Search

The realization of electrochemical nucleic acid amplification tests (NAATs) at the point of care (POC) is highly desirable, but it remains a challenge given their high cost and lack of true portability/miniaturization. Here we show that mass-produced, industrial standardized, printed circuit boards (PCBs) can be repurposed to act as near-zero cost electrodes for self-assembled monolayer-based DNA biosensing, and further integration with a custom-designed and low-cost portable potentiostat. To show the analytical capability of this system, we developed a NAAT using isothermal recombinase polymerase amplification, bypassing the need of thermal cyclers, followed by an electrochemical readout relying on a sandwich hybridization assay. We used our sensor and device for analytical detection of the toxic microalgae

This study aimed to evaluate a clothing prototype that incorporates sensors for the evaluation of pressure, temperature, and humidity for the prevention of pressure injuries, namely regarding physical and comfort requirements. A mixed-method approach was used with concurrent quantitative and qualitative data triangulation. A structured questionnaire was applied before a focus group of experts to evaluate the sensor prototypes. Data were analyzed using descriptive and inferential statistics and the discourse of the collective subject, followed by method integration and meta-inferences. Nine nurses, experts in this topic, aged 32.66 ± 6.28 years and with a time of profession of 10.88 ± 6.19 years, participated in the study. Prototype A presented low evaluation in stiffness (1.56 ± 1.01) and roughness (2.11 ± 1.17). Prototype B showed smaller values in dimension (2.77 ± 0.83) and stiffness (3.00 ± 1.22). Embroidery was assessed as inadequate in terms of stiffness (1.88 ± 1.05) and roughness (2.44 ± 1.01). The results from the questionnaires and focus groups’ show low adequacy as to stiffness, roughness, and comfort. The participants highlighted the need for improvements regarding stiffness and comfort, suggesting new proposals for the development of sensors for clothing. The main conclusions are that Prototype A presented the lowest average scores relative to rigidity (1.56 ± 1.01), considered inadequate. This dimension of Prototype B was evaluated as slightly adequate (2.77 ± 0.83). The rigidity (1.88 ± 1.05) of Prototype A + B + embroidery was evaluated as inadequate. The prototype revealed clothing sensors with low adequacy regarding the physical requirements, such as stiffness or roughness. Improvements are needed regarding the stiffness and roughness for the safety and comfort characteristics of the device evaluated.

In recent years, we have seen major advances in the field of liquid biopsy and its implementation in the clinic, mainly driven by breakthrough developments in the area of molecular biology. New developments have seen an integration of microfluidics and also biosensors in liquid biopsy systems, bringing advantages in terms of cost, sensitivity and automation. Without a doubt, the next decade will bring the clinical validation and approval of these combined solutions, which is expected to be crucial for the wide implementation of liquid biopsy systems in clinical routine.

Introduction: Metastasis involves a set of complex events that finally result in malignant cells from primary tumors invading distant tissues by the pass of circulant tumor cells (CTC) through the blood circulation. To authentically comprehend the complex chain of events and even enable new therapeutic approaches in mCRC patients, ex-vivo functional models are an unmet research need. Experimental Plan: PROMISE project is combining bioengineering solutions such as bioprinting biomaterials and microfluidics to mimic the tumor microenvironment (TME) of metastatic sites in mCRC. In this project, we are developing a bioprinted hydrogel-based microfluidic device that recapitulates this TME. Then, CTCs are isolated from mCRC patients, cultured, and characterized by microdroplet technology. Those CTCs are then infused on the biodegradable polymer that mimics vascular channels and surrounding stromal cells, through microfluidic connection, in order to mimic CTC-endothelial interactions, study the procedures of extravasation (the invasiveness potential of extravasated patient-derived CTCs into TME) and intravasation (the process of CTCs-derived and biopsy-derived tumor spheroids from the TME to the vascular channel). The photopolymerizable hydrogel mimics vascular structures, supporting the growth of HUVEC cells, and is printed in a cost-effective manner (30 minutes) using a semi-direct printing method with visible light. The CROSS chip is used for fast and efficient isolation of CTCs from unprocessed blood of mCRC patients. In parallel, multi-omics of mCRC are done in circulant tumor DNA (ctDNA) and tumor biopsy of mCRC, therefore correlating the phenotypic and genetic profile of the patient cohorts studied. A first pilot test has been carried out successfully obtaining blood from mCRc refractory patients, and excellent coordination between the multidisciplinary consortium has been achieved to draw blood from the patient, isolate CTCs and infuse them into the hydrogel. Extravasation and intravasation studies are ongoing. Conclusion: This multidisciplinary project, which brings together bioengineering, microfluidic, and oncology experts’ teams, is developing a successful ex-vivo, in vitro, and dynamic model that will permit a better study and understanding of the metastatic process in mCRC, from the process of intravasation to extravasation of CTCs, passing thought the better multi-omic characterization of CTCs and ctDNA. Citation Format: Nadia Saoudi González, Francesc Salvà Ballabrera, Ariadna García, Javier Gonzalo-Ruiz, Iosune Baraibar, Javier Ros, Marta Rodriguez, María García Díaz, Marta Falcó Fusté, Melika Parchehbaf Kashani, Carlos Honrado, Alar Ainla, Josep Tabernero, Lorena Diéguez, Elena Martínez Fraiz, Elena Élez. An innovative platform to mimic the tumoral vascular microenvironment (TME) of patients with metastatic colorectal cancer (mCRC) using bioprinted hydrogel microfluidics. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4577.

Product-in-Touch is an ongoing R&D project for developing a virtual reality application for prototyping in-car textures, providing both visual and haptic input to a user. The device will allow designers to virtually prototype textured surfaces, both in terms of visual and haptic perception, without the need for physical prototypes.

Simple Summary Over the last few years, there has been a scientific revolution with the appearance of organ-on-a-chip models that overcome the limitations of conventional 2D systems, while reproducing more faithfully the in vivo features of tissues and organs. The integration of sensors in these systems allows the monitoring of a variety of parameters that could be relevant for the study of diseases. Electrochemical biosensors are ideal candidates for this integration, since they can be miniaturised and are very reliable in real-time continuous measurements of a large panoply of relevant biomarkers. In the context of cancer, these electrochemical cancer-on-a-chip models have the potential to become essential tools for the study of cancer development and drug efficacy. Abstract Currently, conventional pre-clinical in vitro studies are primarily based on two-dimensional (2D) cell culture models, which are usually limited in mimicking the real three-dimensional (3D) physiological conditions, cell heterogeneity, cell to cell interaction, and extracellular matrix (ECM) present in living tissues. Traditionally, animal models are used to mimic the 3D environment of tissues and organs, but they suffer from high costs, are time consuming, bring up ethical concerns, and still present many differences when compared to the human body. The applications of microfluidic-based 3D cell culture models are advantageous and useful as they include 3D multicellular model systems (MCMS). These models have demonstrated potential to simulate the in vivo 3D microenvironment with relatively low cost and high throughput. The incorporation of monitoring capabilities in the MCMS has also been explored to evaluate in real time biophysical and chemical parameters of the system, for example temperature, oxygen, pH, and metabolites. Electrochemical sensing is considered as one of the most sensitive and commercially adapted technologies for bio-sensing applications. Amalgamation of electrochemical biosensing with cell culture in microfluidic devices with improved sensitivity and performance are the future of 3D systems. Particularly in cancer, such models with integrated sensing capabilities can be crucial to assess the multiple parameters involved in tumour formation, proliferation, and invasion. In this review, we are focusing on existing 3D cell culture systems with integrated electrochemical sensing for potential applications in cancer models to advance diagnosis and treatment. We discuss their design, sensing principle, and application in the biomedical area to understand the potential relevance of miniaturized electrochemical hybrid systems for the next generation of diagnostic platforms for precision medicine.

Membrane enclosed intracellular compartments have been exclusively associated with the eukaryotes, represented by the highly compartmentalized last eukaryotic common ancestor. Recent evidence showing the presence of membranous compartments with specific functions in archaea and bacteria makes it conceivable that the last universal common ancestor and its hypothetical precursor, the protocell, may have exhibited compartmentalization. To the authors' knowledge, there are no experimental studies yet that have tested this hypothesis. They report on an autonomous subcompartmentalization mechanism for protocells which results in the transformation of initial subcompartments to daughter protocells. The process is solely determined by the fundamental materials properties and interfacial events, and does not require biological machinery or chemical energy supply. In the light of the authors' findings, it is proposed that similar events may have taken place under early Earth conditions, leading to the development of compartmentalized cells and potentially, primitive division.

Membrane enclosed intracellular compartments have been exclusively associated with the eukaryotes, represented by the highly compartmentalized last eukaryotic common ancestor. Recent evidence showing the presence of membranous compartments with specific functions in archaea and bacteria makes it conceivable that the last universal common ancestor and its hypothetical precursor, the protocell, could have exhibited compartmentalization. To our knowledge, there are no experimental studies yet that have tested this hypothesis. We report on an autonomous subcompartmentalization mechanism for protocells which results in the transformation of initial subcompartments to daughter protocells. The process is solely determined by the fundamental materials properties and interfacial events, and do not require biological machinery or chemical energy supply. In the light of our findings, we propose that similar events could have taken place under early Earth conditions, leading to the development of compartmentalized cells and potentially, primitive division.

Contains fulltext : 193965.pdf (Publisher’s version ) (Closed access)

Potentiometric sensing of ions with ion-selective electrodes (ISEs) is a powerful technique for selective and sensitive measurement of ions in complex matrices. The application of ISEs is generally limited to laboratory settings, because most commercially available ISEs and reference electrodes are large, delicate, and expensive, and are not suitable for point-of-use or point-of-care measurements. This work utilizes cotton thread as a substrate for fabrication of robust and miniaturized ISEs that are suitable for point-of-care or point-of-use applications. Thread-based ISEs selective for Cl-, K+, Na+, and Ca2+ were developed. The cation-selective ISEs were fabricated by coating the thread with a surfactant-free conductive ink (made of carbon black) and then coating the tip of the conductive thread with the ion-selective membrane. The Cl- ISE was fabricated by coating the thread with an Ag/AgCl ink. These sensors exhibited slopes (of electrical potential vs. log concentration of target ion), close to the theoretically-expected values, over four orders of magnitude in concentrations of ions. Because thread is mechanically strong, the thread-based electrodes can be used in multiple-use applications as well as single-use applications. Multiple thread-based sensors can be easily bundled together to fabricate a customized sensor for multiplexed ion-sensing. These electrodes require volumes of sample as low as 200 μL. The application of thread-based ISEs is demonstrated in the analysis of ions in soil, food, and dietary supplements (Cl- in soil/water slurry, K+ and Na+ in coconut water, and Ca2+ in a calcium supplement), and in detection of physiological electrolytes (K+ and Na+ in blood serum and urine, with sufficient accuracy for clinical diagnostics).

In this study, we have systematically investigated the formation of molecular phospholipid films on a variety of solid substrates fabricated from typical surface engineering materials and the fluidic properties of the lipid membranes formed on these substrates. The surface materials comprise of borosilicate glass, mica, SiO2, Al (native oxide), Al2O3, TiO2, ITO, SiC, Au, Teflon AF, SU-8, and graphene. We deposited the lipid films from small unilamellar vesicles (SUVs) by means of an open-space microfluidic device, observed the formation and development of the films by laser scanning confocal microscopy, and evaluated the mode and degree of coverage, fluidity, and integrity. In addition to previously established mechanisms of lipid membrane–surface interaction upon bulk addition of SUVs on solid supports, we observed nontrivial lipid adhesion phenomena, including reverse rolling of spreading bilayers, spontaneous nucleation and growth of multilamellar vesicles, and the formation of intact circular patches of double lipid bilayer membranes. Our findings allow for accurate prediction of membrane–surface interactions in microfabricated devices and experimental environments where model membranes are used as functional biomimetic coatings.

This paper describes the design and fabrication of a "pop-up" electrochemical paper-based analytical device (pop-up-EPAD) to measure beta-hydroxybutyrate (BHB)-a biomarker for diabetic ketoacidosis-using a commercial combination BHB/glucometer. Pop-up-EPADs are inspired by pop-up greeting cards and children's books. They are made from a single sheet of paper folded into a three-dimensional (3D) device that changes shape, and fluidic and electrical connectivity, by simply folding and unfolding the structure. The reconfigurable 3D structure makes it possible to change the fluidic path and to control timing; it also provides mechanical support for the folded and unfolded structures that enables good registration and repeatability on folding. A pop-up-EPAD designed to detect BHB shows performance comparable to commercially available plastic test strips over the clinically relevant range of BHB in blood when used with a commercial glucometer that integrates the ability to measure glucose and BHB (combination BHB/glucometer). With simple modifications of the electrode and the design of the fluidic path, the pop-up-EPAD also detects BHB in buffer using a simple glucometer-a device that is more available than the combination BHB/glucometer. Strategies that use a "3D pop-up"-that is, large-scale changes in 3D structure and fluidic paths-by folding/unfolding add functionality to EPADs (e.g., controlled timing, fluidic handling and path programming, control over complex sequences of steps, and alterations in electrical connectivity) and should enable the development of new classes of paper-based diagnostic devices.

Paper microfluidics and printed electronics have developed independently, and are incompatible in many aspects. Monolithic integration of microfluidics and electronics on paper is demonstrated. This integration makes it possible to print 2D and 3D fluidic, electrofluidic, and electrical components on paper, and to fabricate devices using them.

Defense Threat Reduction Agency; # HDTRA1-14-C-0037, DTRA, Defense Threat Reduction Agency, M. T. Fernández-Abedul thanks the Spanish Ministry of Economy and Competitiveness (project MICINN CTQ2011-25814) for funding her stay at Harvard University. A. Ainla thanks the Swedish Research Council (VR) for a postdoctoral fellowship. A. C. Glavan acknowledges funding by the Bill & Melinda Gates Foundation, award # 51308 and DTRA award # HDTRA1-14-C-0037. M. M. Hamedi acknowledges support from Marie Curie IOF FP7 for project nanoPAD (Grant Agreement Number 330017).

This paper describes the design and characterization of an open-source “universal wireless electrochemical detector” (UWED). This detector interfaces with a smartphone (or a tablet) using “Bluetooth Low Energy” protocol; the smartphone provides (i) a user interface for receiving the experimental parameters from the user and visualizing the result in real time, and (ii) a proxy for storing, processing, and transmitting the data and experimental protocols. This approach simplifies the design, and decreases both the size and the cost of the hardware; it also makes the UWED adaptable to different types of analyses by simple modification of the software. The UWED can perform the most common electroanalytical techniques of potentiometry, chronoamperometry, cyclic voltammetry, and square wave voltammetry, with results closely comparable to benchtop commercial potentiostats. Although the operating ranges of electrical current and voltage of the UWED (±1.5 V, ±180 μA) are more limited than most benchtop commercial potentiostats, its functional range is sufficient for most electrochemical analyses in aqueous solutions. Because the UWED is simple, small in size, assembled from inexpensive components, and completely wireless, it offers new opportunities for the development of affordable diagnostics, sensors, and wearable devices.

Almost all pneumatic and hydraulic actuators useful for mesoscale functions rely on hard valves for control. This article describes a soft, elastomeric valve that contains a bistable membrane, which acts as a mechanical “switch” to control air flow. A structural instability—often called “snap-through”—enables rapid transition between two stable states of the membrane. The snap-upward pressure, Δ P 1 (kilopascals), of the membrane differs from the snap-downward pressure, Δ P 2 (kilopascals). The values Δ P 1 and Δ P 2 can be designed by changing the geometry and the material of the membrane. The valve does not require power to remain in either “open” or “closed” states (although switching does require energy), can be designed to be bistable, and can remain in either state without further applied pressure. When integrated in a feedback pneumatic circuit, the valve functions as a pneumatic oscillator (between the pressures Δ P 1 and Δ P 2 ), generating periodic motion using air from a single source of constant pressure. The valve, as a component of pneumatic circuits, enables (i) a gripper to grasp a ball autonomously and (ii) autonomous earthworm-like locomotion using an air source of constant pressure. These valves are fabricated using straightforward molding and offer a way of integrating simple control and logic functions directly into soft actuators and robots.

Soft, pneumatic actuators that buckle when interior pressure is less than exterior provide a new mechanism of actuation. Upon application of negative pneumatic pressure, elastic beam elements in these actuators undergo reversible, cooperative collapse, and generate a rotational motion. These actuators are inexpensive to fabricate, lightweight, easy to control, and safe to operate. They can be used in devices that manipulate objects, locomote, or interact cooperatively with humans.

Background: Among the various fluidic control technologies, microfluidic devices are becoming powerful tools for pharmacological studies using brain slices, since these devices overcome traditional limitations of conventional submerged slice chambers, leading to better spatiotemporal control over delivery of drugs to specific regions in the slices. However, microfluidic devices are not yet fully optimized for such studies. New method: We have recently developed a multifunctional pipette (MFP), a free standing hydrodynamically confined microfluidic device, which provides improved spatiotemporal control over drug delivery to biological tissues. Results: We demonstrate herein the ability of the MFP to selectively perfuse one dendritic layer in the CA1 region of hippocampus with CNQX, an AMPA receptor antagonist, while not affecting the other layers in this region. Our experiments also illustrate the essential role of hydrodynamic confinement in sharpening the spatial selectivity in brain slice experiments. Concentration-response measurements revealed that the ability of the MFP to control local drug concentration is comparable with that of whole slice perfusion, while in comparison the required amounts of active compounds can be reduced by several orders of magnitude. Comparison with existing method: The multifunctional pipette is applied with an angle, which, compared to other hydrodynamically confined microfluidic devices, provides more accessible space for other probing and imaging techniques. Conclusions: Using the MFP it will be possible to study selected regions of brain slices, integrated with various imaging and probing techniques, without affecting the other parts of the slices.

This article describes a class of robots-"arthrobots"-inspired, in part, by the musculoskeletal system of arthropods (spiders and insects, inter alia). Arthrobots combine mechanical compliance, lightweight and simple construction, and inexpensive yet scalable design. An exoskeleton, constructed from thin organic polymeric tubes, provides lightweight structural support. Pneumatic joints modeled after the hydrostatic joints of spiders provide actuation and inherent mechanical compliance to external forces. An inflatable elastomeric tube (a "balloon") enables active extension of a limb; an opposing elastic tendon enables passive retraction. A variety of robots constructed from these structural elements demonstrate (i) crawling with one or two limbs, (ii) walking with four or six limbs (including an insect-like triangular gait), (iii) walking with eight limbs, or (iv) floating and rowing on the surface of water. Arthrobots are simple to fabricate and are able to operate safely in contact with humans.

This paper describes electrically-activated fluidic valves that operate based on electrowetting through textiles. The valves are fabricated from electrically conductive, insulated, hydrophobic textiles, but the concept can be extended to other porous materials. When the valve is closed, the liquid cannot pass through the hydrophobic textile. Upon application of a potential (in the range of 100–1000 V) between the textile and the liquid, the valve opens and the liquid penetrates the textile. These valves actuate in less than 1 s, require low energy (≈27 µJ per actuation), and work with a variety of aqueous solutions, including those with low surface tension and those containing bioanalytes. They are bistable in function, and are, in a sense, the electrofluidic analog of thyristors. They can be integrated into paper microfluidic devices to make circuits that are capable of controlling liquid, including autonomous fluidic timers and fluidic logic.

We have developed a superfusion method utilizing an open-volume microfluidic device for administration of pharmacologically active substances to selected areas in brain slices with high spatio-temporal resolution. The method consists of a hydrodynamically confined flow of the active chemical compound, which locally stimulates neurons in brain slices, applied in conjunction with electrophysiological recording techniques to analyze the response. The microfluidic device, which is a novel free-standing multifunctional pipette, allows diverse superfusion experiments, such as testing the effects of different concentrations of drugs or drug candidates on neurons in different cell layers with high positional accuracy, affecting only a small number of cells. We demonstrate herein the use of the method with electrophysiological recordings of pyramidal cells in hippocampal and prefrontal cortex brain slices from rats, determine the dependence of electric responses on the distance of the superfusion device from the recording site, document a multifold gain in solution exchange time as compared to whole slice perfusion, and show that the device is able to store and deliver up to four solutions in a series. Localized solution delivery by means of open-volume microfluidic technology also reduces reagent consumption and tissue culture expenses significantly, while allowing more data to be collected from a single tissue slice, thus reducing the number of laboratory animals to be sacrificed for a study.

A few-component network of biologically relevant, organic reactions displays bistability and oscillations, without an enzymatic catalyst. Dissipative chemical reaction networks are systems that operate away from equilibrium and exhibit features such as continuous regeneration of components and autonomous regulation. The biological cell is one such out-of-equilibrium chemical network. Until now it has not been possible to recreate this type of dynamic behavior using simple organic molecules relevant to prebiotic Earth. Now, George Whitesides and colleagues demonstrate a few-component system of interacting organic species that, combined into a reaction network, displays autocatalytic and oscillatory features. All of the organic components are relatively simple and do not require enzymatic catalysis to react. Networks of organic chemical reactions are important in life and probably played a central part in its origin1,2,3. Network dynamics regulate cell division4,5,6, circadian rhythms7, nerve impulses8 and chemotaxis9, and guide the development of organisms10. Although out-of-equilibrium networks of chemical reactions have the potential to display emergent network dynamics11 such as spontaneous pattern formation, bistability and periodic oscillations12,13,14, the principles that enable networks of organic reactions to develop complex behaviours are incompletely understood. Here we describe a network of biologically relevant organic reactions (amide formation, thiolate–thioester exchange, thiolate–disulfide interchange and conjugate addition) that displays bistability and oscillations in the concentrations of organic thiols and amides. Oscillations arise from the interaction between three subcomponents of the network: an autocatalytic cycle that generates thiols and amides from thioesters and dialkyl disulfides; a trigger that controls autocatalytic growth; and inhibitory processes that remove activating thiol species that are produced during the autocatalytic cycle. In contrast to previous studies that have demonstrated oscillations and bistability using highly evolved biomolecules (enzymes15 and DNA16,17) or inorganic molecules of questionable biochemical relevance (for example, those used in Belousov–Zhabotinskii-type reactions)18,19, the organic molecules we use are relevant to metabolism and similar to those that might have existed on the early Earth. By using small organic molecules to build a network of organic reactions with autocatalytic, bistable and oscillatory behaviour, we identify principles that explain the ways in which dynamic networks relevant to life could have developed. Modifications of this network will clarify the influence of molecular structure on the dynamics of reaction networks, and may enable the design of biomimetic networks and of synthetic self-regulating and evolving chemical systems.

Hydrodynamically confined flow device technology is a young research area with high practical application potential in surface processing, assay development, and in various areas of single cell research. Several variants have been developed, and most recently, theoretical and conceptual studies, as well as fully developed automated systems, were presented. In this article we review concepts, fabrication strategies, and application areas of hydrodynamically confined flow (HCF) devices.

Current methods of monitoring breathing require cumbersome, inconvenient, and often expensive devices; this requirement sets practical limitations on the frequency and duration of measurements. This article describes a paper-based moisture sensor that uses the hygroscopic character of paper (i.e. the ability of paper to adsorb water reversibly from the surrounding environment) to measure patterns and rate of respiration by converting the changes in humidity caused by cycles of inhalation and exhalation to electrical signals. The changing level of humidity that occurs in a cycle causes a corresponding change in the ionic conductivity of the sensor, which can be measured electrically. By combining the paper sensor with conventional electronics, data concerning respiration can be transmitted to a nearby smartphone or tablet computer for post-processing, and subsequently to a cloud server. This means of sensing provides a new, practical method of recording and analyzing patterns of breathing.

Nafion®–PBI composites were prepared by diffusing synthesized PBI from solution phase into Nafion® membranes, using different concentrations and drying temperatures. In some cases, Nafion® was treated with diethyl amine to screen the –SO3H groups and thereby avoid the strong acid–base interactions between the polymers during diffusion. The presence of PBI in the membranes was characterized with FT–IR spectroscopy. The performance of the membranes was studied by in-plane conductivity and methanol permeability. The performance ratio (the ratio between conductivity and methanol permeability compared to Nafion®) increased by up to 50% for the composite membranes compared to Nafion®.

Here, we report on a novel approach for the study of single-cell intracellular enzyme activity at various temperatures, utilizing a localized laser heating probe in combination with a freely positionable microfluidic perfusion device. Through directed exposure of individual cells to the pore-forming agent α-hemolysin, we have controlled the membrane permeability, enabling targeted delivery of the substrate. Mildly permeabilized cells were exposed to fluorogenic substrates to monitor the activity of intracellular enzymes, while adjusting the local temperature surrounding the target cells, using an infrared laser heating system. We generated quantitative estimates for the intracellular alkaline phosphatase activity at five different temperatures in different cell lines, constructing temperature-response curves of enzymatic activity at the single-cell level. Enzymatic activity was determined rapidly after cell permeation, generating five-point temperature-response curves within just 200 s.

We present a process for fabrication of free-standing SU-8 probes, with a dry, mechanical release of the final micro-devices. The process utilizes the thermal release tape, a commonly used cleanroom material, for facile heat-release from the sacrificial layer. For characterization of the SU-8 microfluidic probes, two liquid interfaces were designed: a disposable interface with integrated wells and an interface with external liquid reservoirs. The versatility of the fabrication and the release procedures was illustrated by further developing the process to functionalize the SU-8 probes for impedance sensing, by integrating metal thin-film electrodes. An additional interface scheme which contains electronic components for impedance measurements was developed. We investigated the possibilities of introducing perforations in the SU-8 device by photolithography, for solution sampling predominantly by diffusion. The SU-8 processes described here allow for a convenient batch production of versatile free-standing microfluidic devices with well-defined tip-geometry.

CORRIGENDUM: Lab on a Biomembrane: Rapid prototyping and manipulation of 2D fluidic lipid bilayer circuits

Lipid bilayer membranes are among the most ubiquitous structures in the living world, with intricate structural features and a multitude of biological functions. It is attractive to recreate these structures in the laboratory, as this allows mimicking and studying the properties of biomembranes and their constituents and to specifically exploit the intrinsic two-dimensional fluidity. Even though diverse strategies for membrane fabrication have been reported, the development of related applications and technologies has been hindered by the unavailability of both versatile and simple methods. Here we report a rapid prototyping technology for two-dimensional fluidic devices, based on in-situ generated circuits of phospholipid films. In this “lab on a molecularly thin membrane”, various chemical and physical operations, such as writing, erasing, functionalization and molecular transport, can be applied to user-defined regions of a membrane circuit. This concept is an enabling technology for research on molecular membranes and their technological use.

We report the application of a microfluidic device for semi-contact temperature measurement in picoliter volumes of aqueous media. Our device, a freely positionable multifunctional pipette, operates by a hydrodynamic confinement principle, i.e., by creating a virtual flow cell of micrometer dimensions within a greater aqueous volume. We utilized two fluorescent rhodamines, which exhibit different fluorescent responses with temperature, and made ratiometric intensity measurements. The temperature dependence of the intensity ratio was calibrated and used in a model study of the thermal activation of TRPV1 ion channels expressed in Chinese hamster ovary cells. Our approach represents a practical and robust solution to the specific problem of measuring temperature in biological experiments in vitro, involving highly localized heat generation, for example with an IR-B laser.

We present here a novel platform combination, using a multifunctional pipette to individually electroporate single-cells and to locally deliver an analyte, while in their culture environment. We demonstrate a method to fabricate low-resistance metallic electrodes into a PDMS pipette, followed by characterization of its effectiveness, benefits and limits in comparison with an external carbon microelectrode.

Microfluidic technology is a fast growing branch of microdevice development, which has its origin in the dot matrix printing principle demonstrated by R. Elmqvist of the German Siemens AG in the 1950s. In this “ink jet” concept, droplets are formed from a stream of ink ejected from a microscale opening. The drop formation is characterized by a stream breakup mechanism discovered nearly a century earlier by Lord Rayleigh1. It took another twenty years from the initial proof of principle until the first commercial inkjet printer was produced and marketed by IBM in the mid 1970s. It has to be noted that the inkjet printer technology remains the most successful commercial microfluidic application to date.

Microfluidics has emerged as a powerful laboratory toolbox for biologists, allowing manipulation and analysis of processes at a cellular and sub-cellular level, through utilization of microfabricated features at size-scales relevant to that of a single cell. In the majority of microfluidic devices, sample processing and analysis occur within closed microchannels, imposing restrictions on sample preparation and use. We present an optimized non-contact open-volume microfluidic tool to overcome these and other restrictions, through the use of a hydrodynamically confined microflow pipette, serving as a multifunctional solution handling and dispensing tool. The geometries of the tool have been optimised for use in optical microscopy, with integrated solution reservoirs to reduce reagent use, contamination risks and cleaning requirements. Device performance was characterised using both epifluorescence and total internal reflection fluorescence (TIRF) microscopy, resulting in ∼200 ms and ∼130 ms exchange times at ∼100 nm and ∼30 μm distances to the surface respectively.

We report on a free-standing microfluidic pipette made in poly(dimethylsiloxane) having a circulating liquid tip that generates a self-con-fining volume in front of the outlet channels. The method is flexible and scalable as the geometry and the size of the recirculation zone is defined by pressure, channel number, and geometry. The pipette is capable of carrying out a variety of complex fluid processing operations, such as mixing, multiplexing, or gradient generation at selected cells in cell and tissue cultures. Using an uptake assay, we show that it is possible to generate dose response curves in situ from adherent Chinese hamster ovary cells expressing proton-activated human transient receptor potential vanilloid (hTRPV1) receptors. Using confined superfusion and cell stimulation, we could activate hTRPV1 receptors in single cells, measure the response by a patch-clamp pipette, and induce membrane bleb formation by exposing selected groups of cells to formaldehyde/dithiothreitol-containing solutions, respectively. In short, the microfluidic pipette allows for complex, contamination-free multiple-compound delivery for pharmacological screening of intact adherent cells.

We demonstrate that pulse width flow modulation (PWFM) can be used to design fast, accurate, and precise multistage dilution modules for microfluidic devices. The PWFM stage unit presented here yields 10-fold dilution, but several PWFM stages can be connected in series to yield higher-order dilutions. We have combined two stages in a device thus capable of diluting up to 100-fold, and we have experimentally determined a set of rules that can be conveniently utilized to design multistage diluters. Microfabrication with resist-based molds yielded geometrical channel height variances of 7% (22.9(16) microm) with corresponding hydraulic resistance variances of approximately 20%. Pulsing frequencies, channel lengths, and flow pressures can be chosen within a wide range to establish the desired diluter properties. Finally, we illustrate the benefits of on-chip dilution in an example application where we investigate the effect of the Ca(2+) concentration on a phospholipid bilayer spreading from a membrane reservoir onto a SiO(2) surface. This is one of many possible applications where flexible concentration control is desirable.

Molecular Dynamics (MD) techniques have been used to study the structure and dynamics of hydrated Li- and Na-Nafion membranes. The membranes were generated using a Monte Carlo-approach for Nafion 117 oligomers of Mw = 1100 and with water contents of 7.5 and 20 % by weight, equivalent to 5 and 15 water molecules per sulfonate group, respectively. After equilibration, local structural properties and dynamical features such as coordination, cluster stability, solvation and ion conductivity were studied. In a comparison between the two cationic systems, it is shown that the Na-Nafion system is more sensitive than Li-Nafion to the level of hydration, and also show higher ion conductivity. The ionic conductivity is shown to increase with higher level of hydration.

Lipid bilayer membranes are among the most ubiquitous structures in the living world, with intricate structural features and a multitude of biological functions. It is attractive to recreate these structures in the laboratory, as this allows mimicking and studying the properties of biomembranes and their constituents, and to specifically exploit the intrinsic two-dimensional fluidity. Even though diverse strategies for membrane fabrication have been reported, the development of related applications and technologies has been hindered by the unavailability of both versatile and simple methods. Here we report a rapid prototyping technology for two-dimensional fluidic devices, based on in-situ generated circuits of phospholipid films. In this “lab on a molecularly thin membrane”, various chemical and physical operations, such as writing, erasing, functionalization, and molecular transport, can be applied to user-defined regions of a membrane circuit. This concept is an enabling technology for research on molecular membranes and their technological use.View Large Image | View Hi-Res Image | Download PowerPoint Slide