A Spring in Performance: Silica Nanosprings Boost Enzyme Immobilization in Microfluidic Channels.

Enzyme microreactors are important tools of miniaturized analytics and have promising applications in continuous biomanufacturing. A fundamental problem of their design is that plain microchannels without extensive static internals, or packings, offer limited exposed surface area for immobilizing the enzyme. To boost the immobilization in a manner broadly applicable to enzymes, we coated borosilicate microchannels with silica nanosprings and attached the enzyme, sucrose phosphorylase, via a silica-binding module genetically fused to it. We showed with confocal fluorescence microscopy that the enzyme was able to penetrate the ∼70 µm-thick nanospring layer and became distributed uniformly in it. Compared with the plain surface, the activity of immobilized enzyme was enhanced 4.5-fold upon surface coating with nanosprings and further increased up to 10-fold by modifying the surface of the nanosprings with sulfonate groups. Operational stability during continuous-flow biocatalytic synthesis of α-glucose 1-phosphate was improved by a factor of 11 when the microreactor coated with nanosprings was used. More than 85% of the initial conversion rate was retained after 840 reactor cycles performed with a single loading of enzyme. By varying the substrate flow rate, the microreactor performance was conveniently switched between steady states of quantitative product yield (50 mM) and optimum productivity (19 mM min-1) at a lower product yield of 40%. Surface coating with silica nanosprings thus extends the possibilities for enzyme immobilization in microchannels. It effectively boosts the biocatalytic function of a microstructured reactor limited otherwise by the solid surface available for immobilizing the enzyme.

Mild and Selective C-H Activation of COC Microfluidic Channels Allowing Covalent Multifunctional Coatings.

Plastics, such as cyclic olefin copolymer (COC), are becoming an increasingly popular material for microfluidics. COC is used, in part, because of its (bio)-chemical resistance. However, its inertness and hydrophobicity can be a major downside for many bioapplications. In this paper, we show the first example of a surface-bound selective C-H activation of COC into alcohol C-OH moieties under mild aqueous conditions at room temperature. The nucleophilic COC-OH surface allows for subsequent covalent attachments, such as of a H-terminated silane. The resulting hybrid material (COC-Si-H) was then modified via a photolithographic hydrosilylation in the presence of ω-functionalized 1-alkenes to form a new highly stable, solvent-resistant hybrid surface.

Mild Photochemical Biofunctionalization of Glass Microchannels.

The ability to locally modify the inside of microfluidic channels with bioactive molecules is of ever-rising relevance. In this article, we show the direct photochemical coupling of a N-hydroxysuccinimide-terminated ω-alkene onto hydrogen-terminated silicon oxide, and its subsequent functionalization with a catalytically active DNAzyme. To achieve this local attachment of a DNAzyme, we prepared hydrogen-phenyl-terminated glass (H-Φ-glass) by the reaction of glass with H-SiPhCl2. The presence of a radical-stabilizing substituent on the Si atom (i.e., phenyl) enabled the covalent modification of bare glass substrates and of the inside of glass microchannels with a functional organic monolayer that allowed direct reaction with an amine-functionalized biomolecule. In this study, we directly attached an NHS-functionalized alkene to the modified glass surface using light with a wavelength of 328 nm, as evidenced by SCA, G-ATR, XPS, SEM, AFM and fluorescence microscopy. Using these NHS-based active esters on the surface, we performed a direct localized attachment of a horseradish peroxidase (HRP)-mimicking hemin/G-quadruplex (hGQ) DNAzyme complex inside a microfluidic channel. This wall-coated hGQ DNAzyme effectively catalyzed the in-flow oxidation of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) [ABTS] in the presence of hydrogen peroxide. This proof-of-concept of mild biofunctionalization will allow the facile preparation of modified microchannels for myriad biorelevant applications.

A generic microfluidic biosensor of G protein-coupled receptor activation-monitoring cytoplasmic [Ca(2+)] changes in human HEK293 cells.

Cell lines expressing recombinant G-protein coupled receptors (GPCRs) are activated by specific ligands resulting in transient [Ca(2+)] rises that return to basal levels in 30-60s. Yellow Cameleon 3.6 (YC3.6) is a genetically encoded calcium indicator which can be co-expressed to monitor these cytosolic [Ca(2+)] changes in real-time using Förster (Fluorescence) resonance energy transfer (FRET). On this basis, we designed the prototype of a generic microfluidic biosensor of GPCR activation, imaging [Ca(2+)] changes in recombinant human HEK293 cells, which express a combination of a GPCR (Neurokinin 1) and YC3.6. An internal reference for non-specifically induced [Ca(2+)] changes were YC3.6 cells without GPCR but expressing a red fluorescent protein (mCherry) for identification. These cell lines were grown as a mixed population in a flow cell with a volume of ~50µl and a flow cell surface of 170mm(2). Cells were activated by brief exposures to specific and non-specific analytes using an injection valve with a flexible sample volume (tested range 5-100µl) at a flow speed of 100µl/min. A flow cell surface of 0.2mm(2) with 50 cells was imaged every 2-4s to obtain signal kinetics. The lower limit of detection was 30pM Substance P (SP, 2pg/50µl), and reproducible responses to repeated injections every 3min were obtained at 1nM SP. This biosensor was designed for ~50 cells for statistical reasons, but at a lower limit of 1 receptor- and 1 reference-cell, specific ligand detection is still feasible.

Zebrafish embryo development in a microfluidic flow-through system.

The zebrafish embryo is a small, cheap, whole-animal model which may replace rodents in some areas of research. Unfortunately, zebrafish embryos are commonly cultured in microtitre plates using cell-culture protocols with static buffer replacement. Such protocols are highly invasive, consume large quantities of reagents and do not readily permit high-quality imaging. Zebrafish and rodent embryos have previously been cultured in static microfluidic drops, and zebrafish embryos have also been raised in a prototype polydimethylsiloxane setup in a Petri dish. Other than this, no animal embryo has ever been shown to undergo embryonic development in a microfluidic flow-through system. We have developed and prototyped a specialized lab-on-a-chip made from bonded layers of borosilicate glass. We find that zebrafish embryos can develop in the chip for 5 days, with continuous buffer flow at pressures of 0.005-0.04 MPa. Phenotypic effects were seen, but these were scored subjectively as 'minor'. Survival rates of 100% could be reached with buffer flows of 2 µL per well per min. High-quality imaging was possible. An acute ethanol exposure test in the chip replicated the same assay performed in microtitre plates. More than 100 embryos could be cultured in an area, excluding infrastructure, smaller than a credit card. We discuss how biochip technology, coupled with zebrafish larvae, could allow biological research to be conducted in massive, parallel experiments, at high speed and low cost.

Microchip capillary electrophoresis for point-of-care analysis of lithium.

BACKGROUND: Microchip capillary electrophoresis (CE) is a promising method for chemical analysis of complex samples such as whole blood. We evaluated the method for point-of-care testing of lithium. METHODS: Chemical separation was performed on standard glass microchip CE devices with a conductivity detector as described in previous work. Here we demonstrate a new sample-to-chip interface. Initially, we took a glass capillary as a sample collector for whole blood from a finger stick. In addition, we designed a novel disposable sample collector and tested it against the clinical standard at the hospital (Medisch Spectrum Twente). Both types of collectors require < 10 microL of test fluid. The collectors contain an integrated filter membrane, which prevents the transfer of blood cells into the microchip. The combination of such a sample collector with microchip CE allows point-of-care measurements without the need for off-chip sample treatment. This new on-chip protocol was verified against routine lithium testing of 5 patients in the hospital. RESULTS: Sodium, lithium, magnesium, and calcium were separated in < 20 s. The detection limit for lithium was 0.15 mmol/L. CONCLUSIONS: The new microchip CE system provides a convenient and rapid method for point-of-care testing of electrolytes in serum and whole blood.

Rapid inorganic ion analysis using quantitative microchip capillary electrophoresis.

Rapid quantitative microchip capillary electrophoresis (CE) for online monitoring of drinking water enabling inorganic ion separation in less than 15 s is presented. Comparing cationic and anionic standards at different concentrations the analysis of cationic species resulted in non-linear calibration curves. We interpret this effect as a variation in the volume of the injected sample plug caused by changes of the electroosmotic flow (EOF) due to the strong interaction of bivalent cations with the glass surface. This explanation is supported by the observation of severe peak tailing. Conducting microchip CE analysis in a glass microchannel, optimized conditions are received for the cationic species K+, Na+, Ca2+, Mg2+ using a background electrolyte consisting of 30 mmol/L histidine and 2-(N-morpholino)ethanesulfonic acid, containing 0.5 mmol/L potassium chloride to reduce surface interaction and 4 mmol/L tartaric acid as a complexing agent resulting in a pH-value of 5.8. Applying reversed EOF co-migration for the anionic species Cl-, SO42- and HCO3- optimized separation occurs in a background electrolyte consisting of 10 mmol/L 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) and 10 mmol/L HEPES sodium salt, containing 0.05 mmol/L CTAB (cetyltrimethylammonium bromide) resulting in a pH-value of 7.5. The detection limits are 20 micromol/L for the monovalent cationic and anionic species and 10 micromol/L for the divalent species. These values make the method very suitable for many applications including the analysis of abundant ions in tap water as demonstrated in this paper.

Microchip analysis of lithium in blood using moving boundary electrophoresis and zone electrophoresis.

The determination of inorganic cations in blood plasma is demonstrated using a combination of moving boundary electrophoresis (MBE) and zone electrophoresis. The sample loading performed under MBE conditions is studied with the focus on the quantitative analysis of lithium. A concentration adjustment takes place when the sample components migrate into the chip during the sample loading step. Using a heart-cutting method, a diluted sample plug is subsequently separated with capillary zone electrophoresis. The excessive dispersion that is typical of the samples with a high ionic strength is thereby prevented. The method can be easily applied to commercially available capillary electrophoresis microchips under the condition that the electroosmotic flow is suppressed. For the first time the lithium concentration is determined in the blood plasma from a patient on lithium therapy without sample pretreatment. Using a microchip with conductivity detection, a detection limit of 0.1 mmol/L is obtained for lithium in a 140 mmol/L sodium matrix.

Direct measurement of lithium in whole blood using microchip capillary electrophoresis with integrated conductivity detection.

The direct measurement of lithium in whole blood is described. Using microchip capillary electrophoresis (CE) with defined sample loading and applying the principles of column coupling, alkali metals were determined in a drop of whole blood. Blood collected from a finger stick was mixed with anticoagulant and transferred onto the chip without extraction or removal of components. The electrokinetic transport of red blood cells inside the channels was studied to find sample loading conditions suitable for the analysis of lithium without injecting cells into the separation channel. Both bare glass chips and chips coated with polyacrylamide were used showing the behavior of the cells under different electroosmotic flow conditions. In serum a detection limit for lithium of 0.4 mmol/L was reached. Proteins quickly contaminated untreated chip surfaces but devices with coating gave reproducible electropherograms. In addition, potassium and sodium were also detected in the same separation run. To our knowledge, this is the first device to directly measure ions in whole blood with the use of capillary zone electrophoresis on a microchip.