Patent Application: US-8932006-A

Abstract:
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 traps cells of desired sizes focused at nodes of a standing pressure wave in the microchannel .

Description:
fig1 shows a preferred embodiment of the present invention . the apparatus contains a microchannel 100 having at least one cell trapping region 104 . the cell trapping region 104 contains an acoustic transducer 102 , preferably at the bottom of the microchannel as shown , and a reflector 106 , preferably a glass reflector . in certain embodiments , multiple cell trapping regions are located along the flow path of the microchannel 100 , where each flow path aggregates a different cell size . 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 . fig2 shows a schematic of a multilayer ultrasonic transducer 102 containing a transducer element 206 with an external silver electrode 200 connected to the circuitry on a printed circuit board 202 with conductive silver epoxy 204 . the board 202 is covered with epoxy 206 . other ultrasonic transducers may also be appropriate for the present invention , including those described in the &# 39 ; 750 , &# 39 ; 541 , or &# 39 ; 137 patent . 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 fig6 ). the standing pressure wave 600 contains a node 602 and antinodes 604 that trap the desired cell particles . in a most preferred embodiment , the thickness t of material above the channel is an odd number of ¼ wave length [( 2n + 1 ) λ , where n is a whole number ], and the height h of the channel is ½ wavelength ( λ / 2 ). the selectivity of the system may be tuned a described below . 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 fig1 . as such , the chip , having the microchannel therein , does not have to be fabricated with an attached and expensive transducer . it is important to properly design the microfluidic chip so that when it is placed on top of a transducer , acoustic radiation can be delivered into the microchannel through a thin glass . fig7 illustrates this embodiment where the microfluidic chip 700 sits on top of a transducer 702 , where a bottom layer 704 of the chip 700 separates the microchannel 706 from the transducer 702 . for proper function and delivery of the acoustic radiation to the channel , it is critical that the thickness t 2 of the bottom layer 704 should be odd number of ¼ wavelength [( 2n + 1 ) λ , where n is a whole number ]. in this embodiment , the microfluidic chip is not physically attached to the transducer , but is only placed on top of the transducer when it is in operation . 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 fig3 . in this device , the buffer is introduced to the main channel 310 through the buffer inlet 300 and sample introduced at the sample inlet 302 . cells are trapped in the trapping region 312 . trapped cells are collected by initiating flow from side channel inlet 304 to side channel outlet 306 , where trapped material is collected for further sample processing . the untrapped material is collected at the main channel outlet 308 . 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 . 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 &# 39 ; 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 &# 39 ; 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 &# 39 ; 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 , fig5 shows the trapping of bacteria from a mock sexual assault sample in the antinode of the transducer . 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 : 1 ) a glass chip is fabricated to have a channel depth that corresponds to half a wavelength of the desired working frequency of the ade ( at current working frequency of 12 . 4 mhz that is 61 μm ). the configuration of the microchannel above the transducer does not need to be straight walled , and can have the u - shaped channels commonly found in etched glass devices . the reflective surface above the transducer needs however to be planar to ensure a good reflected wave . 2 ) the transducer chip , fabricated by the method previously reported 31 is bonded to the chip by the use of a hydrogel as an adhesive . the chip contains the transducers and the electrical wiring to actuate the transducers at the desired frequency . 3 ) one approach to ensure a tight fit the transducer chip and the glass channels is to hold them together with a brass fixture . however , this would not be needed if any one of a number of bonding processes were carried out to adhere the transducer chip to the glass . 4 ) valves can be incorporated into the microfluidic architecture to control the flow of solutions and cells through specific , predefined fluidic paths for spatial separation and capture of cell and fluid fractions . there are a number of different valving approaches that could be used for this including physical valving , 38 , 39 electrokinetic valving , 40 passive valving as detailed in duffy et al ., 41 and passive flow control with fluidic diodes , capacitors , inductors and band pass filters . 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 : 1 ) cells obtained from forensic evidence ( examples include but are not limited to vaginal swabs and bedsheets ) in an elution buffer ( i . e ., phosphate buffered saline , gill buffer , or other liquid ) are perfused into the microdevice channels using a syringe pump or other pumping means . 2 ) the sperm cells are trapped in the standing wave of the transducer . 3 ) if desired , the trapped sperm cells can be washed by infusing buffer or water through the microchannel . 4 ) after the desired cells are trapped , flow in the cross - channel can be initiated , the standing wave turned off , and the cells released . the flow in the cross - channel directs the released cells into the outlet of interest , for collection or further manipulation on - chip . this collection of the trapped materials can be completed with or without on - chip valving to aid in sample collection . 5 ) the non - trapped cells can be collected from the outlet reservoir throughout the perfusion of sample and sample washing . this can be accomplished by various means , including but not limited to attaching tubing to the outlet reservoir and collecting the flow - through in an attached receptacle . 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 : ( 1 ) woolley , a . t . ; hadley , d . ; landre , p . ; demello , a . j . ; mathies , r . a . ; northrup , m . a . anal . chem . 1996 , 68 , 4081 - 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