Patent Application: US-67088208-A

Abstract:
distinctive components that enable high - throughput , whole - animal screening are described . these components can be used individually or in various combinations . a staging chip strains off the excess fluid that the input animals are immersed in , increasing their density and rapidly bringing them close to other fluidic components . a microfluidic sorter is adapted to isolate and immobilize a single , physiologically active animal in a selected geometry . a multiplexed micro - chamber chip receives single animals and the microchamber chip includes individually addressable screening chambers for imaging , incubation and exposure of individual animals to selected chemical compounds . an imaging structure generates sub - cellular , high - resolution images of the physiologically active animals . a well - plate interface chip is used to deliver elements from a compound library to a single output of the chip .

Description:
the present invention utilizes small animals for high - throughput screening of biologically active materials . a preferred small animal is a nematode such as c . elegans or the embryos or larva of a zebrafish . other animals that may be used in embodiments of the invention include , saccharomyces cerevisiae ( yeast ), drosophilla ( eggs ), and other nematodes . the present disclosure will focus on c . elegans , but it should be noted that wherever c . elegans is mentioned , other small animals may be used . c . elegans is a preferred animal for several reasons . this animal , approximately 50 μm in diameter , can be micro - manipulated inside microfluidic chips and can be directly exposed to harsh ambient environments . this animal can survive a wide range of environmental stress , temperature ranges , ph conditions , and salinity . these animals can be kept alive for months without feeding . the c . elegans worm is optically transparent so that the fate of every single cell type in the worm can be tracked through bright - field and fluorescent microscopy in vivo . the animals can be easily genetically engineered for fluorescent labeling of specific types of neurons . these animals possess all of the major neuronal cell types found in higher organisms including acetylcholine , glutamate , gaba , serotonin , and dopamine neurons . models of alzheimer &# 39 ; s and parkinson &# 39 ; s diseases exist in the c . elegans system . unknown cell - specific toxins can be classified by identifying the type of cells that they affect by both behavioral and morphological assays . c . elegans has a simple enough neuronal network that there is a one - to - one map between behavioral response of the worm and the viability of specific cells . genetic and behavioral compensatory mechanisms due to redundant pathways that exist in higher organisms are not present in c . elegans . thus , the phenotypic effects are very easy to detect . the c . elegans organism is self - fertilizing ( hermaphrodite ) such that every worm has an identical genetic sequence ( apart from any low - probability mutations ). therefore , quantitative assays can be performed correlating measurements at different time points and environments . simple genetic manipulation techniques allow individual genes controlling specific pathways to be turned on and off selectively . this manipulation can allow rapid detection of specific biological pathways . c . elegans also develops quickly ( three days ) and has a long life span of up to three months . with reference to fig9 , a staging area is composed of multiple suction channels and a plurality of microfluidic valves . accumulating multiple animals in the staging area allows simplified tracking . with reference to the sorter designs of fig1 , animals still occupying the main chamber when one animal is captured by the single suction valve can be washed back to the staging area , ensuring all animals are processed and can be tracked . in the preferred embodiment , input to the staging area can be accomplished using the interface device of fig4 , or simply by aspirating from a reservoir or a well in a multi - well plate . the aspiration tip can be placed either manually or automatically using a computer - controlled positioning apparatus . with reference now to fig1 a , a sorter 10 according to one embodiment of the invention includes an inlet 12 through which pass c . elegans worms 14 . fluid flow through the sorter 10 is controlled by valves labeled a , b , c , d , e and f . fig1 b illustrates an open and closed valve that utilizes a flexible membrane to close the valve ( 18 ). returning to fig1 a , when the valve a is opened worms flow into the sorter 10 . suction is applied through a microchannel 13 controlled by a valve c so that when the valve c is open a worm may be captured . the use of the single microchannel 13 eliminates the potential problem of simultaneously capturing multiple animals . once an animal 14 is captured , valves b and e are opened establishing a flow that will sweep any other animals out of the sorter , either by continuing the flow out of the chamber , or flowing the animals back towards the input area or staging area . upon completion of this flushing operation , valve c is closed and valve d is opened to additional suction so that the animal 14 comes to rest on a surface 16 formed by an array of suction channels . the worm 14 will be immobilized in a straight position as shown . at this point , the worm is imaged through a transparent glass or polymer substrate using high - resolution optics for phenotype analysis ( see element 24 in fig5 a .) fig2 a is an image of the on - chip sorter described above in conjunction with fig1 a . the scale bar is 500 μm . fig2 b shows a single worm trapped by multiple suction channels . a combined white - light and fluorescence image is taken by a cooled ccd camera ( roper scientific ) with 6 . 5 μm pixels and a 100 ms exposure time through a 0 . 45 na 10 × objective lens ( nikon ). clearly visible are mec - 4 :: gfp - expressing touch neurons and their processes ( scale bar 10 μm ). fig2 c shows touch neurons plml / r and alml / r ( l , left ; r , right ), which extend processes along the anterior and posterior half of the worm and contribute to mechanosensation in these regions . the cell bodies are shown as black dots . three - dimensional cross - sectioning by two - photon microscopy can also be used at the expense of sorting speed . to achieve greater immobilization , a microfluidic valve layer can be placed above the main chamber of the sorter . fig7 shows this process . once the worm is held by the suction channels , the valve line above the sorter is pressurized . this causes expansion of the membrane separating the layers , which immobilizes the animal against the multiple suction channels . fig7 b and fig7 c show photomicrographs of an animal immobilized by an embodiment of this concept . the microfluidic - chip sorter 10 of fig1 a has flow and control layers , and is permanently bonded onto a glass substrate to allow optical access , though it could also be bonded to another layer of transparent polymer . flow layers are made by casting a room - temperature - vulcanizing dimethylsiloxane polymer ( rtv615 , ge silicones ) using a mold including patterned layers of negative photoresist ( su8 - 2025 , microchem ) and positive photoresist ( sipr - 7123 , shin - etsu ) on a silicon wafer . it is preferred that the main flow layer channels have a width in the range of 250 - 500 μm and are 80 - 110 μm high . the channels are rounded by reflowing the developed photoresist at 150 ° c . in a preferred embodiment , the flow layer includes suction channels defined using a layer of negative photoresist and that the channels are 25 μm high and 15 μm wide to allow capturing of animals . control layers are made by casting from a mold including a patterned layer of negative photoresist ( su8 - 2050 , microchem ) on a silicon wafer . control channels are 70 - 80 μm high and the membrane that separates the two layers is 10 - 20 μm thick . pdms chips cost significantly less than current flow - through animal - screening machines and can be easily incorporated into a variety of microscopy systems . the speed of the sorter 10 depends on the actuation speed of the valves , the concentration of animals at the input , the flow speed of the worms , and the image acquisition and processing times . the technique of immobilizing worms by lowering pressure in a micro - channel is fast because the actuation speed of the valves is less than 30 milliseconds . because of continuous re - circulation at the input , animals can be flowed at high concentration without clogging the chip . the speed of image acquisition and recognition of sub - cellular features is fundamentally limited by the fluorescence signal - to - noise ratio and the complexity of the features being recognized . the entire worm can be imaged in a single frame using a low magnification , high - na objective lens . cellular and sub - cellular features ( touch - neuron axons , etc .) can be detected by wide - field epi - fluorescence wherein the exposure times are limited by the brightness of the fluorescent markers . using a cooled ccd camera allows image acquisition at speeds exceeding one frame every one hundred milliseconds when imaging neurons labeled with green fluorescent protein ( gfp ). with reference now to fig3 a , b , c and d a multiplexed micro - chamber chip 18 includes a plurality of micro - chambers 20 . inputs to the chambers 20 are controlled through multiplexed control lines and valves . the same inputs can be used to deliver both worms and compounds by flushing the lines with clean media . the micro - chambers 20 include barriers or posts 22 preferably arranged in a curved or circular pattern for capturing and immobilizing the worm 14 that enters the micro - chamber chip 18 upon discharge from the sorter 10 . the posts 22 may be arranged in a straight configuration . the posts 22 are preferably arranged in a curved pattern to conserve space to accommodate high - throughput screening . fig3 c shows a close - up of a post or barrier 22 and also shows a gfp - labeled fluorescent touch neuron in an animal 14 . sorted worms from the sorter 10 are delivered to the micro - chambers 20 by opening valves via multiplexed control lines ( 14 ). pressure in the control lines is switched on and off with external electronically controlled valves ( numatics tm series actuators ). since the number of control lines required to independently address n incubation chambers scales only with log ( n ) ( 14 ), micro - chamber chips based on this design can in theory be readily scaled for large - scale screening applications . because of the millimeter scale of the micro - chambers , thousands of micro - chambers can be integrated on a single chip . as with the sorter , an additional valving layer can be placed above the individual chambers to improve the immobilization . each incubation chamber in this embodiment contains posts or barriers 22 arranged in an arc . to image animals , a gentle flow is used to push the animals toward the posts 22 . this flow restrains the animals for sub - cellular imaging without using anesthetics . the arc arrangement of the posts reduces the size of the chambers and also positions the animals in a well - defined geometry to reduce the complexity and processing time of image - recognition algorithms . the media in the chambers can be exchanged through the microfluidic channels for complex screening strategies . thus , precisely timed exposures to biochemicals ( e . g ., neurotoxins ) can be performed . this capability is useful both for identifying mechanisms that rely on the action of more than one compound , and for combinatorial assays involving multiple drug targets . the use of the microfluidic technology disclosed herein also reduces the cost of whole - animal assays by reducing the required volumes of compounds used . interfacing microfluidics to existing large - scale rnai and drug libraries in standard multi - well plates represents a significant challenge . it is impractical to deliver compounds to thousands of micro - chambers on a single chip through thousands of external fluidic connectors . to address this problem , the inventors herein have designed a microfluidic interface chip shown in fig4 a . the device includes an array of aspiration tips that can be lowered into the wells of micro - well plates shown in fig4 b . the chip is designed to allow minute amounts of library compounds to be collected from the wells by suction , routed through multiplexed flow lines one at a time , and delivered to the single output of the device . the output of the interface chip would then be connected to the microfluidic - chamber device 18 shown in fig3 for sequential delivery of compounds to each micro - chamber 20 . combining this multi - well - plate interface chip with existing robotic multi - well - plate handlers will allow large libraries to be delivered to microfluidic chips . the same device can also be used to dispense worms into multi - well plates , simply by running it in reverse . since the sorter 10 and micro - chambers 20 disclosed herein are designed to immobilize and release animals repeatedly within few seconds , the on - chip screening technology will allow high - throughput whole - animal assays at sub - cellular resolution and with time - lapse imaging . it is possible to automate a variety of assays by combining these devices in different configurations . for example , mutagenesis screens can be performed using the microfluidic sorter 10 in combination with the microfluidic dispenser to dispense sorted animals at high speeds into the wells of multi - well plates as shown in fig5 a . large scale rnai and drug screens with time - lapse imaging 24 can be performed by combining the sorter , integrated microchambers , and multi - well plate interface chips as shown in fig5 b . although c . elegans is self fertilizing , and has one of the lowest phenotypic variability of multi - cellular organisms ( 4 ), variations among assayed animals are still present , reducing the robustness of current large - scale screens . sorting technology can be used to select animals with similar phenotypes ( such as fluorescent - marker expression levels ) prior to large - scale assays to significantly reduce initial phenotypic variations ( 4 , 15 ). the micro - chamber technology disclosed herein can be used with feature - extraction algorithms to screen thousands of animals on a single chip . an interface to multi - well plates can be used to deliver large compound libraries to the micro - chambers . the system disclosed herein will allow hundreds of micro - chambers to be independently and simultaneously conditioned and monitored . the multiple - input flow control enables both combinatorial and sequential delivery of compounds to individual chambers to allow complex screening strategies to be designed . for example , both prior to and following toxin exposure multiplexed at various stages , rnai gene silencing can be used to turn off genes to identify biochemical pathways involved in toxicity . the microfluidic - chip system disclosed herein will be continuously kept in a stage - mounted temperature - controlled co 2 / o 2 incubator to allow long - term incubation of animals . the microfluidic worm - sorter disclosed herein will also allow one to screen mutants from a large number of worm populations mutagenized and / or degenerated by toxins . one of the greatest advantages of the worm - sorter disclosed herein is that it can be interfaced with high - resolution optics easily , allowing sorting of worms accurately and with much finer phenotype analysis . several studies indicate that dopaminergic neurons are the neurons most vulnerable to environmental neurotoxins and are especially vulnerable to toxins that target mitochondrial proteins . in particular , it is widely accepted that sporadic parkinson &# 39 ; s disease results from dopaminergic - neuron death induced by environmental toxins . no organism other than c . elegans allows non - invasive in vivo imaging of its dopaminergic neurons . thus , observation of dopaminergic neurons in c . elegans is very appealing for assessment of neurotoxicity . the inventors have performed an experiment that demonstrates on - chip detection of neurotoxicity due to the well known neurotoxin 6 - ohda . the inventors used genetically engineered worms that express gfp ( green fluorescent protein ) under the control of dopamine - neuron - specific promoters . degeneration of neurons by loss of fluorescence after neurotoxin exposure is easily detectable through the transparency of the chip . in order to automatically analyze hundreds of micro - chambers , simple recognition algorithms can be used to track fates of these fluorescently labeled dopaminergic neurons . fig6 illustrates the use of electroporation to facilitate toxin introduction . this is done by applying an electrical potential across the worm to increase uptake of a toxin or plasmid . the electrical potential can be applied using electrodes patterned onto the substrate , via wires threaded through a pin or using the pins themselves . the devices disclosed herein generally consist of multiple thin layers of poly ( dimethyl siloxane ) ( pdms ) fabricated by soft lithography ( 19 ). the pdms layers are each fabricated from a separate mold . to fabricate a flow layer suitable for smaller animals , including c . elegans , the inventors used a mold with two photoresist layers . first a 15 μm - thick layer of su8 - 2025 negative photoresist ( microchem ) was spin - coated and patterned to define the aspiration channels . next , a 100 μm - thick layer of sipr - 7123 positive photoresist ( micro - si ) was spin - coated and patterned to create the remaining parts of the flow - layer mold . the positive part of the flow layer is rounded by reflowing the developed photoresist at 150 ° c . the press - down and control - layer molds were created from 65μm - and 75 μm - thick layers of su8 - 2050 ( microchem ), respectively . from these molds , rtv - 615 pdms ( ge silicones ) was cast . this material was deposited either by pouring ( for the immobilization layer ) or spinning ( for the control and flow layers ). following this , the layers were cured for one hour at 80 ° c ., then bonded together thermally for 36 hours . large animals , including the embryos and larva of zebrafish , can have dimensions on the order of a few millimeters . to create molds of these dimensions , channels are milled into a rigid substrate using a milling machine or laser cutter . for regions requiring rounded channels , including sections containing microfluidic valves , a ball mill with a round tip can be used . a semi - rigid plastic layer can be cast from this mold . once cured and peeled from the rigid layer , this plastic layer can serve as a mold to create pdms devices with channels having large dimensions . flow , control , and immobilization layers can be created in this fashion . with reference now to fig7 ( a ), a 100μm - tall flow channel 30 contains multiple 15 μm - tall aspiration channels 32 that capture / align the animals in a linear position when the pressure in the aspiration channels 32 is lowered . this aspiration immobilizes animals only partially , and it is not sufficient to completely restrict their motion . in order to fully immobilize the animals , a seal is created around them that restricts their motion completely . this sealing is done by using a 15 - 25 μm - thick flexible sealing membrane 34 that separates a press down channel 36 from the flow channel 30 underneath . the press down channel 36 can be rapidly pressurized to expand the thin membrane 34 downwards , as in the microfluidic valves discussed above . the membrane 34 flexes on top of the captured animals , wrapping around them and forming a tight seal which completely restrains their motion , holding them in a linear orientation . although the animals are constrained by the pdms membrane 34 from the top and bottom , they still have access to liquid media by way of the multiple aspiration channels 32 on the left side . fig7 ( b ) shows an image of an immobilized adult animal in the device and fig1 ( c ) shows superimposed brightfield and fluorescent images taken at high magnification . as discussed above , zebrafish have been recognized as a suitable model for drug screening and discovery . zebrafish is a vertebrate animal model in which high - throughput studies can be performed due to its small size and optical transparency . comparison of the human and zebrafish genomes suggests that a majority of human neurological disease genes and pathways also exist in zebrafish . zebrafish models of human cardiovascular , endocrine , and brain disorders have been recently demonstrated . this animal is widely recognized as a powerful vertebrate model for dissecting the processes of neural development , neural regeneration , neural transmission and chemical toxicity . the genetic pathways involved in neural development , function and signaling pathways are highly conserved between zebrafish and humans . the use of zebrafish therefore permits otherwise impossible genetic manipulation and screening techniques in a vertebrate model . fig8 ( a ) and ( b ) illustrate an embodiment of the invention particularly adapted for immobilizing zebrafish in a well - defined orientation for cellular - resolution studies . fig8 ( a ) illustrates the microfluidic chip layout . the chip includes a bottom flow layer where animals are routed and a control layer that turns on and off the flow in the bottom flow layer utilizing membrane valves . in a first stage , animals are loaded into the chip and in stage 2 , a single zebrafish is captured by a micro - aspirator 2 while the rest of animals in the channel are removed / recycled by flushing the channel . in the next stage 3 , the yoke of an animal is pushed against a wide but shallow channel by aspiration while the freely moving tail of the animal aligns toward the aspiration port . the animal is immobilized as shown in fig8 ( b ). thereafter , in a stage 4 , the animal is released and sent to multi - well plates for incubation . as shown in fig8 ( b ) after the animal is aligned in stage 3 as shown in fig8 ( a ), flexible pdms membranes on top of the animal are pressurized to immobilize the animal completely . the transparent animals can then be imaged and injured at cellular resolution through a glass cover slide or polymer layer bonded to the bottom of the chip . this immobilizer can also allow micro - injection into both larvae and embryos . a pulled quartz pipette can be inserted through a molded hole in the flexible membrane on the side of the immobilizer chip . the pipette can be bonded / sealed to the membrane if desired . the flexing of the membrane permits limited motion of the pipette inside the chip . behavioral studies in small animals , including both zebrafish and c . elegans , are important for insight into a wide variety of biological processes . the high - throughput fluidic devices described above can enable many advanced behavioral studies to be performed on - chip . behavioral responses to light , exposure to chemicals , electric shock , and mechanical stimulation can all be studied with little to no modifications to the devices discussed . another method to study some of these responses , including response to electric shock and fluidic / air pressure , is to create a small element that can be inserted into an individual well of a multi - well plate . this is especially well suited for studies involving larger animals whose dimensions are on the order of individual wells . 1 . kamath , r . s ., fraser , a . g ., dong , y ., poulin , g ., durbin ., r ., gotta , m ., kanapin , a ., le bot , n ., moreno , s ., sohrmann , m ., welchman , d . p ., zipperlen , p . & amp ; ahringer , j . 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( 2004 ) nature 432 , 822 . 17 . yanik , m . f ., cinar , h ., cinar , h . n ., chisholm , a ., jin , y . & amp ; ben - yakar , a . ( 2006 ) ieee journal of quantum electronics 12 , 1283 - 1291 . 18 . rohde , c . et al . ( 2007 ) pnas , vol . 104 , no . 35 , 13891 - 13895 . 19 . zeng , f . et al . ( 2008 ) lab chip , 8 , 653 - 656 . it is recognized that modifications and variations of the invention disclosed herein will be apparent to those of ordinary skill in the art . it is intended that all such modifications and variations be included within the scope of the appended claims .