Silicon Nitride-Based Micro-Apertures Coated with Parylene for the Investigation of Pore Proteins Fused in Free-Standing Lipid Bilayers.

In this work, we present a microsystem setup for performing sensitive biological membrane translocation measurements. Thin free-standing synthetic bilayer lipid membranes (BLM) were constructed in microfabricated silicon nitride apertures (<100 µm in diameter), conformal coated with Parylene (Parylene-C or Parylene-AF4). Within these BLMs, electrophysiological measurements were conducted to monitor the behavior of different pore proteins. Two approaches to integrate pore-forming proteins into the membrane were applied: direct reconstitution and reconstitution via outer membrane vesicles (OMVs) released from Gram-negative bacteria. The advantage of utilizing OMVs is that the pore proteins remain in their native lipid and lipopolysaccharide (LPS) environment, representing a more natural state compared to the usage of fused purified pore proteins. Multiple aperture chips can be easily assembled in the 3d-printed holder to conduct parallel membrane transport investigations. Moreover, well defined microfabricated apertures are achievable with very high reproducibility. The presented microsystem allows the investigation of fast gating events (down to 1 ms), pore blocking by an antibiotic, and gating events of small pores (amplitude of approx. 3 pA).

Rapid lipid bilayer membrane formation on Parylene coated apertures to perform ion channel analyses.

We present a chip design allowing rapid and robust lipid bilayer (LBL) membrane formation using a Parylene coated thin silicon nitride aperture. After bilayer formation, single membrane channels can be reconstituted and characterized by electrophysiology. The ability for robust reconstitution will allow parallelization and enhanced screening of small molecule drugs acting on or permeating across the membrane channel. The aperture was realized on a microfabricated silicon nitride membrane by using standard clean-room fabrication processes. To ensure the lipid bilayer formation, the nitride membrane was coated with a hydrophobic and biocompatible Parylene layer. We tested both Parylene-C and Parylene-AF4. The contact angle measurements on both Parylene types showed very good hydrophobic properties and affinity to lipids. No precoating of the Parylene with an organic solvent is needed to make the aperture lipophilic, in contradiction to Teflon membranes. The chips can be easily placed in an array utilizing a 3D printed platform. Experiments show repetitive LBL formation and destruction (more than 6 times) within a very short time (few seconds). Through measurements we have established that the LBL layers are very thin. This allows the investigation of the fusion process of membrane proteins i.e. outer membrane protein (OmpF) in the LBL within a few minutes.

Evaluation of Microfluidic Ceiling Designs for the Capture of Circulating Tumor Cells on a Microarray Platform.

The capture of circulating tumor cells (CTCs) is still a challenging application for microfluidic chips, as these cells are rare and hidden in a huge background of blood cells. Here, different microfluidic ceiling designs in regard to their capture efficiency for CTCs in model experiments and more realistic conditions of blood samples spiked with a clinically relevant amount of tumor cells are evaluated. An optimized design for the capture platform that allows highly efficient recovery of CTCs from size-based pre-enriched samples under realistic conditions is obtained. Furthermore, the viability of captured tumor cells as well as single cell recovery for downstream genomic analysis is demonstrated. Additionally, the authors' findings underline the importance of evaluating rational design rules for microfluidic devices based on theoretical models by application-specific experiments.

Miniature 3D-Printed Centrifugal Pump with Non-Contact Electromagnetic Actuation.

We present a miniature 3D-printed dynamic pump using the centrifugal operating principle. Dynamic pumps typically yield higher flow rates than displacement pumps at reasonable output pressure. Realizing smaller devices suitable for millifluidic and microfluidic applications brings challenges in terms of design, fabrication and actuation. By using microstereolithography printing we have reduced the overall size to an effective pumping volume of 2.58 mL. The free-moving rotor consists of an impeller and permanent magnets embedded during the printing process, which allow for non-contact electromagnetic actuation. The pump is driven by periodically switching the current through stator coils, controlled by a custom built circuit using a Hall effect sensor. It achieves a maximum flow rate of 124 mL/min and a hydrostatic pressure of up to 2400 Pa.

Optimization of on-chip bacterial culture conditions using the Box-Behnken design response surface methodology for faster drug susceptibility screening.

Optimized culture conditions are essential for the investigation of biological processes. In this work, on-chip optimization of bacterial culture conditions by combining microfluidics with the Box-Behnken design response surface methodology is presented. With this methodology, the effects of several cultivation variables and their interactions were investigated enabling very fast drug susceptibility screening. The proposed measurement protocol for the determination of minimum inhibitory concentration (MIC) consist of three steps: i) single factor experiments to determine the effect of pH, nutrient concentration, and temperature on the bacterial culture; ii) analyses of the relationship between variables and the effect of the individual variables by means of the Box-Behnken design and response surface methodology (BBD-RSM) optimization; and iii) bacterial susceptibility screening of drugs and drug combinations. BBD-RSM is efficient to determine the optimal growth conditions of bacteria species with a strongly reduced amount of required experiments. On top of that, these experiments can in principle all be performed at the same time, yielding significant time-savings. The found optimized culture conditions of E. coli were applied to determine the MIC values of the drugs penicillin-streptomycin and baicalein, and combinations of those. MIC values were obtained within 8-14 h, including the 6-8 h required to determine the optimal growth parameters. The microfluidic BBD-RSM method results in a significant time reduction compared to the standard 2-4 days required to determine MIC values and is, therefore, a potential alternative in the management of bacterial infections.

3D Printing Solutions for Microfluidic Chip-To-World Connections.

The connection of microfluidic devices to the outer world by tubes and wires is an underestimated issue. We present methods based on 3D printing to realize microfluidic chip holders with reliable fluidic and electric connections. The chip holders are constructed by microstereolithography, an additive manufacturing technique with sub-millimeter resolution. The fluidic sealing between the chip and holder is achieved by placing O-rings, partly integrated into the 3D-printed structure. The electric connection of bonding pads located on microfluidic chips is realized by spring-probes fitted within the printed holder. Because there is no gluing or wire bonding necessary, it is easy to change the chip in the measurement setup. The spring probes and O-rings are aligned automatically because of their fixed position within the holder. In the case of bioanalysis applications such as cells, a limitation of 3D-printed objects is the leakage of cytotoxic residues from the printing material, cured resin. This was solved by coating the 3D-printed structures with parylene-C. The combination of silicon/glass microfluidic chips fabricated with highly-reliable clean-room technology and 3D-printed chip holders for the chip-to-world connection is a promising solution for applications where biocompatibility, optical transparency and accurate sample handling must be assured. 3D printing technology for such applications will eventually arise, enabling the fabrication of complete microfluidic devices.

PDMS-free microfluidic cell culture with integrated gas supply through a porous membrane of anodized aluminum oxide.

Microfluidic cell cultures are often used in academic research but only rarely in pharmaceutical research because of unsuitable designs, inappropriate choice of materials or incompatibility with standard equipment. In particular, microfluidic cell cultures to control the gaseous microenvironment rely on PDMS despite its disadvantages. We present a novel concept for such a cell culture device that addresses these issues and is made out of hard materials instead of PDMS. Our device contains two microfluidic chambers that are separated by a porous membrane of anodized aluminum oxide. Because of the small pore sizes but high porosity, this design allows a gas supply from one chamber to the other while leakage of the medium is avoided. Furthermore, the cells can be cultured directly on the membrane which induces the same advantageous cell response as cultivation on very soft materials. Furthermore, the chip, made out of silicon and glass, is fabricated with clean-room technologies and thus allows mass production. The interfaces to the outer world are small reservoirs which are accessible with conventional pipettes so that the setup does not require any pump. The fabricated chip is characterized regarding its diffusion characteristics. HaCaT-cells are cultivated successfully up to 14 days inside the chip but can be also removed for further processes. The presented chip is a step to bring cell cultivation with controlled gas supply from academic to industrial applications.

A Fungus Spores Dataset and a Convolutional Neural Network Based Approach for Fungus Detection.

Fungus is enormously notorious for food, human health, and archives. Fungus sign and symptoms in medical science are non-specific and asymmetrical for extremely large areas resulting into a challenging task of fungal detection. Various traditional and computer vision techniques were applied to meet the challenge of early fungus detection. On the other hand, features learned through the convolutional neural network (CNN) provided state-of-the-art results in many other applications of object detection and classification. However, the large amount of data is an essential prerequisite for its effective application. In pursuing this idea, we present a novel fungus dataset of its kind, with the goal of advancing the state of the art in fungus classification by placing the question of fungus detection. This is achieved by gathering various images of complex fungal spores by extracting samples from contaminated fruits, archives, and laboratory-incubated fungus colonies. These images primarily consisted of five different types of fungus spores and dirt. An optical sensor system was utilized to obtain these images, which were further annotated to mark fungal spores as a region of interest using specially designed graphical user interface. As a result, 40,800 labeled images were used to develop the fungus dataset to aid in precise fungus detection and classification. The other main objective of this research was to develop a CNN-based approach for the detection of fungus and distinguish different types of fungus. A CNN architecture was designed, and it showed the promising results with an accuracy of 94.8%. The obtained results proved the possibility of early detection of several types of fungus spores using CNN and could estimate all possible threats due to fungus.

A Gas Chromatographic System for the Detection of Ethylene Gas Using Ambient Air as a Carrier Gas.

Ethylene gas is a naturally occurring gas that has an influence on the shelf life of fruit during their transportation in cargo ships. An unintentional exposure of ethylene gas during transportation results in a loss of fruit. A gas chromatographic system is presented here for the detection of ethylene gas. The gas chromatographic system was assembled using a preconcentrator, a printed 3D printed gas chromatographic column, a humidity sensor, solenoid valves, and an electrochemical ethylene gas sensor. Ambient air was used as a carrier gas in the gas chromatographic system. The flow rate was fixed to 10 sccm. It was generated through a mini-pump connected in series with a mass flow controller. The metal oxide gas sensor is discussed with its limitation in ambient air. The results show the chromatogram obtained from metal oxide gas sensor has low stability, drifts, and has uncertain peaks, while the chromatogram from the electrochemical sensor is stable and precise. Furthermore, ethylene gas measurements at higher ppb concentration and at lower ppb concentration were demonstrated with the electrochemical ethylene gas sensor. The system separates ethylene gas and humidity. The chromatograms obtained from the system are stable, and the results are 1.2% repeatable in five similar measurements. The statistical calculation of the gas chromatographic system shows that a concentration of 2.3 ppb of ethylene gas can be detected through this system.

Microfluidic Platform for the Long-Term On-Chip Cultivation of Mammalian Cells for Lab-On-A-Chip Applications.

Lab-on-a-Chip (LoC) applications for the long-term analysis of mammalian cells are still very rare due to the lack of convenient cell cultivation devices. The difficulties are the integration of suitable supply structures, the need of expensive equipment like an incubator and sophisticated pumps as well as the choice of material. The presented device is made out of hard, but non-cytotoxic materials (silicon and glass) and contains two vertical arranged membranes out of hydrogel. The porous membranes are used to separate the culture chamber from two supply channels for gases and nutrients. The cells are fed continuously by diffusion through the membranes without the need of an incubator and low requirements on the supply of medium to the assembly. The diffusion of oxygen is modelled in order to find the optimal dimensions of the chamber. The chip is connected via 3D-printed holders to the macroscopic world. The holders are coated with Parlyene C to ensure that only biocompatible materials are in contact with the culture medium. The experiments with MDCK-cells show the successful seeding inside the chip, culturing and passaging. Consequently, the presented platform is a step towards Lab-on-a-Chip applications that require long-term cultivation of mammalian cells.

An Impedance-Based Mold Sensor with on-Chip Optical Reference.

A new miniaturized sensor system with an internal optical reference for the detection of mold growth is presented. The sensor chip comprises a reaction chamber provided with a culture medium that promotes the growth of mold species from mold spores. The mold detection is performed by measuring impedance changes with integrated electrodes fabricated inside the reaction chamber. The impedance change in the culture medium is caused by shifts in the pH (i.e., from 5.5 to 8) as the mold grows. In order to determine the absolute pH value without the need for calibration, a methyl red indicator dye has been added to the culture medium. It changes the color of the medium as the pH passes specific values. This colorimetric principle now acts as a reference measurement. It also allows the sensitivity of the impedance sensor to be established in terms of impedance change per pH unit. Major mold species that are involved in the contamination of food, paper and indoor environments, like Fusarium oxysporum, Fusarium incarnatum, Eurotium amstelodami, Aspergillus penicillioides and Aspergillus restrictus, have been successfully analyzed on-chip.

An Infrared Absorbance Sensor for the Detection of Melanoma in Skin Biopsies.

An infrared (IR) absorbance sensor has been designed, realized and tested with the aim of detecting malignant melanomas in human skin biopsies. The sensor has been designed to obtain fast measurements (80 s) of a biopsy using a small light spot (0.5 mm in diameter, typically five to 10 times smaller than the biopsy size) to investigate different biopsy areas. The sensor has been equipped with a monochromator to record the whole IR spectrum in the 3330-3570 nm wavelength range (where methylene and methyl stretching vibrations occur) for a qualitative spectral investigation. From the collected spectra, the CH2 stretch ratio values (ratio of the absorption intensities of the symmetric to asymmetric CH2 stretching peaks) are determined and studied as a cancer indicator. Melanoma areas exhibit different spectral shapes and significantly higher CH2 stretch ratios when compared to healthy skin. The results of the infrared investigation are compared with standard histology. This study shows that the IR sensor is a promising supportive tool to improve the diagnosis of melanoma during histopathological analysis, decreasing the risk of misdiagnosis.

A compact and integrated immunoassay with on-chip dispensing and magnetic particle handling.

We present a compact diagnostic platform for a rapid and sensitive detection of plasma biomarkers. The platform consists of a disposable microfluidic polymer chip, a processing device including a lens-free and cost efficient sensor system and a setup for dispersion of magnetic particles. The biomarkers of interest are quantified by magnetic bead based immunoassays with chemiluminescent readout technology. With a novel system for dispersion and manipulation of the magnetic particles in combination with chemiluminescence detection, the sensitivity of the immunoassay is improved and enables a rapid assay in a microfluidic format. In the disposable chip, extra chambers for storage and dispensing of biomarker specific reagents are integrated, which reduce the need of external dosing devices and thereby the cost of the platform is decreased. Plasma biomarkers for monitoring of sepsis could be quantified at 10 pg/mL concentrations within a total time of 30 min by the present system. This contribution is a fundamental step towards the development of an automatic and compact Point-of-Care testing device for monitoring of patients at the intensive care unit.

A Versatile Microarray Platform for Capturing Rare Cells.

Analyses of rare events occurring at extremely low frequencies in body fluids are still challenging. We established a versatile microarray-based platform able to capture single target cells from large background populations. As use case we chose the challenging application of detecting circulating tumor cells (CTCs)--about one cell in a billion normal blood cells. After incubation with an antibody cocktail, targeted cells are extracted on a microarray in a microfluidic chip. The accessibility of our platform allows for subsequent recovery of targets for further analysis. The microarray facilitates exclusion of false positive capture events by co-localization allowing for detection without fluorescent labelling. Analyzing blood samples from cancer patients with our platform reached and partly outreached gold standard performance, demonstrating feasibility for clinical application. Clinical researchers free choice of antibody cocktail without need for altered chip manufacturing or incubation protocol, allows virtual arbitrary targeting of capture species and therefore wide spread applications in biomedical sciences.

Hydrogel-based microfluidic incubator for microorganism cultivation and analyses.

This work presents an array of microfluidic chambers for on-chip culturing of microorganisms in static and continuous shear-free operation modes. The unique design comprises an in-situ polymerized hydrogel that forms gas and reagent permeable culture wells in a glass chip. Utilizing a hydrophilic substrate increases usability by autonomous capillary priming. The thin gel barrier enables efficient oxygen supply and facilitates on-chip analysis by chemical access through the gel without introducing a disturbing flow to the culture. Trapping the suspended microorganisms inside a gel well allows for a much simpler fabrication than in conventional trapping devices as the minimal feature size does not depend on cell size. Nutrients and drugs are provided on-chip in the gel for a self-contained and user-friendly handling. Rapid antibiotic testing in static cultures with strains of Enterococcus faecalis and Escherichia coli is presented. Cell seeding and diffusive medium supply is provided by phaseguide technology, enabling simple operation of continuous culturing with a great flexibility. Cells of Saccharomyces cerevisiae are utilized as a model to demonstrate continuous on-chip culturing.

Sizing of metallic nanoparticles confined to a microfluidic film applying dark-field particle tracking.

We present Brownian motion-based sizing of individual submicron and nanoparticles in liquid samples. The advantage of our approach is that particles can freely diffuse in a 10 µm thin liquid film and are therefore always within the focal depth of a low numerical aperture objective. Particles are visualized with dark-field microscopy, and the resulting diffraction-limited spots are tracked over a wide field of view of several hundred micrometers. Consequently, it is ascertained that long 2D trajectories are acquired, which leads to significantly increased particle sizing precision. The hydrodynamic diameters of metal particles with nominal sizes ranging from 70 to 200 nm in aqueous solution were determined by tracking for up to 2 min, and it was investigated if the diffusion characteristics were influenced by the proximity of substrates. This was not the case, and the estimated diameters were in good agreement with the values obtained by electron microscopy, thus validating the particle sizing principle. Furthermore, we measured a sample mixture to demonstrate the distinction of close particle sizes and performed the conjugation of a model protein (BSA) on the nanoparticle surface. An average increase in the radius of 9 nm was determined, which corresponds to the size of the BSA protein.

Single-step design of hydrogel-based microfluidic assays for rapid diagnostics.

For the first time we demonstrate a microfluidic platform for the preparation of biosensing hydrogels by in situ polymerization of polyethyleneglycol diacrylate (PEG-DA) in a single step. Capillary pressure barriers enable the precise formation of gel microstructures for fast molecule diffusion. Parallel arrangement of these finger structures allows for macroscopic and standard equipment readout methods. The analyte automatically fills the space in between the gel fingers by the hydrophilic nature of the gel. Introducing the functional structures in the chip fabrication allows for rapid assay customization by making surface treatment, gel curing mask alignment and washing steps obsolete. Simple handling and functionality are illustrated by assays for matrix metalloproteinase, an important factor in chronic wound healing. Assays for total protein concentration and cell counts are presented, demonstrating the possibilities for a wide range of fast and simple diagnostics.

Miniaturized mass-spectrometry-based analysis system for fully automated examination of conditioned cell culture media.

We present a fully automated setup for performing in-line mass spectrometry (MS) analysis of conditioned media in cell cultures, in particular focusing on the peptides therein. The goal is to assess peptides secreted by cells in different culture conditions. The developed system is compatible with MS as analytical technique, as this is one of the most powerful analysis methods for peptide detection and identification. Proof of concept was achieved using the well-known mating-factor signaling in baker's yeast, Saccharomyces cerevisiae. Our concept system holds 1 mL of cell culture medium and allows maintaining a yeast culture for, at least, 40 hours with continuous supernatant extraction (and medium replenishing). The device's small dimensions result in reduced costs for reagents and open perspectives towards full integration on-chip. Experimental data that can be obtained are time-resolved peptide profiles in a yeast culture, including information about the appearance of mating-factor-related peptides. We emphasize that the system operates without any manual intervention or pipetting steps, which allows for an improved overall sensitivity compared to non-automated alternatives. MS data confirmed previously reported aspects of the physiology of the yeast-mating process. Moreover, matingfactor breakdown products (as well as evidence for a potentially responsible protease) were found.

Optofluidic micro-sensors for the determination of liquid concentrations.

We present a novel optofluidic device for non-invasive and label-free determination of liquid concentrations. A microfluidic channel filled with the sample solution is hit by laser light in an angle close to the critical angle for total internal reflection. Due to the intentionally defined divergence of the incident beam, parts of the rays will experience total internal reflection while another part will be transmitted. Both reflected and transmitted light signals are recorded and the ratio of these signals is used for sample characterization. The stability compared to single signal analyses is significantly improved, resulting in a resolution of approximately 40 mmol L(-1). The typical working range of the device under investigation is between a few tens of mmol L(-1) and 5 mol L(-1) making it useful for applications in the food industry, for example to determine the amount of phosphates in liquid products.

Detection of Dissolved Lactose Employing an Optofluidic Micro-System.

In this work, a novel optofluidic sensor principle is employed for a non-invasive and label-free characterization of lactose containing liquid samples. Especially for medicine and food industry, a simple, fast and accurate determination of the amount of lactose in various products is highly desirable. The presented system exploits the impact of dissolved molecules on the refractive index for sample characterization. On the optofluidic chip, a microfluidic channel filled with the analyte is hit by slightly diverging laser light. The center incident angle of the beam on-chip is set close to the critical angle for total internal reflection. Both the reflected and the transmitted light signals are recorded at the solid-liquid interface. The ratio of those two signals is then used as representative value for the analyte. Using this principle, lactose containing samples were differentiated based on their concentrations at a step size of 10 mmol/L. The use of the signals ratio instead of a single signal approach improves the stability of the system significantly, allowing for higher resolutions to be achieved. Furthermore, the fabrication of the devices in PDMS ensures biocompatibility and provides low absorbance of light in the visible range.