ultrasound imaging is 
 one of the most inexpensive , safe , and commonly 
 used diagnostic tools for imaging soft tissues and vasculature .
the use of microbubble contrast agents enables 
 visualization of microvasculature which can not be seen directly with 
 doppler ultrasound .
microbubbles composed of gaseous cores covered 
 with stabilizing agents can drastically enhance the ultrasound signal 
 because of their large compressibility , which leads to enhanced scattering 
 of ultrasound .
the echogenicity of microbubbles 
 coupled with their physical interactions with acoustic energy can 
 also be used for triggered release of active agents or for conversion 
 of acoustic energy to thermal energy to enable therapeutic applications . 

for example , recent studies have shown that the insonation of microbubbles 
 with low - intensity ultrasound can lead to a localized temperature 
 increase , which in turn disrupts tumor vasculature ( also known as 
 antivascular ultrasound therapy ) , enabling a minimally invasive procedure 
 to disrupt cancerous tissues .
these properties of microbubbles make 
 them ideal candidates for theranostics ; that is , the same microbubble 
 agents can be used for diagnostics and therapeutic applications .
currently available commercial agents consist 
 of polydisperse microbubbles 
 with size distributed over a broad range of diameters .
studies have 
 shown that the effectiveness of these agents can be significantly 
 enhanced by making the size distribution narrow for molecular imaging 
 and therapeutic applications . although some methods to fractionate microbubbles 
 to enhance the uniformity of size have been reported , these techniques 
 inevitably lead to loss of significant fraction of bubbles .
another important factor that significantly affects microbubble 
 properties for ultrasound - related applications is the surfactant that 
 is used to stabilize microbubbles .
an approach to control the molecular 
 structure and properties of these surfactants would be highly beneficial 
 because their structure affects the surface functionality and the 
 echogenicity of microbubbles .
the generation of monodisperse microbubbles 
 that are stabilized with surfactants that can be precisely designed 
 and controlled would lead to microbubbles that have ideal functionality 
 for ultrasound imaging and novel therapeutic approaches such as targeted 
 drug delivery and antivascular ultrasound therapy ( avust ) . in this study , we present a method to create stable protein - shelled 
 microbubbles using a microfluidic flow focusing device that uses an 
 air - actuated membrane valve , which enables the production of highly 
 monodisperse sub-10 m microbubbles . although other studies 
 have shown that monodispserse bubbles can be generated based on microfluidic 
 techniques , the size range of microbubbles that can be generated from such devices 
 is somewhat limited .
a method based on the dissolution of highly soluble 
 gas such as co2 in a long microfluidic channel has shown 
 to generate monodisperse bubbles of varying sizes .
a method that enables the formation of bubbles over a wide 
 range of size without using soluble gas and long channels would provide 
 a complementary method that can further expand the use of microfluidic 
 techniques to generate monodisperse microbubbles .
the air - actuated 
 membrane valve enables precise control over the size of microbubbles 
 while producing highly monodisperse microbubbles . to stabilize the 
 microbubbles generated by the microfluidic technique
unlike common proteins that have been used to stabilize microbubbles , oleosin potentially provides 
 versatility in imparting additional functionality via recombinant 
 protein technology .
we demonstrate an example of 
 such modularity by expressing and incorporating fluorescent oleosin 
 into the microbubble shell .
we demonstrate that careful tuning of 
 the composition of the stabilizing agents is critical in the formation 
 of highly stable and monodisperse microbubbles that are echogenic 
 under ultrasound insonation .
microfluidic flow focusing 
 devices with expanding nozzle design ( figure 1a ) are fabricated using single layer soft lithography in poly(dimethylsiloxane ) 
 ( pdms ) .
negative photoresist su-8 2010 ( microchem , 
 newton , ma ) , thinned to a 3:1 ratio with su-8 developer , is spin - coated 
 onto a clean silicon wafer to a thickness of 5 m and patterned 
 to uv light through a transparency photomask ( cad / art service , bandon , 
 or ) using a karl suss ma4 mask aligner ( suss microtec inc . , sunnyvale , 
 ca ) .
to incorporate an air - actuated valve , we use single - layer membrane 
 valves , which exist in the same plane 
 as the microfluidic channel , allowing us to fabricate the entire microfluidic 
 device in a single layer mold .
sylgard 184 poly(dimethylsiloxane ) 
 ( dow corning , midland , mi ) is mixed with cross - linker ( ratio 12:1 ) , 
 degassed thoroughly and poured onto the photoresist pattern , and cured 
 for 1 h at 65 c to make the membrane highly compliant . the pdms 
 replica are peeled off the wafer and bonded to a pdms membrane fabricated 
 by spin - coating pdms on a glass slide after oxygen plasma activation 
 of both surfaces . having a microchannel fully enclosed in pdms allows 
 for more efficient use of the valve membrane .
( a ) schematic illustration 
 of a pdms microfluidic device used to 
 generate monodisperse microbubbles of different sizes . ( b ) cross - sectional 
 geometry of the nozzle ( see figure s1 for 
 junction dimensions ) .
( c ) schematic of a microbubble stabilized with 
 a mixture of oleosin and ( peo)n-(ppo)m-(peo)n triblock 
 copolymer .
multiple rounds of pcr are used to create 
 the oleosin gene 42 - 30g-63 and egfp-30g-63 .
the genes are inserted 
 into the expression vector pbamuk , a pet series derivative constructed 
 by the duyne laboratory ( som , penn ) .
pbamuk adds a 6-histidine tag to the c - terminus of the protein for 
 imac purification .
protein is expressed in the e. coli strain bl21 de3 ( stratagene ) controlled by the lac 
 promoter .
cultures are grown at 37 c in luria bertani ( lb ) with 
 kanamycin ( 50 g ml ) until od600  0.70.9 .
protein expression is induced with isopropyl 
 -d-1-thiogalactopyranoside ( iptg ) to a final concentration 
 of 1.0 mm .
cells are harvested by centrifugation , and cell pellets 
 are frozen at 20 c prior to purification .
b - per protein 
 extraction agent ( fisher scientific ) is used for protein purification . 
 42 - 30g-63 is expressed in inclusion bodies whereas egfp-42 - 30g-63 
 is expressed in the soluble fraction of the cell .
42 - 30g-63 is purified 
 according to the b - per protocol for inclusion bodies , and egfp-42 - 30g-63 
 is purified according to the protocol for soluble proteins .
the concentration of purified protein is measured 
 with a nano - drop 1000 ( thermo scientific ) .
all analysis is completed in pbs unless otherwise noted . 
 to establish the purity of the proteins
, sds / page gels are run on 
 nupage novex 412% bis - tris mini gels ( invitrogen ) in mes buffer . 

the gel is destained overnight in water and imaged 
 with a kodak gel logic 100 imaging system .
sample spots are created with 0.5 l 
 protein in 1 pbs and 0.5 l of saturated sinapinic acid 
 solution ( 50/50 acetonitrile / water + 0.1% tfe ) .
spectra are collected 
 on an ultraflextreme maldi - tof ( bruker , billerica , ma ) ( see figure s5 for egfp spectra ) . to measure the protein 
 secondary structure , far - uv cd spectra
are collected at 25 c 
 on an aviv 410 spectrometer ( aviv biomedical inc . ) using a 1 mm quartz 
 cell .
naf is used to replace nacl due to the strong absorbance of 
 the cl ion .
the liquid 
 phase containing the shell material consists of oleosin or a solution 
 containing oleosin proteins and ( peo)78-(ppo)30-(peo)78 or ( peo)100-(ppo)65-(peo)100 diluted in phosphate - buffered saline ( pbs ) ( ph 7.2 , sigma - aldrich , 
 st .
the liquid phase consisting 
 of oleosin and ( peo)n-(ppo)m-(peo)n triblock copolymers 
 at the optimal concentration dispersed in pbs is supplied to the device 
 using a syringe pump ( harvard apparatus phd ultra ) at flow rates between 
 500 and 1000 l h. to connect the channels 
 to syringes , polyethylene tubing with an i.d . of 0.38 mm and an o.d . 
 of 1.09 mm ( bb31695-pe/2 , scientific commodities inc , lake havasu 
 city , az ) is used .
the gas phase consists of 99.999% pure nitrogen 
 gas ( n2 , gts welco , richmond , va ) or octafluorocyclobutane 
 ( c4f8 ) ( synquest laboratories , alachua , fl ) 
 supplied to the device using a pressure regulator ( type 700 , controlair 
 inc . ,
polyethylene 
 tubing with an i.d . of 0.86 mm and an o.d . of 1.32 mm ( bb31695-pe/5 , 
 scientific commodities inc , lake havasu city , az )
the membrane valve is actuated 
 using a dual - valve pressure controller ( pcd-100psig - d - pcv10 , alicat 
 scientific , tucson , az ) at pressure between 0 and 40 psi .
microbubbles 
 are produced by first applying a small pressure to the gas inlet ( 24 
 psi ) immediately followed by injecting the liquid phase at the desired 
 flow rate ( 5001000 l h ) .
the gas 
 pressure is then increased slowly until steady state of bubble generation 
 is reached .
images of microbubbles production are captured using an 
 inverted microscope ( nikon diaphot 300 ) connected to a high speed 
 phantom v7 camera . for microbubbles that remain stable during generation 
 and collection ,
water interface in 35 mm petri dishes , 
 acquiring images under an upright microscope ( carl zeiss axio plan 
 ii ) connected to a qimaging retiga 2000r camera .
microbubbles diameter 
 variation over time is measured and images are analyzed using imagej 
 ( v 1.47v , nih ) .
a nicolet 
 8700 ft - ir spectrophotometer ( thermo scientific ) is used to obtain 
 the ft - ir spectra of microbubbles and their constituent solutions 
 on znse windows ( phoenix infrared , lowell , ma ) .
samples are prepared 
 by placing a small aliquot of solution on top of the window and are 
 fully dried before measurements are performed .
the spectra are taken 
 between 5000 and 600 cm , at 1.93 cm wavenumber resolution .
microbubbles 
 for ultrasonic imaging 
 are collected and imaged directly in 16 mm membrane dialysis bag , 
 which is prefilled with buffer solution and sealed at one end .
after 
 a desired amount of bubbles is collected , the tube is sealed at the 
 other end , carefully avoiding formations of air pockets .
the collected 
 microbubbles are imaged using a clinical ultrasound scanner hdi 5000 
 ( phillips / atl , bothell , wa ) which is equipped with a broadband high - frequency 
 ultrasound transducer at 715 mhz .
grayscale b - mode images 
 are acquired with a mechanical index ( mi ) of 0.37 and 0.47 with focus 
 between 0.51.5 and 12 cm , respectively .
for a variety of applications 
 that involve microbubbles and ultrasound , 
 the size distribution of microbubble agents drastically influences 
 the efficacy of the image contrast enhancement and therapeutic methods . 

to enable formation of microbubbles with high monodispersity and , 
 at the same time , tunable size , we use an expanding nozzle flow - focusing 
 microfluidic device with a single - layer membrane valve at the orifice 
 as schematically illustrated in figure 1 .
a 
 previous study has shown that the size of liquid emulsion droplets 
 produced by a flow - focusing microfluidic device can be controlled 
 by changing the size of the orifice via the actuation of the valve .
likewise , this design gives us the flexibility 
 to tune the size of gas microbubbles in the same chip without changing 
 the continuous phase or gas flow rates , by only changing the size 
 of the orifice through the application of pressure to the valve .
furthermore , 
 the use of the single - layer membrane valve overcomes the low resolution 
 that is typically achieved by using polymeric photomasks ( smallest 
 feature 10 m ) . for the initial testing of the 
 microfluidic device to control the 
 size of microbubbles
, we use nitrogen gas and a common surfactant , 
 sodium dodecyl sulfate ( sds , sigma - aldrich , st .
louis , mo ) , at a concentration 
 of 20 mg ml in the aqueous phase to stabilize 
 microbubbles .
we are able to produce monodisperse microbubbles with 
 radius ranging from approximately 2 to 10 m for several hours 
 without changes in the bubble size .
an advantage of this microfluidic 
 device is that the size of microbubbles that can be generated from 
 a single microfluidic device can be controlled over a wide range , 
 unlike most flow - focusing microfluidic devices that have limited range 
 of size control . by increasing the pressure that 
 is applied to the single - layer valve
, we can control the size of the 
 nozzle and , in turn , the size of microbubbles as shown in figure 2 .
we observe that the diameter of the microbubbles , db , decreases linearly with the width of the 
 nozzle , wn .
interestingly , the microbubble 
 generation frequency ( f = the number of microbubbles 
 generated per second ) is inversely proportional to the volume of microbubbles 
 as shown in the inset of figure 2b ( f  db ) .
such a trend indicates that the gas 
 flow rate , calculated to be qg  
 62 l h (  = 8.4 l 
 h ) , remains more or less constant under varying 
 nozzle size .
the constant gas flow rate under varying nozzle width 
 may be attributed to the change in the cross - sectional shape of the 
 channel , from a horizontal slit to a square or hourglass shape .
although 
 sds enables the investigation of microfluidic device performance , 
 microbubbles formed using sds are not stable upon collection .
( a1a9 ) 
 series of micrographs of the microfluidic device 
 during the generation of microbubbles using a solution containing 
 sds at a concentration of 20 mg ml in the aqueous 
 phase . by changing the size of the nozzle , which is controlled by 
 an air - actuated valve placed at the orifice
the inset shows the microbubbles generation 
 frequency ( f ) vs volume of microbubbles ( db ) .
the linear relationship 
 between the two quantities indicates that the gas flow rates remains 
 more or less constant under varying nozzle size . to produce stable microbubbles with high monodispersity , 
 size tunability , 
 and structural modularity
the protein has a natural amphiphilic 
 structure with n- and c - terminal hydrophilic arms and a central hydrophobic 
 core containing a proline knot forcing the protein into a hairpin 
 structure .
oleosin has been used in various biotechnology 
 and biomedical applications exploiting its amphiphilic properties . in its native state ,
eliminating a large portion of the hydrophobic domain and removing 
 the majority of the secondary structure in the protein backbone have 
 been shown to yield a oleosin mutant that becomes highly soluble in 
 water and naturally self - assembles into micelles .
the soluble oleosin mutant is named 42 - 30 - 63 defining the 
 number of amino acids in each domain : the n - terminal hydrophilic arm , 
 the central hydrophobic core , and the c - terminal hydrophilic arm , 
 respectively .
this molecule is produced by truncating the wild - type 
 molecule without changes in the sequence of amino acids .
the 42 - 30 - 63 
 oleosin mutant is further modified by inserting five glycines into 
 the hydrophobic core ( see supporting information for protein sequences ) creating a mutant we refer to as 42 - 30g-63 .
the protein is expressed in the escherichia 
 coli strain bl21 ( de3 ) with isopropyl -d-1-thiogalactopyranoside 
 ( iptg ) induction .
protein is purified using immobilized metal affinity 
 chromatography through a 6-histidine tag on the c - terminus of the 
 protein , leading to highly purified products ( figure 3 ) .
protein molecular weight is confirmed with sds - polyacrylamide 
 gel electrophoresis ( sds - page ) and matrix - assisted laser desorption / ionization - time - of - flight 
 ( maldi - tof ) mass spectroscopy ( figure 3 ) .
the 
 addition of the five glycines to the 42 - 30 - 63 mutant increases the 
 protein expression , stability , and solubility while abolishing secondary 
 structure , as shown by circular dichroism ( figure 3 ) .
in contrast , the cd spectra of wild - type ( wt ) oleosin shows 
 -sheet character as previously reported ( see supporting information for detailed analysis ) .
( a ) sds - page gel showing > 95% purity for 42 - 30g-63 . 

( b ) madli - tof 
 spectra confirming the molecular weight for 42 - 30g-63 ( expected : 15  027 ; 
 measured : 15  025 ) . ( c )
far - ultraviolet circular dichroism ( uv 
 cd ) spectra of 42 - 30g-63 and wild - type oleosin .
when we produce microbubbles using oleosin , at concentrations 
 between 
 1 and 2 mg ml , we can only stabilize bubbles with 
 radius above 10 m . during the generation of microbubbles with 

radii smaller than 10 m , bubbles are observed to undergo coalescence 
 within and outside of the microfluidic device ( figure s2 ) .
in addition , the relatively high surface tension 
 between the liquid and the gas phases makes the generation of such 
 microbubbles challenging , often resulting instability of microbubbles 
 in the microfluidic device .
interestingly , a number of microbubble 
 systems that are currently 
 being investigated ( e.g. , phospholipid - stabilized microbubbles ) often 
 have extra components such as poly(ethylene glycol)-based surfactants 
 such as amphiphilic triblock copolymers to enhance the microbubble 
 stability and generation process .
thus , we add widely used poly(ethylene 
 glycol)-b - poly(propylene glycol)-b - poly(ethylene glycol ) triblock copolymers ( ( peo)n-(ppo)m-(peo)n where n and m denote the 
 number of ethylene oxide and propylene oxide repeat units , respectively ; 
 these polymers are also known as pluronic and polxamer ) to the oleosin 
 solution to test whether the production of microbubbles can be facilitated .
we test two different types of ( peo)n-(ppo)m-(peo)n triblock copolymers : ( peo)100-(ppo)65-(peo)100 and ( peo)78-(ppo)30-(peo)78 . when we use a mixture containing 12 
 mg ml oleosin and 520 mg ml ( peo)100-(ppo)65-(peo)100 ( average 
 molecular weight 12  600 ) , we are able to consistently generate 
 monodisperse microbubbles at the nozzle ; however , these microbubbles 
 undergo significant coalescence upon collection .
in contrast , when 
 we add ( peo)78-(ppo)30-(peo)78 ( average 
 molecular weight 8400 ) to oleosin solutions , we are able to generate 
 microbubbles at the nozzle and very limited coalescence is observed 
 upon collection .
we find that the optimal concentration for stable 
 microbubble formation requires an aqueous phase containing 1 mg ml of oleosin and 10 mg ml of ( peo)78-(ppo)30-(peo)78 .
( peo)78-(ppo)30-(peo)78 is known to be more effective 
 in stabilizing gas bubbles than ( peo)100-(ppo)65-(peo)100 , which may explain the effectiveness of the 
 former in facilitating the microbubble production . to further understand the possible role of ( peo)78-(ppo)30-(peo)78 in facilitating the 
 formation of microbubbles 
 and the role of oleosin in imparting long - term stability to microbubbles , 
 we perform ft - ir spectroscopy of four different samples : pure oleosin , 
 pure ( peo)78-(ppo)30-(peo)78 , a mixture 
 of oleosin and ( peo)78-(ppo)30-(peo)78 with the same composition as the solution used for microbubble generation 
 ( 1:18 mole ratio ) , and microbubbles .
ft - ir spectra remarkably show 
 that the composition of microbubble shell is very different from that 
 of the solution as shown in figure 4 .
the concentration 
 of oleosin present in the microbubble shell is significantly higher 
 than that of the original solution used for microbubble generation , 
 as evidenced by the prominent presence of peaks associated with pure 
 oleosin in the microbubble spectrum ( e.g. , peaks found around 1535 , 
 1650 , and 3290 cm ) . although it is not straightforward 
 to quantify the composition of the microbubble shell based on ft - ir , 
 the comparison of the four spectra shows that oleosin seems to be 
 the major species that is stabilizing microbubbles .
these results 
 suggest that ( peo)78-(ppo)30-(peo)78 present in the solution facilitates microbubble production by lowering 
 the surface tension and rapidly covering the microbubbles upon breakup 
 at the nozzle . once microbubbles are generated and flow through the 
 channel , oleosin starts to adsorb and possibly displace some of ( peo)78-(ppo)30-(peo)78 that are on the microbubble 
 surface .
the spectra of pure oleosin and microbubbles 
 are amplified by factors of 2.5 and 5 , respectively , to clearly show 
 the features .
micrographs of microbubbles 
 produced using a solution containing 
 1 mg ml oleosin and 10 mg ml ( peo)78-(ppo)30-(peo)78 .
( a ) a small number of large bubbles are present upon collection 
 via plastic tubing .
( b ) big bubbles disappear 24 h after collection , 
 leaving monodisperse microbubbles . in the samples that are collected through polyethylene tubing
, 
 we typically observe that there are a small number of fairly large 
 bubbles ( > 20 m in diameter ) .
although the physical mechanism 
 behind the appearance of these large bubbles is not known , their number 
 fraction is extremely small , typically less than 1% .
interestingly , 
 these large bubbles disappear completely approximately 24 h after 
 collection , leaving behind a collection of highly monodisperse microbubbles 
 as shown in figure 5 .
since we do not see any major coalescence 
 between microbubbles occurring within the pdms microfluidic device , 
 we believe these large bubbles likely form during transfer of the 
 microbubbles from nozzle to a container via polyethylene tubing .
possibly , 
 abrupt changes in dimensions and relative shear stress experienced 
 by microbubbles between the pdms device and the collection tube as 
 well as the lower speed at which the microbubbles travel in the polyethyelene 
 tube before being released in a petri dish may lead to collision between 
 bubbles and eventual coalescence .
another possibility is that these 
 large bubbles have slightly different surface composition since they 
 are observed to undergo dissolution when they are stored for an extended 
 period , whereas the monodisperse bubbles that were originally generated 
 at the nozzle do not dissolve completely over a long period of time . 

interestingly , we are able to collect highly monodisperse microbubbles 
 without any large bubbles if we collect the produced bubbles straight 
 into a well that is position in the same plane as the microfluidic 
 channel ( figure 6 ) .
the high monodispersity 
 of microbubbles is illustrated by their ability to pack into hexagonal 
 array , which indicates that the coefficient of variation ( cv ) is less than 5% around the 
 average bubble size , consistent with our optical microscopy - based 
 analysis .
these results show that even small perturbations can lead 
 to disruption of microbubbles that are generated using microfluidic 
 devices , and extra care must be taken in collecting microbubbles for 
 clinical applications since large bubbles in blood vessels can lead 
 to serious problems such as embolism .
micrograph of monodisperse microbubbles 
 produced using a solution 
 containing 1 mg ml oleosin and 10 
 mg ml ( peo)78-(ppo)30-(peo)78 and collected into a well in the pdms device without the 
 use of plastic tubing .
 ,  , and cv in the inset represent the average 
 ( in m ) , standard deviation ( in m ) , and coefficient of 
 variation , respectively .
microbubbles generated using the mixture of oleosin and ( peo)78-(ppo)30-(peo)78 ( molar ratio of oleosin : triblock 
 copolymer = 1:18 ) are remarkably stable once they are collected .
when 
 microbubbles are collected and stored in water ( microbubbles reside 
 at the air  water interface due to their buoyancy ) , microbubble 
 radius decreases by about 13% during the first few days and eventually 
 ceases to shrink further .
these microbubbles remain stable at least 
 for 4 weeks , and their size does not show any changes after 5 days 
 as shown in figure 7 , suggesting that these 
 microbubbles will not undergo dissolution even after 4 weeks .
the 
 stability of these microbubbles does not depend on whether n2 or c4f8 is used as the gas phase .
in contrast , 
 microbubbles generated solely with ( peo)78-(ppo)30-(peo)78 do not exhibit such excellent stability .
these 
 results indicate that oleosin plays a critical role in stabilizing 
 the shell of microbubbles , which likely consists of a mixture of oleosin 
 and ( peo)78-(ppo)30-(peo)78 , to prevent 
 complete dissolution or coalescence of microbubbles upon their collection . 

similar examples , in which shells suppresses the dissolution of microbubbles , 
 have been observed in microbubbles that have been stabilized with 
 other types of proteins , nanoparticles , or synthetic polymers .
micrographs 
 showing microbubbles stability over time for microbubbles 
 produced using a solution containing 1 mg ml oleosin and 10 mg ml ( peo)78-(ppo)30-(peo)78 .
( b ) microscope images of 24 days after collection . as discussed briefly above ,
one 
 of the unique aspects of oleosin 
 is that the molecular structure and thus the properties of the monolayer 
 that contains this molecule can be engineered using recombinant protein 
 technology .
recombinant protein technology allows for precise molecular 
 engineering of proteins generated from microorganisms such as bacteria 
 and thus can be used to generate oleosin species with different functionality 
 and properties . to demonstrate proof - of - principle 
 that this molecule has such modularity
, we express a green fluorescent 
 protein mutant oleosin by fusing enhanced green fluorescent protein 
 ( egfp ) to the n - terminus of the 42 - 30g-63 oleosin .
the modified oleosin 
 genes are constructed using standard molecular biology techniques 
 and cloned into the expression vector pbamuk .
it is 
 evident that the microbubbles produced with the blend of the two oleosin 
 species ( pure at 1 mg ml , mutant at 0.05 mg ml ) along with 10 mg ml ( peo)78-(ppo)30-(peo)78 have the egfp mutant 
 species incorporated in the bubble shell , whereas the microbubbles 
 generated without the egfp mutant species do not show surface fluorescence 
 ( figure 8) .
also , fluorescence intensity is 
 observed to be fairly uniform on the surface of the bubbles .
our results 
 clearly indicate that that oleosin with different functionalities 
 can be generated and incorporated into the microbubble shell and that 
 oleosin distributes uniformly on the surface of microbubbles .
confocal fluorescent 
 microscopy images of bubbles produced with 
 ( a , b ) oleosin and ( c , d ) with a blend containing the egfp mutant . 
 in both cases
a solution containing 1 mg ml oleosin and 10 mg ml ( peo)78-(ppo)30-(peo)78 is used to produce microbubbles . 

echogenicity measurements are carried out using microbubbles 
 generated 
 with a solution containing 1 mg ml oleosin and 
 10 mg ml ( peo)78-(ppo)30-(peo)78 .
we collect microbubbles directly in a 3 
 cm long dialysis tubing with a diameter of 16 mm , which is sealed 
 at one end and prefilled with pbs solution containing 10 mg ml ( peo)78-(ppo)30-(peo)78 .
microubbles are transferred directly into the dialysis tube from 
 the pdms device outlet using polyethylene tubing , which is submerged 
 in the pbs solution . after collecting a desired amount of microbubbles , 

the tube is sealed on the other end to avoid introducing any air pockets 
 and is stored in 50 ml centrifuge tubes filled with pbs solution containing 
 10 mg ml ( peo)78-(ppo)30-(peo)78 .
the tube is kept on a spinning wheel rotating 
 at 60 rpm to induce continuous motions of the microbubbles and more 
 importantly to remove large bubbles that may have been collected . 

since antivascular and other microbubble - based therapies are monitored 
 using high - frequency ultrasound , the echogenicity 
 of the microbubbles is tested using a broadband high - frequency ultrasound 
 transducer at 715 mhz in brightness mode ( b - mode ) .
the microbubbles 
 are acoustically active along the entire length of the dialysis tube 
 as shown in figure 9 .
in contrast , a pbs solution 
 containing 10 mg ml ( peo)78-(ppo)30-(peo)78 without any microbubbles does not show 
 any acoustic signal , indicating that the oleosin - stabilized microbubbles 
 are highly echogenic .
microbubbles remain acoustically responsive 
 30 min after the initial measurement and even 1 week after the first 
 measurement , showing nondetectable changes in the signal brightness 
 ( figure 9 ) .
these results clearly indicate 
 that these microbubbles stabilized with oleosin are highly stable 
 and echogenic and thus could have significant potential for theranostic 
 applications .
ultrasound sonography images of c4f8 microbubbles 
 generated with a solution containing 1 mg ml oleosin and 10 mg ml ( peo)78-(ppo)30-(peo)78 . ultrasound images of microbubbles 
 ( a , b ) 12 h after generation and ( c , d ) 30 min and ( e , f ) 
 7 days after initial imaging .
ultrasound images of control samples 
 are reported in panels g and h. the microbubbles have a radius of 
 about 4 m .
we have shown that a recombinant mutant oleosin , 
 in combination 
 with a triblock copolymer , ( peo)78-(ppo)30-(peo)78 , can be used to successfully produce stable and monodisperse 
 microbubbles with high echogenicity .
we demonstrate that the use of 
 a pdms microfluidic device with an air - actuated valve is an effective 
 method to control the size of microbubbles while maintaining narrow 
 size distribution .
microbubbles incorporating oleosin show high stability 
 and can be further functionalized using recombinant protein technology , 
 which we demonstrated by the incorporation of egfp mutant oleosin 
 into microbubbles .
we envisage that the combination of microfluidic 
 generation and oleosin - based stabilization of microbubbles will represent 
 a promising platform for ultrasound - related applications . in particular , 
 by functionalizing oleosin with specific targeting ligands via recombinant 
 protein techniques , it will be possible to enable 
 localized microbubble - based ultrasound therapy . also , by varying the 
 molecular structure of oleosin ( e.g. , controlling the structure of 
 hydrophobic domain ) , microbubble shells with different rheological 
 properties could be generated .
 microbubbles are used as contrast 
 enhancing agents in ultrasound 
 sonography and more recently have shown great potential as theranostic 
 agents that enable both diagnostics and therapy . 
 conventional production 
 methods lead to highly polydisperse microbubbles , which compromise 
 the effectiveness of ultrasound imaging and therapy . stabilizing microbubbles 
 with surfactant molecules that can impart functionality and properties 
 that are desirable for specific applications would enhance the utility 
 of microbubbles . here 
 we generate monodisperse microbubbles with a 
 large potential for functionalization by combining a microfluidic 
 method and recombinant protein technology . 
 our microfluidic device 
 uses an air - actuated membrane valve that enables production of monodisperse 
 microbubbles with narrow size distribution . 
 the size of microbubbles 
 can be precisely tuned by dynamically changing the dimension of the 
 channel using the valve . 
 the microbubbles are stabilized by an amphiphilic 
 protein , oleosin , which provides versatility in controlling the functionalization 
 of microbubbles through recombinant biotechnology . 
 we show that it 
 is critical to control the composition of the stabilizing agents to 
 enable formation of highly stable and monodisperse microbubbles that 
 are echogenic under ultrasound insonation . 
 our protein - shelled microbubbles 
 based on the combination of microfluidic generation and recombinant 
 protein technology provide a promising platform for ultrasound - related 
 applications . 
Introduction
Materials
and Methods
Results and Discussion
Conclusions
and Outlook