A Diagnostic Chip for the Colorimetric Detection of Legionella pneumophila in Less than 3 h at the Point of Need.

Legionella pneumophila has been pinpointed by the World Health Organization as the highest health burden of all waterborne pathogens in the European Union and is responsible for many disease outbreaks around the globe. Today, standard analysis methods (based on bacteria culturing onto agar plates) need several days (~12) in specialized analytical laboratories to yield results, not allowing for timely actions to prevent outbreaks. Over the last decades, great efforts have been made to develop more efficient waterborne pathogen diagnostics and faster analysis methods, requiring further advancement of microfluidics and sensors for simple, rapid, accurate, inexpensive, real-time, and on-site methods. Herein, a lab-on-a-chip device integrating sample preparation by accommodating bacteria capture, lysis, and DNA isothermal amplification with fast (less than 3 h) and highly sensitive, colorimetric end-point detection of L. pneumophila in water samples is presented, for use at the point of need. The method is based on the selective capture of viable bacteria on on-chip-immobilized and -lyophilized antibodies, lysis, the loop-mediated amplification (LAMP) of DNA, and end-point detection by a color change, observable by the naked eye and semiquantified by computational image analysis. Competitive advantages are demonstrated, such as low reagent consumption, portability and disposability, color change, storage at RT, and compliance with current legislation.

Integrated Plastic Microfluidic Device for Heavy Metal Ion Detection.

The presence of heavy metal ions in soil, air and water constitutes an important global environmental threat, as these ions accumulate throughout the food chain, contributing to the rise of chronic diseases, including, amongst others, cancer and kidney failure. To date, many efforts have been made for their detection, but there is still a need for the development of sensitive, low-cost, and portable devices able to conduct on-site detection of heavy metal ions. In this work, we combine microfluidic technology and electrochemical sensing in a plastic chip for the selective detection of heavy metal ions utilizing DNAzymes immobilized in between platinum nanoparticles (PtNPs), demonstrating a reliable portable solution for water pollution monitoring. For the realization of the microfluidic-based heavy metal ion detection device, a fast and easy-to-implement fabrication method based on the photolithography of dry photosensitive layers is proposed. As a proof of concept, we demonstrate the detection of Pb2+ ions using the prototype microfluidic device.

A High-Performance Antibacterial Nanostructured ZnO Microfluidic Device for Controlled Bacterial Lysis and DNA Release.

In this work, the antibacterial properties of nanostructured zinc oxide (ZnO) surfaces are explored by incorporating them as walls in a simple-to-fabricate microchannel device. Bacterial cell lysis is demonstrated and quantified in such a device, which functions due to the action of its nanostructured ZnO surfaces in contact with the working fluid. To shed light on the mechanism responsible for lysis, E. coli bacteria were incubated in zinc and nanostructured ZnO substrates, as well as the here-investigated ZnO-based microfluidic devices. The unprecedented killing efficiency of E. coli in nanostructured ZnO microchannels, effective after a 15 min incubation, paves the way for the implementation of such microfluidic chips in the disinfection of bacteria-containing solutions. In addition, the DNA release was confirmed by off-chip PCR and UV absorption measurements. The results indicate that the present nanostructured ZnO-based microfluidic chip can, under light, achieve partial inactivation of the released bacterial DNA via reactive oxygen species-mediated oxidative damage. The present device concept can find broader applications in cases where the presence of DNA in a sample is not desirable. Furthermore, the present microchannel device enables, in the dark, efficient release of bacterial DNA for downstream genomic DNA analysis. The demonstrated potential of this antibacterial device for tailored dual functionality in light/dark conditions is the main novel contribution of the present work.

Closed-Loop Microreactor on PCB for Ultra-Fast DNA Amplification: Design and Thermal Validation.

Polymerase chain reaction (PCR) is the most common method used for nucleic acid (DNA) amplification. The development of PCR-performing microfluidic reactors (µPCRs) has been of major importance, due to their crucial role in pathogen detection applications in medical diagnostics. Closed loop (CL) is an advantageous type of µPCR, which uses a circular microchannel, thus allowing the DNA sample to pass consecutively through the different temperature zones, in order to accomplish a PCR cycle. CL µPCR offers the main advantages of the traditional continuous-flow µPCR, eliminating at the same time most of the disadvantages associated with the long serpentine microchannel. In this work, the performance of three different CL µPCRs designed for fabrication on a printed circuit board (PCB) was evaluated by a computational study in terms of the residence time in each thermal zone. A 3D heat transfer model was used to calculate the temperature distribution in the microreactor, and the residence times were extracted by this distribution. The results of the computational study suggest that for the best-performing microreactor design, a PCR of 30 cycles can be achieved in less than 3 min. Subsequently, a PCB chip was fabricated based on the design that performed best in the computational study. PCB constitutes a great substrate as it allows for integrated microheaters inside the chip, permitting at the same time low-cost, reliable, reproducible, and mass-amenable fabrication. The fabricated chip, which, at the time of this writing, is the first CL µPCR chip fabricated on a PCB, was tested by measuring the temperatures on its surface with a thermal camera. These results were then compared with the ones of the computational study, in order to evaluate the reliability of the latter. The comparison of the calculated temperatures with the measured values verifies the accuracy of the developed model of the microreactor. As a result of that, a total power consumption of 1.521 W was experimentally measured, only ~7.3% larger than the one calculated (1.417 W). Full validation of the realized CL µPCR chip will be demonstrated in future work.

Isothermal Recombinase Polymerase Amplification (RPA) of E. coli gDNA in Commercially Fabricated PCB-Based Microfluidic Platforms.

Printed circuit board (PCB) technology has been recently proposed as a convenient platform for seamlessly integrating electronics and microfluidics in the same substrate, thus facilitating the introduction of integrated and low-cost microfluidic devices to the market, thanks to the inherent upscaling potential of the PCB industry. Herein, a microfluidic chip, encompassing on PCB both a meandering microchannel and microheaters to accommodate recombinase polymerase amplification (RPA), is designed and commercially fabricated for the first time on PCB. The developed microchip is validated for RPA-based amplification of two E. coli target genes compared to a conventional thermocycler. The RPA performance of the PCB microchip was found to be well-comparable to that of a thermocycler yet with a remarkably lower power consumption (0.6 W). This microchip is intended for seamless integration with biosensors in the same PCB substrate for the development of a point-of-care (POC) molecular diagnostics platform.

Towards PCB-Based Miniaturized Thermocyclers for DNA Amplification.

In recent years, printed circuit board (PCB)-based microfluidics have been explored as a means to achieve standardization, seamless integration, and large-scale manufacturing of microfluidics, thus paving the way for widespread commercialization of developed prototypes. In this work, static micro polymerase chain reaction (microPCR) devices comprising resistive microheaters integrated on PCBs are introduced as miniaturized thermocyclers for efficient DNA amplification. Their performance is compared to that of conventional thermocyclers, in terms of amplification efficiency, power consumption and duration. Exhibiting similar efficiency to conventional thermocyclers, PCB-based miniaturized thermocycling achieves faster DNA amplification, with significantly smaller power consumption. Simulations guide the design of such devices and propose means for further improvement of their performance.

Three-dimensional (3D) hierarchical oxygen plasma micro/nanostructured polymeric substrates for selective enrichment of cancer cells from mixtures with normal ones.

The enrichment of cancer cell population when in mixtures with normal ones is of great importance for cancer diagnosis. In this work, poly(methyl methacrylate) films have been processed applying different oxygen plasma conditions to fabricate surfaces with structure height ranging from 22 to more than 2000 nm. The surfaces were then evaluated with respect to adhesion and proliferation of both normal and cancer human cells. In particular, normal skin and lung fibroblasts, and four different cancer cell lines, A431 (skin cancer), HT1080 (fibrosarcoma), A549 (lung cancer), and PC3 (prostate cancer), have been employed. It was found that adhesion and proliferation of cancer cells was favored when cultured onto the hierarchical micro/nanostructured surfaces as compared to untreated ones with the maximum values obtained for substrates treated at -100 V for 3 min. On the other hand, although the adhesion of normal fibroblasts was not influenced by the micro/nanostructured surfaces, their morphology and proliferation was significantly impaired, especially after 3-day culture on these surfaces. The reduced proliferation rate of adherent fibroblasts was linked to reduced focal points formation, as it was verified through vinculin staining, and not to apoptosis. The micro/nanostructured surfaces prepared with plasma treatment at -100 V for 3 min (hierarchical topography with mean height of ∼800 nm) were selected as substrates for normal and cancer cell co-culture experiments. It was found that 25-80 times enrichment of cancer over the normal cells was achieved on the nanostructured surfaces after 3-day culture, while it was 5-8 times lower on the untreated ones. It should be noticed that this is the first time such high enrichment ratios are achieved without implementing surfaces modified with binding molecules specific for cancer cells. Thus, the nanostructured surfaces hold a strong promise as culture substrates for separation and enrichment of cancer cells from mixtures with normal ones that should find application in cancer diagnostics.

Ultrafast, low-power, PCB manufacturable, continuous-flow microdevice for DNA amplification.

The design and fabrication of a continuous-flow µPCR device with very short amplification time and low power consumption are presented. Commercially available, 4-layer printed circuit board (PCB) substrates are employed, with in-house designed yet industrially manufactured embedded Cu micro-resistive heaters lying at very close distance from the microfluidic network, where DNA amplification takes place. The 1.9-m-long microchannel in combination with desirably high flow velocities (for fast amplification) challenged the robustness of the sealing that was overcome with the development of a novel bonding method rendering the microdevice robust even at extreme pressure drops (12 bars). The proposed fabrication methods are PCB compatible, allowing for mass and reliable production of the µPCR device in the established PCB industry. The µPCR chip was successfully validated during the amplification of two different DNA fragments (and with different target DNA copies) corresponding to the exon 20 of the BRCA1 gene, and to the plasmid pBR322, a commonly used cloning vector in E. coli. Successful DNA amplification was demonstrated at total reaction times down to 2 min, with a power consumption of 2.7 W, rendering the presented µPCR one of the fastest and lowest power-consuming devices, suitable for implementation in low-resource settings. Detailed numerical calculations of the DNA residence time distributions, within an acceptable temperature range for denaturation, annealing, and extension, performed for the first time in the literature, provide useful information regarding the actual on-chip PCR protocol and justify the maximum volumetric flow rate for successful DNA amplification. The calculations indicate that the shortest amplification time is achieved when the device is operated at its enzyme kinetic limit (i.e., extension rate). Graphical abstract.

3D Plasma Nanotextured® Polymeric Surfaces for Protein or Antibody Arrays, and Biomolecule and Cell Patterning.

Plasma micro-nanotexturing is a generic technology for topographical and chemical modification of surfaces and their implementation in microfluidics and microarrays. Nanotextured surfaces with desirable chemical functionality (and wetting behavior) have shown excellent biomolecule immobilization and cell adhesion. Specifically, nanotextured hydrophilic areas show (a) strong binding of biomolecules and (b) strong adhesion of cells, while nanotextured superhydrophobic areas show null adsorption of (a) proteins and (b) cells. Here we describe the protocols for (a) biomolecule adsorption control on nanotextured surfaces for microarray fabrication and (b) cell adhesion on such surfaces. 3D plasma nanotextured® substrates are commercialized through Nanoplasmas private company, a spin-off of the National Centre for Scientific Research Demokritos.

Micro-nano-bio acoustic system for the detection of foodborne pathogens in real samples.

The fast and efficient detection of foodborne pathogens is a societal priority, given the large number of food-poisoning outbreaks, and a scientific and technological challenge, given the need to detect as little as 1 viable cell in 25 gr of food. Here, we present the first approach that achieves the above goal, thanks to the use of a micro/nano-technology and the detection capability of acoustic wave sensors. Starting from 1 Salmonella cell in 25 ml of milk, we employ immuno-magnetic beads to capture cells after only 3 h of pre-enrichment and subsequently demonstrate efficient DNA amplification using the Loop Mediated Isothermal Amplification method (LAMP) and acoustic detection in an integrated platform, within an additional ½ h. The demonstrated 4 h sample-to-analysis time comes as a huge improvement to the current need of few days to obtain the same result. In addition, the work presents the first reported Lab-on-Chip platform that comprises an acoustic device as the sensing element, exhibiting impressive analytical features, namely, an acoustic limit of detection of 2 cells/µl or 3 aM of the DNA target and ability to detect in a label-free manner dsDNA amplicons in impure samples. The use of food samples together with the incorporation of the necessary pre-enrichment step and ability for multiple analysis with an internal control, make the proposed methodology highly relevant to real-world applications. Moreover, the work suggests that acoustic wave devices can be used as an attractive alternative to electrochemical sensors in integrated platforms for applications in food safety and the point-of-care diagnostics.

How to Achieve Reversible Electrowetting on Superhydrophobic Surfaces.

Collapse (Cassie to Wenzel) wetting transitions impede the electrostatically induced reversible modification of wettability on superhydrophobic surfaces, unless a strong external actuation (e.g., substrate heating) is applied. Here we show that collapse transitions can be prevented (the droplet remains suspended on the solid roughness protrusions) when the electrostatic force, responsible for the wetting modification, is smoothly distributed along the droplet surface. The above argument is initially established theoretically and then verified experimentally.

Three-dimensional (3D) plasma micro-nanotextured slides for high performance biomolecule microarrays: Comparison with epoxy-silane coated glass slides.

Glass slides coated with a poly(methyl methacrylate) layer and plasma micro-nanotextured to acquire 3D topography (referred as 3D micro-nanotextured slides) were evaluated as substrates for biomolecule microarrays. Their performance is compared with that of epoxy-coated glass slides. We found that the proposed three-dimensional (3D) slides offered significant improvements in terms of spot intensity, homogeneity, and reproducibility. In particular, they provided higher spot intensity, by a factor of at least 1.5, and significantly improved spot homogeneity when compared to the epoxy-silane coated ones (intra-spot and between spot coefficients of variation ranging between 5 and 15% for the 3D micro-nanotextured slides and between 25 and 85% for the epoxy-silane coated ones). The latter was to a great extent the result of a strong "coffee-ring" effect observed for the spots created on the epoxy-coated slides; a phenomenon that was severely reduced in the 3D micro-nanotextured slides. The 3D micro-nanotextured slides offered in addition higher signal to noise ratio values over a wide range of protein probe concentrations and shelf-life over one year without requirement for specific storage conditions. Finally, the protocols employed for protein probe immobilization were extremely simple.

Durable superhydrophobic and superamphiphobic polymeric surfaces and their applications: A review.

Wetting control is essential for many applications, such as self-cleaning, anti-icing, anti-fogging, antibacterial action as well as anti-reflection and friction control. While significant effort has been devoted to fabricate superhydrophobic/superamphiphobic surfaces (repellent to water and other low surface tension liquids), very few polymeric superhydrophobic/superamphiphobic surfaces can be considered as durable against various externally imposed stresses (e.g. application of heating, pressure, mechanical forces, chemical, etc.). Therefore, durability tests are extremely important for applications especially when such surfaces are made of "soft" materials. Here, we review the most recent and promising efforts reported towards the realization of durable, superhydrophobic/superamphiphobic, polymeric surfaces emphasizing the durability tests performed, and some important applications. We compare and put in context the scattered durability tests reported in the literature, and present conclusions, perspectives and challenges in the field.

Is There a Threshold in the Antibacterial Action of Superhydrophobic Surfaces?

The realization of antibacterial surfaces is an important scientific problem, which may be addressed by the use of superhydrophobic surfaces, reducing bacterial adhesion. However, there are several limitations and contradicting reports on the antibacterial efficacy of such surfaces. Moreover, achieving antibacterial action through minimization of adhesion does not ensure complete protection against bacteria. Here, we identify the important factors affecting antibacterial action on superhydrophobic surfaces, emphasizing the role of bacterial concentration, and observing an upper concentration threshold above which antibacterial action of any surface is compromised. Finally, we propose metal enriched, superhydrophobic surfaces, as the "ultimate" "hybrid" antibacterial surfaces for in vitro applications.

The lab-on-PCB approach: tackling the µTAS commercial upscaling bottleneck.

Commercialization of lab-on-a-chip devices is currently the "holy grail" within the µTAS research community. While a wide variety of highly sophisticated chips which could potentially revolutionize healthcare, biology, chemistry and all related disciplines are increasingly being demonstrated, very few chips are or can be adopted by the market and reach the end-users. The major inhibition factor lies in the lack of an established commercial manufacturing technology. The lab-on-printed circuit board (lab-on-PCB) approach, while suggested many years ago, only recently has re-emerged as a very strong candidate, owing to its inherent upscaling potential: the PCB industry is well established all around the world, with standardized fabrication facilities and processes, but commercially exploited currently only for electronics. Owing to these characteristics, complex µTASs integrating microfluidics, sensors, and electronics on the same PCB platform can easily be upscaled, provided more processes and prototypes adapted to the PCB industry are proposed. In this article, we will be reviewing for the first time the PCB-based prototypes presented in the literature to date, highlighting the upscaling potential of this technology. The authors believe that further evolution of this technology has the potential to become a much sought-after standardized industrial fabrication technology for low-cost µTASs, which could in turn trigger the projected exponential market growth of µTASs, in a fashion analogous to the revolution of Si microchips via the CMOS industry establishment.

Plasma micro-nanotextured polymeric micromixer for DNA purification with high efficiency and dynamic range.

We present a polymeric microfluidic chip capable of purifying DNA through solid phase extraction. It is designed to be used as a module of an integrated Lab-on-chip platform for pathogen detection, but it can also be used as a stand-alone device. The microfluidic channels are oxygen plasma micro-nanotextured, i.e. randomly roughened in the micro-nano scale, a process creating high surface area as well as high density of carboxyl groups (COOH). The COOH groups together with a buffer that contains polyethylene glycol (PEG), NaCl and ethanol are 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. DNA is subsequently eluted in water. The chip is able to isolate DNA with high recovery efficiency (96± 11%) in an extremely large dynamic range of prepurified Salmonella DNA as well as from Salmonella cell lysates that correspond to a range of 5 to 1.9 × 108 cells (0.263 fg to 2 × 500 ng). The chip was evaluated via absorbance measurements, polymerase chain reaction (PCR), and gel electrophoresis.

Micro-Nano-Bio Diagnostic System for Food Pathogen Detection Revolutionizes Food Safety Management & Protects Consumers Health.

The development of integrated, fast and affordable platforms for pathogen detection is an emerging area where a multidisciplinary approach is necessary for designing microsystems employing miniaturized devices; these new technologies promise a significant advancement of the current state of analytical testing leading to improved healthcare. In this work, the development of a lab-on-chip microsystem platform for the genetic analysis of Salmonella in milk samples is presented. The heart of the platform is an acoustic detection biochip, integrated with a microfluidic module. This detection platform is combined with a micro-processor, which, alongside with magnetic beads technology and a DNA micro-amplification module, are responsible for performing sample pre-treatment, bacteria lysis, nucleic acid purification and amplification. Automated, multiscale manipulation of fluids in complex microchannel networks is combined with novel sensing principles developed by some of the partners. This system is expected to have a significant impact in food-pathogen detection by providing for the first time an integrated detection test for Salmonella screening in a very short time. Finally, thanks to the low cost and compact technologies involved, the proposed set-up is expected to provide a competitive analytical platform for direct application in field settings.

Superamphiphobic polymeric surfaces sustaining ultrahigh impact pressures of aqueous high- and low-surface-tension mixtures, tested with laser-induced forward transfer of drops.

Superamphiphobic, (quasi-)ordered plasma-textured surfaces, coated with a perfluorinated monolayer, exhibit extreme resistance against drop-pinning for both water-like and low-surface-tension mixtures (36 mN m(-1)). The highest values reported here are 36 atm for a water-like mixture, 5 times higher than previously reported in the literature, and 7 atm for a low-surface-tension mixture, the highest ever reported value for lotus-leaf-inspired surfaces.

Plasma micro-nanotextured, scratch, water and hexadecane resistant, superhydrophobic, and superamphiphobic polymeric surfaces with perfluorinated monolayers.

Superhydrophobic and superamphiphobic toward superoleophobic polymeric surfaces of polymethyl methacrylate (PMMA), polyether ether ketone (PEEK), and polydimethyl siloxane (PDMS) are fabricated in a two-step process: (1) plasma texturing (i.e., ion-enhanced plasma etching with simultaneous roughening), with varying plasma chemistry depending on the polymer, and subsequently (2) grafting of self-assembled perfluorododecyltrichlorosilane monolayers (SAMs). Depending on the absence or not of an etch mask (i.e., colloidal microparticle self-assembly on it), random or ordered hierarchical micro-nanotexturing can be obtained. We demonstrate that stable organic monolayers can be grafted onto all these textured polymeric surfaces. After the monolayer deposition, the initially hydrophilic polymeric surfaces become superamphiphobic with static contact angles for water and oils>153°, for hexadecane>142°, and hysteresis<10° for all surfaces. This approach thus provides a simple and generic method to obtain superamphiphobicity on polymers toward superoleophobicity. Hydrolytic and hexadecane immersion tests prove that superamphiphobicity is stable for more than 14 days. We also perform nanoscratch and post nanoscratch tests to prove the scratch resistance of both the texture and the SAM and demonstrate lower coefficient of friction of the SAM compared to the uncoated surface. Scanning electron microscope observation after the nanoscratch tests confirms the scratch resistance of the surfaces.

Superior performance of multilayered fluoropolymer films in low voltage electrowetting.

The requirement for low operational voltage in electrowetting devices, met using thin dielectrics, is usually connected with serious material failure issues. Dielectric breakdown (visible as electrolysis) is frequently evident slightly beyond the onset of the contact angle saturation. Here, plasma-enhanced chemical vapor deposition (PECVD) is used to deposit thin fluorocarbon films prior to the spin-coating of Teflon® amorphous fluoropolymer. The resulting multilayered hydrophobic top coating improves the electrowetting performance of the stack, by showing high resistance to dielectric breakdown at high applied voltages and for continuous long term application of DC and AC voltage. Leakage current measurements during electrowetting experiments with the proposed composite coating showed that current remains fairly constant at consecutive electrowetting tests in contrast to plain Teflon® coating in which material degradation is evident by a progressive increase in the leakage current after multiple electrowetting tests. Since the proposed composite coating demonstrates increased resistance to material failure and to dielectric breakdown even at thin configurations, its integration in electrowetting devices may impact their reliability, robustness, and lifetime.