Patent Application
20110033922
The advent of microdevice technology for biochemical and chemical analysis has begun to revolutionize the analytical measurement sciences. While the microchip revolution is rooted in ultrafast separations, recent forays seek to move laborious and time-intensive steps for sample collection, lysis, extraction, and reaction to microchips1-5. A number of emerging “lab-on-a-chip” systems have been described to address sample preparation issues, this has been extended to the field of cellomics—the manipulation of cells, and even single cells, in microfluidic devices. Developments in this area will be key to the achieving a more complete micro-total-analysis system (μ-TAS).
Cell manipulation on microdevices has been demonstrated extensively. For example, Ramsey and colleagues demonstrated transport and lysis of cells on microfluidic devices.6 Cell lysis, induced chemically or with an electric field, was utilized to release cell lysate for further analysis. Similarly, Harrison et al. developed a microchip-based method for detection of cell lysate including the use of enzymatic reactions.7 Cell separations on microdevices have been demonstrated by a number of means, including the use of electric fields or other physical means. Sorting of sub-cellular components has been demonstrated on a microdevice using isoelectric focusing, in which the pH gradient is set up across a channel and the flow (containing electric field have also been described extensively, using electroosmotic flow for fluid flow throughout the device.9, 10 Additionally, dielectrophoresis has been shown effective for separation and manipulation of cell and bacteria in microdevices.11, 12, Arai, 2001 #56 13 Micro-fluorescence activated cell sorters have been developed for sorting fluorescently-labeled cells in microdevices.14-16 A microdevice cell separation method by application of an electric field and introduction of a filter, without damage to the cells, has been invented by Yasuda.17 Filters, in particular, have been utilized extensively for trapping various cell types, which utilize the adhesiveness of white blood cells to further enhance the separation.18, 19 A separation based upon differential settling and adsorption of sperm and epithelial cells in microfluidic devices has been demonstrated.20
Particles subjected to acoustic waves are influenced by acoustic radiation forces, which are particularly strong in standing wave fields21. The forces can be divided into axial and transverse components of the primary radiation force, and secondary particle-particle interactions due to scattering of incident waves22. The acoustic properties of the particulate material as compared to the surrounding medium determine whether the primary radiation force is directed towards the pressure nodes or antinodes in a standing wave. The magnitude of the radiation force is proportional to the acoustic frequency22 and for particle manipulation it is therefore advantageous to increase the frequency to the ultrasonic region. Consequently ultrasonic has successfully been used to manipulate particles or biological material, e.g. as acoustic tweezers23 and for particle separation from continuous fluid flow in macro-24 and microscale devices.25 Two-dimensional trapping and manipulation of microorganisms has been performed using orthogonal standing waves.26 Size-selective ultrasonic trapping of microbeads in capillaries has also been investigated in order to allow separation of immunocomplexes for trace-amount protein detection.27, 28 Other bio-related applications making use of acoustic forces include separation of fat from blood during cardiovascular surgery29 and the retention of mammalian cells in cell culture fermentations.30 The combination of acoustic trapping and microsystems has been examined for development of bead based bioassays.31, 32
Patents using ultrasonic radiation to separate cells have been shown in U.S. Pat. Nos. 6,332,541 to Coakley et al. (the '541 patent); 6,929,750 to Laurell et al. (the '750 patent); and 7,108,137 to Lal et al. (the '137 patent); the disclosures of which are incorporated herein by reference. The '541 and '750 patents are drawn to cell separation by applying an ultrasonic wave in a direction orthogonal to the direction of flow. This system separates the cells but does not trap it at a particular location within the channel. The '137 patent apply acoustic radiation in a longitudinal direction, and therefore, does aggregate cells at various locations along the flow path rather than at a single define position directly above the transducer.
With an interest in creating a μ-TAS for totally-integrated analysis of DNA evidence from forensic sample, a focused effort has been invested in the development of microminiaturized fluidic device for isolating sperm cells from sexual assault evidence. The benefit of such an invention is that the separation of cells, such as sperm from cell mixtures, in the conventional analysis of sexual assault evidence is time-consuming, labor-intensive and results in relatively poor separation efficiency.33 Conventional differential extraction (DE)—the separation of male (sperm cell) and female (epithelial cell) DNA is the currently accepted method and is based on that proposed by Gill34 which has been morphed into the various protocols used today in crime laboratories. This method yields separate fractions of male and female DNA, essential to obtaining DNA profiles from both a victim and perpetrator in a forensic sexual assault case. Conventional DE is a chemical process that induces the differential lysis of the cells by exploiting the differences in the stability of the cell membranes on sperm and epithelial cells. This method is initiated by lysing the vaginal epithelial cells under mild conditions that allow the sperm cells to remain intact. The intact sperm cells (predominately the heads, as tails are solubilized under mild lysis conditions) are pelleted by centrifugation, allowing the now released DNA from the epithelial cells to be removed in the supernatant. The pelleted sperm cells are then resuspended and lysed in a buffer that contains dithiothreitol (DTT), a reagent that reduces disulfide bonds on the sperm cell surface, and the DNA is extracted independently. This method typically requires a minimum of 3 hours, and is often allowed to incubate overnight. While this method has been used for a number of years, the presence of female DNA in the sperm cell fraction leads to co-amplification of the female alleles which complicate the genetic fingerprint provided by the male DNA.33 This creates difficulties with the interpretation of evidence and compromises the effectiveness of connecting the DNA fingerprint to the perpetrator (lowers the probabilities when presenting statistical data) in court proceedings. Aspects of various embodiments of the present invention described herein seek to improve the purity of the male and female fractions obtained, so as to improve forensic DNA analysis of sexual assault evidence.
The use of acousto-trapping for isolation of sperm cells from the biological mixture (sexual assault evidence) significantly reduces analysis times and, perhaps most importantly, increases sample purity. While this technology is described for cell selection/capture in fluidic microdevices known for manipulation of nanoliter-picoliter volumes, the mechanism for sperm cell capture from a cell mixture it selective under conditions that allow for high volumetric flow rate and, hence, milliliter volume samples. This concept is supported by the already demonstrated application of lipid removal from recovered blood during thoracic surgery35 is highly amenable to multiplexing, which allows for analysis of sample sizes ranging from hundreds of microliters to milliliters in minutes. Moreover, with the microscalar fluidic structures used in these devices, multisample capability can easily be added. Greater purity of the sperm cell fraction results in an increased likelihood for identification and prosecution of the perpetrator in sexual assault casework. In addition, greatly enhancing the rate of evidence analysis will diminish the backlog of cases awaiting DNA analysis in many criminal laboratories.
An aspect of various embodiments of the present invention is to, but not limited thereto, utilize acoustic standing waves in a fluid-filled microchannel in an analytical microchip device to create a chip-based acoustic differential extraction (ADE) microdevice. This device will allow for the selective isolation of cell, preferably sperm cells, from small or large volumes flowing streams containing the cells and cellular material obtained, for example from forensic evidence.
The advantages of the current system include: 1) the rapid manner in which cells can be trapped in the near field of the ultrasonic transducer, 2) its ease of use, 3) the effectiveness for isolating a pure cell fraction from evidentiary material in forensic samples, 4) the effectiveness relative to separation of sperm cells from other biological material in sexual assault evidence by conventional means, 5) the ability to separate free DNA from a cellular mixture, 6) its versatility in handling microliter or milliliter scale samples (hence, large volume reduction), 7) its tenability for selective cell capture, 8) the concentrating effect that stems from its ability to capture cells from large volumes (milliliters) and release them in extremely small volumes (microliters-nanoliters), 9) amenability to multi-sample analysis (multiplexing), 10) the ability of the microchip to prevent contamination of evidentiary material, 11) the ability define configuration wherein the transducer is part of a platform and not part of the chip and 12) the low cost and disposability of the chip.
The present invention provides a method and apparatus for separating by size a mixture of different size particles using ultrasound. The apparatus contains a microchannel having an acoustic transducer thereon. As a mixture of cells having different sizes flows down the microchannel, the ultrasonic radiation, applied in a direction perpendicular to the flow, traps cells focused at nodes of a standing pressure wave in the microchannel, directly above the transducer. The size selection of the cell to be trapped is based on the trapping force of the ultrasonic radiation which can be tuned to trap the desired cell size.
Further objects, features and advantages of the invention will be apparent from the following detailed description when taken in conjunction with the accompanying drawings.
In an embodiment, the ultrasonic transducer 102 is fabricated using a screen-printed PZT-multilayer device. The detailed description of the actuator fabrication and means of contacting is given in Lilliehorn et al.31, which is incorporated herein by reference.
During use a cell mixture, preferably in a fluid media, flows into the microchannel 100 using, for example a pump. An acoustic radiation is applied the direction of the axis of the microchannel 100 generating a standing pressure wave 600 (see
The standing wave is set by the distance between the transducer surface and the reflector surface, which defines the fundamental acoustic resonance mode of a half wavelength standing wave in the microchannel. In the acoustic trapping device for the present invention the half wavelength distance is preferably approximately 61 μm, which corresponds to a 12.4 MHz fundamental resonance criterion.
In one embodiment, the microchip can be designed such that the transducer element is part of a separate platform that does not come into fluidic contact with the forensic sample. In this embodiment, when trapping cells, the microchip substrate (e.g. glass) would be positioned in contact (either permanently or temporarily) with the transducer element, thus decreasing the chances of sample contamination, while making the microchip more cost-effective and, perhaps, disposable. In this case, the microchip is separate from the transducer and does not form the bottom of the microchannel as illustrated in
The dimension of the channel (transducer to reflector distance) defines the fundamental resonance of the resonator. The acoustic trapping force is directly proportional to the standing wave frequency and thus with a reduced distance between the transducer and the reflector the higher the fundamental resonance frequency will be and consequently a higher acoustic trapping force is obtained. The width of the microchannel, a priori, is not a limiting factor and, thus, if a higher capacity is needed more material can be trapped by a wider transducer. On the other hand, channels that are too wide may eventually compromise the benefits of a microfluidic format.
Preliminary experiments show that the cells are initially clustered in a monolayer. Others have reported the same observation in macroscopic acoustic traps.36 When operating the device, the particles and/or cells are collected in a single layer, enclosing several hundred or even thousands of particles/cells (of course depending of the spatial size of the trapping region). As the trap becomes overloaded, multiple layers and aggregates are formed.37 This is significant as it pertains to the effectiveness of this method for trapping cells from forensic or biological samples. The predominately planar accumulation of cells decreases the potential for contamination of the collected cells with other biomolecules. For example, as it pertains to the isolation of sperm cells from sexual assault evidence, any free DNA from lysed female cells (e.g., epithelial cells or white blood cells) is less likely to be nonspecifically trapped in a planar, monolayer-like grouping of cells than would be expected with a three-dimensional cluster of cells (where much opportunity would exist for trapping of free DNA. Moreover, the planar collection of cells can be washed extensively with whatever reagents are desired in order to diminish any trapping of free DNA. This latter embodiment describes the acousto-differential extraction (ADE) device. A generalized description of the apparatus used is seen in
One embodiment of a method for ADE involves the step of the conventional DE prior to injecting the sample into the microchannel. Vaginal epithelial cells would be selectively lysed (e.g., by the procedure described by Gill et al.34) and, thus, the sperm cells trapped from a biological mixture containing epithelial cell lysate. Sperm cells (and other particulate matter) are trapped in the standing wave of the ultrasonic transducer, while DNA from the lysed cells is not trapped, but carried with the fluid flow in the channel. Once the epithelial cells are lysed, according using the Gill buffer or other means, sample is flowed (using a syringe pump or other means depending upon sample volume) into the microchannel, where flow is directed over the transducer(s).
A second embodiment of a method for ADE does not require that the cells be lysed but, instead, separates them from the sperm cells intact by trapping at a second transducer. In this embodiment, various cell types could be trapped by a series of transducers. The force acting upon the particle, as described in equation 1, illustrates the utility of the method for trapping particles of various physical properties in the various standing waves.
Where P0=the applied acoustic pressure amplitude
Vc=particle volume
βw=compressibility of the liquid
βc=compressibility of the particle
λ=wave length of the sound wave
z=particle distance to the node
δc=density of the particle
δW=density of the liquid
Φ(β, ρ)=the acoustic contrast factor
Fr=the acoustic radiation force
As previously described, the trapping force is dependent of the distance between the transducer surface and the reflector a smaller distance yields a higher trapping force. This is a fundamental approach to control the trapping efficiency (a smaller channel height results in a higher resonance frequency and thus a better trapping force). The force is also highly dependent of the size of the particle to be trapped and is, for each cell-type, essentially a fixed parameter. The next factor in equation 1 to take into account is the Φ-factor (commonly referred to as the ‘acoustic contrast factor’), which is defined by the densities of the carrier fluid, the particle and the ratio of the compressibility's between the carrier fluid and the particle (equation 2). The parameters to modulate, from an engineering perspective, involve defining the carrier fluid with respect to compressibility and density. In ultracentrifugation work, the carrier media is selected with respect to suitable density. Likewise, in the acoustic trapping experiments, selection can be made in a similar manner with respect to fluid density, but now, fluid compressibility is an additional parameter that can be used optimize the trapping capability of the system. Another alternative is to use the much stronger forces acting on the larger cells (e.g., epithelial cells) to induce a selective trapping. This could be achieved by finding the threshold where the magnitude of the acoustic forces are strong enough to trap epithelial cells but don't effect smaller cells (e.g., sperm cells). Consequently, as it pertains to the separation of epithelial cells from sperm cells, epithelial cells would be trapped in the standing wave generated by one transducer, while sperm cells are trapped in the standing wave generated by a second transducer in a spatially-distinct part of the microfluidic architecture. Such selectivity can be obtained by tuning the amplitude output of the waveform generator with the physical properties of the cell types.
Another embodiment of this method involves the trapping of cells, as described earlier, and release of cells for further processing on the microdevice, including, but not limited to, cell lysis and DNA extraction. In this embodiment, the cell trap of the present invention can be used with other existing microfluidic apparatus including those disclosed in U.S. Patent Application Publication Nos. 2006/0084185, 20050287661, 20040131504, all to Landers et al. and are incorporated herein by reference.
Other than the cell trapping site, microfluidic devices may also include micromachined fluid networks. Fluid samples and reagents are brought into the device through entry ports and transported through channels to a reaction chamber, such as a thermally controlled reactor where mixing and reactions (e.g., synthesis, labeling, energy-producing reactions, assays, separations, or biochemical reactions) occur. The biochemical products may then be moved, for example, to an analysis module, where data is collected by a detector and transmitted to a recording instrument. The fluidic and electronic components are preferably designed to be fully compatible in function and construction with the reactions and reagents.
There are many formats, materials, and size scales for constructing microfluidic devices. Common microfluidic devices are disclosed in U.S. Pat. Nos. 6,692,700 to Handique et al.; 6,919,046 to O'Connor et al.; 6,551,841 to Wilding et al.; 6,630,353 to Parce et al.; 6,620,625 to Wolk et al.; and 6,517,234 to Kopf-Sill et al.; the disclosures of which are incorporated herein by reference. Typically, a microfluidic device is made up of two or more substrates or layers that are bonded together. Microscale components for processing fluids are disposed on a surface of one or more of the substrates. These microscale components include, but are not limited to, reaction chambers, electrophoresis modules, microchannels, fluid reservoirs, detectors, valves, or mixers. When the substrates are bonded together, the microscale components are enclosed and sandwiched between the substrates.
Other cells of forensic importance (and often encountered in evidentiary material) include microorganisms. In another embodiment of the method described, these cells may also be isolated by trapping or selectively not trapping these cells. For example,
A number of designs can be envisioned for the ADE chip and, accordingly, there are a number of different approaches that could effectively lead to recovery of the trapped sperm cells can be from the forensic sample.
A potential protocol for assembling an ADE microdevice, as represented by a glass microfluidic chip bonded to the transducer chip, is as follows:
A method trapping sperm cells from a biological sample with an ADE microdevice, as represented by a transducer bonded to, e.g., a glass microfluidic chip, is as follows:
The removal of cells, materials, analytes, etc., from these devices should be appreciated by and are with in the capability of those skilled in the art.
Although certain presently preferred embodiments of the invention have been specifically described herein, it will be apparent to those skilled in the art to which the invention pertains that variations and modifications of the various embodiments shown and described herein may be made without departing from the spirit and scope of the invention. Accordingly, it is intended that the invention be limited only to the extent required by the appended claims and the applicable rules of law.
The following references are hereby incorporated by reference herein in their entirety:
This application claims the priority of U.S. Provisional Patent Application Ser. Nos. 60/723,551, filed Oct. 4, 2005 and 60/776,751, filed Feb. 24, 2006.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US2006/038943 | 10/4/2006 | WO | 00 | 6/30/2010 |
| Number | Date | Country | |
|---|---|---|---|
| 60723551 | Oct 2005 | US | |
| 60776751 | Feb 2006 | US |
