Continuous Electroformation of Gold Nanoparticles in Nanoliter Droplet Reactors.

Gold nanoparticles (AuNPs) are employed in numerous applications, including optics, biosensing and catalysis. Here, we demonstrate the stabilizer-free electrochemical synthesis of AuNPs inside nanoliter-sized reactors. Droplets encapsulating a gold precursor are formed on a microfluidic device and exposed to an electrical current by guiding them through a pair of electrodes. We exploit the naturally occurring recirculation flows inside confined droplets (moving in rectangular microchannels) to prevent the aggregation of nanoparticles after nucleation. Therefore, AuNPs with sizes in the range of 30 to 100 nm were produced without the need of additional capping agents. The average particle size is defined by the precursor concentration and droplet velocity, while the charge dose given by the electric field strength has a minor effect. This method opens the way to fine-tune the electrochemical production of gold nanoparticles, and we believe it is a versatile method for the formation of other metal nanoparticles.

Single-cell protein profiling in microchambers with barcoded beads.

Single-cell profiling provides insights into cellular behaviour that macroscale cell cultures and bulk measurements cannot reveal. In the context of personalized cancer treatment, the profiling of individual tumour cells may lead to higher success rates for therapies by rapidly selecting the most efficacious drugs. Currently, genomic analysis at the single-cell level is available through highly sensitive sequencing approaches. However, the identification and quantification of intracellular or secreted proteins or metabolites remains challenging. Here, we introduce a microfluidic method that facilitates capture, automated data acquisition and the multiplexed quantification of proteins from individual cells. The microfluidic platform comprises 1026 chambers with a volume of 152 pL each, in which single cells and barcoded beads are co-immobilized. We demonstrated multiplexed single-cell protein quantification with three different mammalian cell lines, including two model breast cancer cell lines. We established on-chip immunoassays for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), galectin-3 (Gal-3) and galectin-3 binding protein (Gal-3bp) with detection limits as low as 7.0 × 104, 2.3 × 105 and 1.8 × 103 molecules per cell, respectively. The three investigated cell types had high cytosolic levels of GAPDH and could be clearly differentiated by their expression levels of Gal-3 and Gal-3bp, which are important factors that contribute to cancer metastasis. Because it employed commercially available barcoded beads for this study, our platform could be easily used for the single-cell protein profiling of several hundred different targets. Moreover, this versatile method is applicable to the analysis of bacteria, yeast and mammalian cells and nanometre-sized lipid vesicles.

Cultivation and quantitative single-cell analysis of Saccharomyces cerevisiae on a multifunctional microfluidic device.

Here, we present a multifunctional microfluidic device whose integrative design enables to combine cell culture studies and quantitative single cell biomolecule analysis. The platform consists of 32 analysis units providing two key features; first, a micrometer-sized trap for hydrodynamic capture of a single Saccharomyces cerevisiae (S. cerevisiae) yeast cell; second, a convenient double-valve configuration surrounding the trap. Actuating of the outer valve with integrated opening results in a partial isolation in a volume of 11.8 nL, i.e. the cell surrounding fluid can be exchanged diffusion-based without causing shear stress or cell loss. Actuation of the inner ring-shaped valve isolates the trapped cell completely in a small analysis volume of 230 pL. The device was used to determine the growth rate of yeast cells (S. cerevisiae) under under optimum and oxidative stress conditions. In addition, we successfully quantified the cofactor beta-nicotinamide adenine dinucleotide phosphate (NAD(P)H) in single and few cells exposed to the different microenvironments. In conclusion, the microdevice enables to analyze the influence of an external stress factor on the cellular fitness in a fast and more comprehensive way as cell growth and intracellular biomolecule levels can be investigated.

An integrated field-effect microdevice for monitoring membrane transport in Xenopus laevis oocytes via lateral proton diffusion.

An integrated microdevice for measuring proton-dependent membrane activity at the surface of Xenopus laevis oocytes is presented. By establishing a stable contact between the oocyte vitelline membrane and an ion-sensitive field-effect (ISFET) sensor inside a microperfusion channel, changes in surface pH that are hypothesized to result from facilitated proton lateral diffusion along the membrane were detected. The solute diffusion barrier created between the sensor and the active membrane area allowed detection of surface proton concentration free from interference of solutes in bulk solution. The proposed sensor mechanism was verified by heterologously expressing membrane transport proteins and recording changes in surface pH during application of the specific substrates. Experiments conducted on two families of phosphate-sodium cotransporters (SLC20 & SLC34) demonstrated that it is possible to detect phosphate transport for both electrogenic and electroneutral isoforms and distinguish between transport of different phosphate species. Furthermore, the transport activity of the proton/amino acid cotransporter PAT1 assayed using conventional whole cell electrophysiology correlated well with changes in surface pH, confirming the ability of the system to detect activity proportional to expression level.

A novel method for determining residence time distribution in intricately structured microreactors.

A precise characterisation of microreactors can be achieved by determining the residence time distribution as one of the most important flow characteristics. An approach specially designed for microreactor applications was developed, which employs a tracer 'injection' using the optical activation of a caged fluorescent dye. Furthermore, the effect of the laminar flow on the determination of the residence time distribution in microreactors has been taken into account during the measurements and their interpretation to fulfill the requirements of the so-called 'mixing-cup-problem' on the microscale. Residence time distributions for an intricately structured thin microreactor were determined for different velocities. The ideality of the stimulus signal generated by the newly introduced technique is demonstrated for an analytically well-defined straight channel and compared with a signal derived from deconvolution of non-ideal input signals.

micro-Hotplate enhanced optical heating by infrared light for single cell treatment.

In this study we present a simple approach for fast and localised heating that relies on the strong absorbance of infrared light by microsized patterned surfaces ("micro-hotplates"). Two different materials, micro-arrays of carbon and gold, were tested with respect to their absorbance of the 830 nm diode laser light and their applicability. Both materials were found to be suitable for inducing controlled heating to a temperature increase of more than 10 degrees C within less than a second. The effect of optical heating on living cells (colon cancer cell line SW 480) was investigated with a modified fluorescence microscope. The temperature was controlled by varying the intensity and the exposure time of the laser light. Depending on temperature, induced death of cells in direct contact with the absorbent material was observed, or otherwise cells were kept alive. Cells survive the direct exposure of IR light without the use of the micro-hotplates. In contrast to common heating systems, the optical heating does not need direct contact to a temperature control device. Therefore, it is a very flexible method that can easily be implemented within any microchip. We believe that it will be a versatile tool for initiation and modulation of biochemical or cellular reactions, reversible cell membrane opening, and for control of cell growth.