A microreactor with phase-change microvalves for batch chemical synthesis at high temperatures and pressures.

We present a simple microreactor with dimethyl sulfoxide (DMSO) phase-change valves suitable for performing batch organic chemistry under high temperature and pressure conditions. As a proof of principle, we demonstrate a radiofluorination reaction important in the synthesis of [(18)F]FAC, a new positron emission tomography biomarker for immune system monitoring and prediction of chemotherapy response. We achieved high radioactivity recovery (97 ± 1%, n = 3) and conversion efficiency (83 ± 1%, n = 3), comparable to that achieved with macroscale systems, but with a volume 30× smaller. This platform overcomes the limitations of previously reported phase-change valves in terms of compatibility with organic chemistry, and extends the range of reaction conditions for carrying out harsh batch chemistry at the microscale.

Plug-and-play modules for flexible radiosynthesis.

We present a plug-and-play radiosynthesis platform and accompanying computer software based on modular subunits that can easily and flexibly be configured to implement a diverse range of radiosynthesis protocols. Modules were developed that perform: (i) reagent storage and delivery, (ii) evaporations and sealed reactions, and (iii) cartridge-based purifications. The reaction module incorporates a simple robotic mechanism that removes tubing from the vessel and replaces it with a stopper prior to sealed reactions, enabling the system to withstand high pressures and thus provide tremendous flexibility in choice of solvents and temperatures. Any number of modules can rapidly be connected together using only a few fluidic connections to implement a particular synthesis, and the resulting system is controlled in a semi-automated fashion by a single software interface. Radiosyntheses of 2-[(18)F]fluoro-2-deoxy-d-glucose ([(18)F]FDG), 1-[(18)F]fluoro-4-nitrobenzene ([(18)F]FNB), and 2'-deoxy-2'-[(18)F]fluoro-1-ß-d-arabinofuranosyl cytosine (d-[(18)F]FAC) were performed to validate the system and demonstrate its versatility.

Reusable electrochemical cell for rapid separation of [¹8F]fluoride from [¹8O]water for flow-through synthesis of ¹8F-labeled tracers.

A brass-platinum electrochemical micro-flow cell was developed to extract [(18)F]fluoride from an aqueous solution and release it into an organic-based solution, suitable for subsequent radio-synthesis, in a fast and reliable manner. This cell does not suffer electrode erosion and is thus reusable while operating faster by enabling increased voltages. By optimizing temperature, trapping and release potentials, flow rates, and electrode materials, an overall [(18)F]fluoride trapping and release efficiency of 84 ± 5% (n=7) was achieved. X-ray photoelectron spectroscopy (XPS) was used to analyze electrode surfaces of various metal-metal systems and the findings were correlated with the performance of the electrochemical cell. To demonstrate the reactivity of the released [(18)F]fluoride, the cell was coupled to a flow-through reactor and automated synthesis of [(18)F]FDG with a repeatable decay-corrected yield of 56 ± 4% (n=4) was completed in < 15 min. A multi-human dose of 5.92GBq [(18)F]FDG was also demonstrated.

In situ microarray fabrication and analysis using a microfluidic flow cell array integrated with surface plasmon resonance microscopy.

Surface Plasmon Resonance Microscopy (SPRM) is a promising label-free analytical tool for the real-time study of biomolecule interactions in a microarray format. However, flow cell design and microarray fabrication have hindered throughput and limited applications of SPRM. Here we report the integration of a microfluidic flow cell array (MFCA) with SPRM enabling in situ microarray fabrication and multichannel analysis of biomolecule probe-target interactions. We demonstrate the use of the MFCA for delivery of sample solutions with continuous flow in 24 channels in parallel for rapid microarray creation and binding analysis while using SPRM for real-time monitoring of these processes. Label-free measurement of antibody-antibody interactions demonstrates the capabilities of the integrated MFCA-SPRM system and establishes the first steps of the development of a high-throughput, label-free immunogenicity assay. After in situ probe antibody immobilization, target antibody binding was monitored in real time in 24 channels simultaneously. The limit of detection for this particular antibody pair is 80 ng/mL which is approximately 6 times lower than the industry recommended immunogenicity assay detection limit. The integrated MFCA-SPRM system is a powerful and versatile combination for a range of array-based analyses, including biomarker screening and drug discovery.

"Spot and hop": internal referencing for surface plasmon resonance imaging using a three-dimensional microfluidic flow cell array.

We have developed a novel referencing technique for surface plasmon resonance imaging systems referred to as "spot and hop." The technique enables internal referencing for individual flow cells in a parallel processing microfluidic network. Internal referencing provides the ability to correct for nonspecific binding and instrument drift, significantly improving data quality at each region of interest. The performance of a 48-flow-cell device was demonstrated through a series of studies, including "rise and fall" time, ligand preconcentration, ligand immobilization, analyte binding, and regeneration tests. Interfacing parallel processing fluidics with imaging systems will significantly expand the throughput and applications of array-based optical biosensors while retaining high data quality.

Parallel determination of phenotypic cytotoxicity with a micropattern of mutant cell lines.

This work presents a novel tool, the Continuous Flow Microspotter (CFM) and its use in patterning cellular microarrays of multiple cell types into the bottom of a tissue culture well. The CFM uses a system of isolated microfluidic channels to make an array of localized microspots of adhesion dependent cells in the bottom of a conventional tissue culture well. With this device we have created micropatterns of multiple cell lines in a single tissue culture well and used this system to conduct simultaneous cytotoxicity tests and recover dose survival curves in a parallel study. This mechanism of parallel testing allows the researcher to employ the use of positive and negative controls, as well as compare the chemical response of phenotypes in a tightly controlled microenvironment. For the experiments presented in this paper we have fabricated a CFM with a set of ten microchannels (five inlet channels and five outlet channels) to pattern a row of five microspots consisting of four cellular microspots and one empty spot for background measurements. Micropatterns containing a set of four different Chinese hamster ovarian cell (CHO) mutant phenotypes were deposited into the bottom of commercially available tissue culture wells then interrogated with mitomycin C, a chemotherapeutic agent. This study shows statistically significant (P < 0.05) hypersensitivity of the UV20 CHO mutant to a DNA interstrand cross-linking agent (mitomycin C). Because the CFM is also capable of depositing proteins and other biomolecules to the individual microspots of the array we foresee capabilities of the 48 microspot CFM to multiplex 48 cell types with 48 chemical reagents all within the confines of a 60 mm(2) area.

Improved continuous-flow print head for micro-array deposition.

Limitations in depositing ligands using conventional micro-array pin spotting have hindered the application of surface plasmon resonance imaging (SPRi) technology. To address these challenges we introduce a modification to our continuous-flow micro-spotting technology that improves ligand deposition. Using Flexchip protein A/G and neutravidin capturing surfaces, we demonstrate that our new microfluidic spotter requires 1000 times less concentrated antibodies and biotinylated ligands than is required for pin spotting. By varying the deposition flow rate, we show that the design of our tip overlay flow cell is efficient at delivering sample to the substrate surface. Finally, contact time studies show that it is possible to capture antibodies and biotinylated ligands at concentrations of less than 0.1 ug/ml and 100 pM, respectively. These improvements in spotting technology will help to expand the applications of SPRi systems in areas such as antibody screening, carbohydrate arrays, and biomarker detection.