figure 1 represents the schematic diagram 
 of our solid - state , label - free plasmonic biosensor fabrication for 
 mir detection .
chemically synthesized gold nanoprisms ( figure 1a ) , which displayed their lspr at 797 nm upon attachment to solid substrate immersed in 
 pbs buffer , were selected as nanoantennas for our biosensor fabrication 
 because ( 1 ) their lspr peak position ( in the 700900 
 nm wavelength range ) is particularly suitable for biomolecule detection 
 because of negligible background scattering and adsorption of endogeneous 
 chromophores from physiological media such as blood and plasma ; ( 2 ) they have strong electromagnetic ( em ) field enhancement at the 
 sharp tips ; ( 3 ) they exhibits a strong lspr 
 response to small changes in their surrounding environment ; ( 4 ) their atomically smooth surface allows formation of a self - assembled 
 monolayer ( sam ) of receptors with both 
 a tightly packed lower layer of alkylthiols and a more loosely packed 
 upper layer that provide the required space for duplex formation with 
 complementary mir strands ; ( 5 ) gold is nontoxic and stable under extreme 
 physiological conditions ; and ( 6 ) the 
 gold  sulfur bond is very stable with thiol - modified receptors 
 making a strong covalent bond with the gold surface .
details describing 
 the synthesis of gold nanoprisms and their attachment onto the silanized 
 glass substrate are provided in supporting information .
recently , we have shown that a molecular 
 sensor fabricated using an 35 nm average edge - length gold 
 nanoprisms displayed an unprecedentedly large 21 nm reversible shift 
 upon a minor 0.6 nm increase in the thickness of the local dielectric 
 environment .
therefore , gold nanoprisms of this size and geometry 
 are unique and should provide extremely high sensitivity if plasmonic 
 biosensors are fabricated using them , which is the scope of this letter . 

this letter provides the first example of lspr - based mirs sensing 
 in physiological media .
design of plasmonic biosensors and detecting 
 mir - x in various physiological 
 media .
( a ) chemically synthesized and freshly prepared gold nanoprisms 
 were covalently attached onto a 3-mercaptopropyltriethoxysilane - functionalized 
 glass coverslip ( substrate ) .
( b ) surface of gold nanoprisms was chemically 
 modified with a 1.0 m 1:1 mixture of sh - c6-ssdna - x and peg6-sh in pbs buffer ( ph 7.4 ) to prepare the plasmonic biosensor .
( c ) incubation of 
 sensor in mir - x solution and formation of dna duplex .
( d ) schematic 
 of the extinction spectrum of the biosensor collected in pbs buffer 
 after modification with a 1.0 m 1:1 mixture of sh - c6-ssdna - x 
 and peg6-sh ( blue curve ) .
the extinction spectrum was again 
 collected after incubation in mir - x solution and careful rinsing with 
 pbs buffer to determine the new peak position ( red curve ) .
the extent 
 of lspr dipole peak shift ( lspr ) depends 
 on the concentration of mir - x used during the incubation in ( c ) , which 
 ranged from 100 nm to 50 fm .
( e ) plot of lspr versus log of mir - x concentrations used to determine the limit of 
 detection .
our targets were mir-21 
 and mir-10b , 
 because we have shown by locked nuclei acid  based in situ hybridization 
 that they are overexpressed in pancreatic cells ( pccs ) within the 
 tumor mass and that circulating mir-10b 
 may serve as biomarker for diagnosis of pdac .
we designed the sensing strategy based on the hybridization between 
 complementary probes ( -c6-ssdna - x , x = 21 and 10b ) attached to gold 
 nanoprisms and their target mirs .
the introduction of spacers in - between 
 the dna probes was included to reduce steric hindrance between the 
 probes and the mirs and therefore to enhance the hybridization and 
 ultimately the sensitivity . as shown in figure 1b , poly(ethylene glycol)6-thiols ( peg6-sh ) 
 were used as spacers because they avoid nonspecific adsorption of 
 extraneous materials onto the nanoprism s surface and are not 
 reactive toward mirs or other biological constituents present in plasma . 

previously , we demonstrated that functionalization of a nanoantenna s 
 surface with an equal mole ratio of receptor and spacer provided the 
 best sensitivity and lowest limit of detection ( lod ) .
therefore , a 1:1 ratio of hs  c6-ssdna - x : peg6-sh was used to prepare the plasmonic biosensors ( figure 1b ) .
all the mirs and oligonucleotides sequences 
 used in these studies are provided in supporting 
 information , table s1 . as illustrated in figure 2a
, we used uv  vis 
 spectroscopy to monitor the changes in lspr of the 
 gold nanoprisms at different functionalization steps . the functionalization 
 of substrate - bound nanoprisms with 1:1 ratio of hs 
c6-ssdna-21:peg6-sh resulted in an 20.5  3.2 nm red - shift of 
 lspr as a result of the increase in local refractive 
 index , which suggested the attachment of both molecular species onto 
 the nanoprism s surface .
these plasmonic biosensors were utilized 
 for mir detection by incubating mir-21 ( obtained from sigma - aldrich , 
 u.s.a . ) with concentration ranging from 100 nm to 50 fm in pbs buffer , 
 40% bovine plasma , or 40% human plasma . the lspr response of gold nanoprisms for each mir-21 concentration
was measured 
 where the highest 18.8  1.9 nm lspr red shift 
 was observed for 100 nm mir-21 ( figure 2a , 
 blue ) in pbs buffer .
we hypothesize that the lspr red - shift is due to hybridization between ssdna-21 and mir-21 .
it 
 was found that the magnitude of the lspr shift was 
 concentration dependent , where 50 fm mir-21 caused a 3.7  0.3 
 nm lspr red shift in pbs buffer .
figure 2b illustrates the magnitude of the lspr shift ( lspr ) upon dna / rna duplex formation 
 for various mir-21 concentrations in the three different media .
evidently 
 higher concentrations of mir-21 induced a larger number of ssdna-21 
 strands to convert to dna / rna duplexes and consequently a larger change 
 in the local refractive index around the nanoprisms , which results 
 in a larger value of lspr .
( a ) monitoring 
 lspr dipole peak ( lspr ) changes by uv  visible 
 absorption spectra of gold nanoprisms during various functionalization 
 steps : before ( black , lspr = 800 nm ) and after functionalized 
 with a 1 m/1 m ratio of hs - c6-ssdna-21/peg6-sh ( red , lspr = 821 nm ) , and after incubation 
 with 100 nm mir-21 solution in pbs buffer ( blue , lspr = 839 nm ) .
( b ) average lspr peak shift ( lspr ) of gold nanoprisms functionalized with a 1 m/1 
 m ratio of hs - c6-ssdna-21/peg6-sh after incubation 
 in different concentrations of mir-21 in pbs buffer ( red triangles ) , 
 40% human plasma ( black squares ) , and 40% bovine plasma ( blue diamonds ) . 

the lspr were calculated by taking the difference 
 between the lspr peak position of the plasmonic 
 biosensor after and before the hybridization with mir-21 in the various 
 media .
( c ) average lspr of the plasmonic 
 biosensor functionalized with a 1 m/1 m ratio of hs - c6-ssdna-10b / peg6-sh after hybridization with different mir-10b concentrations 
 in pbs buffer ( red triangles ) , in 40% human plasma ( black squares ) , 
 and in 40% bovine plasma ( blue diamonds ) .
all extinction spectra recorded 
 after mir - x incubation were measured in pbs buffer after rinsing with 
 pbs buffer .
the highest red shift 
 of 18.8  1.9 nm we observed for 100 
 nm mir-21 incubation with our plasmonic biosensor is significant considering 
 only an 5% change in the refractive index upon ssdna / rna duplex 
 formation .
we believe such a high sensitivity 
 of our plasmonic biosensors is because of the unique lspr properties 
 of our gold nanoprisms and the possibility of electron delocalization 
 as the ssdna forms the duplex and becomes double - strand . the atomically 
 flat surface , extremely small height ( 8 nm ) , and sharp tips 
 of our nanoprisms display strong em field enhancement near their surface 
 and therefore are expected to be extremely sensitive to small changes 
 of their local dielectric environment .
moreover , 
 transformation of ssdna into double - strand dna significantly changes 
 the refractive index because of the high charge density and polarizability 
 of the dnas .
the duplex dna is capable 
 of long - range charge transfer and alters the electron density around 
 the nanoprisms thus influencing their lspr properties .
this interesting 
 phenomenon requires further scientific study , which is currently under 
 our investigation .
our sensing mechanism is based on the hypothesis 
 that the attachment 
 of complementary target mirs to our plasmonic biosensor will shift 
 the lspr to higher wavelength ( figure 1c ) . the total shift ( lspr )
depended 
 on the mir concentration ( figure 1d ) and could 
 be used to determine the limit of detection ( lod ) ( figure 1e ) .
the lods calculated for mir-21 in three different 
 media were found to be in the range of 2335 fm , which was 
 more than 1000 and 3 fold lower than with the label - free microring 
 resonator ( 150 fmol ) and the nanopore 
 based ( 100 fm ) mir sensors , respectively . 

importantly , these techniques detected mirs in pbs buffer whereas 
 we have demonstrated here for the first time a sensing approach in 
 physiological media . utilizing our same direct hybridization - based 
 detection approach , plasmonic biosensors were constructed with of 
 c6-ssdna-10 , while keeping other parameters constant .
the 
 lod for mir-10b in the above media was determined over a concentration 
 range from 100 nm to 50 fm .
the average lspr and lods for mir-10b in three diverse media are shown in figure 2c .
the principle underlying the actions of 
 plasmonic biosensors is 
 based on the successful hybridization between mirs and ssdna attached 
 to nanoprisms , where a higher number of duplex formations will result 
 in a larger change in the refractive index surrounding the nanoprisms 
 resulting in larger lspr and higher sensitivity . 

therefore , it would be expected that functionalization of gold nanoprisms 
 with 100% hs  c6-ssdna - x ( without the peg6-thiol 
 spacer ) should reduce the lod values because of steric hindrance and 
 low attachment of mirs . to investigate this ,
gold nanoprisms were 
 functionalized with 100% c6-ssdna-21 resulting in an 15.0 
  1.8 nm lspr red shift ( figure 3a ) .
the sensor was then incubated in different concentrations 
 of mir-21 prepared in 40% human plasma . as illustrated in figure 3a , an 9.6  1.1 nm red shift was observed 
 for a 100 nm mir-21 concentrations and the lowest concentration that 
 can be repeatedly detected was 10 pm from a lspr of 3.4  0.5 nm . figure 3b shows the 
 lspr versus concentration plot .
evidently , 
 functionalization of the nanoprism s surface with 100% c6-ssdna-21 
 resulted in a 200-fold increase in detection limit in comparison to 
 the 1:1 ratio c6-ssdna-21/peg6-sh mixed functionalization 
 ( supporting information table s2 ) .
these 
 experimental data further highlight our rationale for using spacers 
 that increase the likelihood of hybridization .
we believe fully covered 
 gold nanoprisms were obtained when 100% c6-ssdna-21 was used 
 for functionalization , which creates steric hindrance and does not 
 allow the maximum number of mir-21 strands to come into close proximity 
 with c6-ssdna-21 for hybridization .
therefore , not all the 
 c6-ssdna-21 attached on the gold nanoprisms surface 
 was hybridized with mir-21 strands resulting in low sensing response . 
 thus
, if we introduce a spacer between the c6-ssdna-21 , it 
 will allow the maximum -ssdna-21 strands to be freely available for 
 hybridization without any interference and ultimately enhance the 
 sensitivity of the biosensor .
accordingly , for the remaining of our 
 investigation , we used a 1:1 mixed c6-ssdna - x : peg6-sh to functionalize the gold nanoprisms .
( a ) uv  visible 
 extinction spectra monitoring the lspr dipole peak ( lspr ) of gold nanoprisms attached to silanized glass substrate before 
 ( black , lspr = 796 nm ) and after ( red , lspr = 811 nm ) functionalization with 1 m of hs - c6-ssdna-21 
 without peg6-sh spacers and after incubation in 100 nm 
 mir-21 solution in 40% human plasma ( blue , lspr = 
 822 nm ) .
( b ) average lspr of these hs - c6-ssdna-21 
 functionalized gold nanoprisms upon hybridization with different mir-21 
 concentrations in 40% human plasma .
the lspr were calculated by taking the difference between the lspr peak position of the nanoprisms after and before the incubation 
 with mir-21 in pbs buffer . in order to confirm the hybridization of mir - x with c6-ssdna - x 
 that resulted in the lspr
, the enzyme rnase 
 h was used to selectively cleave the dna : rna duplex and potentially 
 reverse the lspr .
initially , the plasmonic 
 biosensor for mir-21 was incubated in a 100 nm solution of mir-21 , 
 which resulted in red - shift of lspr potentially 
 reflecting hybridization .
the biosensor was then immersed in 15 units 
 of rnase h solution for 2 h. afterward the lspr showed 
 a blue shift and reverted back to its original position before mir-21 
 incubation ( figure 4a ) . when the 1:1 ratio 
 c6-ssdna-21:peg6-sh mixed functionalized biosensor 
 was incubated with rnase h solution alone overnight , no noticeable 
 change in lspr value was observed ( supporting information figure s7 ) .
these experimental results 
 validate our previous observation that the lspr blue 
 shift was due to the cleavage of heteroduplex done by rnase h. the 
 biosensors were rinsed with rnase free water to pbs buffer and again 
 incubated in 100 nm mir-21 solution for rehybridization where an 14 
 nm red shift of the lspr was observed .
these experiments 
 confirm our working hypothesis that hybridization between the nanoprism s 
 surface ligands ( c6-ssdna - x ) and the mir - x resulted in changes 
 in the local dielectric environment around the nanoprisms causing 
 wavelength shift .
as shown in figure 4b , the 
 lspr responses were identical for several cycles 
 due to hybridization and dehybridization of mir-21 over a period of 
 6 days .
the same experiments were done for the mir-10b biosensor and 
 similar results were observed , underscoring the long - term stability 
 of the sensors and their potential for being developed into cost - effective 
 point of care diagnostic tools .
( a ) uv  visible extinction spectra of gold nanoprisms functionalized 
 with a 1 m/1 m ratio of hs - c6-ssdna-21/peg6-sh ( red , lspr = 818 nm ) attached to silanized 
 glass , after incubation with 100 nm of mir-21 ( blue , lspr = 832 nm ) , after treatment with 15 units of rnase h for 2 h ( red 
 dotted , lspr = 818 nm ) , and after rehybridized with 
 100 nm of mir-21 ( blue dotted , lspr = 832 nm ) .
( b ) 
 changes in lspr dipole peak position ( lspr ) of gold 
 nanoprisms functionalized with a 1 m/1 m ratio of hs - c6-ssdna-21/peg6-sh upon hybridization and dehybridization with mir-21 for 
 several cycles .
the lspr peak shifts back and forth 
 upon sensor regeneration with rnase h by cleaving dna / rna duplex and 
 rehybridization after incubation into 100 nm mir-21 in 40% human plasma . 

after each of the dehybridization steps , the plasmonic biosensors 
 were thoroughly rinsed with pbs buffer to completely remove enzyme 
 rnase h. the hybridization takes place 
 at the 5 end of c6-ssdna-21 
 and the 3 end of mir-21 , which evidently increased the refractive 
 index .
additionally such hybridization would also increase the thickness 
 of the local dielectric environment of the nanoprisms .
together , a 
 significantly large lspr was generated for 
 both mir-21 and mir-10b .
atomic force microscopy ( afm ) analysis was 
 conducted to characterize our plasmonic biosensors and also to verify 
 the change in surface area caused by mir-21 incubation with mixed 
 c6-ssdna-21 and peg6-sh - functioanlized gold nanoprisms . 
 after analyzing 40 different nanoprisms ( figure 5 and supporting information figure s8 ) 
  the exact same area of four different sections of the sensor 
 -
an average 2.4  10 m increase 
 in surface area was observed by afm .
thus , attachment of mirs to plasmonic 
 biosensors has increased the thickness of local dielectric environment 
 around the gold nanoprisms and influenced their lspr properties .
ultrasensitive 
 refractive index - induced lspr response of nanoprisms allows us to 
 fabricate label - free plasmonic biosensor .
atomic force 
 microscopy images of gold nanoprisms bound to silanized glass substrate 
 ( a ) after functionalization with 1:1 ratio of hs - c6-ssdna-21/peg6-sh ( b ) and after hybridization with100 nm mir-21 in 40% human 
 plasma ( c ) .
( d ) changes in 
 the surface area of gold nanoprisms after each functionalization steps . 

the successful implementation 
 of plasmonic biosensors for use with 
 real biological samples mandates documentation of their specificity 
 toward target mirs in that patient samples containing multiple mir 
 species .
the mix functionalized ( c6-ssdna-21 and peg6-sh ) biosensors were incubated overnight in 40% human plasma solution 
 containing 100 nm each of mir-16 , mir-122 , mir-126 , and mir-141 because 
 these mirs are commonly present in human plasma .
the lspr response was measured before and after incubation ( supporting information figure s9 ) and resulted in an 2.5 
  0.3 nm lspr red shift , which is within the 
 instrument noise level and/or minor nonspecific adsorption of extraneous 
 materials present in human plasma . in another control experiment , 
 gold nanoprisms attached as before to glass substrate were functionalized 
 with 100% peg6-sh by incubation in a 1 m aqueous 
 solution , and after rinsing with large amounts of water , incubated 
 in a 40% human plasma solution of 100 nm mir-21 for 12 h. this procedure 
 resulted in only an 0.9  0.7 nm lspr red shift ( supporting information figure 
 s10 ) , confirming that the plasmonic biosensors we designed are highly 
 specific toward the target mirs .
pancreatic cancer is the fourth - leading cause of 
 cancer death in the united states with an annual mortality of nearly 
 40  000 and a dismal five - year survival rate of 6% .
pdac is characterized by chemotherapeutic resistance 
 and by the absence of an effective screening procedure for early disease . 

it is generally accepted that early diagnosis could reduce mortality 
 rates substantially and thus a noninvasive early pdac test must be 
 developed . several mirs ( such as mir-21 , -10b , -103 , -155 , -196a , 
 210 , and -221 )
were found to be overexpressed in pdac . given their resistance to degradation , 
 plasma mirs have the potential to serve as biomarkers for the noninvasive 
 diagnosis of pdac .
previously , nanopore sensors were used to detect 
 mirs in lung cancer patients , but to the best of our knowledge no 
 sensors have been developed to date to detect pdac - related mirs in 
 human plasma . utilizing our plasmonic biosensors we detected 
 mir-21 and mir-10b in plasma from pdac patients .
total 
 plasma rnas including mirs were extracted from 100 l of each 
 plasma sample using a trizol kit with a final elution volume of 28 
 l .
next , 14 l volumes were used for mir quantification 
 by the plasmonic biosensor and the remaining 14 l were used 
 in the qrt - pcr assay .
the plasmonic biosensors were fabricated as 
 described before for both mir-21 and mir-10b detection .
the extracted 
 human mir-21 or mir-10b samples were diluted in pbs buffer and incubated 
 with the biosensors were for 12 h , followed by rinsing with pbs buffer 
 and measurement of the lspr response in pbs buffer . 

the observed lspr shift for each mir-21 and mir-10b 
 sample was converted into the corresponding concentration using the 
 calibration curve derived for mir-21 or 10b under optimized conditions 
 and compared with the value from normal control subjects ( figure 6a , c ) .
the concentrations of mir-21 and mir-10b determined 
 from plasmonic biosensors were also compared with the values obtained 
 from the qrt - pcr assay ( figure 6b and supporting information figure s16 ) .
importantly , 
 for the first time through a label - free technique we have shown that 
 mir-10b concentration is nearly 4-fold higher than the mir-21 level 
 in patient samples .
inasmuch as both mir-21 and mir-10b are overexpressed 
 in pdac , it is possible that mir-10b is released more efficiently 
 by pancreatic cancer cells than mir-21 , allowing it to achieve higher 
 levels in the circulation .
it is therefore possible that mir-10b levels 
 are also increased within the pancreatic tumor microenvironment , where 
 it could be acting to enhance pdac biological aggressiveness .
( a ) the average lspr peak shifts 
 of gold nanoprisms functionalized with a 1:1 ratio of hs - c6-ssdna-21/peg6-sh upon hybridization with mir-21 from the total rnas extracted 
 from plasma samples of pdac patients ( blue diamonds ) and normal control 
 subjects ( blue squares ) .
the respective lspr peak 
 shifts were converted to concentrations using the calibration curve 
 established for mir-21 in pbs buffer as shown in figure 2b [ pdac patients 
 ( red triangles ) , and for normal control subjects ( red circles ) ] .
( b ) 
 comparison of mir-21 concentration for six pdac patients determined 
 using plasmonic biosensors ( blue diamond ) and qrt - pcr ( red square ) . 

( c ) similar experiments were conducted to detect mir-10b where the 
 lspr peak shifts and concentrations for pdac patients 
 are shown in blue diamonds and red triangles , respectively .
the lspr peak shifts ( blue squares ) and concentrations ( red circles ) 
 for normal controls are shown for comparison .
( d ) the average lspr peak shifts ( blue diamonds ) and concentration ( red triangles ) 
 for the mir-21 in plasma samples from pdac patients without any purification .
we also detected mir-21 levels 
 directly in human plasma samples 
 collected from pdac patients without rnas extraction .
thus , human 
 plasma ( 50 l / sample ) from six pancreatic cancer patients were 
 diluted in pbs buffer followed by incubation with our plasmonic biosensors 
 for 12 h. the lspr response of each sample was measured 
 through uv  vis spectroscopy and showed a steady increase in 
 concentration from sample 6 to 1 ( figure 6d ) . 

both plasmonic biosensor and qrt - pcr results indicated that mir-10b 
 levels were higher in pdac patients compared to normal control subjects 
 and that the levels of mir-21 and mir-10b can be quantified with high 
 accuracy using our gold nanoprism - based plasmonic biosensor without 
 any modification , amplification , or labeling .
importantly , the mir-21 
 concentration in extracted samples was at least 2-fold lower than 
 in the pure plasma samples .
we believe this is due to loss of mirs 
 during the rna extraction process , which requires multiple steps for 
 rna purification .
therefore , the most widely used qrt - pcr method to 
 determine the concentration of mirs in patients may not accurately 
 represent the actual concentration
. this limitation and imprecise 
 quantification can be avoided by using our newly developed plasmonic 
 biosensors , which can provide a unique opportunity as potential diagnostic 
 and prognostic markers in pdac , other cancers , and potentially other 
 disease states .
we have designed , fabricated , 
 and characterized 
 a plasmonic biosensor that was able to detect pdac relevant mirs in 
 human plasma without using rnas extraction , which opens a new avenue 
 for the direct detection and quantification of mir levels in clinical 
 samples without any form of sample preparation . to our knowledge , 

this is the first lspr - based , label - free , direct hybridization method 
 for mir detection , which eliminates all the current drawbacks such 
 as labeling , tagging , amplification , use of highly toxic chemical , 
 and further modification of the sensor .
furthermore , it vastly simplifies 
 the detection approach without requiring detailed knowledge of the 
 electron or energy transfer processes involved as in other more complicated 
 techniques .
additionally , this ultrasensitive , plasmonic - based , direct 
 hybridization - controlled detection approach is applicable to any type 
 of mirs that are relevant to various diseases .
it was found that our 
 plasmonic biosensor can be regenerated through several cycles and 
 is stable for several days without compromising its sensitivity and 
 selectivity , which should enable the development of simple , cost - effective 
 tools for the early detection of mirs and thus facilitate the early 
 diagnosis of various cancers .
finally , the large em - field enhancement 
 at the nanoprism s sharp tips will 
 enhance the raman scattering intensity of the analytes . in theory , 

therefore , nanoprisms can be used to design effective substrates for 
 surface - enhanced raman spectroscopy - based detection and quantification 
 of multiple mirs simultaneously through integration of their spectral 
 characteristic with the lspr shifts .
all synthetic dna probes and 
 micrornas were purchased from sigma - aldrich ( u.s.a . ) .
pbs buffer prepared 
 with rnase - free water was used to dilute oligonucleotides and mirs 
 solutions .
patient plasma was obtained from the indiana university 
 simon cancer center solid tissue bank ( indianapolis , indiana ) .
the gold nanoprism - based 
 mir sensors were designed using our published 
 procedure with modification .
the attachment 
 of gold nanoprisms on silanized glass substrates is described in the supporting information file .
the substrate - bound 
 nanoprisms were incubated in pbs buffer solution containing 1 m 
 each of hs  c6-ssdna - x and peg6-sh overnight and 
 rinsed with pbs buffer .
the initial lspr peak position of each sensing 
 platform was determined using uv  visible spectroscopy in pbs 
 buffer and then was incubated in the different concentrations of mir 
 solutions , for exmaple , either in pbs buffer , 40% bovine plasma , or 
 40% human plasma for 12 h at room temperature . the plasmonic biosensors 
 were thoroughly washed with pbs buffer to remove any nonspecifically 
 adsorbed species .
the mir bound biosensor was then placed in pbs buffer 
 for 10 min before the lspr peak position was determined . for uv 
vis 
 extinction spectra measurement , one particular solvent was chosen 
 to avoid the solvent dielectric constant effect , which is known to 
 shift the lspr peak .
total rna was isolated from plasma samples that were obtained from 
 the indiana university simon cancer center solid tissue bank ( indianapolis , 
 in , u.s.a . ) using trizol - ls reagent ( life technologies , carlsbad , 
 ca , u.s.a . ) .
cdna was generated using 10 ng of rna and mir-10b , mir-21 , 
 or mir-4255p rt primers and a mir reverse transcription kit 
 ( life technologies ) as per the manufacturer s recommendations . 

expression levels as determined by qpcr were normalized 
 to mir-425 - 5p , since this mir was expressed at similar levels in all 
 samples and exhibited < 1 cycle threshold ( ct ) difference across 
 all samples .
after normalization to mir-425 - 5p ( ct ) , the ct 
 values for mirs in controls were averaged and subtracted from the 
 ct values of each individual sample ( ct ) .
the gold nanoprism - based 
 mir sensors were designed using our published 
 procedure with modification .
the attachment 
 of gold nanoprisms on silanized glass substrates is described in the supporting information file .
the substrate - bound 
 nanoprisms were incubated in pbs buffer solution containing 1 m 
 each of hs  c6-ssdna - x and peg6-sh overnight and 
 rinsed with pbs buffer .
the initial lspr peak position of each sensing 
 platform was determined using uv  visible spectroscopy in pbs 
 buffer and then was incubated in the different concentrations of mir 
 solutions , for exmaple , either in pbs buffer , 40% bovine plasma , or 
 40% human plasma for 12 h at room temperature . the plasmonic biosensors 
 were thoroughly washed with pbs buffer to remove any nonspecifically 
 adsorbed species . the mir bound biosensor was then placed in pbs buffer 
 for 10 min before the lspr peak position was determined . for uv 
vis 
 extinction spectra measurement , one particular solvent was chosen 
 to avoid the solvent dielectric constant effect , which is known to 
 shift the lspr peak .
total rna was isolated from plasma samples that were obtained from 
 the indiana university simon cancer center solid tissue bank ( indianapolis , 
 in , u.s.a . ) using trizol - ls reagent ( life technologies , carlsbad , 
 ca , u.s.a . ) .
cdna was generated using 10 ng of rna and mir-10b , mir-21 , 
 or mir-4255p rt primers and a mir reverse transcription kit 
 ( life technologies ) as per the manufacturer s recommendations . 

expression levels as determined by qpcr were normalized 
 to mir-425 - 5p , since this mir was expressed at similar levels in all 
 samples and exhibited < 1 cycle threshold ( ct ) difference across 
 all samples .
after normalization to mir-425 - 5p ( ct ) , the ct 
 values for mirs in controls were averaged and subtracted from the 
 ct values of each individual sample ( ct ) .
 micrornas ( mirs ) are small noncoding 
 rnas that regulate mrna stability 
 and/or translation . because of their release into the circulation 
 and their remarkable stability , mir levels in plasma and other biological 
 fluids can serve as diagnostic and prognostic disease biomarkers . 
 
 however , quantifying mirs in the circulation is challenging due to 
 issues with sensitivity and specificity . 
 this letter describes for 
 the first time the design and characterization of a regenerative , 
 solid - state localized surface plasmon resonance ( lspr ) sensor based 
 on highly sensitive nanostructures ( gold nanoprisms ) that obviates 
 the need for labels or amplification of the mirs . 
 our direct hybridization 
 approach has enabled the detection of subfemtomolar concentration 
 of mir - x ( x = 21 and 10b ) in human plasma in pancreatic cancer patients . 
 
 our lspr - based measurements showed that the mir levels measured directly 
 in patient plasma were at least 2-fold higher than following rna extraction 
 and quantification by reverse transcriptase - polymerase chain reaction . 
 through lspr - based measurements 
 we have shown nearly 4-fold higher 
 concentrations of mir-10b than mir-21 in plasma of pancreatic cancer 
 patients . 
 we propose that our highly sensitive and selective detection 
 approach for assaying mirs in plasma can be applied to many cancer 
 types and disease states and should allow a rational approach for 
 testing the utility of mirs as markers for early disease diagnosis 
 and prognosis , which could allow for the design of effective individualized 
 therapeutic approaches . 
Fabrication of the Plasmonic Biosensor for miRs Detection
Detection of miR Levels
in in Plasma from Pancreatic Cancer
Patients
Conclusion
Materials and
Methods
Fabrication
of LSPR-Based miR Sensors and Detection
Total RNA Extraction and Quantification of MicroRNA by qRT-PCR
Supplementary Material