Microfluidic Gradient Generator Research Articles

Chemical gradient‐mediated melting curve analysis for genotyping of SNPs

This report describes a microfluidic solid-phase chemical gradient-mediated melting curve analysis method for SNP analysis. The method is based on allele-specific denaturation to discriminate mismatched (MM) from perfectly matched (PM) DNA duplexes upon exposure to linear chemical gradient. PM and MM DNA duplexes conjugated on beads are captured in a microfluidic gradient generator device designed with dams, keeping the beads trapped perpendicular to a gradient generating channel. Two denaturants, formamide and urea, were tested for their ability to destabilize the DNA duplex by competing with Watson-Crick pairing. Upon exposure to the chemical gradient, rapid denaturing profile was monitored in real time using fluorescence microscopy. The results show that the two duplexes exhibit different kinetics of denaturation profiles, enabling discrimination of MM from PM DNA duplexes to score SNP.

Lab-on-a-print: from a single polymer film to three-dimensional integrated microfluidics

With the major advances in soft lithography and polymer materials, use of microfluidic devices has attracted tremendous attention recently. A simple and fast micromachining process is highly in demand to prototype such a device efficiently and economically. In this paper, we first reported an out-of-cleanroom printing-based integrated microfabrication process, referred to as the lab-on-a-print (LOP), for rapid-prototyping three-dimensional microfluidics. Using this lab-on-a-print process, we demonstrated the potential to accomplish an entire design-to-fabrication cycle within an hour, including about 70 microm resolution of direct-lithography patterning, well-controlled polyimide wet etching, three-dimensional pattern alignment and multilayer wax thermal-fusion packaging. A microfluidic gradient generator was prepared and tested for validation of the lab-on-a-print microfabrication process.

Generalized serial dilution module for monotonic and arbitrary microfluidic gradient generators

In this paper, we propose a generalized serial dilution module for universal microfluidic concentration gradient generators including N cascaded-mixing stages in a stepwise manner. Desired concentrations were generated by means of controlled volumetric mixing ratios of two merging solutions in each stage. The flow rates were adjusted by controlling channel length, which is proportional to fluidic resistance in each channel. A generalized mathematical model for generating any complex concentration and output flow rate gradients is presented based on the fact that there is an analogy between microfluidic circuits and electrical circuits. The pressure drop corresponds to a voltage drop, the flow rate to an electrical current, and the flow resistance to an electrical resistance. A simple equivalent electrical circuit model was generalized, and in the model each channel segment was represented by an electrical resistance. As a result of the mathematical modelling, the only variable parameter in the generalized serial dilution module was the channel length. By the use of the generalized serial dilution module with N = 4, three types of microfluidic gradient generators for linear, logarithmic and Gaussian gradients were successfully designed and tested. The proposed strategy is capable of generating universal monotonic gradients with a single module or arbitrary gradients with multiple modules ranging from linear to complex non-linear shapes of concentration gradients as well as arbitrary output flow rate gradients in a stepwise manner. The simple universal gradient generation technology using the generalized serial dilution module will find widespread use in the greater chemical and biological community, and address many challenges of gradient-dependent phenomena.

Microfluidic system with integrated electroosmotic pumps, concentration gradient generator and fish cell line (RTgill-W1)—towards water toxicity testing

This study presents a microfluidic system that incorporates electroosmotic pumps, a concentration gradient generator and a fish cell line (rainbow trout gill) to perform toxicity testing on fish cells seeded in the system. The system consists of three mechanical components: (1) a toxicity testing chip containing a microfluidic gradient generator which creates a linear concentration distribution of toxicant in a cell test chamber, (2) an electroosmotic (EO) pump chip that controls the flow rate and operation of the toxicity chip, and (3) indirect reservoirs that connect the two chips allowing for the toxicant solution to be pumped separately from the electroosmotic pump solution. The flow rate and stability of the EO pumps was measured and tested by monitoring the gradient generator using fluorescence microscopy. Furthermore, a lethality test was performed with this system setup using a rainbow trout gill cell line (RTgill-W1) as the test cells and sodium dodecyl sulfate as a model toxicant. A gradient of sodium dodecyl sulfate, from 0 to 50 microg mL(-1), was applied for 1 hr to the attached cells, and the results were quantified using a Live/Dead cell assay. This work is a preliminary study on the application of EO pumps in a living cell assay, with the potential to use the pumps in portable water quality testing devices with RTgill-W1 cells as the biosensors.

Generation of complex concentration profiles by partial diffusive mixing in multi-stream laminar flow

This paper proposes novel microfluidic concentration gradient generator (CGG) devices that are capable of constructing complex profiles of chemical concentrations by laterally combining the constituent profiles (e.g., linear and bell-shaped) generated in simple Y- or psi-shaped mixers. While the majority of currently existing CGG devices are based on complete mixing of chemical species, our design harnesses partial diffusive mixing in multi-stream laminar flow, and hence, features simple network structures and enhanced device reliability. An iterative simulation approach that incorporates our previous system-level models of CGG networks is developed to locate best-matched combinations of geometrical and operating parameters (e.g., inlet flow rates and inlet sample concentrations) for the device design. Microfluidic CGG chips are fabricated and experimentally characterized using optimal layout and operating conditions selected by the design process. The experimental results not only serve as a benchmark for model verification but also establish the feasibility of concentration gradient generation based on partial mixing for a variety of microfluidic applications.

Optimisation and analysis of microreactor designs for microfluidic gradient generation using a purpose built optical detection system for entire chip imaging

This paper presents and fully characterises a novel simplification approach for the development of microsystem based concentration gradient generators with significantly reduced microfluidic networks. Three microreactors are presented; a pair of two-inlet six-outlet (2-6) networks and a two-inlet eleven-outlet (2-11) network design. The mathematical approach has been validated experimentally using a purpose built optical detection system. The experimental results are shown to be in very good agreement with the theoretical predictions from the model. The developed networks are proven to deliver precise linear concentration gradients (R(2) = 0.9973 and 0.9991 for the (2-6) designs) and the simplified networks are shown to provide enhanced performance over conventional designs, overcoming some of the practical issues associated with traditional networks. The optical measurements were precise enough to validate the linearity in each level of the conventional (2-6) networks (R(2) ranged from 0.9999 to 0.9973) compared to R(2) = 1 for the theoretical model. CFD results show that there is an effective upper limit on the operating flow rate. The new simplified (2-11) design was able to maintain a linear outlet profile up to 0.8 microl/s per inlet (R(2) = 0.9992). The proposed approach is widely applicable for the production of linear and arbitrary concentration profiles, with the potential for high throughput applications that span a wide range of chemical and biological studies.

Biomolecular gradients in cell culture systems

Biomolecule gradients have been shown to play roles in a wide range of biological processes including development, inflammation, wound healing, and cancer metastasis. Elucidation of these phenomena requires the ability to expose cells to biomolecule gradients that are quantifiable, controllable, and mimic those that are present in vivo. Here we review the major biological phenomena in which biomolecule gradients are employed, traditional in vitro gradient-generating methods developed over the past 50 years, and new microfluidic devices for generating gradients. Microfluidic gradient generators offer greater levels of precision, quantitation, and spatiotemporal gradient control than traditional methods, and may greatly enhance our understanding of many biological phenomena. For each method, we outline the salient features, capabilities, and applications.

Characterization of transport in microfluidic gradient generators

We present a two-dimensional model that describes the concentration profile of a class of previously reported microfluidic devices which are of particular interest in cellular taxis research. The devices generate stable concentration gradients by mixing and dividing two or more external inputs into a large number of discrete streams. This study focuses specifically on modeling the confluence of the discrete streams in a long chamber. We derive a closed-form solution for gradient generators with any arbitrary number of sampling streams. By relating the physical dimensions to the Peclet number, we create a model independent of flow rate and therefore dependent only on the specific nature of the boundary condition provided by the upstream network. As a result, the modeling method we propose may help evaluate the effectiveness of competing gradient generation schemes. Finally, our analytical work introduces a framework for developing simple design rules of interest to experimentalists working with these devices.

Generation of linear and non-linear concentration gradients along microfluidic channel by microtunnel controlled stepwise addition of sample solution

The ability to generate stable chemical gradients in microfluidics has important applications, since such gradients are useful in both chemical and biological studies. Growing evidence reveals that many cellular responses are specific to non-linear spatial gradients, hence a need to control complex concentration gradient profiles with and within microfluidics. In this paper, we present a structure-based approach to generate linear and non-linear chemical gradients, with profiles controlled by microtunnels fabricated alongside two main channels. Using single-step photolithography, microtunnels and main channels were fabricated at different heights thus having different fluidic resistance. Through these microtunnels, sample solutions were stepwise dispensed into the buffer stream to generate a chemical gradient profile. By varying the lengths of microtunnels that dictated the volume of sample solutions being dispensed, complex gradient profiles were generated. We have successfully demonstrated the formation of linear, convex and concave gradient profiles and a simple mathematical expression was established to approximate the profiles produced in our microfluidic gradient-generators.

Systematic modeling of microfluidic concentration gradient generators

This paper presents a systematic modeling methodology for microfluidic concentration gradient generators. The generator is decomposed into a system of microfluidic elements with relatively simple geometries. Parameterized models for such elements are analytically developed and hold for general sample concentration profiles and arbitrary flow ratios at the element inlet; hence, they are valid for concentration gradient generators that rely on either complete or partial mixing. The element models are then linked through an appropriate set of parameters embedded at the element interfaces. This yields a systematic, lumped-parameter representation of the entire generator in terms of a network of gradient-generation elements. The system model is verified by numerical analysis and experimental data and accurately captures the overall effects of network topologies, element sizes, flow rates and reservoir sample concentrations on the generation of sample concentration gradient. Finally, this modeling methodology is applied to propose a novel and compact microfluidic device that is able to create concentration gradients of complex shapes by juxtaposing simple constituent profiles along the channel width.

A three-channel microfluidic device for generating static linear gradients and its application to the quantitative analysis of bacterial chemotaxis

We have developed a prototype three-channel microfluidic chip that is capable of generating a linear concentration gradient within a microfluidic channel and is useful in the study of bacterial chemotaxis. The linear chemical gradient is established by diffusing a chemical through a porous membrane located in the side wall of the channel and can be established without through-flow in the channel where cells reside. As a result, movement of the cells in the center channel is caused solely by the cells chemotactic response and not by variations in fluid flow. The advantages of this microfluidic chemical linear gradient generator are (i) its ability to produce a static chemical gradient, (ii) its rapid implementation, and (iii) its potential for highly parallel sample processing. Using this device, wildtype Escherichia coli strain RP437 was observed to move towards an attractant (e.g., l-asparate) and away from a repellent (e.g., glycerol) while derivatives of RP437 that were incapable of motility or chemotaxis showed no bias of the bacteria's distribution. Additionally, the degree of chemotaxis could be easily quantified using this assay in conjunction with fluorescence imaging techniques, allowing for estimation of the chemotactic partition coefficient (CPC) and the chemotactic migration coefficient (CMC). Finally, using this approach we demonstrate that E. coli deficient in autoinducer-2-mediated quorum sensing respond to the chemoattractant l-aspartate in a manner that is indistinguishable from wildtype cells suggesting that chemotaxis is insulated from this mode of cell-cell communication.

Photopolymerization in Microfluidic Gradient Generators: Microscale Control of Substrate Compliance to Manipulate Cell Response

Photopolymerization in Microfluidic Gradient Generators: Microscale Control of Substrate Compliance to Manipulate Cell Response