Proceedings of the 4th European Conference on Microfluidics - Microfluidics Limerick, December 10-12, 2014

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1 µflu14-89 A MICROFLUIDICS TOOL FOR HIGH-THROUGHPUT, REAL-TIME MULTIMODAL IMAGING OF NANOPARTICLE-CELL INTERACTIONS C. A. Cunh-Mtos 1, O. M. Millington 2, A. W. Wrk 3, M. Zgnoni 4* 1 Deprtment of Biomedicl Engineering, University of Strthclyde, UK 2 Centre for Biophotonics, SIPBS, University of Strthclyde, UK 3 Centre for Moleculr Nnometrology, Dept. Pure nd Applied Chemistry, University of Strthclyde, UK 4 Centre for Microsystems nd Photonics, EEE Dept., University of Strthclyde, UK KEY WORDS single-cell, gold nnoprticles, nnotoxicity, high-throughput ssy ABSTRACT The incresing use of nnomterils for biomedicl pplictions hs rised the need for efficient, robust nd low-cost high-throughput ssessment of nnotoxicity nd cell-nnoprticle interctions. Microfluidics provides the tools for high-throughput single-cell functionl monitoring, while gold nnorods hve unique potentil for intrcellulr trcking nd cn simultneously be used s drug crriers. Presented here is miniturised pltform tht integrtes these fetures with multimodl pproch to cell imging. A microfluidic device llows for trpping of n rry of singlecells, followed by the controlled delivery of nnoprticles into the cell rry nd subsequent rel-time multimodl imging of cellulr interctions with functionlised nnoprticles. This system hs been successfully used to ssess cellnnoprticle interctions t the single-cell level. Figure 1: Schemtic overview of the proposed system. Dendritic cells under confocl fluorescence microscopy (membrne stining with choler-toxin-b, in green, nd nucleus stining with DAPI, in blue). Scle br is 20μm. SEM imge of nnorods with longitudinl λ mx of 765nm. Scle br is 200nm. Schemtic of the microfluidic device nd SEM imge of the PDMS microtrp rry. Chnnels re 25μm deep nd trp interior is 20μm wide. Scle br is 40μm. 1. INTRODUCTION Single-cell microfluidic pproches enble experimentl sttisticl dt to be cquired within single device with throughput comprble to tht of stndrd flow cytometry, with the dvntges of using reduced smple volumes nd llowing for long-term monitoring of individul cells [1,2]. By bringing these fetures together with multiple opticl imging nd spectroscopy techniques nd enhnced signls from functionlized gold nnorods [3], intrcellulr ctivity nd cell-nnoprticle interctions cn be monitored in rel-time nd down to the single nnoprticle level, n re of incresing relevnce with the growing use of * Corresponding uthor (michele.zgnoni@strth.c.uk) 1

2 Reltive intensity (.u.) Proceedings of the 4 th Europen Conference on Microfluidics - Microfluidics Limerick, December 10-12, 2014 nnomterils [4]. This solution gretly improves upon previous work bsed on cells pre-incubted with gold nnoprticles [5] nd offers new mens to study cell-nnoprticle dynmics which re menble to drug nd vccine delivery pplictions. A schemtic representtion of the system is presented in Fig EXPERIMENTAL A microfluidic device tht enbles trpping of lrge number of single-cells in n rry formt ws fbricted [1] using conventionl soft-lithogrphy methods [6] (Fig.1). The device is comptible with vrious nlyticl methods, nmely bright-field nd fluorescence microscopy, confocl microscopy nd surfce enhnced Rmn scttering (SERS) spectroscopy. Gold nnorod opticl probes were produced through n dpttion [7] of n estblished seed-medited synthesis pproch [8], followed by coting with Rmnctive molecules, polyelectrolyte lyers nd proteins. These custom-designed gold nnorods re visible vi multiple opticl imging techniques (e.g. fluorescence microscopy nd SERS), with verstile opticl properties nd surfce chemistry tht cn be fine-tuned for specific intrcellulr trcking nd delivery pplictions [9]. Primry (bone mrrow-derived) dendritic cells were used, which brings dditionl chllenges to cell trpping when compred to work done with cell lines [1,2], but llows for more relible ssessment of cell function nd heterogeneity. Cells were trpped in the microfluidic device, which ws kept in microscope stge incubtor with constnt medium perfusion to gurntee biocomptible conditions for the durtion of the experiments (up to 24 hours). A grdient of nnoprticle concentrtions ws delivered onto the rry of trpped cells with subsequent rel-time monitoring of the cell response to different nnoprticle concentrtions (cell motility nd fluorescent mrkers of poptosis nd necrosis), using bright-field nd fluorescence microscopy nd SERS. Trpped cells were lso fixed within the device for further nlysis. As control experiment, off-chip evlution of cell-nnoprticle interctions (cell vibility, nnoprticle uptke nd ctivtion sttus) ws performed by incubting cells with nnorods nd using flow cytometry nd confocl microscopy. 3. RESULTS AND DISCUSSION 3.1 Microfluidic cell trpping nd controlled nnoprticle delivery Cells were successfully trpped nd kept live within the microfluidic environment. Controlled dispensing of nnoprticles onto the cell rry in grdient formt ws chieved (Fig.2). Multimodl imging of trpped cells (bright-field, fluorescence microscopy nd SERS) nd ssessment of nnoprticle uptke ws ccomplished, n exmple of which is shown in Fig.2b-c. b c Rmn shift (cm -1 ) Figure 2: Controlled delivery of nnoprticles to trpped cells. ) Fluorescence imge obtined when generting concentrtion grdient of nnoprticles cross the width of the trp rry. The intensity profile shows incresing nnoprticle fluorescence t 633 nm excittion. Scle br is 50 μm. b) Confocl bright-field imge of trpped dendritic cell. Intrcellulr nnoprticle identifiction is shown in red. Scle br is 10 μm. c) Rmn signl from PDMS chmber bckground (blue) nd the trpped cell (red), showing notbly higher intensity for specific nnorod signl within the cell. The figure insert is schemtic of the polymer-wrpped nnorod-dye conjugte. Spectr hve been verticlly offset for clrity. 2

3 3.2 High-throughput ssessment of cell-nnoprticle interctions in the microfluidic device Vibility dyes were successfully used in long-term experiments to detect poptotic (Annexin-V FITC) nd necrotic (Sytox Blue) cell behviour in response to the nnoprticles. Fig.3 shows n re of trpped cells nd n exmple of the fluorescence informtion extrcted in rel-time for ech trp. A certin degree of nonspecific dsorption of the nnorods to the PDMS wlls ws observed, n issue tht needs further investigtion to define the miniml resolution of nnoprticle detection. b Figure 3: Rel-time ssessment of nnoprticle toxicity in trpped cells. ) Field of view of the microtrp rry with trpped cells. Averge single/double-cell coverge ws pproximtely 80%. Scle br is 100 μm. b) Microscopy imges of nnoprticle uptke nd cell vibility, from top to bottom: bright-field imge, gold nnorod fluorescence (633 nm excittion), necrosis mrker (Sytox Blue), poptosis mrker (Annexin-V FITC). Scle br is 50 μm. Uptke of nnorods by dendritic cells within the microfluidic rry ws qulittively estimted by monitoring the increse in nnorod-specific fluorescence intensity during nnoprticle delivery (Fig.4). Results show cler difference in this increse between the trcked cells nd the bckground, indicting tht the cells were tking up the nnorods. The initil dely of the fluorescent signl (0-15 minutes) is due to lg time between ctivtion of the flow of nnorods nd their presence in the rry due to the complince of the system (syringe-pdms). Future work includes, on one side, relting different degrees of uptke to cell motility nd ctivtion sttus, nd on the other side, enhncing control of uptke by mnipulting the nnorod surfce chemistry, ultimtely iming t intrcellulr trgeting nd single-nnorod trcking. b Figure 4: Qulittive estimtion of nnorod uptke by dendritic cells in the microfluidic device. ) Representtive frme from video obtined during nnoprticle delivery to cells in the microfluidic rry, showing the fluorescence intensity signl of five trcked cells (blue circles) nd three trcked bckground res (red circles) during nnoprticle delivery. Scle br = 50 m. b) Temporl progression of the verge MFI vlue of the three bckground regions nd the five cells trcked from Fig.4. Error brs represent stndrd devition. 3

4 Proceedings of the 4 th Europen Conference on Microfluidics - Microfluidics Limerick, December 10-12, 2014 Assessment of cell-nnoprticle interctions performed off-chip using flow cytometry nd confocl microscopy ws successful in the detection of nnoprticle uptke for different concentrtions of nnoprticles nd times of exposure (Fig.4). Results obtined on nd off-chip showed no significnt toxicity of the nnoprticles t the concentrtions nd exposure times used, nd ech microfluidic device ws cpble of providing lrge sets of dt (round 400 cells nlysed in only hlf of the trp rry). This shows the potentil of this tool to work simultneously for high-throughput nd high-resolution cell monitoring, with the dded feture of llowing for dynmic rel-time imging of individul cells, s opposed to popultion end-point mesurements s obtined with flow cytometry. b Figure 5: Nnoprticle uptke by dendritic cells mesured using flow cytometry nd confocl fluorescence microscopy. ) Cells were incubted in sttic conditions with different nnoprticle concentrtions in duplictes nd the fluorescence intensity t 633 nm excittion ws obtined using flow cytometer t different time points. The grph shows the verge of the two smples for ech condition, with error brs representing stndrd devition. b) Confocl fluorescence imge of dendritic cells stined with choler-toxin-b (membrne, in green) nd DAPI (nucleus, in blue) with nnoprticle fluorescence t 633 nm excittion shown in red. Scle br = 20 m. 4. CONCLUSION Results show proof-of-concept of n integrted system for rel-time, high-throughput testing of nnomteril cytotoxicity, which is menble to different imging methods nd offers the opportunity to ssess nnoprticle uptke t the single-cell level. Current work is focused on understnding the cellulr response to different formultions nd concentrtions of nnorods, specificlly through nlysis of cell motility nd vibility. By bringing together high-resolution imging techniques, high-throughput microfluidics nd highly specific nnoprticle probes, this work ims to provide new insight into the immunologicl behviour of dendritic cells relted to intrcellulr, nnoprticle-medited vccine delivery, s well s providing verstile tool with numerous cell monitoring, drug screening nd nnomteril testing pplictions. ACKNOWLEDGEMENTS This work ws funded by Bridging The Gp scheme (University of Strthclyde) nd the EPSRC Centre for Doctorl Trining in Medicl Devices. REFERENCES [1] Wlodkowic, D., Fley, S., Zgnoni, M., Wikswo, J. P. nd Cooper, J. M. (2009). Microfluidic Single- Cell Arry Cytometry for the Anlysis of Tumor Apoptosis. Anlyticl Chemistry, 81, [2] Crlo, D. D., Wu, L. Y. nd Lee, L. P. (2006). Dynmic single cell culture rry. Lb on Chip, 6,

5 [3] Murphy, C. J., Sn, T. K., Gole, A. M., Orendorff, C. J., Go, J. X., Gou, L., Hunydi, S. E. nd Li, T. (2005). Anisotropic metl nnoprticles: Synthesis, ssembly, nd opticl pplictions. Journl of Physicl Chemistry B, 109, [4] Vlenci, P. M., Frokhzd, O. C., Krnik, R. nd Lnger, R. (2012). Microfluidic technologies for ccelerting the clinicl trnsltion of nnoprticles. Nture Nnotechnology, 7, [5] Syme, C. D., Sirimuthu, N. M. S., Fley, S. L. nd Cooper, J. M. (2010). SERS mpping of nnoprticle lbels in single cells using microfluidic chip. Chemicl Communictions, 46, [6] Si S. K. nd Whitesides, G. M. (2003). Microfluidic devices fbricted in poly(dimethylsiloxne) for biologicl studies. Electrophoresis, 24, [7] McLintock, A., Hunt, N. nd Wrk, A. W. (2011). Controlled side-by-side ssembly of gold nnorods nd dye molecules into polymer-wrpped SERRS-ctive clusters. Chemicl Communictions, 47, [8] Su, T. K. nd Murphy, C. J. (2004). Seeded high yield synthesis of short Au nnorods in queous solution. Lngmuir, 20, [9] McLintock, A., Cunh-Mtos, C.A., Zgnoni, M., Millington, O.M. nd Wrk, A.W. (2014). Universl Surfce-Enhnced Rmn Tgs: Individul Nnorods for Mesurements from the Visible to the Infrred ( nm). ACS Nno, 8,