Peter Koltay

Rapid prototyping of microfluidic chips in COC

We present a novel, cost-efficient process chain for fast tooling and small-lot replication of high-quality, multi-scale microfluidic polymer chips within less than 5 days. The fabrication chain starts with a primary master which is made by well-established cleanroom processes such as DRIE or negative SU-8 resist based surface micromachining. The formation of undercuts in the master which would complicate demolding is carefully avoided. Secondary PDMS masters or epoxy-based masters which are more suitable for common polymer replication schemes such as soft-embossing, hot-embossing or injection molding are subsequently cast from the primary masters. The polymer replica are mainly made of COC and show excellent fidelity with the conventionally micromachined master while displaying no degeneration, even after more than 200 cycles. The use of other polymers such as PMMA is also possible. The process chain further includes surface modification techniques for overall, long-term stable hydrophilic coatings and for local hydrophobic patches as well as a durable sealing based on thermal bonding.

Completely Superhydrophobic PDMS Surfaces for Microfluidics

This study presents a straightforward two-step fabrication process of durable, completely superhydrophobic microchannels in PDMS. First, a composite material of PDMS/PTFE particles is prepared and used to replicate a master microstructure. Superhydrophobic surfaces are formed by subsequent plasma treatment, in which the PDMS is isotropically etched and PTFE particles are excavated. We compare the advancing and receding contact angles of intrinsic PDMS samples and composite PTFE/PDMS samples (1 wt %, 8 wt %, and 15 wt % PTFE particle concentration) and demonstrate that both the horizontal and vertical surfaces are indeed superhydrophobic. The best superhydrophobicity is observed for samples with a PTFE particle concentration of 15 wt %, which have advancing and receding contact angles of 159° ± 4° and 158° ± 3°, respectively.

Multi-layer SU-8 lift-off technology for microfluidic devices

This paper reports on a novel multilayer SU-8 lift-off technology which allows for low cost rapid prototyping of microfluidic devices. The process presented is based on a multi-layer structure of SU-8 which can be released from the substrate after processing and enables the creation of through holes. The lift-off is accomplished during the development by making use of the volume shrinkage of the SU-8 during postbaking and by modification of the adhesion to the substrate. To demonstrate the technology, prototypes of a multichannel microdispenser according to the Dispensing Well Plate (DWP™) principle (Koltay et al 2004 Sensors Actuators A 116 472, 483) were fabricated. The samples contain 24 parallel dispensing units with 100 µm through holes and a dosage volume of 60 nl. For the first time all functional structures such as reservoirs, channels and through holes (nozzles) of the DWP™ were realized exclusively in the photodefinable epoxy SU-8. To assess the quality of the SU-8 process the geometry of the presented prototypes is characterized by profiler measurements and scanning electron microscopy. Furthermore, the dispensing performance is studied experimentally by gravimetrical measurements. A reproducibility of the dosage volume of 1% and a homogeneity within individual droplet arrays of 3.6% were achieved.

Technologies for Single-Cell Isolation

The handling of single cells is of great importance in applications such as cell line development or single-cell analysis, e.g., for cancer research or for emerging diagnostic methods. This review provides an overview of technologies that are currently used or in development to isolate single cells for subsequent single-cell analysis. Data from a dedicated online market survey conducted to identify the most relevant technologies, presented here for the first time, shows that FACS (fluorescence activated cell sorting) respectively Flow cytometry (33% usage), laser microdissection (17%), manual cell picking (17%), random seeding/dilution (15%), and microfluidics/lab-on-a-chip devices (12%) are currently the most frequently used technologies. These most prominent technologies are described in detail and key performance factors are discussed. The survey data indicates a further increasing interest in single-cell isolation tools for the coming years. Additionally, a worldwide patent search was performed to screen for emerging technologies that might become relevant in the future. In total 179 patents were found, out of which 25 were evaluated by screening the title and abstract to be relevant to the field.

Increasing μDMFC efficiency by passive CO2 bubble removal and discontinuous operation

A new concept that enables fully passive CO2 gas bubble removal in micro direct methanol fuel cells (μDMFCs) is presented. The original concept behind the presented degassing structure (flowfield) is based on microchannels with a T-shaped cross section. These channels have defined tapering angles over their cross section (α) and along their axis (β). The tapered channel design creates an intrinsic transport mechanism that removes the gas bubbles from the electrodes by capillary forces only. Computational fluid dynamic (CFD) simulations have been used to determine applicable opening angles of α = 5° and β = 1.5°. The experimental verification was done by using a transparent flowfield to show the passive bubble removal as well as with a fully operational μDMFC. During the operation, the fuel cell delivered an output of up to 8 mW cm−2 without the need for external pumping in short-term measurements. During the long-term measurements, discontinuous pumping showed the highest fuel cell efficiency compared to the continuously pumped fuel supply.

PipeJet: A Simple Disposable Dispenser for the Nano- and Microliter Range

This paper reports on a simple, disposable non-contact dispenser for the nano- and microliter range. In contrast to other known dispensers manufactured by silicon micromachining1-4 the new device simply consists of an elastic polymer tube with a circular cross section. Actuation is done by a piezostack driven piston, squeezing the tube at a defined position near the open end by a significant fraction of the cross section. In contrast to drop-on-demand devices based on an acoustic actuation principle,5 the squeezing of the tube leads to a significant mechanical displacement of the liquid. Our experiments tested a large number of media in the viscosity range from 1 to 27 mPas. Some of our experiments tested up to approximately 2,000 mPas. Frequency characteristics showed an independent dosage volume for water up to a frequency of 15 Hz for tubes with an inner diameter of approximately 200 um. Standard deviation within 1,000 shots resulted in an excellent CV (standard deviation/dosage volume) of less than 2% of the dosage volume. Using tubes with an inner diameter of approximately 1,000 um and a print frequency of 340 Hz, a flow rate of less than or equal to 143 μL/s could be reached. Beyond the possibility to dispense pure liquids, emulsion paints with particles that have a diameter of approximately 40 μm have also been printed successfully.

Single-Cell Printer: Automated, On Demand, and Label Free

Within the past years, single-cell analysis has developed into a key topic in cell biology to study cellular functions that are not accessible by investigation of larger cell populations. Engineering approaches aiming to access single cells to extract information about their physiology, phenotype, and genotype at the single-cell level are going manifold ways, meanwhile allowing separation, sorting, culturing, and analysis of individual cells. Based on our earlier research toward inkjet-like printing of single cells, this article presents further characterization results obtained with a fully automated prototype instrument for printing of single living cells in a noncontact inkjet-like manner. The presented technology is based on a transparent microfluidic drop-on-demand dispenser chip coupled with a camera-assisted automatic detection system. Cells inside the chip are detected and classified with this detection system before they are expelled from the nozzle confined in microdroplets, thus enabling a “one cell per droplet” printing mode. To demonstrate the prototype instrument’s suitability for biological and biomedical applications, basic experiments such as printing of single-bead and cell arrays as well as deposition and culture of single cells in microwell plates are presented. Printing efficiencies greater than 80% and viability rates about 90% were achieved.

Single-cell PCR of genomic DNA enabled by automated single-cell printing for cell isolation

Single-cell analysis has developed into a key topic in cell biology with future applications in personalized medicine, tumor identification as well as tumor discovery (Editorial, 2013). Here we employ inkjet-like printing to isolate individual living single human B cells (Raji cell line) and load them directly into standard PCR tubes. Single cells are optically detected in the nozzle of the microfluidic piezoelectric dispenser chip to ensure printing of droplets with single cells only. The printing process has been characterized by using microbeads (10µm diameter) resulting in a single bead delivery in 27 out of 28 cases and relative positional precision of ±350µm at a printing distance of 6mm between nozzle and tube lid. Process-integrated optical imaging enabled to identify the printing failure as void droplet and to exclude it from downstream processing. PCR of truly single-cell DNA was performed without pre-amplification directly from single Raji cells with 33% success rate (N=197) and Cq values of 36.3±2.5. Additionally single cell whole genome amplification (WGA) was employed to pre-amplify the single-cell DNA by a factor of >1000. This facilitated subsequent PCR for the same gene yielding a success rate of 64% (N=33) which will allow more sophisticated downstream analysis like sequencing, electrophoresis or multiplexing.

Towards a “Sample-In, Answer-Out” Point-of-Care Platform for Nucleic Acid Extraction and Amplification: Using an HPV E6/E7 mRNA Model System

The paper presents the development of a “proof-of-principle” hands-free and self-contained diagnostic platform for detection of human papillomavirus (HPV) E6/E7 mRNA in clinical specimens. The automated platform performs chip-based sample preconcentration, nucleic acid extraction, amplification, and real-time fluorescent detection with minimal user interfacing. It consists of two modular prototypes, one for sample preparation and one for amplification and detection; however, a common interface is available to facilitate later integration into one single module. Nucleic acid extracts (n = 28) from cervical cytology specimens extracted on the sample preparation chip were tested using the PreTect HPV-Proofer and achieved an overall detection rate for HPV across all dilutions of 50%–85.7%. A subset of 6 clinical samples extracted on the sample preparation chip module was chosen for complete validation on the NASBA chip module. For 4 of the samples, a 100% amplification for HPV 16 or 33 was obtained at the 1 : 10 dilution for microfluidic channels that filled correctly. The modules of a “sample-in, answer-out” diagnostic platform have been demonstrated from clinical sample input through sample preparation, amplification and final detection.

Microfluidics in silicon/polymer technology as a cost-efficient alternative to silicon/glass

We investigate TMMF photopolymer as a cost-efficient alternative to glass for the leak-tight sealing of high-density silicon microchannels. TMMF enables low temperature sealing and access to structures underneath via lamination and standard UV-lithography instead of costly glass machining and anodic bonding. TMMF is highly transparent and has a low autofluorescence for wavelengths larger than 400 nm. As the photopolymer is too thin for implementing bulky world-to-chip interfaces, we propose adhesive bonding of cyclic olefin copolymer (COC) modules. All materials were tested according ISO 10993-5 and showed no cytotoxic effects on the proliferation of L929 cells. To quantify the cost efficiency of the proposed techniques, we used an established silicon/Pyrex nanoliter dispenser as a reference and replaced structured Pyrex wafers by TMMF laminates and COC modules. Thus, consumable costs, manpower and machine time related to sealing of the microchannels and implementing the world-to-chip interface could be significantly reduced. Leak tightness was proved by applying a pressure of 0.2 MPa for 5 h without delamination or crosstalk between neighboring microchannels located only 100 µm apart. In contrast to anodic bonding, the proposed techniques are tolerant to surface inhomogeneities. They enable manufacturing of silicon/polymer microfluidics at lower costs and without compromising the performance compared to corresponding silicon/glass devices.