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.

We have developed a simple and direct method to fabricate paper-based microfluidic devices that can be used for a wide range of colorimetric assay applications. With these devices, assays can be performed within minutes to allow for quantitative colorimetric analysis by use of a widely accessible iPhone camera and an RGB color reader application (app) to measure color intensity. In the described laboratory experiment, students design and create their own microfluidic devices with common laboratory supplies such as Kimwipes, Parafilm, and a thermal laminator, and gain hands-on experience in the analysis of Fe 2+ and Cu 2+ by colorimetric determination.

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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.

Novel means of fabricating polymeric microfluidic devices are presented. An SU-8 master is applied in two-stage embossing, followed by vaporized organic solvent bonding. The primary master is created by standard photolithography; the inexpensive SU-8 primary master is used in a two-stage process to generate microfeatures in hard polymers. A vaporized solvent bonding technique that readily produces complete microfluidic chips, without the need of a sacrificial layer to prevent channel deformation, was used to form complete multilayer microfluidic devices. This technique provides a more direct method to generate hard polymer microfluidic chips than classical techniques and therefore is highly amenable to rapid prototyping. The technique lends itself readily to many polymers, facilitating device production for a variety of applications, even permitting hybrid polymer chips, and provides a rapid, cost-effective, simple, and versatile approach to the production of polymer-based microdevices. The fabrication technique was tested to build microchips to perform several analyses, including chromatographic separations and a quantitative indicator assay. High separation efficiencies of 10,000-45,000 plates/m were obtained using the fabricated liquid chromatography (LC) microchip. The fabrication method was also tested in building a passive micromixer that contained high-density microfeatures and required three polymer layers. A glycine assay using o-phthaldialdehyde (OPA) was performed in the micromixer. With glycine concentrations ranging from 0.0 to 2.6 microM, a linear calibration plot was obtained with a detection limit of 0.032 microM.

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