10.1109/ULTSYM.2011.0040
Parallel Output, Liquid Flooded Flow-Focusing Microfluidic Device for Generating Monodisperse Microbubbles within a Catheter Johnny L. Chen1, Ali H. Dhanaliwala1,2, Shiying Wang1, and John A. Hossack1,2 1
Dept. of Biomedical Engineering, 2R.M. Berne Cardiovascular Research Center University of Virginia, Charlottesville, VA, 22903, USA
[email protected]
[12], and gravity / flotation [13]. However, these steps add to production complexity, rarely achieve a fine degree of monodispersity, and decrease yield. In contrast, flow-focusing microfluidic devices (FFMDs) [14–16] can produce monodisperse populations of MBs in a continuous manner but have low production rates [8]. Expanding nozzle flow-focusing microfluidic devices, the underlying approach used in our FFMDs, produce MBs by creating a velocity and shear gradient at the tip of the gas cone. Due to the length scales of their design, microfluidic devices are typically characterized by laminar flow and low Reynolds numbers. As a result of these conditions, a co-flow is formed where the gas and liquid phases meet. The expanding nozzle design helps generate MBs by applying a high shear stress, via a high velocity gradient, to the gas-liquid co-flow at a single point [17]. As a result, MB diameter is a function of nozzle dimensions (width, length, height), liquid flow rate, gas flow rate (or the easier measured pressure), and gas and liquid properties such as viscosity. One common model [18] reduces MB diameter to a function of nozzle diameter and gas and liquid flow rate:
Abstract— A new method for supplying the liquid phase to a flow focusing microfluidic device (FFMD), designed for the production of monodisperse microbubbles (MBs), is introduced. The FFMD is coupled to a pressurized liquid-filled chamber, avoiding the need for dedicated liquid phase tubing or interconnects from the external field to the microfluidics device. This method significantly reduces the complexity of FFMD fabrication and simplifies the parallelization of FFMDs – an important consideration for increasing MB production rate. Using this new method, flooded FFMDs were fabricated and MB diameter and production rate were measured. The minimum MB size produced was 7.1±0.5 µm. The maximum production rate from a single nozzle FFMD was 333,000, MB/s. Production was increased 1.5-fold using a two nozzle, parallelized device, for a maximum production rate of approximately 500,000 MB/s. In addition to increased production, the flooded design allows for miniaturization, with the smallest FFMD measuring 14.5 x 2.8 x 2.3 mm. Finally, B-mode and intravascular ultrasound images were obtained, highlighting the potential for flooded FFMDs to generate microbubbles in situ in a catheter and immediately thereafter image the same MBs in a target blood vessel.1 Index Terms— Flow Focusing Microfluidic Devices Monodisperse Microbubbles, Parallel Device Production, Flooded Liquid Input
= 1.1
I. INTRODUCTION
(1)
Where db is MB diameter, D is nozzle diameter, and Qg and Ql are gas and liquid flow rates respectively. To the best of the authors’ knowledge, no model has been developed to predict MB production rate from a FFMD. Low production rates from FFMDs have resulted in limited adoption of microfluidic methods for MB production because large quantities of MBs are required to compensate for losses during intravenous administration and vascular circulation. Administration through syringes results in MB destruction [19], and systemic delivery can lead to loss through filtration such as in the lung [20]. MB production from an FFMD can be increased by increasing the gas and liquid flow rate, at the expense of monodispersity [16], [8] or through parallelization, which can require a complex manifold of gas and liquid inlets in order to deliver equal pressures to each channel [21]. In order to increase the production rate of MBs from a FFMD while maintaining monodispersity, we have developed a new method for distributing the gas and liquid phases. Instead of using an array of tubing to supply every individual liquid inlet, or a 3D maze of long microfluidic channels to supply multiple
Microbubbles (MBs) as ultrasound contrast agents are shell stabilized gas bubbles that are less than 10 µm in diameter and have high acoustic reflectivity. In addition to providing contrast for diagnostic imaging applications, MBs have the potential for molecular targeting [1], [2] and drug delivery [3], [4]. While there are several methods of MB production [5], the most common is the agitation method – either sonication [6] for large batches or mechanical [7] for smaller batches – which results in production rates of several billion MBs per batch. MBs produced by agitation methods are characterized by a large size polydispersity, while monodisperse MB populations are preferred for imaging [8] and drug-delivery applications [9]. Polydisperse populations can be sorted into semi-monodisperse sub-populations via various size sorting techniques including differential centrifugation [10], microfluidic flow devices [11], This work was supported by NHLBI NIH R0I HL90700. AHD is supported by the University of Virginia Cardiovascular Training Grant (CVTG) and the Medical Scientist Training Program (MSTP).
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floow-focusing nozzles, we haave coupled the t FFMD to o a preessurized liquid d flooded comp partment. This removes the neeed forr complex tubin ng interconneccts, minimizes the distance from thee liquid inlet to o the flow-focu using nozzle, and a more reliab bly asssures equal prressure at all liquid inlets. This design also a sim mplifies parallelization as additional a liqu uid inlets can be addded without the need for addiitional tubing or o interconnectts. t In addition to improved MB producction rates, this sim mplification also allows fo or significant miniaturization, creeating the poteential for catheter-based FFM MDs. By pairin ng a cattheter capablee of producing g MBs with an intravascu ular ulttrasound cathetter, it may be possible p to pro oduce, image, and a desstroy MBs near the site of intterest and as a result, r fewer MBs M maay be necessary y to achieve th he desired imaging contrast and a biooeffects. II.
Aparatus). The gas pressure syringe pump (PHD20000, Harvard A was varried between 344.5 – 103 kPa ((5-15 psi) usinng a regulator and a diigital manometter. For a selected gas ppressure, the liqquid flow rate was stepped w rate that woould produce from thhe lowest to thhe highest flow MBs. A At each point m microbubble diaameter and prooduction rate were m measured opticaally using an innverted microsscope (IX71, Olympuus, Center Vaalley, PA) coonnected to a high speed framingg camera (SIM MD24, Specialiised Imaging, Simi Valley, CA) cappable of capturring up to 330 million framess per second.
ME ETHODS
A A. Microfluidiic Device Fabrrication F Flow-focusing g microfluidic devices (FFM MDs) were castt in poly-dimethyl-silloxane (PDMS S) (Sylgard 184, Dow Corning, w Miidland, MI) frrom a custom fabricated mold. Molds were prooduced using a negative phottoresist (SU8-3 3025, Microcheem, Neewton, MA), a custom maask (Quartz, 0.1µm 0 spot siize, Miicrotronics Incc, Newtown PA A), and microp photolithography. Tw wo different mo old designs weere used: a sing gle nozzle dev vice andd a two nozzlle parallel dev vice (Fig. 1 e--f). Both desig gns shaared the follow wing common features: 7 µm m nozzle width h, 7 µm m filters at gas and liquid inleets, 36 µm and d 160 µm postss to preevent channel collapse, 35 µm µ gas chann nel, 50 µm liquid chaannel, and 20 µm µ height. Oncce the PDMS cured, liquid inllets weere opened wiith a blunt needle (18 GA A, Zephyrtroniics, Pomona CA) and a epoxy (H Hysol RE2039 9, Henkel Co orp. FE, Moooresville, NC), was used to bond tubing (microbore PTF Coole Parmer, Verrnon Hills, IL) to the gas inleet device. Two types off flooded FFM MDs were prod duced. Externaally floooded FFMDs (eFFMDs) were w plasma bo onded (March II, Noordson March, Concord, CA A) directly to the outside of a polystyrene cuveette [22] (1 min n at 60W) with h pre drilled ho oles h the liquid inllets. Internally y flooded FFM MDs thaat aligned with (iF FFMDs) were plasma p bonded to a thin layer of PDMS (30 s at 25W W). The devicce was then alig gned inside a 3 mm inner width squuare glass tubee with a pre-drrilled bubble outlet o and bond ded witth epoxy. Finaally, epoxy was used to seaal any remaining openings in the cuvette c or glasss tube (Fig 1d)..
Fig. 1: Scchematic and im mage of (a,b) exterrnally flooded FF FMD (eFFMD) and (c,d d) internally floooded FFMD (iFFMD). The liqu uid phase was delivered d to the flooded c hamber via a syrringe pump, whilee the gas phase was contrrolled using a reggulator and digitaal manometer. (e) Mold for a two nozzle paarallel FFMD. (f) Mold for a singlee channel FFMD..
C. U Ultrasonic imagging An eFFM MD was insertted into a gelatiin phantom [233], consisting of 6% ggelatin (Type A A, Fisher Scienntific) and 1% Agar (Fisher Scientiffic), which coontained a 1.779 cm diametter wall-less lumen. An oscillatoryy flow (peak poositive flow raate of 8 cm/s; peak neegative flow rrate of 2 cm/ss) was establisshed using a peristalttic pump (U Unispense 3440, Wheaton Industries, Millvillle, NJ). MB prroduction insidde the eFFMD was verified optically ly before the ddevice was placced inside the vessel. MBs were im maged inside thhe vessel – imm mediately upon production – using eiither a 6.6 MH Hz, 128 elemeent linear arrayy (Sonix RP, Ultrasonnix, Richmondd, British Colum mbia, Canada) or a 45 MHz single element intrravascular (IV VUS) catheteer (Volcano
B B. Microbubble production M MBs were produced using a liquid phase co onsisting of either 3 m mg/mL PEG-4 40-stearate (Sigma-Aldrich, St. Louis MO), 10% glycerol (F Fisher Scientiffic, Pittsburgh h PA), and 10 0% proopylene glycoll (Fisher Scien ntific) in puriffied water from ma Miillipore system m (PEG40s-G GPW) or 2% Tween (Fisher Sciientific). Priorr to use, PEG G40s-GPW waas sonicated and a filttered through 0.45 0 µm syringe filters to disp perse any micellles andd remove any large aggregattes. The gas ph hase consisted d of 99.998% pure nittrogen gas (GT TS Welco Rich hmond, VA). The T 0 µL/min using g a liqquid flow rate was varied beetween 10-110
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Reevolution, Volccano San Diego, CA) conneccted to a Volcaano IV VUS imaging sy ystem (Fig 2). III. I
tubing bbonded directlyy to each liquidd and gas inlet), suggesting that fllooded FFMD Ds have sim milar fluid ddynamics to non-floooded devices.
RESULTS AND A DISCUSSION N Table I: P Production rate aat smallest MB diiameter and diam meter at highest productioon rate for eFFM MD and iFFMD. Max Raate Size @ Device Liquid Smallest Raate @ (µm) Max rate Sm mallest (MB/s) (µm) (M MB/s) eFFMD PEG40s-GPW 7.1±0.5 53 3,000 333,000 11.3±0.6
The smallest flooded f FFMD manufactured d measured 14.5 x 2.88 x 2.3 mm, and both externally and in nternally flood ded FF FMDs were ablle to produce MBs M (Table 1). The diameterr of thee smallest MB produced by an eFFMD was 7.1 ± 0.5 µm, µ whhile the highestt throughput was w 333,000 MB/s. The smalllest diaameter MB pro oduced by an iF FFMD was 15..2 ± 0.1µm, wh hile thee highest throughput was 530 0 MB/s. W While eFFMD Ds stably prod duced MBs at high production rattes for several hours, iFFMD Ds only producced microbubb bles forr approximately y 10 minutes - longer for low wer flow rates. The T volume of liquid d entering the iFFMDs, wass greater than the uid exiting th he device, leeading to liqu uid volume of liqu T acccumulation insside the device and an increassed pressure. This preessure compressed the co ompliant micrrofluidic deviice, lim miting flow through t the microchannelss and lowering thrroughput. By bonding the FFMD to thee exterior of the cuvvette, we weere able mitig gate this prob blem while still s maaintaining the advantages off the flooded design – i.e. no liqquid inlet tubes, a single gaas inlet tube, and a potential for miiniaturized desiign.
iFFMD
Tween
15.2±0.1
28 80
530
20.4±0.9
To fuurther increase production ratte, a two nozzlee parallelized eFFMD D was fabricateed. The parallelized device haad a 1.5-fold increasee in MB prodduction rate w without the need for any additionnal tubing or innterconnects. T The smallest M MB produced by this ddevice was 7.33 ± 0.3 µm whille a peak produuction rate of approxiimately 500,0000 MB/s was reealized (Fig 4)..
Fig. 3: M MB diameter and d throughput oveer a range of flow rates for an eFFMD. Liquid phase = P PEG40s-GPW.
Figg. 2: Schematic of ultrasonic imag ging apparatus. B-mode B images were w acq quired using a 128 8 element 6.6 MH Hz linear array atttached to a resea arch enaabled clinical scan nner. Intravascullar ultrasound im mages were acquiired usin ng a 45 MHz sing gle element IVUS catheter.
20 µm
A Although we found f that the FFMD had to o be bound to the outtside of the preessurized cham mber to preventt collapsing of the miicrochannels, this t may poten ntially be allev viated by using g a MS riggid substitute (ee.g. glass, metaal, or plastic) in place of PDM andd will be invesstigated in the future. f A characterizaation of an eFF FMD (Fig 3) exhibits a trend d of deccreased MB sizze as productio on rate increasees. The flow raates weere limited to th hose that stably y produced MB Bs with a diameeter lesss than the heiight of the FFM MD to avoid the t production n of verrtically constraained, cylinderr shaped, MBss. The throughp put andd MB diameterr curves in Fig 3 follow a sim milar pattern as the thrroughput and MB diameterr of non-flood ded devices (i.e. (
Fig. 4: H High speed imagess of a parallelized d eFFMD. Maxim mum combined productioon rate = 500,0000 MB/s. Left im mage shows MB generation at nozzle 1 and right image shows MB generration at nozzle 22. Both nozzles shared th he same gas inputt and each liquid inlet was attacheed to a common pressurizzed liquid comparrtment.
Finallly, an eFFMD was imaged ussing a 6.6 MHzz linear array (Fig 4a)) and a 45 MHzz IVUS catheteer (Fig 4b). Strrong acoustic reflectivvity from the MBs was obsserved, and thee MBs were stable eenough to survvive the oscillaatory flow alonng the entire length oof the vessel (1 3 cm). The MB Bs could be seeen forming in the eFF FMD and stronggly enhanced tthe lumen of thhe vessel (Fig
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[3]
5b). The MBs produced by the eFFMD exhibited the same characteristics of MBs produced using lipid based agitation methods. They could be used to perform Doppler velocity measurements, could be pushed away from the top of the vessel wall using primary radiation force (1 MHz continuous wave), and could be ruptured at power settings known to rupture lipid based MBs. Even at 45 MHz, eFFMD generated MBs produced good contrast (Fig 5d), where the vessel wall can be seen in the upper right and the MBs in the lumen in the lower left.
[4] [5] [6]
[7]
[8] [9]
[10] [11]
[12] [13] [14] Fig. 5: Ultrasound images of an eFFMD producing MBs in a 1.79 cm diameter wall-less vessel. Flow rate = 8 cm/s. Top row: 6.6 MHz B-mode 128 linear array; (a) vessel, (b) vessel with MBs. Scale bar = 0.25 cm, dynamic range = 80 dB. Bottom Row: 45 MHz IVUS image; (c) vessel, (d) vessel with MBs.
IV.
[15] [16]
CONCLUSIONS
We have successfully demonstrated the fabrication and operation of a flooded flow-focusing microfluidic device. By coupling the device to a pressurized flooded compartment, this new method for supplying liquid (i.e. shell material) to a FFMD eliminates complex manifolds and tubing interconnects. This simplifies FFMD parallelization and miniaturization, which helps us towards our goal to develop a therapeutic intravascular ultrasound catheter capable of in situ MB production, imaging, and destruction.
[17] [18]
[19] [20]
ACKNOWLEDGMENTS
[21]
The authors would like to thank Dr. Arthur Weston Lichtenberger for his advice on photolithography. Volcano Corp provided a loan of equipment and IVUS catheters. [22]
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