Heterogeneous integration of gels into microfluidics using a mesh carrier.

The incorporation of hydrogels inside microfluidics is a promising method for localizing receptors inside microfluidic structures for many bio-analytical applications as well as for working with cells. However, current methods rely on the in situ polymerization of hydrogels and therefore necessitate optical masks and extensive post-polymerization steps for example for washing uncrosslinked gel precursors and receptors. Here, we report a simple and efficient method for the integration of hydrogels to microfluidic chips. Small volumes of poly(ethylene)glycol-based acrylamide (PEGACA) hydrogels are photopolymerized on a mesh, rinsed, partially dried and transferred to microfluidic structures by simple contact. The gels can be derivatized before transfer with receptors such as streptavidin, antibodies, or can entrap beads as small as 200 nm. We detail the role of meshes relative to the mesh density and wettability and demonstrate how hydrogels can be transferred into capillary-driven microfluidic chips, which are easily sealed using a dry-film resist. By analogy to microfabrication strategies wherein critical components are produced separately and then combined, our method introduces the concept of heterogeneous integration of critical (bio)chemicals to microfluidic chips using an intermediate mesh carrier.

Label-free recognition of drug resistance via impedimetric screening of breast cancer cells.

We present a novel study on label-free recognition and distinction of drug resistant breast cancer cells (MCF-7 DOX) from their parental cells (MCF-7 WT) via impedimetric measurements. Drug resistant cells exhibited significant differences in their dielectric properties compared to wild-type cells, exerting much higher extracellular resistance (Rextra ). Immunostaining revealed that MCF-7 DOX cells gained a much denser F-actin network upon acquiring drug resistance indicating that remodeling of actin cytoskeleton is probably the reason behind higher Rextra , providing stronger cell architecture. Moreover, having exposed both cell types to doxorubicin, we were able to distinguish these two phenotypes based on their substantially different drug response. Interestingly, impedimetric measurements identified a concentration-dependent and reversible increase in cell stiffness in the presence of low non-lethal drug doses. Combined with a profound frequency analysis, these findings enabled distinguishing distinct cellular responses during drug exposure within four concentration ranges without using any labeling. Overall, this study highlights the possibility to differentiate drug resistant phenotypes from their parental cells and to assess their drug response by using microelectrodes, offering direct, real-time and noninvasive measurements of cell dependent parameters under drug exposure, hence providing a promising step for personalized medicine applications such as evaluation of the disease progress and optimization of the drug treatment of a patient during chemotherapy.

A supramolecular approach to enzyme immobilization in micro-channels.

A supramolecular assembly scheme is developed to enable the facile in-situ immobilization of enzymes in a microfluidic channel system. A combination of orthogonal supramolecular interactions of host (ß-cyclodextrin)-guest (adamantane) and biotin-Streptavidin (SAv) interactions are employed to generate reusable homogeneous enzyme layers in microchannels. The structural integrity and catalytic activity of the immobilized enzyme calf-intestine alkaline phosphatase (AlkPh) is demonstrated. From the kinetic analysis of a dephosphorylation reaction, the specificity constant k(cat)/K(M) for immobilized alkaline phosphatase in the channels is on the order of 10(5) M(-1) s(-1) and comparable to known literature values in other environments. These observations are ascribed to the good access of the substrate to favorably oriented enzymes across the microchannel. Therefore, this study demonstrates the great potential for adopting a supramolecular assembly scheme to immobilize enzymes in microfluidic devices.

A microfluidic-based frequency-multiplexing impedance sensor (FMIS).

We present a novel technology for the simultaneous and simple impedimetric screening of multiple microfluidic channels with only one electrode pair. We have exploited the frequency dimension to distinguish between up to three channels. Each 'sub-sensor' possesses its corresponding measurement frequency where the sample-specific dielectric properties can be probed. We have shown the validity of our frequency-multiplexing impedance sensor (FMIS) by comparison with conventional 'single sensors'. Our highly sensitive FMIS was proven suitable for life science applications through usage as a cell-based toxicology platform. We are confident that our technology might find great utility in parallelized cell-based analysis systems as well as in biomedical devices where size limitations and spatially distributed probing are important parameters.

A supramolecular sensing platform for phosphate anions and an anthrax biomarker in a microfluidic device.

A supramolecular platform based on self-assembled monolayers (SAMs) has been implemented in a microfluidic device. The system has been applied for the sensing of two different analyte types: biologically relevant phosphate anions and aromatic carboxylic acids, which are important for anthrax detection. A Eu(III)-EDTA complex was bound to ß-cyclodextrin monolayers via orthogonal supramolecular host-guest interactions. The self-assembly of the Eu(III)-EDTA conjugate and naphthalene ß-diketone as an antenna resulted in the formation of a highly luminescent lanthanide complex on the microchannel surface. Detection of different phosphate anions and aromatic carboxylic acids was demonstrated by monitoring the decrease in red emission following displacement of the antenna by the analyte. Among these analytes, adenosine triphosphate (ATP) and pyrophosphate, as well as dipicolinic acid (DPA) which is a biomarker for anthrax, showed a strong response. Parallel fabrication of five sensing SAMs in a single multichannel chip was performed, as a first demonstration of phosphate and carboxylic acid screening in a multiplexed format that allows a general detection platform for both analyte systems in a single test run with µM and nM detection sensitivity for ATP and DPA, respectively.

Distinguishing drug-induced minor morphological changes from major cellular damage via label-free impedimetric toxicity screening.

We present a novel perfusion-based microfluidic platform for label-free drug toxicity screening which can single out non-lethal morphological changes from cellular death using electrical impedance spectroscopy. Minor cellular changes such as cell-cell contacts and major cell injury were identified via impedance phase angle analysis and follow-up of impedance magnitude at different frequencies. Having exposed HepG2/C3A cells to acetaminophen (AP), we showed that continuous drug perfusion caused a time and concentration-dependent impedance decrease. Moreover, perfusion of repeated doses revealed altered dielectric properties of the cell culture after recovery from AP exposure. This study highlights the possibility to sense cellular changes long before cellular death takes place, pointing out the remarkable sensitivity advantage of this technique over standard endpoint viability tests and its interest for toxicology.

ENZYMATIC POLYMERIZATION OF PHENOLS IN ROOM TEMPERATURE IONIC LIQUIDS.

Soybean peroxidase (SBP) was used to catalyze the polymerization of phenols in room-temperature ionic liquids (RTILs). Phenolic polymers with number average molecular weights ranging from 1200 to 4100 D were obtained depending on the composition of the reaction medium and the nature of the phenol. Specifically, SBP was highly active in methylimidazolium-containing RTILs, including 1-butyl-3-methylimidazolium tetrafluoroborate (BMIM(BF(4))), and 1-butyl-3-methylpyridinium tetrafluoroborate (BMPy(BF(4))) with the ionic liquid content as high as 90% (v/v); the balance being aqueous buffer. Gel permeation chromatography and MALDI-TOF analysis indicated that higher molecular weight polymers can be synthesized in the presence of higher RTIL concentrations, with selective control over polymer size achieved by varying the RTIL concentration. The resulting polyphenols exhibited high thermostability and possessed thermosetting properties.

Enzyme-carbon nanotube conjugates in room-temperature ionic liquids.

Room-temperature ionic liquids (RTILs) are intriguing solvents, which are recognized as "green" alternatives to volatile organics. Although RTILs are nonvolatile and can dissolve a wide range of charged, polar, and nonpolar organic and inorganic molecules, there remain substantial challenges in their use, not the least of which is the solvents' high viscosity that leads to potential mass transfer limitations. In the course of this work, we discovered that the simple adsorption of the bacterial protease, proteinase K, onto single-walled carbon nanotubes (SWNTs) results in intrinsically high catalytic turnover. The high surface area and the nanoscopic dimensions of SWNTs offered high enzyme loading and low mass transfer resistance. Furthermore, the enzyme-SWNT conjugates displayed enhanced thermal stability in RTILs over the native suspended enzyme counterpart and allowed facile reuse. These enzyme-SWNT conjugates may therefore provide a way to overcome key operational limitations of RTIL systems.