BIOMEMES

Compact Disc Microfluidics

See videos and respective publications.

CD Microfluidics is a sub-field of microfluidics that deals with the behavior, precise control and manipulated of fluids in the micro-domain. CD Microfluidics takes advantage of centrifugal forces for fluid propulsion. Fluidic channels and reservoirs are embedded in a CD-like plastic substrate and the whole platform is spun on a motor in order to manipulate fluids. A whole range of fluidic functions have been designed and implemented which include valving, decanting, calibration, mixing, metering, sample splitting, and separation. Those fluidic functions have been combined with analytical measurement techniques, such as optical imaging, absorbance, and fluorescence spectroscopy and mass spectrometry, to make the centrifugal platform a powerful solution for medical and clinical diagnostics and high throughput screening (HTS) in drug discovery. Applications of a compact disc (CD) based centrifuge platform include two-point calibration of an optode-based ion sensor, an automated immunoassay platform, multiple parallel screening assays, and cellular-based assays.

Advantages of the microfluidic CD

  • Multiplex
  • Automation
  • Simple
  • Compatible with wide range of samples
  • High throughput
  • Mass Producible
  • Fast Development (Rapid Prototyping)
  • Low Cost (< $1/disc)

In order to perform complex fluidic manipulations, required for Lab on CD, our group has developed an optimized various capabilities, including valving (capillary valve, Coriolis valve, syphon valve, serial syphon valve), reciprocal mixing, separation (such as DEP on the CD). These technologies are described below.

Valving Technologies (capillary, hydrophobic, siphon)

Valving is a most important function in any type of fluidic platform. Both hydrophobic and capillary valves have been integrated into the CD platform. Hydrophobic valves feature an abrupt decrease in the hydrophobic channel cross-section, i.e., a hydrophobic surface prevents further fluid flow. In contrast, in capillary valves, liquid flow is stopped by a capillary pressure barrier at junctions where the channel diameter suddenly expands.

In hydrophobic valving, for liquid to move beyond these pressure barriers, the CD must be rotated above a critical speed, at which point the centripetal forces exerted on the liquid column overcome the pressure needed to move past the valve.

Capillary valves have been implemented frequently on CD fluidic platforms. The physical principle involved is based on the surface tension, which develops when the cross section of a hydrophilic capillary expands abruptly.

One of the main challenges to integration for sample-to-answer microfluidic systems is the requirement for serial valving to allow the sequential release of fluids in a temporally and spatially controlled manner. The advantages offered by centrifugal microfluidic platforms make them excellent candidates for integration of biological analysis steps, yet they are limited by the lack of robust serial valving technologies. This is especially true for the majority of centrifugal microfluidic devices that rely on hydrophilic surfaces, where few passive serial valving techniques function reliably. Building on the useful functionality of centrifugal microfluidic siphoning previously shown, a novel serial siphon valve is introduced that relies on multiple, inline siphons to provide for a better controlled, sequential release of fluids.

The serial siphon is shown to be robust and reproducible, with variability caused by the dependence on contact angle, rotation velocity, and fluidic properties (viz., surface tension) significantly reduced compared to current microfluidic, centrifugal serial valving technologies. This design withholds fluids through multiple high and low speed operations and allows the fluids to be subsequently distributed to areas of interest. The serial siphon valve has numerous potential applications, and will be especially useful in the development of IVD systems for NA analysis. Integrated microfluidic NA diagnostic devices can be quite complex, and the avail- ability of a simple and passive serial valve will eliminate the need for many of the more complex valving solutions.


Applications

Reciprocating Centrifugal Microfluidics Mixer

Proper mixing of reagents is of paramount importance for an efficient chemical reaction. While on a large scale there are many good solutions for quantitative mixing of reagents, as of today, efficient and inexpensive fluid mixing in the nanoliter and microliter volume range is still a challenge. Complete mixing is of special importance in any small-scale analytical application where the scarcity of analytes and the low volume of the reagents demand that all of the available analytes and reagents are efficiently used. We have demonstrated the design and fabrication of a novel centrifugal force-based unit for fast mixing of fluids in the nanoliter to microliter volume range (Noroozi et.al, Review of Scientific Instruments, vol. 80, no. 7, pp. 075102-8(2009)). The device consists of a number of chambers (including two loading chambers, one pressure chamber and one mixing chamber) that are connected through a network of microchannels, and is made by bonding a slab of polydimethylsiloxane (PDMS) to a glass slide. The PDMS slab was cast using a SU-8 master mold fabricated by a two-level photolithography process. This microfluidic mixer exploits centrifugal force and pneumatic pressure to reciprocate the flow of fluid samples in order to minimize the amount of sample and the time of mixing. Our results suggest that quantitative mixing was achieved in less than three min. This device can be employed as a stand-alone mixing unit or may be integrated into a disk-based microfluidic system where, in addition to mixing, several other sample preparation steps may be included.

[go to] Z. Noroozi, et al., "Reciprocating flow-based centrifugal microfluidics mixer," Review of Scientific Instruments, vol. 80, p. 075102, 2009.



Sample Lysis

Tissue homogenization and cell lysis are the first steps in virtually all molecular biology and molecular diagnostic techniques. The purpose of cell lysis is to disrupt the cells and release genomic and proteomic material to enable downstream processing. Cell lysis and subsequent nucleic acid or protein purification represent a considerable obstacle commonly encountered in molecular biology sample preparation protocols. Although easily attainable with certain cells and microorganisms, many cells of interest pose significant challenges due to increased cell wall structural integrity. Molecular biology sample preparation kits and equipment are part of a rapidly growing multi-billion dollar market. It has been estimated that up to 15% of researchers’ time is consumed with the simple and redundant task of sample preparation.

The most efficient and elegant method of mechanical cell lysis is bead-beating. Bead-beating is accomplished by combining a liquid or solid sample with milling beads in a closed container and exposing the entire mixture of grinding media, buffer and tissue to intense mixing by rapid and abrupt motion of the container. If a component of angular motion is introduced into a bead-beating system, then additional lysis results from impaction and friction between cells and the lysing matrix as a result of the Coriolis effect. The lysing matrix directly contributes to lysis again during centrifugation, as observed previously in a centrifugal CD-based homogenizer.

No currently commercially available sample lysis and preparation system can handle samples in the microliters volume range. The novel magnetohydrodynamic disk-based homogenization system described herein successfully combines microfluidics, magnetically actuated cell lysis, and lysate clarification by centrifugation in a single device. The device consists of a simple rotary magnetic field superimposed on a rotor-based, disposable CD-like disk containing sample chambers preloaded with lysing matrix and a ferromagnetic blade. The device yields clarified lysates from even the most difficult to lyse microorganisms utilizing up to 70 ’L of substrate. Thus, the magnetohydrodynamic disk-based homogenization system is a feasible solution for efficient lysis on a microscale. Based on results with E. coli and S. cerevisiae we demonstrate that this system can be utilized for semi-automated molecular biology sample preparation on a micro and nano scale sample volume to provide genomic materials. The CD-like magnetically actuated centrifugal lysis system described herein has the additional advantage of facile integration with other CD- based microfluidics analytical systems, and as such promotes the novel concept of an entire molecular diagnostics microfluidics laboratory on a disk.

Separation using Dielectrophoresis on the CD

Dielectrophoresis (DEP) enables the selective manipulation of a targeted particle, or population of particles, using the interaction of a non-uniform electric field with the induced effective dipole moment of the particle(s). DEP is advantageous over other particle separation techniques such as FACS (Fluorescence-activated cell sorting) and MACSÒ (Magnetic-activated cell sorting) because discrimination between different particles is based solely on their intrinsic physical properties. These physical properties determine the particle’s dielectric properties and give it a characteristic dielectric phenotype. The use of DEP eliminates functionalized magnetic beads or fluorophores required by other separation techniques such as magnetophoresis and flow cytometry. The elimination of these often expensive labels reduces assay complexity, time and costs which can expand the availability of diagnostic tests, such as for HIV, and make therapies for cancer or degenerative diseases available to a broader number of patients.

The use of centrifugal forces for fluid pumping and fluid manipulation improves traditional DEP platforms in terms of footprint, cost, robustness and practicality. 3D carbon-electrode DEP, or carbon-DEP, offers key advantages over the use of other DEP techniques including a simple and inexpensive fabrication process. The implemented modularity allows for the use of interchangeable disposable chips depending on the type of assay or study to be conducted. Other electrokinetic-based applications besides filtering are targeted including cell, microorganism and biomolecule sorting and cell electroporation for DNA release and transfection.

[go to] R. Martinez-Duarte, et al., "The integration of 3D carbon-electrode dielectrophoresis on a CD-like centrifugal microfluidic platform," Lab on a Chip, vol. 10, pp. 1030-1043, 2010.


Alternative Pumping/Propulsion Technologies

Enabling technologies described above are utilized in the several diagnostic applications which are developed by our group. One example of an enabling technology is pneumatic pumping.

Centrifugal microfluidics platform, despite its many advantages, has a size limitation due to the centripetal pumping mechanism in which fluids can only be moved from the center of the disc to the rim. This limits the footprint of the microfluidic network to one radius of the disc, and this in turn limits the amount of space available to embed complex assays. In order to overcome this space limitation problem, we are developing new techniques to pump fluids back toward the center of the disc as to allow greater path lengths for the fluidic network.

One novel pumping mechanism for centrifugal microfluidics utilizes a combination of centrifugation and pneumatic compression. Pneumatic energy is stored during high-speed centrifugation with sample fluids trapping then compressing air in specially designed chambers. The accumulated pneumatic energy is released by spinning down, which expands the trapped air and thus pumps liquids back toward the center of the CD. This newly developed method overcomes current limitations of centripetal pumping avoiding external manipulation or surface treatments.

We have applied pneumatic pumping to siphon priming. In CD microfluidics, siphoning is often used to pump fluids; for example, to define sample volumes. Additionally, siphon designs in centrifugal microfluidics provide a valving solution for CD applications that require high spin speeds during initial steps of the operation (e.g., plasma separation from whole blood). In a pneumatic pump design, fluid is pumped toward the CD center as a result of the release of stored energy created during rotation. Spinning the CD propels sample fluids toward the CD rim causing the intake sub-compartment to fill. As the fluid continues to flow, the sample liquid traps air in a compression sub-compartment. In case the CD rotation speeds are kept low, the fluid loads the pneumatic chamber without any compression of the trapped air and immediately primes the siphon. However, if rotational speeds are high enough, the centrifugal force exerted on the fluid pressurizes the trapped air. At a critical rotation frequency, the fluid levels in the two sub-compartments nearly equalize and therefore at that point the maximum air compression is achieved. If the rotational frequency is reduced now, the centrifugal force on the sample fluids becomes lower and fluid is pumped back toward the center of the disc due to the relaxation of the pressured air in the compression reservoir. By continued spinning at low speeds, the fluid overcomes the siphon crest and primes the siphon. Slightly increasing the rotational frequency breaks the capillary valve at the end of the siphon capillary and causes fluid to enter into the collection chamber. Continued centrifugation at a yet higher frequency completely empties the pneumatic pumping chamber.

Anthrax Detection on the CD

Anthrax is an acute and deadly disease caused by the bacterium Bacillus anthracis. If not quickly detected and treated, humans can die within days. With renewed bio-terrorism concerns, there is a need for a rapid and automated system capable of sample (respiratory or blood) to answer (positive infection) so that quarantine procedures and treatment can be administered immediately. This project aims to combine the previously developed Cell Lysis CD and PCR Card to create a microfluidic sample-to-answer system capable of processing raw respiratory samples in under 1 hour. Real-time PCR is being utilized for amplification and detection along with a bead-beating cell lysis step. In addition, the necessary CD spin-stand hardware (e.g., heaters and fluorescence detection system) are being developed and automated. In collaboration with Texas A&M, critical aspects of the device are being modeled to optimize the entire system. This project promises to show the true feasibility of a completely integrated CD system. This project is funded by the MF3 center at UCI.

Microfluidics for In-Vitro Diagnostic (IVD) Analysis

During the past ten years, research on developing nucleic acid- (NA) and protein-based IVD tests using microfluidics has skyrocketed and has been primarily driven by the rapid progress in molecular biology and molecular diagnostics. The complex challenges involved in producing microfluidic systems for molecular diagnostics are usually addressed by developing separately the various pieces of equipment that handle specific individual steps within the total sample-to-answer process. However, this development approach places little or no emphasis on sample preparation, and does not consider how and from where a sample is obtained.

The neglect of sample preparation is one of the most significant pitfalls that have prevented the widespread use of portable, integrated, microfluidic systems for IVDs and nucleic acid IVDs in particular. In order to design successfully a sample-to-answer system for nucleic acid IVDs, the desired system's specifications and characteristics must be established. Doing so requires a fundamental understanding of both the engineering and biological sides of the system, particularly when discussing the sample preparation and amplification steps.

Microfluidic platforms have the potential to tackle and integrate the NA diagnostics steps including sample collection, sample preparation, amplification and detection. There are numerous demonstrations of the benefits gained by moving from a typical wet-bench set-up to a microfluidic device. Such benefits include reduced reagent use, significantly decreased total processing time, increased abilities for parallel processing, and reduced process variability via automation.

When developing any fluidic devices, the main concern is how to get liquids to and from the areas of interest in a controlled manner. This general problem can be encapsulated by the need for two related technologies: pumps and valves. The centrifugal platform based on the CD format provides simple and effective modes for pumping and valving. Centrifugally induced pressure on the fluid as the CD spins causes fluid propulsion on the CD. Centrifugal pumping forces on the CD provide many advantages compared with other alternative pumping methods, such as syringe, peristaltic, and electroosmotic pumping. While pressure-driven syringe and peristaltic pumps provide good control over large flow rates, they can be unwieldy when trying to miniaturize and/or conduct parallel processing. In addition, the pressures needed to move fluids through the microchannels do not scale well at 1/r4 so they become very large in the micro domain, which makes implementing the pumps into small, high-throughput platforms difficult.

 

Novel Liquid Handling and Storage

A novel active valving technique, whereby paraffin wax plugs in microchannels on a centrifugal microfluidic platform are actuated using focused infrared (IR) radiation was developed by our team. Microchannels can be simultaneously or sequentially opened using a stationary IR source by forming wax plugs with similar or differing melting points. Key advantages of these techniques include a less involved fabrication procedure, a simpler actuation process, and the ability to multiplex experiment with active valves. In addition, we developed a new technique for automated liquid reagent storage and release on the microfluidic disc platform, based on the formation and removal of a wax layer.

 



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