Microfluidics, Lab- & Organ-on-a-Chip – Research

Fabrication technology

We use a great variety of materials for the fabrication of microfluidic devices developed in house. We have adopted replica molding for the rapid prototyping of PDMS-based microfluidic components and devices. In addition, we have demonstrated a mass production amenable technology for fabrication and surface modification of plastic disposable microfluidic devices, namely direct lithography on the plastic substrate followed by deep polymer etching of materials such as Poly(dimethyl siloxane) PDMS, Poly(methyl methacrylate) PMMA, poly(ether ether ketone) PEEK, Polyimide PI, etc. To complement microfluidics fabrication, bonding processes have been developed based on plasma activation and functionalization of the substrates. We have developed a novel process for irreversible bonding of polymeric substrates including PDMS, PMMA, PS and SU8. More recently, a technology has been pioneered by our group for the fabrication of microfluidic devices on flexible and rigid printed circuit boards (PCB) allowing the mass fabrication of industrially produced microfluidic devices.

  • Wetting and biofunctionalization control of microfluidic surfaces

In microfluidics, the control of wall surface properties is very crucial to the performance of the fabricated devices. For this reason, our group has paid special attention on the control of wetting properties of all materials implemented in microfluidics fabrication, as well as on the adsorption of biomolecules on such surfaces. Both plasma or wet chemistries have been used for the chemical modification of polymeric microfluidic surfaces, in order to enhance immobilization or prevent adsorption of biomolecules on surfaces, depending on the application. More specifically, selective protein adsorption has been demonstrated on various materials (SiO2, glass), significantly enhanced protein adsorption has been observed on plasma nanotextured polymeric surfaces and has been exploited for the sensitive detection of biomarkers (e.g. CRP-protein) and efficient bacteria capture on microfluidic surfaces. In addition, surface properties have been tuned to minimize biomolecule adsorption on microchannel walls in order to prevent inhibition of bioreactions. Finally, wettability of microfluidic walls has been also tuned to allow capillary pumping or provide hydrophobic valving in microfluidics.

Microfluidic devices

Funding: EU-FP5-Growth, GSRT-PENED, GSRT-Synergasia

Microtechnology allows the realization of miniaturized planar devices of micrometer-sized channels for performing small volume chemistry and biology, thus revolutionizing the life-sciences, medicine and diagnostics. Individual microfluidic components and devices, either as stand-alone or as parts of integrated lab-on-a-chip systems, have been designed, fabricated, and tested in our laboratories. Examples include digital microfluidics for electrowetting on dielectric-based transport of biomolecule-containing droplets in Si devices, PDMS-based microfluidic add-ons for sensors, microdevices for DNA amplification, micromixing, bacteria capturing, and DNA purification, on polymeric and printed circuit board (PCB) substrates. The group pioneered the implementation of flexible and rigid printed circuit board (PCB) approach that emerges as a very promising microfluidics manufacturing technology.

  • Electrowetting on dielectrics (EWOD)

Electrowetting on dielectrics (EWOD) has gained considerable attention for its capacity of transporting smallest volumes of liquids especially in biochips and BioMEMS approaches. Optimized fluorocarbon films have been developed by our group as the hydrophobic top layer of droplet-based microfluidic devices, where droplet actuation and transport has been achieved via electrowetting on dielectric (EWOD). We have demonstrated droplet transport on an open microfluidic device with a series of sequentially activated microelectrodes.

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  • PDMS-based microfluidic components

Replica molding and rapid prototyping of PDMS-based components and devices has been adopted by our group for the fabrication of microfluidic modules and devices integrated with sensors, in collaboration with other groups. In particular, a gas chromatography microcolumn combined with a gas sensor has been shown to enhance the separation efficiency of the sensor. In addition, a multichannel microfluidic module has been integrated on a surface acoustic wave (SAW) sensor to allow for parallel biological multisample analysis (in collaboration with E. Gizeli, K. Mitsakakis, Univ. Crete & FORTH).


  • Microfluidics for chromatographic separations

Chromatographic microcolumns on Si or PMMA substrates have been developed by our group to separate phosphopeptides, in collaboration with Biomedical Research Foundation, Academy of Athens. The microcolumns consisted of 32 parallel microchannels with common input and output and were fabricated by direct lithography and plasma etching on Silicon and polymeric substrates, and sealed with a lamination film. TiO2-ZrO2 or TiO2 films, used as stationary phase in the affinity column, were deposited with rf magnetron sputtering, or with liquid phase deposition.


  • DNA amplification microdevices

Most molecular diagnostics rely on nucleic acid testing, with DNA amplification comprising an indispensable process with expanding applications in health, food safety and environmental monitoring. The most common DNA amplification method is the Polymerase Chain Reaction (PCR), which enables the detection of trace amounts of DNA within a biological sample. We have developed two generations of continuous flow microfluidic device for DNA amplification (μPCR), integrated with microresistors on commercially available substrates and processes compatible with the PCB industry. In the first generation of such devices, microchannels were formed on flexible polyimide (PI) substrates through the implementation of photopatternable layers by means of lithography. The small thermal mass of the chip, in combination with the low thermal diffusivity of PI substrate on which the heating elements reside, has yield an unprecedented low power consumption PCR chip with fast amplification rates (within few minutes compared to nearly 1 hour needed in conventional thermocyclers). In the second generation of continuous flow μPCR, commercially available, 4-layer printed circuit board (PCB) substrates have been employed, with in-house designed yet industrially manufactured embedded Cu micro-resistive heaters lying at very close distance from the microfluidic network. We demonstrated successful DNA amplification at total reaction times down to 2 min, with a power consumption of 2.7 W, rendering our μPCR device one of the fastest and lowest power-consuming devices, suitable for implementation in low-resource settings. Furthermore, we have demonstrated static well DNA amplification devices, implementing various amplification methods requiring either one single temperature (isothermal, i.e. HDA) or up to three different temperatures (thermocycled μPCR). In particular, based on our microfluidics-on-PCB concept, we have developed microfluidic devices that enable isothermal nucleic acid (DNA) amplification methods, such as Rolling Circle Amplification (RCA @ 65◦C), Helicase Dependent Amplification (HDA @ 65◦C), Loop Mediated Amplification (LAMP @ 65◦C) and Recombinase Polymerase Amplification (RPA @ 40◦C) Chaon PCB substrates with embedded microresistors. In all such microdevices, the observed DNA amplification efficiencies were comparable with those on conventional thermocyclers.


  • Micromixers

Mixing is imperative in microfluidics for the efficient performance of reactions in lab-on-chip systems. Passive micromixers (no external energy required) of various geometries have been designed, fabricated and demonstrated by our group. Examples include a micromixer with a zig-zag microchannel to perform simultaneously mixing of a restriction enzyme with DNA and digestion of DNA. When heated at 37 C, the micromixer achieved both complete mixing of the reagents and DNA digestion with restriction enzymes within 2.5 min. A novel, highly enhanced split and merge (SAM) passive micromixer has been also developed, with labyrinth-like microchannels to efficiently mix biomolecular solutions. Enzymatic digestion within 30 s was demonstrated.


  • Microdevices for bacteria capturing

Sample preparation is indispensable functionality of most lab-on-chip devices for rapid pathogen detection at the point of need. We designed, fabricated, and demonstrated a novel sample preparation module comprising bacteria cell capture and thermal lysis on chip with potential applications in food sample pathogen analysis. Plasma nanotexturing of the polymeric substrate allowed enhancement of the surface area of the chip and the antibody binding capacity. The module exhibited 100% efficiency in Salmonella enterica serovar Typhimurium bacteria capture for cell suspensions below 105 cells/ml, and efficiency larger than 50% for 107 cells/ml. Moreover, thermal lysis achieved on chip from as low as 10 captured cells was demonstrated and was shown to compare well with off-chip lysis. Excellent selectivity (over 1:300) was obtained in a sample containing, in addition to S. Typhimurium, E-coli bacteria.


  • DNA purification microdevices

For molecular diagnostics applied in food industry, medicine, forensics and water industry, DNA purification is a procedure of high significance. A polymeric microfluidic chip capable of purifying DNA through solid phase extraction was designed, fabricated and evaluated in our group. Here again, plasma nanotextured surfaces were implemented to create high surface area as well as high density of carboxyl groups (eCOOH) able to bind DNA on the microchannel surface. The chip design incorporates a mixer so that sample and buffer can be efficiently mixed on chip under continuous flow. The chip was able to isolate DNA with high recovery efficiency (96± 11%) in an extremely large dynamic range of pre-purified Salmonella DNA as well as from Salmonella cell lysates that correspond to a range of 5 to 1.9 108 cells.

Lab-on-a-chip (LoC)

Funding: GSRT (Corallia), FP7, H2020, SNF

Since the introduction of the micro total analysis system (μTAS) concept in 1990 and thanks to the extensive research efforts from the increasingly growing lab-on-a-chip (LoC) community since then, the global excitement for this revolutionary technology has been exponentially increasing, owing to its appealing advantages over conventional laboratory tests: rapid response time, miniaturized sample volumes, reduced cost, automation and portability. Leveraging our novel microfluidic devices, we are designing and developing more integrated lab-on-a-chip devices accommodating sample preparation (e.g., bacteria capture, thermal lysis, DNA purification, DNA amplification) and detection (mainly through collaborations; S. Chatzandroulis, E. Gizeli) addressing pathogen screening or quantification for application in food safety, healthcare, and environmental monitoring. Examples include a LoC for foodborne pathogen detection and a Lab-on-Printed Circuit Board (LoPCB) accompanied by a point-of-care platform for molecular diagnosis of urinary tract infections, both at unprecedented short time-to-result.

  • Lab-on-Printed Circuit Board and Point-of-Care for application in Healthcare

Building on the successful fabrication of microfluidics with photopatternable polyimide, we have developed a Lab-On-a-Chip platform on Printed Circuit Board substrates, seamlessly integrating a μPCR module and Si-based biosensors (placed in a hybridization chamber on the PCB), to allow DNA amplification and detection on the same chip. Currently, a lab-on- a-chip where bacterial DNA is amplified rapidly and efficiently, combined with detection of amplified DNA on sensitive graphene-oxide biosensors is being developed. This LoPCB is accompanied by a prototype, automated, portable diagnostic platform (Point-of-Care), for temperature and flow control and sensor read-out. This platform is currently tested for urinary tract infections, but it can be easily extended to other bacterial or viral infections.


  • Lab-on-a-Chip and Point-of-Care for Foodborne Pathogen Detection

Diseases initiated by food-borne pathogens represent an increasing threat for the public health and safety worldwide, therefore, rapid and reliable pathogen detection is of utmost importance. However, the duration of common food analysis practices is typically 2–5 days. Lab-on-chip devices can reduce labor and analysis time to a few hours, proving its beneficial use and rendering the response of food industry as well as public safety agents faster. The aim of our work was the fabrication and evaluation of an integrated Lab-on-Chip platform for sample preparation, accommodating bacteria capture, lysis, DNA purification (where necessary), and DNA amplification, addressing pathogen screening in milk samples. Three generations of LoC devices were developed for rapid detection of bacteria in dairy products, in collaboration with FORTH-Univ. Crete, Pasteur Institute, Jobst Technologies GmbH, SENSeOR SA and Univ. Pardubice; the most advanced device integrated in a single chamber the steps of bacteria capture, lysis and isothermal amplification (LAMP), while detection was achieved on an acoustic (SAW) device, providing a sensitive, highly integrated and user-friendly platform for sample-to-result analysis within 4.5 h.


  • Molecular testing with Colorimetric detection at the Point-of-need for Food and Environmental safety

Food- and water-borne infections are commonly caused by bacteria. Very recently Legionella has been identified by the World Health Organization as the highest health burden of all waterborne pathogens in the European Union. However, the low frequency sampling and the long duration of legionella cultures (up to 10 days) do not allow for prevention of legionella outbreaks. Thus, the development of more efficient water diagnostics for pathogens and faster analyses methods is recognized worldwide. Our group, leveraging the expertise acquired in the molecular testing of food-borne bacteria and in collaboration with Nanoplasmas PC, is realizing a lab-on-a-chip based on colorimetric detection (visual inspection) for ultra-rapid and easy L. pneumophila detection at the point of need.

Organ-on-a-chip

Funding: State Scholarships Foundation (IKY)

Conventional biomedical research models are confined to static cell culture models and animal testing, both of them representing suboptimal preclinical models. Organs-on-chips (OOCs) are novel 3D microfluidic cell culture devices lined with living cells that allow for faithful mimicry of the physiology and function of a vital human organ unit. Currently, we are developing a purely in vitro and scaffold-free bone marrow-on-a-chip, intended for both the generation and sustainment of the hematopoietic niche, to serve as a study platform for the chronic autoimmune disease of systemic lupus erythematosus (SLE, in collaboration with BRFAA, Prof. D. Boumbas).