Analytical Instrumentation : Microfluidics

Analytical Instrumentation Portfolios


New Gradient-Based Cell Labeling Method Maintains Location Information During Downstream Analysis

UW–Madison researchers have developed a new device and method for tagging and monitoring individual cells in a microenvironment.

Concentrated dye solution is placed within molded wells inside a gel, diffusing throughout to form a color-based gradient. This stamp is then placed over the cell culture to be studied, allowing the dye particles to diffuse into the culture and label the cells. When the cells are removed from their environment for further analysis, the technique allows them to be easily identified and their previous location to be tracked.

Streamlined Design for Transferring Analytes

The researchers have now improved their design and developed a microfluidic device that directly integrates with tubes, strip tubes and well plates. In this way a sample can be directly transferred from the device to downstream analysis instruments.

The device comprises a strip of wells that hold various volumes of output fluid. Following sample isolation via the researchers’ previously developed SLIDE technique, the strip containing the sample and output buffer is removed from the SLIDE and applied to a set of strip tubes in the same way that conventional covers would be applied.

Then, by flicking or centrifuging the tubes, the sample is transferred from the cap to the tube. At this point the sample is ready for PCR or other downstream analysis.

Circulating Tumor Cell Assay Using Simple Blood Draw

The researchers now have developed the first fully integrated CTC assay that could eliminate the need for painful biopsies. The device leverages verIFAST technology to capture, purify and molecularly analyze CTCs from a single blood sample.

The sample is deposited in a first well and then magnetically drawn through a second well containing an isolation buffer like oil or wax. The increasingly purified cells are drawn into a final well for extraction or further treatment.

The device includes new technical features and is combined with downstream techniques for staining rare cells like CTCs.

Microwell Moat Prevents Spillover

UW–Madison researchers have developed a new microwell design for use with conventional micropipetting equipment (e.g., a hand-held micropipette or automated pipetting robot). Similar to other microwell designs, the flat device is checkered with nanoliter microwells. However, in the new design, groups of microwells are ringed by deeper moat-like channels that isolate the groups and prevent any spillover when a fluid droplet, e.g., a reagent or cell suspension, is deposited. The moat also keeps fluid from being ‘squished’ and spread out when a when a lid is applied to seal each microwell, avoiding cross-contamination of each experimental condition.

The design enables multiple different reagents/experimental conditions to be tested on the same device using standard pipetting operations. Together, the new features enable easier, more robust, quantitative and massively parallelized single-cell assays for a range of endpoints that can interface with standard pipette equipment.

Low-Waste and Contamination Device for Isolating Cells from a Biological Sample

UW–Madison researchers have developed an improved microfluidic device for isolating a desired fraction without the sample loss or contamination associated with prior designs.

The device features multiple wells through which a magnet (positioned perpendicularly to gravity) is drawn, attracting the fraction from one well to the next while undesired material settles to the bottom. For this to occur the biological sample first is combined with a magnetically attractive reagent that binds to the targeted fraction and forms a solid-phase substrate.

The sample is deposited in the first well and then magnetically drawn through a second well containing an isolation buffer like oil or wax. The increasingly purified cells are drawn into a final well for extraction or further treatment. Surface tension prevents fluid from spilling between wells.

Snap-On Microfluidic Lid for Handheld Diagnostics and Chemical Tests

UW–Madison researchers have developed a microfluidic design and method that supports rapid and simplified handheld diagnostics and assays.

The device is formed in two separate sections— a base and a functionalized lid—that can be snapped together. The base has channels running between two ports. The lid, which is disposable and holds a well prepackaged with selected liquids like a drug or chemical, has a piercing mechanism. When pressed to the base, the membrane covering the well is perforated and the substance induced to flow down into the base, through its channels and back up into the lid’s absorption pad.

The functionalized lid can not only pump, but also can be designed to apply a chemical gradient using wells filled with hydrogel and reagents that diffuse into the channels.

Fast, Flexible Platform for Handheld Microfluidic Cell Assays

UW–Madison researchers have developed a new microfluidic device design, KOALA, which can perform assays in five-minute steps without reagent waste or time-consuming preparation.

The chip comprises a disengaging lid and base. The lid is networked by channels with protruding inputs while the base features multiple fluid wells and an absorbent pad. When the two components are pressed together, fluid from the wells is drawn into the channel by the pad’s capillary action.

Additional functionalities, like creating gradients with a diffusing source, also are achievable given the design’s passive fluid contact at the channel extremities. Packaged with the reagents and cells required of the assay and enabling encapsulation and freezing, KOALA is an eminently accessible and flexible assay tool.

Device for Efficiently Extracting a Fraction Containing Nucleic Acids or Other Desired Material from a Biological Sample

UW–Madison researchers have developed a device and method for extracting and purifying a desired fraction from cultured cells, tissue samples and other biological materials. A biological sample, including both non-desired material and a fraction-bound solid phase substrate, is added to an input zone. The input zone is adjacent to a separation zone that includes an isolation buffer. A force moves the fraction-bound solid phase substrate from the input zone, through the separation zone and into an output zone, leaving the non-desired material behind. The improved purification method is simple, more efficient and produces a higher throughput than prior devices and methods. The device may be configured to allow for quantification of the fraction in the biological sample via labeling of the fraction-bound solid phase substrate.

Simple Microfluidic Device and Method for Determining Single-Cell Adhesion Strength

UW–Madison researchers have developed a new microfluidic device and method for measuring the adhesion strength of individual cells. The method is compatible with existing measurement and imaging approaches found in industrial biology, biomedical and pharmaceutical research labs, including standard Petri dish- and multiwell plate-based procedures on an inverted microscope.

In the device, a local microfluidic chamber is created by positioning a microfluidic top chip with an inlet and outlet port a small distance above a surface. The chip does not make physical contact with the surface. In contrast to a traditional microfluidic channel in which the structure is created by forming a channel in a plastic, glass or silicon substrate, this device produces a microfluidic chamber by simply positioning a structured substrate above another surface.

After the microfluidic chamber is created over a cell, flow is applied to generate shear stress on the cell. The shear stress is linearly proportional to the flow rate and inversely proportional to the square of the channel height. In a typical cell adhesion test, the shear stress on the cell is increased over time by changing either the flow rate or channel height and the cell is monitored using optical or fluorescence microscopy. At some critical point, the cell delaminates. The stress applied to the cell at this time is defined as the shear strength.

Microfluidic Device for Capturing and Analyzing Rare Cells, Including Circulating Tumor Cells

UW-Madison researchers have developed a microfluidic device for concentrating rare cells.  The velocity of flow through the device is manipulated such that particles in suspension, such as cells, are carried to and deposited in a particular location within the device.  The flow in the region where the cells are deposited is slow, so the cells remain in the collection area without any modifications to the surfaces of the device or cells while the fluid that carried them is routed out of the device.  Because particles in suspension enter the collection region but do not leave, many particles may be captured from a relatively dilute suspension.  The cells then can be cultured, stained and imaged for analysis without being removed from the collection area, creating a gentle and efficient way to implement extensive washing and treatment protocols.

Microfluidic Device for Rapid Nucleic Acid Isolation and Purification

UW-Madison researchers have developed a microfluidic device and method for rapidly extracting and purifying nucleic acids from biological materials. The device includes an input zone for receiving the biological sample, a phase-gate zone for holding an isolation buffer and an output zone for receiving a reagent. The isolation buffer consists of oil that acts as a phase-gate and prevents unwanted material from passing through to the output well. The device employs an isolation mechanism using paramagnetic particles (PMPs), which preferentially bind the nucleic acids, and a lysis buffer, which inhibits the polymerase chain reaction until the nucleic acids have been isolated.

The isolation method proceeds through three main steps. First, the lysis buffer and PMPs are added to the biologic sample in the input well. Then, a magnetic field is used to draw the bound nucleic acid/PMPs through the phase-gate and into the output well, a process that lasts around 15 seconds. This simple phase-gate step replaces the multiple washes required in current methods. Finally, the nucleic acid/PMP reagent is collected for processing via polymerase chain reactions.

The device and method are configured to maximize isolation and purification of nucleic acids while maintaining compatibility with existing multiwell plates and liquid-handling robotic systems. Isolation and purification using this invention can be accomplished in approximately five minutes, including separation, device loading and sample collection. This provides a vast improvement over current kits, which require multiple washing steps and 15 to 45 minutes for purification.

Microfluidic Systems and Methods Applicable to In Vitro Fertilization

UW-Madison researchers have developed a microfluidic device and method that are applicable to in vitro fertilization techniques. The device comprises a substrate with a plurality of microchannels placed in the substrate so that each microchannel includes an inlet at one end to receive a cell and an outlet at the opposite end. Each microchannel also includes a restriction near the outlet to allow the cell to be contained as fluid passes through the channel. At least two microchannels make up an inlet on the substrate, which aligns with a fluid-handling device. The device may be constructed using microfabrication techniques such as injection molding, and may include an automatic top-off system using surface tension-based valve. This inexpensive top-off system reduces inaccurate liquid handling in the microfluidic system, which can lead to contamination of the sample.

The invention provides a flexible platform that allows microscale wells to be sized to accommodate a certain species’ cell size, as well as a fabricated insert to allow the specially sized wells to be included in the system. The system may be configured as a ready-to-use culture system tailored for particular applications, allowing improved embryo growth through optimized cell-to-cell communication. An open-closed-open-closed-open (OCOCO) system may be used for efficient cell culture and convenient sample access, which improves yield by eliminating damage to the sample due to over-handling.

Robust Biological and Chemical Detection Method and Microfluidic Device with Liquid Crystal Sensing Element

UW-Madison researchers have now developed an improved method for autonomously generating stabilized liquid crystal thin films and two microfluidic devices that employ the technique for detecting trace amounts of biological agents and chemical compounds.  The handheld microfluidic devices each contain a microchannel defined by grooved polymer materials sandwiched between glass substrates.  Priming the device involves filling the microchannel with liquid crystal material, which fills specific nickel-plated structures in the channel, and flushing the liquid crystals outside the container with the laminar flow of an aqueous solution.  This method allows for automatic formation and rapid regeneration of the stable aqueous/liquid crystal interface. 

In the presence of a target compound the orientation of the liquid crystals changes, altering optical properties of the liquid crystals through a phenomenon known as optical birefringence.  After the analyte has been introduced into the channel, a white light is passed through a first polarizing lens, the microfluidic device, and a second orthogonally oriented polarized lens.  The intensity of the light, determined by the degree of optical birefringence, is detected by a microscope to confirm the presence or absence of the specific target in the aqueous solution.

The microfluidic biological and chemical detection device with a liquid crystal sensing element allows for automatic formation of the sensing interface through its design and operation.  The device design also provides better control of the interaction between the aqueous target containing solution and liquid crystal region.  By providing a robust device and method, as well as reducing the need for advanced technical training, the improved detection apparatus will greatly enhance in-field applicability of biological and chemical sensor technologies.

Coupling Microfluidic Devices Yields Physiologically Relevant Micro-Environment for Cellular Assays

UW-Madison researchers have developed an improved device to create a controllable, physiologically relevant micro-environment for studying cellular interactions and pathways. This device provides a means for coupling two discrete microfluidic channels using only fluid contact. Two microchannels, each having one inlet and one outlet, can be coupled by combining fluid droplets on the outlet port and inlet port of the respective microchannel. This fluid contact method allows channels with two isolated environments to initiate the transfer of signaling molecules by means of diffusion or flow, thus allowing controllable physiological communication between cells. Cells can be exposed to a variety of cellular signals without cell contamination by simply breaking fluid contact with the current microchannel and forming a new fluidic coupling with a new microchannel. This new approach can be particularly useful in the co-culture of cells, where cell contamination can be prevalent and result in skewed data.

The controllable micro-environment implemented by this device also provides improved parameters for cellular assays and is well suited for high throughput screening. The reduction of culture dimensions in this microfluidic system results in a more physiologically relevant cellular micro-environment due to certain physical phenomena and interactions that become more dominant. The scale of microfluidic systems offer more precise control over parameters that affect the cellular micro-environment, including fluid shear stress, diffusion of soluble factors and patterning of cells and extracellular matrix (ECM). These parameters can influence cell development and signaling pathways. For example, shear forces can modulate stem cell differentiation pathways and/or apoptotic activity.

In addition, the fluid reduction that results from using a microscale, rather than a macroscale, culture system allows minimal use of expensive reagents. Less reagent use also increases the repeatability and reliability of assays and reduces the amount of time necessary to move cells and reagents in and out of channels.

Population-Averaged Method to Quantify Cell Motility and Migration

UW-Madison researchers have developed an alternative, microtechnology-based method for conducting cell mobility assays. This technique combines microfluidic gradient generation with micro-patterning to simplify the extraction of important migratory information. Rather than tracking individual cells, it uses parameters from the cell population as a whole.

A population of cells is labeled (e.g., with fluorescent dye) and patterned within a microchannel network so the cells are uniformly dispersed along the channel in the form of a generally rectangular strip. A predetermined medium that includes a migration- or motility-promoting signal is patterned along one sidewall of the channel. Then a first image of the population is obtained. After a predetermined time period, a second image is obtained and compared to the first. Simple mathematical processing of the images yields quantitative measurements that can be used to calculate the average directional migration and motility of the cell population.

Device for Improved Cell Staining and Imaging

A UW-Madison researcher has developed a multichannel microfluidic device that increases the efficiency of sample preparation for cell imaging. The microfluidic device includes a cartridge with a patterned inlet port to contain reagent droplets and avoid cross-channel contamination. The lower surface of the cartridge includes several recesses that define channels for containing cells. Different reagents can be added to each channel. The upper surface of a cover slip is placed against the lower surface of the cartridge, and the cartridge is mechanically clamped to the cover slip to ensure no leakage during an assay. After staining is complete, the cover slip can be removed and traditionally mounted to a glass slide for standard imaging protocols.

Microfluidic Device for High Resolution, In Vitro Monitoring of Neuronal Tissue

UW-Madison researchers have developed an in vitro device for examining and testing a slice of brain tissue, which delivers precise amounts of chemical stimuli to neurons with a high degree of spatial and temporal resolution. The device combines the localized drug delivery capability of microfluidics with multi-channel neural recordings.

The device includes a chamber and microfluidic channels that communicate with the chamber. A support structure within the chamber holds the slice of brain tissue and includes an array of electrodes that engages the slice, allowing for multi-channel electrical recording and stimulation of the slice at each of the electrode sites within the microfluidic channels. The microfluidic channels allow many parallel, but independent, laminar fluid streams to flow across the surface of the brain slice. Special valves enable a highly focused stream of chemicals to flow across the slice at any desired location while being pulsed with high temporal resolution.

Method of Performing Gradient-Based Assays in a Microfluidic Device

UW-Madison researchers have developed a method for performing gradient-based, high throughput assays in a microfluidic device. The method involves passing two fluids through a channel in a microfluidic system. The first fluid contains a predetermined concentration of particles. Particles from this fluid diffuse into the second fluid, creating a concentration gradient of particles in the second fluid as it flows through the channel. The second fluid intersects a series of targets along the channel wall. Because a gradient in a microchannel is equivalent to a multi-well plate containing thousands of wells, this method is simpler, faster, more efficient and less expensive than traditional high throughput assay systems.

Method and Apparatus for Measuring the Environment within a Microfluidic Device

UW-Madison researchers have developed a simple and inexpensive apparatus for monitoring the environment within a microfluidic device. The apparatus displays a positive/negative type of readout and is directly fabricated within the microchannel. The readout structure, which consists of dyes imbedded within a hydrogel matrix, functions as both a sensor and a display unit. When specific agents are present, the hydrogel structure swells and releases the dye molecules. Both the size and color of the readout structure change in response to the presence of a predetermined amount of a specific chemical or agent.

Method and Device for Sensing Microfluidic Flow

UW-Madison researchers have developed an improved flow sensor for microfluidic channels. A cantilever beam is placed inside the microchannel at a slight angle to the channel. The flow of fluid around the beam results in different drag forces on the two sides of the beam and causes the beam to bend. From the amount of beam bending, which can be measured optically, a flow rate is calculated.