Generation Of Chemical Gradients Research Articles

Nanovortex-Driven All-Dielectric Optical Diffusion Boosting and Sorting Concept for Lab-on-a-Chip Platforms.

The ever‐growing field of microfluidics requires precise and flexible control over fluid flows at reduced scales. Current constraints demand a variety of controllable components to carry out several operations inside microchambers and microreactors. In this context, brand‐new nanophotonic approaches can significantly enhance existing capabilities providing unique functionalities via finely tuned light−matter interactions. A concept is proposed, featuring dual on‐chip functionality: boosted optically driven diffusion and nanoparticle sorting. High‐index dielectric nanoantennae is specially designed to ensure strongly enhanced spin−orbit angular momentum transfer from a laser beam to the scattered field. Hence, subwavelength optical nanovortices emerge driving spiral motion of plasmonic nanoparticles via the interplay between curl−spin optical forces and radiation pressure. The nanovortex size is an order of magnitude smaller than that provided by conventional beam‐based approaches. The nanoparticles mediate nanoconfined fluid motion enabling moving‐part‐free nanomixing inside a microchamber. Moreover, exploiting the nontrivial size dependence of the curled optical forces makes it possible to achieve precise nanoscale sorting of gold nanoparticles, demanded for on‐chip separation and filtering. Altogether, a versatile platform is introduced for further miniaturization of moving‐part‐free, optically driven microfluidic chips for fast chemical analysis, emulsion preparation, or chemical gradient generation with light‐controlled navigation of nanoparticles, viruses or biomolecules.

Fatty Acid Fueled Transmembrane Chloride Transport.

Generation of chemical gradients across biological membranes of cellular compartments is a hallmark of all living systems. Herewe report a proof-of-concept prototype transmembrane pumping systemin liposomes. The pump uses fatty acid to fuel chloride transport, thus generating a transmembrane chloride gradient. Addition of fatty acid to phospholipid vesicles generates a transmembrane pH gradient (pHin < pHout), and this electrochemical H+ potential is harnessed by an anionophore to drive chloride efflux via H+/Cl- cotransport. Further addition of fatty acid efficiently fuels the system to continuously drive chloride transport against the concentration gradient, up to [Cl-]in 65 mM | [Cl-]out 100 mM, and is 1400 times more efficient than using an external fuel.Based on our findings from dissecting the H+/Cl-flux process with the use of different liposomal fluorescence assays, and supported by additional liposome-based13C NMR and DLS studies; we proposed that the presence of an anionophore can induce asymmetric distribution of fatty acid, and contribute to another Cl- flux mechanism in this system.

Lab-scale prototyping of polymer based microfluidic devices using gallium as phase-changing sacrificial material

We present a fabrication method for polymer based microfluidic devices using gallium as a sacrificial material during the sealing process based on solvent bonding, allowing for lab-scale prototyping of microfluidic chips. The microfluidic channels are structured by laser engraving of poly(methyl methacrylate) (PMMA) sheets. The devices are temporarily closed by clamping the engraved parts on a flat surface with a thin polymer film in between. Liquid gallium is then injected into the temporarily sealed channels and is subsequently cooled to form a solid sacrificial layer preventing clogging and distortion of the microchannels during the subsequent bonding process. After finally sealing the device, it is warmed up to liquefy the gallium, which is then flushed from the channels. The feasibility of this approach is demonstrated by fabricating a microfluidic flow focusing device and a chemical gradient generator. The proposed method using gallium features distinct advantages compared to previously reported similar approaches that successfully make use of, e.g., wax, hydrogels or water as sacrificial materials.

A compact low-cost low-maintenance open architecture mask aligner for fabrication of multilayer microfluidics devices.

A custom-built mask aligner (CBMA), which fundamentally covers all the key features of a commercial mask aligner, while being low cost and light weight and having low power consumption and high accuracy, is constructed. The CBMA is composed of a custom high fidelity light emitting diode light source, a vacuum chuck, a mask holder, high-precision translation and rotation stages, and high resolution digital microscopes. The total cost of the system is under $7500, which is over ten times cheaper than a comparable commercial system. It produces a collimated ultraviolet illumination of 1.8-2.0 mW cm-2 over an area of a standard 4-in. wafer, at the plane of photoresist exposure, and the alignment accuracy is characterized to be <3 μm, which is sufficient for most microfluidic applications. Moreover, this manuscript provides detailed descriptions of the procedures needed to fabricate multilayered master molds using our CBMA. Finally, the capabilities of the CBMA are demonstrated by fabricating two- and three-layer masters for micro-scale devices, commonly encountered in biomicrofluidic applications. The former is a flow-free chemical gradient generator, and the latter is an addressable microfluidic stencil. Scanning electron microscopy is used to confirm that the master molds contain the intended features of different heights.

The role of magma mixing/mingling and cumulate melting in the Neapolitan Yellow Tuff caldera-forming eruption (Campi Flegrei, Southern Italy)

Understanding the mechanisms responsible for the generation of chemical gradients in high-volume ignimbrites is key to retrieve information on the processes that control the maturation and eruption of large silicic magmatic reservoirs. Over the last 60 ky, two large ignimbrites showing remarkable zoning were emplaced during caldera-forming eruptions at Campi Flegrei (i.e., Campanian Ignimbrite, CI, ~ 39 ka and Neapolitan Yellow Tuff, NYT, ~ 15 ka). While the CI displays linear compositional, thermal and crystallinity gradients, the NYT is a more complex ignimbrite characterized by crystal-poor magmas ranging in composition from trachy-andesites to phonolites. By combining major and trace element compositions of matrix glasses and mineral phases from juvenile clasts located at different stratigraphic heights along the NYT pyroclastic sequence, we interpret such compositional gradients as the result of mixing/mingling between three different magmas: (1) a resident evolved magma showing geochemical characteristics of a melt extracted from a cumulate mush dominated by clinopyroxene, plagioclase and oxides with minor sanidine and biotite; (2) a hotter and more mafic magma from recharge providing high-An plagioclase and high-Mg clinopyroxene crystals and (3) a compositionally intermediate magma derived from remelting of low temperature mineral phases (i.e., sanidine and biotite) within the cumulate crystal mush. We suggest that the presence of a refractory crystal mush, as documented by the occurrence of abundant crystal clots containing clinopyroxene, plagioclase and oxides, is the main reason for the lack of erupted crystal-rich material in the NYT. A comparison between the NYT and the CI, characterized by both crystal-poor extracted melts and crystal-rich magmas representing remobilized portions of a “mature” (i.e., sanidine dominated) cumulate residue, allows evaluation of the capability of crystal mushes of becoming eruptible upon recharge.

In vitro quantitative analysis of Salmonella typhimurium preference for amino acids secreted by human breast tumor

Bacterial therapies have been paid significant attentions by their ability to penetrate deep into the solid tumor tissue and its propensity to naturally accumulate in tumors of living animals. Understanding the actual mechanism for bacteria to target the tumor is therapeutically crucial but is poorly understood. We hypothesized that amino acids released from the specific tumors induced bacteria to those tumors and the experiments for chemotactic response of bacteria toward the cancer secreting amino acids was then performed by using the diffusion based multiple chemical gradient generator constructed by in situ self-assembly of microspheres. The quantitative analysis was carried out by comparison of intensity using green fluorescent protein (GFP) tagged Salmonella typhimurium (S. typhimurium) in the gradient generator, which showed the clear preference to the released amino acids, especially from breast cancer patients. The understanding chemotaxis toward the cancer secreting amino acids is essential for controlling S. typhimurium targeting in tumors and will allow for the development of bacterial therapies.

Formation of Actin Networks in Microfluidic Concentration Gradients

The physical properties of cytoskeletal networks are contributors in a number of mechanical responses of cells including cellular deformation and locomotion, and are crucial for the proper action of living cells. Local chemical gradients modulate cytoskeletal functionality including the interactions of the cytoskeleton with other cellular components. Actin is a major constituent of the cytoskeleton. Introducing a microfluidic-based platform, we explored the impact of concentration gradients on the formation and structural properties of actin networks. Microfluidics-controlled flow-free steady state experimental conditions allow for the generation of chemical gradients of different profiles, such as linear or step-like. We discovered specific features of actin networks emerging in defined gradients. In particular, we analyzed the effects of spatial conditions on network properties, bending rigidities of network links, and the network elasticity.

Microfluidics made easy: A robust low-cost constant pressure flow controller for engineers and cell biologists.

Over the last decade, microfluidics has become increasingly popular in biology and bioengineering. While lab-on-a-chip fabrication costs have continued to decrease, the hardware required for delivering controllable fluid flows to the microfluidic devices themselves remains expensive and often cost prohibitive for researchers interested in starting a microfluidics project. Typically, microfluidic experiments require precise and tunable flow rates from a system that is simple to operate. While many labs use commercial platforms or syringe pumps, these solutions can cost thousands of dollars and can be cost prohibitive. Here, we present an inexpensive and easy-to-use constant pressure system for delivering flows to microfluidic devices. The controller costs less than half the price of a single syringe pump but can independently switch and deliver fluid through up to four separate fluidic inlets at known flow rates with significantly faster fluid response times. It is constructed of readily available pressure regulators, gauges, plastic connectors and adapters, and tubing. Flow rate is easily predicted and calibrated using hydraulic circuit analysis and capillary tubing resistors. Finally, we demonstrate the capabilities of the flow system by performing well-known microfluidic experiments for chemical gradient generation and emulsion droplet production.

A diffusion based long-range and steady chemical gradient generator on a microfluidic device for studying bacterial chemotaxis

Studies on chemotaxis in microfluidics device have become a major area of research to generate physiologically similar environment in vitro. In this work, a novel micro-fluidic device has been developed to study chemo-taxis of cells in near physiological condition which can create controllable, steady and long-range chemical gradients using various chemo-effectors in a micro-channel. Hydrogels like agarose, collagen, etc, can be used in the device to maintain exclusive diffusive flux of various chemical species into the micro-channel under study. Variations of concentrations and flow rates of Texas Red dextran in the device revealed that an increase in the concentration of the dye in the feed from 6 to 18 μg ml−1, causes a steeper chemical gradient in the device, whereas the flow rate of the dye has practically no effect on the chemical gradient in the device. This observation confirms that a diffusion controlled chemical gradient is generated in the micro-channel. Chemo-taxis of E. coli cells were studied under the steady gradient of a chemo-attractant and a chemo-repellent separately in the same chemical gradient generator. For sorbitol and NiSO4·6H2O, the bacterial cells exhibit a steady distribution in the micro channel after 1 h and 30 min, respectively. From the distribution of bacterial population chemo-tactic strength of the chemo-effectors was estimated for E. coli. In a long microfluidic channel, migration behavior of bacterial cells under diffusion controlled chemical gradient showed chemotaxis, random movement, aggregation, and concentration dependent reverse chemotaxis.

A spatiotemporally controllable chemical gradient generator via acoustically oscillating sharp-edge structures.

The ability to generate stable, spatiotemporally controllable concentration gradients is critical for resolving the dynamics of cellular response to a chemical microenvironment. Here we demonstrate an acoustofluidic gradient generator based on acoustically oscillating sharp-edge structures, which facilitates in a step-wise fashion the rapid mixing of fluids to generate tunable, dynamic chemical gradients. By controlling the driving voltage of a piezoelectric transducer, we demonstrated that the chemical gradient profiles can be conveniently altered (spatially controllable). By adjusting the actuation time of the piezoelectric transducer, moreover, we generated pulsatile chemical gradients (temporally controllable). With these two characteristics combined, we have developed a spatiotemporally controllable gradient generator. The applicability and biocompatibility of our acoustofluidic gradient generator are validated by demonstrating the migration of human dermal microvascular endothelial cells (HMVEC-d) in response to a generated vascular endothelial growth factor (VEGF) gradient, and by preserving the viability of HMVEC-d cells after long-term exposure to an acoustic field. Our device features advantages such as simple fabrication and operation, compact and biocompatible device, and generation of spatiotemporally tunable gradients.

An array microhabitat system for high throughput studies of microalgal growth under controlled nutrient gradients.

Microalgae have been increasingly recognized in the fields of environmental and biomedical engineering because of its use as base materials for biofuels or biomedical products, and also the urgent needs to control harmful algal blooms protecting water resources worldwide. Central to the theme is the growth rate of microalgae under the influences of various environmental cues including nutrients, pH, oxygen tension and light intensity. Current microalgal culture systems, e.g. raceway ponds or chemostats, are not designed for system parameter optimizations of cell growth. In this article, we present the development of an array microfluidic system for high throughput studies of microalgal growth under well defined environmental conditions. The microfluidic platform consists of an array of microhabitats flanked by two parallel side channels, all of which are patterned in a thin agarose gel membrane. The unique feature of the device is that each microhabitat is physically confined suitable for both motile and non-motile cell culture, and at the same time, the device is transparent and can be perfused through the two side channels amendable for precise environmental control of photosynthetic microorganisms. This microfluidic system is used to study the growth kinetics of a model microalgal strain, Chlamydomonas reinhardtii (C. reinhardtii), under ammonium (NH4Cl) concentration gradients. Experimental results show that C. reinhardtii follows Monod growth kinetics with a half-saturation constant of 1.2 ± 0.3 μM. This microfluidic platform provides a fast (~50 fold speed increase), cost effective (less reagents and human intervention) and quantitative technique for microalgal growth studies, in contrast to the current chemostat or batch cell culture system. It can be easily extended to investigate growth kinetics of other microorganisms under either single or co-culture setting.

A microfluidic device for generation of chemical gradients

A microfluidic device was developed for generating chemical concentration gradients by integrating PEG-diacrylate (PEGDA) hydrogel separators in a Polydimethylsiloxane (PDMS) chamber. In this device, the linear chemical gradient in the central culture channel is achieved by the diffusion of two side flow streams which are separated by hydrogel separators. Two long serpentine channels feed into a transition zone to balance the pressure of both streams insuring a stable gradient is formed and maintained before it reaches the central culture channel. This device provides a reproducible, controllable, long-term steady and linear chemical concentration gradient without complex supports. Additionally, the concentration gradient can be controlled by varying the hydrogel thickness, thereby changing the amount of diffusion through the hydrogels. This device can be modified to create different chemical gradients for a variety of cell culture applications.

Modulation of alpha-synuclein toxicity in yeast using a novel microfluidic-based gradient generator.

Parkinson's disease (PD) is a common age-associated neurodegenerative disorder. The protein α-synuclein (aSyn) is a key factor in PD both due to its association with familial and sporadic cases and because it is the main component of the pathological protein aggregates known as Lewy bodies. However, the precise cellular effects of aSyn aggregation are still elusive. Here, we developed an elastomeric microfluidic device equipped with a chemical gradient generator and 9 chambers containing cell traps to study aSyn production and aggregation in Saccharomyces cerevisiae. This study involved capturing single cells, exposing them to specific chemical environments and imaging the expression of aSyn by means of a GFP fusion (aSyn-GFP). Using a galactose (GAL) gradient we modulated aSyn expression and, surprisingly, by tracking the behavior of single cells, we found that the response of individual cells in a population to a given stimulus can differ widely. To study the combined effect of environmental factors and aSyn expression levels, we exposed cells to a gradient of FeCl3. We found a dramatic increase in the percentage of cells displaying aSyn inclusions from 27% to 96%. Finally, we studied the effects of ascorbic acid, an antioxidant, on aSyn aggregation and found a significant reduction in the percentage of cells bearing aSyn inclusions from 87% to 37%. In summary, the device developed here offers a powerful way of studying aSyn biology with single-cell resolution and high throughput using genetically modified yeast cells.

Lighting Up Redox Propulsion with Luminol Electrogenerated Chemiluminescence

The development of systems at different scales that are remotely controlled or actuated in a wireless fashion is becoming a fast-developing area at the interface between chemistry and physics. In that framework, self-propelled macro-, microand nano-objects that are able to move in liquids when submitted to external physical fields or chemical fuels have recently been designed. In the latter case, the motion is induced by the conversion of locally available chemicals into mechanical energy via the production of gas bubbles. The mainstream strategy involves a metallic moiety which is able to catalyze the decomposition of hydrogen peroxide, resulting in the interfacial generation of oxygen bubbles. In contrast to this chemically-induced locomotion, physical methods involving electric fields have also been demonstrated. Bipolar electrochemistry (BPE) has recently encountered an important renewal thanks to the development of new applications in the fields of analytical chemistry and chemical sensing, surface science and generation of chemical gradients, catalysis and materials synthesis. The behaviour of a bipolar electrode (BE) is atypical with respect to conventional electrochemistry because it acts as an anode and a cathode at the same time. This asymmetric redox reactivity can also be advantageously used to set in motion conductive particles. The very first example of translational motion was proposed on the basis of an electrochemical self-regeneration with a zinc dendrite. Alternatively, water splitting at both reactive extremities of a BE is responsible for a spatially separated bubble production. This overall reaction generates twice more hydrogen bubbles at the cathodic pole when compared to oxygen at the anodic end. Here, the intrinsic stoichiometry controls the directionality of the resulting motion from the feeder anode to the feeder cathode. Enhancing the speed of motion can be achieved by introducing additive sacrificial redox compounds such as quinone or hydroquinone. In the latter case, the anodic counter reaction is not useful and simply allows to produce bubbles at only one pole of the BE. Interestingly, this recent approach allows as well the addition of new functionalities to dynamic systems like for example the emission of electrogenerated chemiluminescence (ECL). ECL is the light emission resulting from an initial electrochemical reaction. A model ECL system is based on the oxidation of luminol (5-Amino-2,3-dihydro-1,4-phthalazine-dione) in alkaline solution in the presence of hydrogen peroxide in order to promote blue light emission. Luminol and its derivatives are widely used in biochemical and clinical analysis such as enzymatic assays and immunoassays. For example, luminol ECL coupled to oxidasecatalysed reactions generating hydrogen peroxide constitutes a sensitive and versatile analytical strategy. ECL has not only been exploited in combination with BPE for analytical purposes, but also to design the first light emitting electrochemical swimmer involving [Ru(bpy)3] 2+ and tripropylamine (TPrA). The present contribution will show that this methodology can be generalized with another ECL system based on luminol, allowing to tune the wavelength of light emission. Moreover, all previously described BPE-driven motions were reported in neutral or acidic aqueous solutions with cathodic bubbles generation. In contrast, this first example in an alkaline solution proceeds through an alternative mechanism based on H2O2 oxidation and allowing the light emission at the same pole (anode) than the bubble production (Scheme 1). Simultaneous generation of gas bubbles and of ECL could be theoretically achieved when the interfacial difference of potential alongside a BE becomes sufficiently high. Preliminary

Combinatorial Approach for Fabrication of Coatings to Control Bacterial Adhesion

Due to the high importance of bacterial infections in medical devices there is an increasing interest in the design of anti-fouling coatings. The application of substrates with controlled chemical gradients to prevent microbial adhesion is presented. We describe here the co-polymerization of poly(ethylene glycol) dimethacrylate with a hyperbranched multimethacrylate (H30MA) using a chemical gradient generator; and the resulting films were characterized with respect to their ability to serve as coating for biomedical devices. The photo-polymerized materials present special surface properties due to the hyperbranched structure of H30MA and phase separation at specific concentrations in the PEGDM matrix. This approach affords the investigation of cell response to a large range of different chemistries on a single sample. Two bacterial strains commonly associated with surgical site infections, Escherichia coli and Pseudomonas aeruginosa, have been cultured on these substrates to study their attachment behaviour. These gradient-coated samples demonstrate less bacterial adhesion at higher concentrations of H30MA, and the adhesion is substantially affected by the extent of surface phase segregation.

Quantitatively controlled in situ formation of hydrogel membranes in microchannels for generation of stable chemical gradients

The in situ formation of membranes in microfluidic channels has been given attention because of their great potential in the separation of components, cell culture support for tissue engineering, and molecular transport for generation of chemical gradients. Among these, the porous membranes in microchannels are vigorously applied to generate stable chemical gradients for chemotaxis-dependent cell migration assays. Previous work on the in situ fabrication of membranes for generating the chemical gradient, however, has had several disadvantages, such as fluid leaking, uncontrollable membrane thickness, need of extra equipment, and difficulty in realizing stable interfacial layers. In this paper, we report a novel technique for the in situ formation of membranes within microchannels using enzymatically crosslinkable hydrogels and microfluidic techniques. The thickness of the membrane can be controlled quantitatively by adjusting the crosslinking reaction time and velocity of the microfluidics. By using these techniques, parallel dual hydrogel membranes were prepared within microchannels and these were used for the generation of stable concentration gradients. Moreover, the migration of Salmonella typhimurium was monitored to validate the efficacy of the chemical gradients. These results suggest that our in situ membrane system can be used as a simple platform to understand many cellular activities, including cell adhesion and migration directed by chemotaxis or complex diffusions from biological fluids in three-dimensional microstructures.

Versatile gradients of chemistry, bound ligands and nanoparticles on alumina nanoporearrays

Nanoporous alumina (PA) arrays produced by self-ordering growth, using electrochemicalanodization, have been extensively explored for potential applications based upon theunique thermal, mechanical and structural properties, and high surface-to-volume ratio ofthese materials. However, the potential applications and functionality of these materialsmay be further extended by molecular-level engineering of the surface of the pore rims. Inthis paper we present a method for the generation of chemical gradients on the surface ofPA arrays based upon plasma co-polymerization of two monomers. We further extend thesechemical gradients, which are also gradients of surface charge, to those of bound ligandsand number density gradients of nanoparticles. The latter represent a highlyexotic new class of materials, comprising aligned PA, capped by gold nanoparticlesaround the rim of the pores. Gradients of chemistry, ligands and nanoparticlesgenerated by our method retain the porous structure of the substrate, whichis important in applications that take advantage of the inherent properties ofthese materials. This method can be readily extended to other porous materials.

Microfluidic Device with Chemical Gradient for Single-Cell Cytotoxicity Assays

Here, we report the fabrication of a chemical gradient microfluidic device for single-cell cytotoxicity assays. This device consists of a microfluidic chemical gradient generator and a microcavity array that enables entrapment of cells with high efficiency at 88 ± 6% of the loaded cells. A 2-fold logarithmic chemical gradient generator that is capable of generating a serial 2-fold gradient was designed and then integrated with the microcavity array. High density single-cell entrapment was demonstrated in the device without cell damage, which was performed in 30 s. Finally, we validated the feasibility of this device to perform cytotoxicity assays by exposing cells to potassium cyanide (0-100 μM KCN). The device captured images of 4000 single cells affected by 6 concentrations of KCN and determined cell viability by counting the effected cells. Image scanning of the microcavity array was completed within 10 min using a 10× objective lens and a motorized stage. Aligning cells on the microcavity array eases cell counting, observation, imaging, and evaluation of singular cells. Thus, this platform was able to determine the cytotoxicity of chemicals at a single-cell level, as well as trace the cytotoxicity over time. This device and method will be useful for cytotoxicity analysis and basic biomedical research.

Fit-to-Flow (F2F) interconnects: Universal reversible adhesive-free microfluidic adaptors for lab-on-a-chip systems

World-to-chip (macro-to-micro) interface continues to be one of the most complicated, ineffective, and unreliable components in the development of emerging lab-on-a-chip systems involving integrated microfluidic operations. A number of irreversible (e.g., adhesive gluing) and reversible techniques (e.g., press fitting) have attempted to provide dedicated fluidic passage from standard tubing to miniature on-chip devices, none of which completely addresses the above concerns. In this paper, we present standardized adhesive-free microfluidic adaptors, referred to as Fit-to-Flow (F2F) Interconnects, to achieve reliable hermetic seal, high-density tube packing, self-aligned plug-in, reworkable connectivity, straightforward scalability and expandability, and applicability to broad lab-on-a-chip platforms; analogous to the modular plug-and-play USB architecture employed in modern electronics. Specifically, two distinct physical packaging mechanisms are applied, with one utilizing induced tensile stress in elastomeric socket to establish reversible seal and the other using negative pressure to provide on demand vacuum shield, both of which can be adapted to a variety of experimental configurations. The non-leaking performance (up to 336 kPa) along with high tube-packing density (of 1 tube/mm(2)) and accurate self-guided alignment (of 10 μm) have been characterized. In addition, a 3D microfluidic mixer and a 6-level chemical gradient generator paired with the corresponding F2F Interconnects have been devised to illustrate the applicability of the universal fluidic connections to classic lab-on-a-chip operations.

High-pressure on-chip mechanical valves for thermoplastic microfluidic devices

A facile method enabling the integration of elastomeric valves into rigid thermoplastic microfluidic chips is described. The valves employ discrete plugs of elastomeric polydimethylsiloxane (PDMS) integrated into the thermoplastic substrate and actuated using a threaded stainless steel needle. The fabrication process takes advantage of poly(ethylene glycol) (PEG) as a sacrificial molding material to isolate the PDMS regions from the thermoplastic flow channels, while yielding smooth contact surfaces with the PDMS valve seats. The valves introduce minimal dead volumes, and provide a simple mechanical means to achieve reproducible proportional valving within thermoplastic microfluidic systems. Burst pressure tests reveal that the valves can withstand pressures above 12 MPa over repeated open/close cycles without leakage, and above 24 MPa during a single use, making the technology well suited for applications such as high performance liquid chromatography. Proportional valve operation is demonstrated using a multi-valve chemical gradient generator fabricated in cyclic olefin polymer.