Attomolar detection of extracellular microRNAs …

1 NanoscalePAPERCite this:Nanoscale, 2017,9, 17387 Received 19th June 2017,Accepted 7th October 2017 DOI: detection of extracellular microRNAsreleased from living prostate cancer cells by aplasmonic nanowire interstice sensor Siyeong Yang, aHongki Kim, aKyung Jin Lee,aSeul Gee Hwang,b,cEun-Kyung Lim,b,c,dJuyeon Jung,b,cTae Jae Lee,eHee-Sung Park,*aTaejoon Kang*b,c,dand Bongsoo Kim*aProstate cancer (PC) is the second leading cause of cancer death for men worldwide. The serum pros-tate-specific antigen level test has been widely used to screen for PC. This method, however, exhibits ahigh false-positive rate, leading to over-diagnosis and over-treatment of PC patients.

2 ExtracellularmicroRNAs (miRNAs) recently provided valuable information including the site and the status of thecancers and thus emerged as new biomarkers for several cancers. Among them, miR141 and miR375 arethe most pronounced biomarkers for the diagnosis of high-risk PC. Herein, we report an Attomolar detec-tion of miR141 and miR375 released from living PC cells by using a plasmonic nanowire interstice (PNI)sensor. This sensor showed a very low detection limit of 100 aM as well as a wide dynamic range from100 aM to 100 pM for all target miRNAs. In addition, the PNI sensor could discriminate perfectly thediverse single-base mismatches in the miRNAs.

3 More importantly, the PNI sensor successfully detectedthe extracellular miR141 and miR375 released from living PC cell lines (LNCaP and PC-3), proving thediagnostic ability of the sensor for PC. We anticipate that the present PNI sensor can hold great promisefor the precise diagnosis and prognosis of various cancer patients as well as PC (miRNAs) are single-stranded, small, and noncod-ing RNAs that play important roles as regulators in numerousbiological ,2 Recently, it has been reported that mul-tiple miRNAs are dysregulated in several human cancers, sup-porting the hypothesis that miRNAs are involved in theinitiation and progression of ,4 The dysregulatedmiRNAs can be released into biological fluidsviavarious path-ways and thus the expression pattern of miRNAs in cancercells can be mirrored in biological ,6 Consequently.

4 extracellular miRNAs released from cancer cells have been con-sidered as promising noninvasive cancer cancer (PC) is one of the most common cancersand the second leading cause of cancer death for men the serum prostate-specific antigen (PSA) leveltest has been widely used to diagnose PC, the high false-positive rate of this test has limited the accurate diagnosis ,9 The US Preventive Services Task Force even rec-ommended that physicians should not routinely perform PCscreening based on serum PSA , it is highlyimportant to develop a novel PC-specific biomarker detectionmethod.

5 In 2008, it was firstly reported that the level ofmiR141 is upregulated in the serum of metastatic PC com-pared with healthy controls and benign prostatic then, numerous studies have been conductedto investigate miRNA markers for PC in biological fluids and itwas known that the extracellular miR141 and miR375 canprovide valuable information of the status and location of thePC ,13 Moreover, extracellular miR141 and miR375 havebeen the most pronounced biomarkers for high-risk PC,including castrate-resistant PC and metastatic PC, whichaccount for approximately 15% of PC diagnoses and have thepotential to progress to a lethal ,15 Thus, it iscritical to develop a sensor that is capable of detecting extra- Electronic supplementary information (ESI) available.

6 See DOI: These authors contributed equally to this of Chemistry, KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon 34141,Korea. E-mail: Monitoring BioNano Research Center, 125 Gwahak-ro, Yuseong-gu, KRIBB,Daejeon 34141, Korea. E-mail: of Nanobiotechnology, KRIBB School of Biotechnology, UST,217 Gajeong-ro, Yuseong-gu, Daejeon 34113, KoreadBioNano Health Guard Research Center, 125 Gwahak-ro, Yuseong-gu, KRIBB,Daejeon 34141, KoreaeNano-bio Application Team, NNFC, 291 Daehak-ro, Yuseong-gu, Daejeon 34141,KoreaThis journal is The Royal Society of Chemistry 2017 Nanoscale,2017,9,17387 17395 |17387 Published on 10 October 2017.

7 Downloaded by Korea Advanced Institute of Science & Technology / KAIST on 20/11/2017 12:53:26. View Article OnlineView Journal | View Issuecellular miR141 and miR375 with high sensitivity and speci-ficity. Recently, the optical and electrochemical sensors havebeen developed for the detection of miR141 in PC ,17 While these sensors provide high sensitivity formiR141 through signal amplification processes, it is moredesirable to detect the extracellular miRNAs released fromliving PC cells rather than the miRNAs in PC cell lysate for thepractical diagnosis of PC patients. Furthermore, thesimultaneous detection of miR141 and miR375 might lead theaccurate diagnosis of PC , we report a plasmonic nanowire (NW) interstice(PNI)

8 Sensor which can detect the extracellular miR141 andmiR375 released from living PC cells into a culture sensor shows an extremely low detection limit of 100 aMfor both miR141 and miR375, and a wide dynamic range from100 aM to 100 pM, covering the typical concentration range ofextracellular miRNAs in the bloodstreams of ,19 Additionally, the PNI sensor can completely discriminate thesingle-base mismatches of miR141 and miR375. This excellentsensing capability of the PNI sensor enables the simultaneousdetection of miR141 and miR375 released from the living PCcells (LNCaP and PC-3), showing the potential applicability toa novel PC diagnostic method.

9 Since the PNI sensor can beeasily modified for capturing the desired target miRNAs, main-taining the superior sensing ability of miRNAs, we expect thatthe PNI sensor could apply to the diagnosis and prognosis ofthe several miRNA-related sectionMaterialsAu powder ( ) and sodium dodecyl sulfate (SDS) werepurchased from Sigma-Aldrich. All miRNAs, including thetarget miRNAs and single-base mismatched miRNAs, were pur-chased from Bioneer (Daejeon, Korea). The thiolated probelocked nucleic acid (LNA) and Cy5-tagged reporter LNA werepurchased from Eurogentec (Seraing, Belgium). Sequences ofall miRNAs, probe LNA, and reporter LNA used in this researchare described in the ESI (Table S1 ).

10 Saline sodium citrate(SSC) buffer solution and ultrapure water were purchased fromBiosesang (Sungnam, Korea). The miRNeasy serum/plasma kitwas purchased from Qiagen (Germany).InstrumentationThe surface-enhanced Raman scattering (SERS) spectra weremeasured using a custom-built micro-Ramansystem on anOlympus BX41 microscope. A 633 nm He/Ne laser (MellesGriot) was used as an excitation source, and the laser wasfocused on the PNI sensor through a 100 objective (numericalaperture = , Mitutoyo). The polarization of the laser wasdirected by rotating a half-wave plate. The SERS signals wererecorded with a thermodynamically cooled electron-multiply-ing charge-coupled device (Andor) mounted on a spectrometerwith a 1200 groove per mm grating (Dongwoo Optron).