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), capillary electrophoresis, microchip electrophoresis, and immunoblotting. Synthetic Ab peptides samples prepared in buffer and spiked in human CSF were analyzed. Finally, on-chip immunoprecipitation of Ab peptides in human CSF sample was performed. V C 2012 American Institute of Physics. [http://dx

A dynamic coating using methylcellulose (MC) and a nonionic detergent (polysorbate 20) was developed, which controlled protein adsorption onto the surface of microchannels on a microchip made of poly(methyl methacrylate) (PMMA). Optimum concentration of polysorbate 20 in combination with the range of MC concentrations controlled the protein adsorption onto the microchannel surface, and increased the solubility of the protein samples while facilitating the injection of high concentrations of MC solutions into the microchannels. Higher concentrations of nonionic detergent increased the EOF mobility as opposed to the electrophoretic mobility and caused the electrophoresis to fail. Nondenaturing microchip electrophoresis of protein samples with molecular masses ranging from 20 to 100 kDa were completed in 100 s. Also, successful separation of a BSA sample and its complex with anti-BSA mAb ( 220 kDa) was achieved on a PMMA microchip. The separation exhibited high reproducibility in both migration time (RSD = 1%) and peak area (RSD = 10-15%).

In order to be more extensively used outside of research laboratories, lab-on-chip technologies must be mass-produced using low-cost materials such as thermoplastics. Thermoplastics, however, are generally hydrophobic in their native state, which makes them unsuitable for direct use with biological samples in aqueous solution, and thus require surface coating. This coating should be robust, inexpensive and simple to implement, in order not to hinder the industrial advantage of thermoplastic chips. Cyclic Olefin Copolymer (COC) is a particularly appealing polymer, but it is also difficult to functionalize due to its chemical inertness. Here we introduce and compare the performance of two new approaches for COC coating. One relies on the use of a commercial triblock copolymer, Pluronic® F127. The second approach uses new copolymers synthesized by radical polymerization, and consisting of a dimethylacrylamide (DMA) backbone carrying aliphatic side chains (C22). Two DMA-C22 copolymers were synthesized with various C22/DMA ratios: DMA-S at 0.175% and DMA-M at 0.35%. Different physicochemical properties of the polymers such as critical micellar concentration (CMC), water contact angle, electroosmosis were investigated. Coated COC chips were then tested for their ability to reduce the adsorption of proteins, microparticles, and for protein electrophoresis. For each application we found an optimal treatment protocol to considerably improve the performance of the thermoplastic chip. These treatments use physisorption in situ which requires no photografting or chemical reaction and can be performed by a simple incubation either after chip production, or just prior to use.