Vincent T. Remcho

On-chip aptamer-based sandwich assay for thrombin detection employing magnetic beads and quantum dots.

In this paper, we report the development of an on-chip aptamer-based fluorescence assay for protein detection and quantification based on sandwich ELISA principles. Thrombin was selected as a model analyte to validate the assay design, which involves two DNA thrombin aptamers recognizing two different epitopes of the protein. Aptamer-functionalized magnetic beads were utilized to capture the target analyte, while a second aptamer, functionalized with quantum dots, was employed for on-chip detection. The binding of thrombin to the two aptamers via sandwich assay was monitored by fluorescence microscopy. The sandwich assay was performed on disposable microfluidic devices, fabricated on double-sided tapes and polymeric materials using a laser cutting approach. The approach enabled rapid thrombin detection with high specificity. Experimental conditions, such as reagent consumption and incubation time, were optimized in the microchip platform for the lowest limit of detection, highest specificity, and shortest assay time. The analytical performance of the microchip based assay was compared to that in the well plate format (generally utilized for ELISA-based methodologies). The results show that microfluidic chip proved to be a rapid and efficient system for aptamer-based thrombin assays, requiring only minimal (microliter) reagent use. This work demonstrated the successful application of on-chip aptamer-based sandwich assays for detection of target proteins of biomedical importance.

SILICATE ENTRAPPED COLUMNS : NEW COLUMNS DESIGNED FOR CAPILLARY ELECTROCHROMATOGRAPHY

Designed especially for capillary electrochromatography (CEC), silicate‐entrapped columns are made by trapping particles of chromatographic packing material in a network of silica. Once entrapped, the capillary no longer requires frits. This renders a more homogeneous and stable packed bed. Accidental breakage of the fragile frits is not an issue with these robust columns. Columns packed with reverse‐phase material subjected to silicate entrapment demonstrated faster separations of retained analytes and increased efficiencies compared with nonentrapped columns. The method was also used to prepare chiral CEC columns by entrapping a molecular imprinted polymeric (MIP) packing having minimal surface charge density, thus being unable alone to support sufficient electroosmotic flow for CEC.

Aptamers as analytical reagents

Many important analytical methods are based on molecular recognition. Aptamers are oligonucleotides that exhibit molecular recognition; they are capable of specifically binding a target molecule, and have exhibited affinity for several classes of molecules. The use of aptamers as tools in analytical chemistry is on the rise due to the development of the “systematic evolution of ligands by exponential enrichment” (SELEX) procedure. This technique allows high‐affinity aptamers to be isolated and amplified when starting from a large pool of oligonucleotide sequences. These molecules have been used in flow cytometry, biosensors, affinity probe electrophoresis, capillary electrochromatography, and affinity chromatography. In this paper, we will discuss applications of aptamers which have led to the development of aptamers as chromatographic stationary phases and applications of these stationary phases; and look towards future work which may benefit from the use of aptamers as stationary phases.

Peer Reviewed: MIPs as Chromatographic Stationary Phases for Molecular Recognition.

Molecular imprint polymers recognize specific compounds and show promise as separation media, especially for chiral molecules.

Novel monolithic columns with templated porosity.

A new type of monolithic stationary phase was prepared within the confines of fused-silica tubing by in situ polymerization of divinylbenzene or ethylene dimethacrylate either with styrene or butyl methacrylate. The porosity of the monolith was dictated by silica beads packed in the capillary prior to flushing the column with the monomeric solution. Subsequent washing of the polymeric rod with sodium hydroxide rendered a porous monolith that was used for both micro-LC and capillary electrochromatography. The novelty of the approach presented herein lies in preparing the polymer within the confines of a fused-silica capillary. The challenges posed in this new context and their resolution are presented in detail. In addition, this study proposes that in addition to tailoring the pore size, the silica beads, through their surface chemistry, can influence the surface characteristics of the finished polymer monolith. For example, the data suggests that octadecyl modified silica particles interacted with hydrophobic moieties of monomers before initiation of polymerization, thus dictating their orientation in the resulting polymer.

A simple procedure for the preparation of fritless columns by entrapping conventional high performance liquid chromatography sorbents

A rapid and direct method for immobilizing conventional high performance liquid chromatography (HPLC) packing material inside fritless capillaries has been developed. Due to the simple composition of the entrapment matrix (tetraethoxysilane, alkyltriet‐hoxysilane, ethanol and water), straightforward manufacturing procedure and modest equipment requirement, the method can readily be transferred to any laboratory and easily automated. The entrapment procedure has minimal influence on the structure and chromatographic properties of the original reverse‐phase sorbent. Various immobilization solutions have been tested, and a comparison between columns entrapped with different immobilization mixtures and conventional packed capillaries is presented. High effficiency separations were obtained using tert‐butyl‐triethoxysilane entrapped columns in both capillary electrochromatography (reduced plate heights of 1.1—1.4 were measured) and microliquid chromatography (reduced plate heights of 2.2—2.6 were observed) formats. Elimination of frits, stabilization of the packed bed and on‐the‐fly customization of column length render mechanically robust columns that are remarkably stable over time, from which manufacturing imperfections can be removed easily.

Development of a Carbon Dot (C-Dot)-Linked Immunosorbent Assay for the Detection of Human α-Fetoprotein

A sensitive, selective, environmentally friendly, high-throughput, well-plate-based immunosorbent assay was developed to detect human α-fetoprotein (AFP) using carbon dots (C-Dots). Highly fluorescent C-Dots were synthesized using a one-step hydrothermal reaction, with citric acid serving as the carbon source and ethylene diamine acting as the nitrogen source. The reaction conditions were optimized to obtain the desired surface functionality. Then, the C-Dots were used to label one member of the anti-AFP pair (Ab2) via amine-amine coupling using glutaraldehyde. The capture anti-AFP (Ab1) was coated onto polystyrene well plates and bovine serum albumin (BSA) was used to block unsaturated binding sites. AFP was incubated in Ab1-coated wells; unbound AFP was then washed away with Tween-20. Next, the C-Dot-labeled Ab2 was added to form a sandwich immunocomplex with the AFP bound to the Ab1-coated wells. The fluorescence intensities detected from the C-Dots on these sandwich immunocomplexes were positively correlated to the concentrations of AFP antigen. A five-parameter logistic regression calibration curve was established between fluorescence and clinically important AFP concentrations (range: 0-350 ng/mL with a correlation coefficient of R(2) = 0.995). The results from the C-Dot-based immunoassay were in agreement with results from traditional immunoassays, which used horseradish peroxidase (HRP, R(2) = 0.964) and fluorescein isothiocyanate (FITC, R(2) = 0.973). These results indicated that C-Dots have great potential to be applied as biolabels for high-throughput well-plate-based immunoassays.

Synthesis and characterization of low-generation polyamidoamine (PAMAM) dendrimer-sodium montmorillonite (Na-MMT) clay nanocomposites.

Polymer-inorganic nanocomposites are a recently developed class of materials that have altered physical or chemical properties with respect to the pure polymer, inorganic host, or their micro- and macrocomposites. Lower generation (G0.0-2.0) polyamidoamine (PAMAM) dendrimer/sodium montmorillonite (Na-MMT) nanocomposites were synthesized in a solution-phase exfoliation adsorption reaction. These are the first reports of the G0.0/ and G1.0/Na-MMT nanocomposites and of a structurally-ordered G2.0/Na-MMT. The materials were characterized using powder X-ray diffraction (PXRD), thermogravimetric analysis (TGA), and Fourier transform infrared spectroscopy (FTIR). PAMAM characteristics at acidic and basic aqueous media were studied using capillary zone electrophoresis (CZE). Pseudospherical PAMAM dendrimers in aqueous medium attain a highly flattened conformation within the confined space between MMT sheets upon nanocomposite formation. The nanocomposite structure depends on the PAMAM generation and the starting dendrimer/organic composition. G0.0 always forms monolayer structures (d = 0.42 nm), while G2.0 forms monolayer structure, mixed phase, and bilayer structures (d = 0.84 nm) at lower, intermediate, and higher organic content, respectively, showing an interesting monolayer to bilayer transition. G1.0 showed an intermediate behavior, with monolayer to mixed-phase transition at the reactant ratios studied. This monolayer arrangement of PAMAM/clay nanocomposites is reported for the first time. Maximum organic contents of G0.0 monolayer and G2.0 bilayer nanocomposites were ∼7% and ∼14%, respectively. Gallery expansions were similar to those observed with linear polymer intercalates, but the packing fractions (0.31-0.32) were 2-3 times lower. At acidic pH, the nanocomposites forming only monolayer structures are obtained, indicating a stronger electrostatic attraction between MMT and protonated PAMAM, and these nanocomposites formed more slowly and were more ordered. Na(+) ions play a significant role in nanocomposite formation. At high pH, PAMAMs show high mobility, ζ potential, and surface charge densities due to Na(+) complexation in solution. FTIR data indicates that both Na-MMT and PAMAM structural units are preserved in the nanocomposites obtained.

Fabrication of a microfluidic system for capillary electrophoresis using a two-stage embossing technique and solvent welding on poly(methyl methacrylate) with water as a sacrificial layer.

Methods for fabricating poly(methyl methacrylate) microchips using a novel two-stage embossing technique and solvent welding to form microchannels in microfluidic devices are presented. The hot embossing method involves a two-stage process to create the final microchip design. In its simplest form, a mold made of aluminum is fabricated using CNC machining to create the desired microchannel design. In this work, two polymer substrates with different glass transition temperatures (Tg), polyetherimide (PEI) and poly(methyl methacrylate) (PMMA), were used to make the reusable secondary master and the final chip. First, the aluminum mold was used to emboss the PEI, a polymeric substrate with Tg approximately 216 degrees C. The embossed PEI was then used as a secondary mold for embossing PMMA, a polymeric substrate with a lower Tg ( approximately 105 degrees C). The resulting PMMA substrate possessed the same features as those of the aluminum mold. Successful feature transfer from the aluminum mold to the PMMA substrate was verified by profilometry. Bonding of the embossed layer and a blank PMMA layer to generate the microchip was achieved by solvent welding. The embossed piece was first filled with water that formed a solid sacrificial layer when frozen. The ice layer prevented channel deformation when the welding solvent (dichloroethane) was applied between the two chips during bonding. Electrophoretic separations of fluorescent dyes, rhodamine B (Rh B) and fluorescein (FL), were performed on PMMA microchips to demonstrate the feasibility of the fabrication process for microreplication of useful devices for separations. The PMMA micro-chip was tested under an electric field strength of 705 V cm-1. Separations of the test mixture of Rh B and FL generated 55 500 and 66 300 theoretical plates/meter, respectively.

In-line extraction employing functionalized magnetic particles for capillary and microchip electrophoresis

An approach to performing in‐line extraction employing functionalized magnetic particles for CE and microchip electrophoresis is presented. Silica‐coated iron oxide particles were synthesized and used as the solid support. The particles were functionalized with octadecylsilane and used as reverse‐phase sorbents for in‐line SPE followed by electrophoresis. Magnets were used to locally immobilize these sorbents inside the capillary or microchip. Extraction, elution, and detection of the analytes were performed sequentially without interruption or need for sample handling. Mixtures of hydrophobic analytes were successfully extracted from solution using the synthesized magnetic sorbents. CE was able to extract and separate mixture of parabens within 10 min. In‐line extraction was also carried out on a disposable PMMA microfluidic device with LIF detection. Electrophoretic separation of fluorescent dyes, Rhodamine 110 and SulfoRhodamine B, was completed in under a minute. The results demonstrated the feasibility of performing the in‐line extraction/separation technique in a microchip platform enabling rapid analysis, low sorbent consumption, and increased analyte recovery (relative to the capillary format).