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    Us 20130236901 A 1

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    cu») United States ‘US 2013023690141 cz) Patent Application Publication — co) Pub. No.: US 2013/0236901 Al Potier et al. (54) MICROFLUIDIC DEVICE FOR PRODUCTION AND COLLECTION OF DROPLETS OF A. FLUID (75) Inventors: Marie-Claude Potier, Paris (FR): Patrick Tabeling, ay les Roses (FR); Lace Dauphinot, Voulsngis (FR) Nadege Bois, Ceson (FR); Fabrice Monti, Sauls les Charteu (FR) Pascaline Mary, Cambridge, MA (US) (73) Assignee: CENTRE NATIONAL DE LA RECHERCHE SCIEN PARIS Cedex 16 (FR) (21) Appl. No: 137876,960 (22) PCT Filed: Oet.3,2011 (86) PCT Ne. $371 (ol), (2), Date: per p2011/067229 May 28, 2013 G0) xt, 1,2010 (EP) 10290526.2 (43) Pub. Date: Sep. 12, 2013 Publication Classification (1) Ince C9 168 2006.01) @) vs cee C129 1/6806 (2013.01) USPC 435/612; 435/287.2 on ABSTRACT A microluidie device and method for producing and eollect- ing single droplets of a fist Muid, the device including @ microfluidic platform having at east a droplet microchannel ‘wherein is prcuceda flow of single droplets of the fitst fk spersed in a sooond Sid immiscible with he frst Bui, dhe ‘droplet microchannel having at feast one inlet extremity and atleast one oulet extremly Zor distributing the ow of drop- Tes, the deviee futher including: ‘collection device including a plurality of receiving areas ‘adapted to collect atleast one of the droplets, lements for changing the relative postion ofthe collection ‘device and the outlet of the mierofiuiie platform, elements for controlling the flow of droplets, and ‘elements forsynchronizing the low of droplets at theoutlet ‘of the deoplet microchannel and the relative movement ‘of the collection device with regards to the microhsiic platfonm, US 2013/0236901 AI Sep. 12, 2013 Patent Application Publication x me oe Act y k— a Lozi | Loo rd ezL cc ee be (00z ost Ga | ozt || L_ Ce BbE me wl sl eh Lt = Pezer t t Ost Lu be 2 Wt \ I~ | LA vee t + l, a eH gin | bee 3 Aa gout =e Clvon ov 1614 oot }_—______] US 2013/0236901 Al MICROFLUIDIC DEVICE FOR PRODUCTION AND COLLECTION OF DROPLETS OF A FLUID FIELD OF THE INVENTION {0001} The present invention relates 9 a mictofuidic ‘device forthe production and collection of droplets ofa fist ‘sid, said device comprising a microfluidic platorm inchad- ingatleasta droplet iroebiaanel wherein is produced flow ‘ofsingle droplets of sad is uid dispersed ina second Mid Jmmiscible with the first uid, sad droplet mierochannel ‘comprisingat feast one inlt extremity and eomprisingat least ‘one outlet extremity forthe distribution of said low of drop Jets. 10002] _Italso relates tox method for producing andeollec- Jing droplets of first Mui. 10003] The device and method according tothe invention are particularly useful when implemented with a first fd ‘comprising a plurality of panicles, as it allows encapsulation ‘ofa singe panicle in each droplet TECHNOLOGICAL BACKGROUND 10003] A microfluidic device for production of droplets of snagucous solution containing living cells in carrier oil with such a mierofluidic platform is known from the document ‘enliled <-Drople-based microfluidic platforms for the ‘encapsulation and sereening of mammalian ells and mult ‘elluarorganisms>>, published by J.Clausell-Thormos etal in Chemistry and Biology, volume 15, pages 427 to 437 in may 2008. 10005] The use of such a microfluidic platform allows the ‘encapsulation of single cells in microresctors formed by the ‘aqueous droplets. This document shows that these microre> fsctors are adapted to maintain the cells alive and even alow them to proiferate, 10006) In this document, the flow of droplets produced in the microfluidic platform is collected as & whole, thereby orming an emulsion, This emulsion i then broken or rein- Jected in another mierofluidi platform for analysis, [0007] Ics then impossible to collet each ofthe droplets separately, in onder perform lurker reaetions if nccestary, 10008] Ics also know from document WO2010/018465 to tse a <> technology to encapsulate nucleic ‘eid fragments a reagents in a droplet in order to perform 3 polymerase chain reaction (PCR) inthe small volume of the “dplets 10009] ‘Thanks tothe isolated environment ofthe droplet, the products of the reactions are protected from contamina tion and only a very small amount of reagents is needed [0010], Theoutcomeof the PCR aceurtingin each dropletis ‘optically probed while the droplets are stil in the fow of “droplets, Therefore, no collection ofthe dropletsis performed in this document. {0011} A current method for collecting single cells and studying them separately from each others hosed on manual collection and deposition af these cells in the receiving areas ‘ofa microtiter plate, However, such a method is time-co sumingand only allows thestudyof a imited numberof ells SUMMARY OF THE INVENTION 10012] _Thepreseat invention simsat providing adeviceand shed for producing and collecting droplets of first hid Sep. 12, 2013 in anne, allowing theeollee- all [0013] More precisely, the invention relates toa microflu- ‘de device es described inthe intoduction, comprising: 0014] 2 collection device comprising a plorality of receiving areas adupted to collect at least one of id droplets, [0015] means for changing the relative position of the collection device and the outlet ofthe micrauiie pat- form, [0016] means for conolting the flow of droplets, and [0017] means for synchronizing the fow of droplets at the outlet of sid droplet microchannel and the relative movement ofthe eallection device with regards t the microfluidic platform, [0018] Thanks to this device, i is possible to deposit pre- cisely eal of the droplets exiting from the microfluidic plt- orminone of therseeiving areas ofthe collectiondeviee. The droplets are then separated from each otherand ean be treated separately [0019] According to other advantageous and non limitative characteristics ofthe device aecording wo the iavento [0020] said Now contro! means contr atleast the speed ‘of std flow of droplets andar the distance between 0 successive single droplets in said flow of droplets: [W021] said syuchronization means synchronize the flow ‘of droplets a the outlet extromity of sail droplet miero= ‘channel and the movements of the collection device ‘depending onthe positions ofthe roeeiving arcason sid collection devices [0022] said mierofuidic platform comprises at least a first uid microchannel fed with said first uid and a second fuid microchannel fed with said second fluid, both of sad frst fluid microchannel and second uid ‘microchanael intersecting at the inlet extremity of sid droplet microchannel and communicating with said droplet microchannel ia order to merge said frst and second fd and proiace said flow of single droplets oF first id dispersed in said second Mid: 0023] said tow control means comprise means foreon- trolling the pressure of sai is uid in said frst fig ‘mierochanael and means for controlling the pressure of | said second fi in said second fluid microchannel 0024] said microfluidic platform comprises at least @ reagent microchannel fed with a chemical or biologic reagent, the eagent microchannel leading into said est fluid microchannel, upstream from the intersection between this first uid microchannel and said second ‘uid microchannel; [0025] sai ow contro means comprise means for con- trolling the pressure of said reagent in said reagent ‘microchannel, [0026] said droplet microchannel comprises a capillary ‘with which itis Muidly connected, the end of which forms the oulet forthe distribution ofthe ow of drop lots 0027] said microfuidie platform comprises. probing ‘mans that allow the Mentfcation of products contained in each droplet, and preferably said probing means are located inthe vicinity of the droplet microchannel; [0028] said probing means are connected to said syn- >, published by J. Clausell-Thorm etal. in Chemistry and Biology, volume 15, pages 427 19 437 in vay 2008, [0106] It is therefore possible, by decreasing the eoncen- tration of cells 12 inthe frst fluid 11, to encapsulate ze oF ‘one cell 12 in cach droplet 14, meaning thot the probability of encapsulating two or more cells in @ droplet can be greatly limited [0107] Inother words, varying the concentration of cells 12 inthesuspensionallows controling the statistical distribution ‘of the number of ells trapped in each droplet 1. [0108] The concentration of cells inthe ist fuid can either ’e predetermined in the suspension introduced in the fist ‘uid inlet hole 114, or controlled on site. by diluting @sus- pension exhibiting & high concentration of eels when in {duced in the microlluidie platform with an aquects solution introduced in the first fluid microchannel through an addi ‘ional microchannel as deserined before. [0109] The concentration of ells inthe fst fd 11 can then be computercontrole [0110] Thenumber ofcells encapsulated in each droplet 14 ray be probed thanks to probing means 200 while they are still owing inthe droplet microchannel 119. [0111] These probing means 200 are for example phase contrast microscopy “means of Muorescence measuring ‘eats. In this later ease, the membrane or eytoplasn ofthe cel ts to be marked with fluorophores. [0112] ‘The probing means 200 may also comprise other | ypesof analysis means such as absorbance measurements oF ‘conelctance meastrements means. [0113] In a remarkable fashion, said microfluidic device 100 also comprises [0114] 2 collection device 120 for collecting suid drop- lots, [0115] means 130, 181 for changing the relative position ‘of the collection device 120 and the outlet 171 of the nicrouidie platform 110 [0116] means 140, 150,160, 182 forcontolling the ow ‘of droplets 14 and [0117] means 183 for synchronizing the Now of droplets AMatihe outlet 171 of sid microfhidie platform 11nd US 2013/0236901 Al 4 relative movement of the collect ‘regards tothe microfludie platform 110. [0118] The collection device 120 comprises plurality of receiving areas 122 adapted to collect at least anc of said “droplets 14 10119] In the example shown on FIG. 1, this collection device 120 isa microtiter plate 120. It comprises a support 4121 in the form of Mat plate with a plurality of wells 122 ‘opened to a front face 123 of the support 121. This support 121 is typically made of rigid or flexible plastic material. 10120] The microtiter plate 120 typically has 6, 12,24, 06 384 or 1536 wells 122 arranged ina rectangular matrix. The ‘wells 122 cas either exhibit a cicular or square section. device with [0121] Bach well 122 ofthe microtiter plate 120is adapted to hold between tens of nanoliters to sever milliliters of liquid. 10122] The froat fee 123 of the microtiter plate 120 is ‘oriented oward theoutlt 171 ofthe microfluidic device 10, inorder to allow the droplets exiting from this outlet 170 10 fall under the action of the gravity int the wells 122 of the meroiter plate 120, 10123] The schematic view of FIG. 1 shows a simplified relative arrangement ofthe microfluidic platfoets 110 snd the ‘microtiter plate 120, where the capillary’ 170 extends strsight rom the microfluidic platform 11, [0124] However, the capillary 170 is advantageously fex- ‘ble, and allows to bring the droplets in front of the wells 122 ‘of the microtiter plate 120 while the microfluidic platform 110 ays forexample ina plane parallel othe microtiter plate 120, [0125] This capillary 170 is made for example of flexible Plastic material, such as polytetrafiuoroethylene (PTFE), 10126] In the example shown schematically on FIG. 1, these wells 122 are filled with ol prior to the droplet deposi tion. The droplets fll into the well, under the oil phase, then remain isolated from air and from their environment, and thereby are protected from contamination or evaporation 10127] In practice, each droplet remains surrounded by & fil of oil when exiting he capillary 170 10128] | Moreover. ina preferred embodiment ofthe method according to the invention, the outlet 171 ofthe capillary 170 is brought to or under the surface of the oil contined in each ‘well 122, s0 thatthe droplet 14 exiting the eapllary 170 Nod any coatat with ai, [0129] Intheexample shown on FIG. 4, themeans 130,181 Jor changing the relative positon ofthe collection device 120 ‘andthe outlet 170 af the microfluidic platform 110 comprise translation means 130 ofthe microtiter plate 120, 10130] The outlet 170 ofthe microfiuidie device 110 thus Femains fixed, while the msierotter plate 120 is moved along ‘wo perpendicularaxis X.Y in the plane the microtiter plate 120 Bach well 122 can ts be brought successively infront ‘of the outlet 171 of droplet mierockannel 119. [0131] The direction and amplitude of the translation ‘movement imposed tothe microtiter plate 120 by the tas ton means 130 are comutercontrolled by translation con- trol means 181 of the electronic command unit 180. will be described in more details later on 10132] In onder to allow the precise deposition ofa droplet 14 in cach of the wells 122 of the micmiter plate 120, the icroidic device 100 also comprises means 182 for con- telling the low of droplets 14, Sep. 12, 2013 10133] These flow conteol means 182 are implemented by the clectronic control unit 180 that controls the il injections ‘means 160 ofthe oil eater and he frst Hid flow and reagent ow control means 140, 180, [0134] Tae contro of theo injection means 160 allows to regulate the flow rate of oil 10 through the second-id ‘microchannel 116A, 116B and thas to adjust the spocd of the oil carrier flow. The speed of said flow of droplets 14 is thus controled [0138] The contro ofthe first aid flow control means 140 allows to regulate the frequency of opening ad closing ofthe fnlet microchannel ofthe valve VI and subsoquently of frst ‘uid mieroehsanael 17, [0136] This frequency determines the frequency at which the droplets of fist uid 11 are produced, and thus regulates the distance between two suecessve single droplets 14 insaid flow of droplets fora given low rate of cil 10 [0137] The reagent flow contro! means 150 are synchro- od with the first Bui low contol means 140, in order to ‘mix the reagent and suspension just before production of each droplet. [0138] Finally, the electronic control unit 180 implements the synchronization means 183. These. synchronization scans 183 synchronize the flow of droplet M atthe outlet 171 ‘of said mierothidic platform 110 and the movements of the collection device 120 by controlling the droplet flow eoatol ‘means 182 and the translation control means 181 depending ‘on the positions ofthe receiving areas 122 on sad collection device 120, [0139] "More precisely, in the example deseribedhere, these synchronization means 183 ensure that, during the time between the reception ofa first droplet Mina first well 220 the microtiter plate 120 and the reveption ofa second droplet 14, the microtiter plate 120s moved fom a position where ‘the outle 171 ofthe microfluidic plan 110 faces sid frst ‘well 22toapostion where theontlt 171 faces second well 122 distinet from the first well 122 [0140] Ina preferred embodiment of the invention, a single «droplet is deposited in each of the wells ofthe microtiter plate 120. However, one can consider depositing a plurality of opie into given wells or no droplet in some other wells [0141] The synchronization means 183 aso allow the ien- ‘ication ofthe droplet deposited in each well 122 [0142] More precisely the conditions of production of the droplet 14, namely, introduction of reagent, nature of the ‘reagent if several ackitional microchnnels are connected to the frst fluid microchanael 117, concentrations of these reayents if these are varied in time, concentration of cells ia the suspension in the ease where this concentration is adjusted onsite, ean be recorded by the electronic contro uit 180 and associated withthe position of the well where the corresponding droplet wos deposited. [0143] The result ofthe analysis by the probing means 200 for each droplet 14 can also be reconded by the electronic control unit 180 and associated with the position ofthe cor responding well 122 [0144] Invariant, the frst uid comprises a suspension of particles comprising atleast one ofthe following: living oF ‘ead cells, cell omunelles such as mitochondria, ganic oF inorganic beads, for example beads of polymer, micells vesicles liposomes, multiellular organism, meroorganisms such as bacteria, [0145] In these cases, the droplets produced encapsulate a ‘controlled numberof pariles US 2013/0236901 Al 10146], Inthe case where the microfluidic device is used foe collecting and studing cells, mulcelllar onanism o Inicroorgniss, the microfluidic device 100 is preferably located within contlled atmosphere enclosure, Which pro- ides controled conditions such as pressure imiity and ‘enapertie, pein cll survival nd asniiests sks ‘of contamination {147} Is practice, the microidc device 100 is particu Jnry sel when used to analyze the tansripome of exch of the cells initially contained i te suspension fed to the ist ‘hud miewchannel othe microg pltfonn 10. 0148] The followin steps are tortor implemented by the electronic contol wit IND to proce ad collet oat droplets of a eel suspension while performing atthe same time cel lysis and mRNA reverse trnseriptio ofthe mies Senger ribomicleic acids, called MRNA of the comesponding cal 10149] The electronic contro anit 180 controls he feed of the cell suspension 11 to sad first uid microchannel 117 of ‘sid miceoidie platform 110 and the fed of sid engcnt mmirochanne! 118 witha mx of el Isis indcing agent and revere transcriptase eyes. [0180] The regents are mixed withthe ell suspeasion at the intersection Tot the mirochannel, jst before oe during formation of the droplets. More poss, in a refered ‘emdment, the reagents age mixed with thecal sispension between and I sscond before fommaton ofthe droplets [0181] The ysis o the cll isthe perlonued witha afew Second, while the coresponding doplt is produced and ‘atid sway inthe sytem. 10182] The lecroni contro anit 180 controls he feed of the second Mid microchanne! 116A, 1165 of sid mierofi- ‘ie plato 110 with sid cariril 1 and the roicton, inthe droplet mierochanne! 119, of acontoled ow of snale Aroplets I of said mix between the eel suspension and the resent, dispersed inthe carer il, as described before {0183} The droplets hee contin preferably ihe only the reagent, in the eae no cell was eneapssted, ora sation ‘containing the mRNA ofa single cel {0184} Fa cll was eacapolated, the reverse wasseriptase ‘eazymes perform the retranscripion ofthe mRNA at the ‘el hile the droplet produced fowsalong thedroplet ero ‘channel 119. [0155] In order to help the retetanserpson being per formed, the ear oil my’ be heated at 42 degrees Celi that is to say, the temperature at which the reverse tan- serptse enzymes are mos elicieat {0186} The proba means 200 may here compris optical ‘analysis means to determine which doplets contain bidogi- ‘al motel With mRNA and which droplets comprise oly the reagents In his way, the wells 1220 the microtiter pate 120 tht wil be empty fons molecules of interest may Be ‘easily idemtifed and thrown away [0187] The How of droplets 14 is then dstbuted at che ‘outlet 171 of ssid mieroliie plato 110 {0188} The electronic contol device 180 contol the pos ining ofthe miertiter plate 120 under the outlet 171, and sychtonizs th ow of droplets Laeriving tssidoutlt 171 ‘ith te relative movements ofthe microtiter plate 120 in ‘order to deposit one droplet 4 a cach well 122. {0189} Advantageously the time elapsed been the pro- Auetion of a given single droplet 14 and the arrival of this ‘mpl 1 at sid oul 171 of the mio plate 10 can be contlled by the eleewonic contol unit 180. Sep. 12, 2013 [0160] The electronic control unit 180 therefore adjusts the How rate ofthe cari oil in the droplet microchannel 119.4 Jower low rate will slow the progression ofthe droplets 14 in the droplet mierochannel 119 and therefore increase the time spent by the droplets inthe mieroflidie platform 110, [0161] Here only a few seconds is preferably elapsed between the production of the droplet and is deposition into the microtiter plate 120, [0162] The risks of damaging the mRNA ofthe cell, and therefore the risks of losing par ofthe information comprised inthe transriptome ofthe cell are thus decreased [0163] Inthe context ofthe analysis presented above itwas preferred to mix reagents and cell suspension a the time of production ofthe droplets in order to avoid lysis ofthe cell before encapsulation. The applicant demonstrated that it needs Teast 1 to 2 secon for eel to bein conte with the reagents before ysis initiates [0164] However, other reactions may require a longer time between mixing the eagentsand particles ofthe fst uid and producing the droplets [0165] Inthis case, the microfluidic device used comprises ‘reagent microchannel that iaterseets the frst Did miero- ‘channel upstream from the intersection with the second fn channel [0166] tn this way, the electronic control unit may control the time elapsod between the moment when apartieleis putin ‘cont with the reagent by inioduction of said reagent insaid fist Mid microchannel, and the formation af a deoplet com prising said particle by merging of suid fst and second Mud. by controlling the ow rate of first uid inthe first oid snicrochannel, This low rate is related tothe frequency of activation ofthe coresponding fis fi low contol valve. [0167] Thanks othe invention, each droplet 14 containing the trinscriplome of « single cell 12 of the initial sample suspension in the fist uid is collected indvically in one of the wells 122 ofthe microtiter plate 120. They can therefore be subsequently treated, for example for ampiifcaton ofthe genetic material by polymerase chain reaction [0168] Although the present invention has been deseribed in details with respect o certain embexdimens and examples, ‘variations and modifications exis which are within the scope ‘of the present invention as defined inthe follewving claims, 0169} For example, the means for controlling the flow of | droplets comprise here an extemal pressure-acuted valve that opens or closes the first ud microchannel. [0170] Advantageously, the contol of the pressure of at Jeast one of said ist, second or tind fui in the eoerespond- ‘ng microchannel ofthe microifuidie device may also be used to generate droplets ina controlled manner withost restricting the low path of any ofthese fis, second and third Hud [0171] In this case, no pressure-setuated valve needs to be ‘sed. First and third fds are simply loaded in small reser voles or vials, each of them being fluidly connected to the corresponding inlet inthe microfluidic deviee and linked t0 the pressure control means, The socond fluid i for example injected through injection means similar to those desribed above, [0172] The gas pressure applied on top of suid reservoir or vial containing sai frst or third fai v0 introduce this oid into the microfluidic device is then dynamically controlled to pradice the transient pressure variation nthe it ling in the corresponding microchannel. The injection means ofthe second fitid may also be used to prodace such a pressire ‘aration inthe second fui. US 2013/0236901 Al 10173] In such a variant, the production of a droplet may then be triguered by a trasient negative variation of pressure ‘ofsaid second Hid in the second microchannel ofthe miro luidic deve, 10174) In another such variant, the production of a droplet may be triggered by a transient positive variation of pressure ‘of said irs fui and said tind uid, or of said second uid, in the corresponding microchannels of the microfluidic devi 10175]. Thisavoids theuseof valves, which arecomplicated to fabricate and may raise reproducibility and time constant problems, 10176] Transient positive or negative variations of pressure ae for example achieved through a square vai sure ofthe corresponding Mui in time, 10177] Aliertively, the production of the droplets may be ‘controlled by electric, thermal of acoustic means. [0178] -Theelectric conteo! means may comprise electrodes producing an electric field inthe vicinity ofthe intersection between first uid and second oid microchannels. he frst ‘uid comprising « diclectic aqueous solution, when sei vated the electric field may exert foree on the first fig, thereby accelerating it and producing a droplet 10179] The thermal control means may comprise heating ‘means inthe vicinity ofthe intersection between frst uidand second fd microchaanels, When these heating means heat the oil, the oil expands, hereby blocking the production of 2 ‘droplet. When the ofl cools dowa, it shrinks and allows the production of droplet, [0180] -Theacoustic control means comprise means for pro ‘ducing an acoustic wave that induces pressure variation i the fist ud, thereby favoring the production of a droplet. [0181] Othe collection means may he used, such ssa glass plate with a coating comprising hydrophilic zones forming the reception areas and being surrounded by hydrophobic [0182] The micrfMuie platform described here is meant to be disposable, but reusable platform may be achieved in a similar fashion, fom of press “Materials and Methods Pint Example Microfluidic Platform Fabrication [0183] Single-se microfluidic devies were fabricated by softlthograhy. PDMS mieroidie platforms were made ting replica sokling of @SUR-nold patterned by uavio- Je-ithogsaphy. [0184] The height and the width of the channels were 125 Injrometrs except tte pe-sapedjuneton hole with the ‘opillay ofthe droplet microchannel where they were 500 Inrometers and 700 micrometers respectively {0185} The channels were formed by ating two PDMS Parts, cach Comprising the imprint of al ofthe mierochan nets [0186] A thick ayer (about 7 milters) of PDMS as produced by pouring «10:1 mixture of monomer (GE RTV 615 component A) and a hardener (GE RIV 615 component 5) onto fist mold place in Pete dsh an felt at room temperature for 15 minutes to deg [0187] A thin layer (about 3 millimeters) of PDMS was prodhced by pouring» 10-1 mixtie ofa monomer (GERTV 615 component A) and a hardener (GE RTV 615 component Sep. 12, 2013 1) onto a second mold placed in a Petri dish and lef at 0 temperature for 15 mites wo desas [0188] The frst and second molds are aap to inpint bal the sirocanels {0189} The two layers were heated for 4S minutes at 78 degrocs Celsius {0190} Inlet ote forthe injection of ist and second Mids ‘were piched on the ist PDMS pat {0191} The two PDMS parts were hound together with @ doxygen plasms cleaner ad ligne [0192] Thedevicewaslet fora week at 78 deyroos Celsius [0193] 4 polyether eer Ketone tbe, also called PEEK ‘ube, rom Upehurch, wihan ower diameterof S10 microme- temvand an ner diameterof 128 micrometers was aloe atthe pear-shaped junction le {0194} ‘Then-uitaviole-curablephue(NOA 81, Norland Optical Adhesive) was deposited between the PEEK tube ad the PDMS and euredhy union illimination at 368 nanom- cers wavelength wih Hamamatsu LCS lamp. {0195} "Pressur-Actusted Valves Fabrication [0196] Singlesse microfluie valves were abicated by layer sofesitbosrphy. Valve pats were made by ep ing of « SUSsmold and « positive resist mol ( 1275 1V) patted by ultaviole-tihoarapy. [0197] Fach pressure-ctsted valve eosisted of to lev- cls of microchannel. The fst Buid or reagent circles in the bottom ler comprising Mudie channels, while the top chanel, called contol ehatnels, operat he pressire-acht fied valves, {0198} The typical dimensions use fr the valves were 40 ‘nierometer forthe eight and 500 micrometers forthe width ofthe fst tid or reogeatparaoli-shaped channel and 80 ‘micrometer forthe height and 500 micrometers forthe width ofthe rectangular contol chanel [0199] ~A tick liyer of PDMS was produce by pouring 8 5:1 mixtreofa monomer (GE RIV 615 component A)anda Jurdener(GERCTV 615 component onto themold placedin 4 Petr dish an left t room fempsrture for 1S minaes fo degss. PDMS 20:1 mixture was pincoated at 90° rotations Per minute for 60 seconds ont the mold for misking the “sociated conto channels [0200] The two layers were cured for 45 minutes at 78 degrees Celsius. Holes for the contol channels were punched. [0201] Te layers ofthe first Huid or reagent channel were Aligned to those the control channel. The two layers device ‘was cured ovemight Holes for firs id or reagent channels ‘were then punched. The device was eventually sealod onto a pre-cleaned plas side ater a 40 seconds plasma treatment And let overnight at 78 degrees Celsius 10202] Mokds Fabrication [0203] The resist molds used for PDMS casting were ‘oblained using ullnviolet optical liography. The optical masks were designed using the layout editor CleWin and ‘er printed on rsp ting'ohighseon printer [0204] The first mold was made of SUB-2100 photoresist ‘swosteps using the MicroChem! protocol for 120 microme- ters high channels forthe fist mask and 90 micrometers high channels for the second mask, [0205] ‘The second mold was made of SUS-3050 photore- sist in (Wo sleps using the MieroChem® protocol for 50 ‘micrometers high chinnels for the first’ mask and 0 ers high channels forthe second ma US 2013/0236901 Al [0206] The molds corresponding to the valve control chan- tle were farce by opal itopepy inn 8 scr ters hick her of SUB 3080 photoresist Miro Che 10207] Thermal comesponding to the dic channcls was fabricated in 40 micrometers thick yer of Ma 1275 HV heres (Micrreint technology) ad oud at 150 {egres Celso for 15 minutes 0208] -Mierotter Plate Preparation [0209] A microtiter plate from FrameStar dtitude was modified [0210] A ultrviolet-curable glue (NOA 81:8 from Norland ‘Optical Adhesive) was deposited the holes othe plate and ‘cured by a uniform illurination at 365 nanometers wave= Jength using @ Hamamatsu LC8 Lamp, inorder fill any gap that may be present within tbe wells and make them oitight [0211] The microtiter plate was placed on a Mirzhausee translation stage, The translation was contolled by Labview via Tango Desktop Controller. [9212] Bach well of te plate was then filled with MS904 ‘mineral oil rom Sigma 19213] Device Operation {0214} Cell suspension, reagents und oil injections were ‘controlled by a commercially available pressure controller MECS8C from Fluigent Oil was injected in the microfluidic platform oil inlet. Cell suspension and regents were injected Via the pressure-cetvated Valves. [0215] The oil was MS904 Mineral Oil from Sigma 10216) _Thereagent for reverse transcription was composed ‘of 2s first strand synthesis bull, 2 millimoles per liter of 589 10 ¥0 2 ‘00> segumnce: 3 <210> 559 1D wo 2 00> seQumne: 2 <210> 589 1D wo 3 US 2013/0236901 Al 10 Sep. 12, 2013

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