Nanomechanical Microfluidic Mixing and Rapid Labeling of Silica

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Biological and Medical Applications of Materials and Interfaces

Nanomechanical Microfluidic Mixing and Rapid Labelling of Silica Nanoparticles with Allenamide to Thiol Covalent Link for Bioimaging Sivaramapanicker Sreejith, Rahul Kishor, Ata Abbas, Rijil Thomas, Trifanny Yeo, Vivek Damodar Ranjan, Ramanathan Vaidyanathan, Yen Peng Seah, Bengang Xing, Zhenfeng Wang, Li Zeng, Yuanjin Zheng, and Chwee Teck Lim ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b20315 • Publication Date (Web): 09 Jan 2019 Downloaded from http://pubs.acs.org on January 9, 2019

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ACS Applied Materials & Interfaces

Nanomechanical Microfluidic Mixing and Rapid Labelling of Silica Nanoparticles with Allenamide to Thiol Covalent Link for Bioimaging Sivaramapanicker Sreejith†,,*, Rahul Kishor‡,, Ata Abbas¶,, Rijil Thomas‡, Trifanny Yeo#, Vivek Damodar Ranjan£, Ramanathan Vaidyanathan#, Yen Peng Seah€, Bengang Xing¥, Zhenfeng Wang€, Li Zeng £, Yuanjin Zheng‡, *, Chwee Teck Lim†,#,±,*

†Biomedical

Institute for Global Health Research & Technology, National University of

Singapore, 117599, Singapore. ‡School of Electrical and Electronics Engineering, Nanyang Technological University, 639798, Singapore. ¶Department of Chemistry, Graduate School of Science, The University of Tokyo, Bunkyo-ku, 113033, Japan. #Department

of Biomedical Engineering, National University of Singapore, 117543,

Singapore. £National Neuroscience Institute, 11 Jalan Tam Tock Seng, 308433, Singapore.

€Singapore

Institute of Manufacturing Technology, 71 Nanyang Drive,

ACS Paragon Plus Environment

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638075, Singapore. ¥Division of Chemistry and Biological Chemistry, School of Physical and Mathematical Sciences, Nanyang Technological University, 637371, Singapore. ±Mechanobiology

 These

Institute, National University of Singapore, 117411, Singapore.

authors contributed equally.

KEYWORDS: nanoparticles, surface acoustic wave (SAW), allenamide-thiol chemistry, microfluidics, bioimaging.

ABSTRACT: Rapid surface functionalization of nanomaterials using covalent linkage following ‘green chemistry’ remains challenging and the quest for developing simple protocols are persisting. We report a nanomechanical microfluidic approach for the coupling of allenamide functionalized organic derivatives on the surface of thiol modified silica nanoparticles using allenamide-thiol chemistry. The coupling principle involves the use of a microfluidic surface acoustic wave (SAW) device that generates acoustic streaming based chaotic fluid micromixing that enables mixing of laterally flowing fluids containing active components. This approach was used to demonstrate the direct


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surface labeling of thiol modified silica nanoparticles using a selected group of modified fluorescence tags containing allenamide handles and achieved a total efficiency of 8390 %. This green approach enabled a highly efficient surface functionalization in aqueous conditions, with tunable control over the conjugation process via the applied field. The dye labelled silica particles were characterized using various analytical techniques and found to be biocompatible with a potential in live cell bioimaging. It is envisaged that this bioconjugation strategy will find numerous applications in the field of bioimaging and drug delivery.

INTRODUCTION

Chemical modification of nanosurfaces via covalent synthetic protocols is essentials for a multitude of applications ranging from biology to engineering.1–4 For example, in anticancer treatment in order to overcome intrinsic limitations of active molecules, nanoparticles (NP’s) are often considered as platforms for direct anchoring of vectors such as drug molecules and fluorescent labels.5,6 Last decades witnessed the development of various synthetic methods for micro/nano surface modifications capable


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of efficient bio-functionalization.7 Most often, reactions such as carbodiimode coupling (EDC), maleamide coupling, azide-alkyne coupling, disulfide bridges, bio-recognition reactions are employed for nanosurface bio-functionalization or conjugation for specific molecules especially fluorescent tags.8 However, the usage of common organic reactions for covalent modification nano-surfaces suffers mainly due to non-uniform coupling, stability of the formed chemical bonds, pH dependent variations and difficulty in removing side products after reactions (for example removal of Cu after catalyzed click-chemistry). Thus, the quest for developing robust and green synthetic protocols is continuing especially in versatile research frontiers such as nanobiotechnology, biosensor

design,

anticancer

therapy

applications

etc.

In

this

context,

microelectromechanical technological advances offer a controllable, sustainable, fast and commercially viable conditions for chemical processes involving organic reactions, nanosynthesis and surface modification procedures.9 Again, factors such as identification of safe reaction methods, compatibility of reagents with the active components inside fabricated device as well as safer reaction conditions remain challenging.


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Recently, microfluidics has been used for the synthesis10 and clinical translation of nanoparticles,11 functionalization of polymeric nanoaggregates,12 shape controlled synthesis of metal nanostructures13 and fluorescent organic dots using self-assembly.14 Typically, a microfluidic laminar flow based mixing relies on diffusion which is a slow and demands long channel lengths to achieve efficient mixing (passive mixing). However, the extent of mixing depends on the device configuration and design which offers no external control on mixing process.15,16,17 To enhance mixing efficiency, active mixing inside channels could be achieved by providing active external energy sources. Active

mixing

generate

disturbances

inside

the

channels

using

pressure,

dielectrophoretic, electrodynamics, magnetohydrodynamic, and optics.17,18 Microfluidic approaches, serving as a nano-conjugation platform, has already demonstrated its synergistic effects in the field of biomedicine. Despite the great potential, several microfluidic based nano-conjugation approaches suffer from (i) ready diffusion that can affect the conjugation efficiency, (ii) solvent compatibility, and (iii) need for additional purification steps for subsequent particle characterization.9

In this regard, Surface

acoustic wave (SAW) powered microdevices offer simple alternative to traditional


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laminar flow based devices. SAW generated on a piezoelectric substrate activates mixing of fluid inside microchannels by the acoustic streaming effect and enables tunable control of the fluid mixing process by simple manipulating the applied field.19,20 A high mixing efficiency of >90 % within a few milliseconds were achieved to date, which makes

the

SAW

integrated

device

ideal

for

surface

functionalization

in

microchannels.21,22

A

myriad

of

site-selective

chemical

modification

methodologies

were

experimented and widely employed in protein modification and surface modification in mild reaction conditions.23 Herein, we introduce the potential of a new covalent heterogeneous nano-surface functionalization method using allenamide-thiol linkage and demonstrated a SAW mediated rapid surface functionalization of nanoparticles in mild aqueous reaction conditions (Scheme 1). To demonstrate the potential of this method, we selected thiol modified silica nanoparticles (SiNP’s) and allenamide-thiol chemistry24 to demonstrate surface modification (Scheme 1a). Reaction of free thiol residue with allenamide modified derivatives following a “green chemistry” procedure in


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aqueous buffer triggers formation of a stable carbon-sulphur bond, thus leading to efficient surface labeling and modification. A nanomechanical mixing of allenamide functionalized dye derivatives and thiol modified silica nanoparticles was performed in a lab-on-a-chip device equipped with SAW mixer which consisted of interdigitated electrodes on either side of a microchannel (Scheme 1b & c). The acoustic streaming induced by SAW coupled to microchannel on the surface triggers acoustic turbulence and a rapid fluid mixing, which leads to efficient formation of allenamide thiol link on the surface of nanoparticles.


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SiNP’s = silica nanoparticles.


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Scheme 1. Nanomechanical mixing and rapid labelling of SiNP’s using allenamide thiol covalent linkage. (a) Scheme of a model reaction of SiNP-SH with 1 to obtain derivate SiNP-1. (b) Surface modification using fluorescent dyes (2 & 3) with allenamide handles using nanomechanical microfluidic mixing method, and (c) Device configuration and schematic illustration of microfluidic in situ mixing.

RESULTS AND DISCUSSION Prior to microfluidic coupling experiments, two pilot reactions were performed by selecting a reaction between model allenamide, 1 with thiol modified SiNP (SiNP-SH) (Scheme 1a) as well as cysteine methyl ester hydrochloride (Scheme S1). The progress of allenamide-thiol reaction in solid-state was traced using

13C

cross-polarization magic

angle spinning solid-state nuclear magnetic resonance (13C CP-MAS NMR) spectroscopy. First, a reaction between cysteine methyl ester hydrochloride and allenamide 1 in methanol/phosphate buffered saline (MeOH/PBS) at pH 7.4 yielded a modified derivative 1a (See experimental section for the details of the procedure).

13C

NMR of 1 & 1a were measured in chloroform (CDCl3). Figure S1 represents 13C NMR of


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1 with a characteristic allenamide carbon peak at  211.90 ppm, while no characteristic peak was observed for derivative for 1a (Figure S2). Solid-state

13C

CP-MAS NMR

would provide direct proof for the formation of allenamide-thiol adduct on SiNP surface. To demonstrate the feasibility of surface functionalization using allenamide-thiol covalent linkage, SiNP-SH (Scheme 1a) was allowed to react with 1 in a 1:1 weight equivalence for 2 h in MeOH/PBS buffer at pH 7.2. The reaction was monitored using thin-layer chromatography of 1 and halted upon complete consumption of 1. The reaction mixture was then centrifuged at 6000 rpm for 30 min to obtain functionalized SiNP-1.

13C

CPMAS NMR of SiNP-SH, a physical mixture of SiNP-SH & 1 and SiNP-1

were obtained separately (Figure 1). A physical mixture of SiNP-SH & 1 (black curve) showed a characteristic peak at  213.20 ppm, indicating the presence of allenamide unsaturated carbon. Upon formation of the allenamide-thiol link, SiNP-1 (blue curve) showed peaks at  128.03 ppm indicating the presence of benzene units on the surface, whilst the absence of any peak at  213.20 ppm suggested the completion of adduct formation on the surface. This data confirms the formation of allenamide-thiol linkage and illustrates the feasibility of this for heterogeneous functionalization on SiNP surface.


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SiNP-1

SiNP-SH & 1 (physical mixture)

SiNP-SH 250

200

150

100

50

0

δ (ppm)

Figure 1.

13C

CP-MAS NMR of SiNP-SH (red), a physical mixture of SiNP-SH & 1

(black) and SiNP-1 (blue). To investigate the potential for on-chip functionalization of nanoparticles using desired surface chemistry, we used our expertise in SAW integrated microfluidic system design25,26 and developed a compact device with a microfluidic nanomechanical mixing channel (Scheme 1b). The device comprised of two components mainly, (i) a SAW device with a pair of interdigitated electrodes (IDT) fabricated on the surface of a piezoelectric substrate, and (ii) a polydimethylsiloxane (PDMS) microfluidic channel


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embedded on the substrate for efficient fluid mixing. Figure 2a & b represent graphical illustrations of various device components and working scheme, respectively. Figure 2c represents the device setup equipped with a microchannel containing inlet and outlet tubes (Details of device fabrication can be found in experimental section). The designated microfluidic channel was 200 µm wide and 100 µm high, which allowed continuous flow of aqueous solutions. The in situ surface labelling of SiNP-SH’s was demonstrated using fluorescent dyes with allenamide handles, 2 & 3, which enabled the clearly visualization of the mixing process and evaluation of the extent of mixing in the channel. The derivative 2 with allenamide handle conjugated with fluorescein isothiocyanate (FITC) was selected and used to demonstrate nanomechanical mixing with SiNP-SH. During the SAW mediated nanomechanical mixing experiment, a solution of 2 and SiNP-SH (0.5 mg/mL) in PBS buffer were introduced through inlet 2 and inlet 1, respectively into the device (Figure 2b, 2c & S4) at a flow rate of 2 µL/min. The strong fluorescence of 2 inside the channel at 525 nm was monitored to study the extent of mixing and the in-situ process was observed using a reflection microscope equipped


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with a dichoric mirror/beam splitter for 490 nm and emission filter for 525 nm. The images were then captured using a CMOS (complementary metal oxide semiconductor) camera. Initially, laminar flow of both liquids was observed prior to inducing mechanical mixing via SAW activation. Figure 2d shows images corresponding to laminar flow of 2 and SiNP-SH particles inside the microchannel when excited using a 490 nm laser. Upon initiating acoustic streaming using SAW at a voltage of 37 V, rapid mixing of 2 and SiNP-SH was observed (See Movie S1). Figure 2d (2-12) depicts stages of mixing at different time intervals in 0-10 s time period. A reference flow was maintained as control by externally mixing the two reactants (Figure 2d (1)). Labelled SiNP-2 was then collected through the outlet and centrifuged for further material characterization. The mixing efficiency quantification was carried out by image analysis using MATLAB programming (ROI, Figure 2d (1-12) marked as white dotted lines). This was done by selecting a region of interest (ROI), in this case the section parallel to the SAW aperture (Supporting Information Figure S5).


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Figure 2. Nanomechanical microfluidic mixing. Schematic illustration of (a) various components of the nanomechanical mixer, (b) SAW nanomechanical actuation to functionalize SiNP-SH particles in situ, (c) Snapshot of the SAW integrated nanomechanical mixing set up, (d) Representative fluorescence images (2-12) of microchannel taken at different time of SAW mixing of 2 and SiNP-SH’s with an RF


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signal voltage of 37 Vpp and , reference image (1) obtained by flowing through an externally mixed solution of 2 and SiNP-SH. The dotted white boxes are the ROI used for mixing efficiency evaluation. See Movie S1 also. The efficiency of mixing inside the microchannel was quantified by extracting the intensity values of pixels from the captured images at various time intervals. Similarly, for the calculation of mixing efficiency by avoiding diffusive mixing present in the interface of the two fluids, we used a rectangular section as offset from center of the channel at 20 µm from the channel boundary (Figure S5 c & d). The pixel intensity (Ii) was normalized based on the intensity value at the same point for the unmixed (Iunmix) (t=0) and the fully mixed (Imix) state. Therefore, mixing efficiency (σ) was further calculated as:

  1

1 N

N

 i 1

where  i 

2 i

(1)

I i  I mix and N is the total number of pixels. The mixing efficiency I unmix  I mix

varies from one for complete mixing to zero for an unmixed state. During each experiment, the SAW acoustic streaming was switched on for 10 to 20 s and frames


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representative of mixing at different time intervals were recorded to calculate the mixing efficiency. As can be seen in Figure 3a, a maximum mixing efficiency of 87% was observed for an applied voltage of 37 Vpp. However, the mixing efficiency at lower voltages even for longer SAW actuation periods did not result in any enhancement, which was attributed to the lower acoustic streaming at such voltages that resulted in lesser number of interactions between SiNP-SH and 2. A quadratic relation between mixing efficiency and applied voltages in the range of 15 to 30 V was observed (Figure 3b). It was reported previously that such behavior could be attributed to the proportionality relationship between acoustic streaming and square of applied voltage relationship. Also, at 37.5 V, the mixing efficiency of the device reached its maximum of 92.5% with a concomitant saturation for voltages above 30 V. Further high voltages induced bubble formation inside the microchannel which caused turbulence and irregular mixing patterns. We then estimated the Péclet number (Pe), a ratio (equation 2) between the advective and diffusive transport rate, which governs the flow characteristics in the channel (a higher Pe value indicates lesser extend of mixing).


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Pe 

UW D

(2)

where U is the flow velocity and W is the width of the channel. The diffusion coefficient (D) of the dye 2 in the current channel dimension was 0.64 × 10-9 m2/s. Thus, a Pe value of 5.2 × 103 was calculated and found to be an ideal flow characteristic suitable for mixing. Figure 3c shows the UV-Vis absorption spectra of labelled silica particles (SiNP2) at different SAW mixing voltages ranging from 14.2 to 37 V in comparison with a stock solution of SiNP-SH and 2. A broadening of absorption peak at 495 nm indicated surface labelling with a hyper chromic effect upon increasing voltage inputs. The mixing efficiency estimated for reference prepared via external vortexing (Figure 2d) was found to be lower, compared to labelled particles obtained at low voltage (10 V) SAW excitations. The labelled particles SINP-2 and SiNP-3 obtained from SAW excitation at 37 V was centrifuged and re-dispersed in deionized water and subjected to photophysical characterization experiments. A characteristic broad absorption spectrum was observed for SiNP-2 and SINP-3, with absorption maxima at 492 nm and 650 nm, respectively (Figure 3d). The observed blue shift in the absorption maxima could be a


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result of aggregation on the particle surfaces. Emission spectra of SiNP-2 and SiNP-3 in comparison with SiNP-SH is shown in Figure 3c, d.


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Figure 3. Analysis and characterization of nanomechanical mixing of 2 with SiNP-SH. (a) Plot demonstrating mixing efficiency as a function of time for different RF input voltage excitation of the SAW-IDT. (b) Estimated mixing efficiency as a function of applied voltage under a constant flow rate of 2µL/min. (c) UV-Vis absorption spectra showing SiNP-2 particles upon different SAW-RF excitation voltages in comparison with bare sample and an externally mixed control sample, (d) Comparison of UV-vis absorption spectra of freshly prepared SiNP-2 & SiNP-3 with SiNP-SH. (e, f) Emission spectra of (e) SiNP-2 (exc 490 nm) and (f) SiNP-3 (exc at 600 nm), respectively. The labelled nanoparticles SiNP-2 and SiNP-3 were then characterized using Transmission

electron

microscopy

(TEM),

Field-Emission

Scanning

Electron

Microscopy (FE-SEM) and stochastic optical reconstruction microscopy (STORM) techniques. TEM (Figure 4a) and FE-SEM images (Figure 4b) of SiNP-2 confirmed formation of spherical nanoparticles with no structural deformation during SAW actuation in the microchannel. Effective peripheral labeling of SiNP’s with 2 & 3 were further confirmed by STORM super resolution imaging. For this, labelled nanoparticles were deposited on glass coverslips using buffer solution (OxyFluor) for preventing any photo bleaching during imaging. Figure 4c shows STORM images of a section


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containing five individuals (pointed by red arrows) and three cluster units (white dotted squares) of SiNP-2 particles. A resolved and localized single NP’s labeled (Figure 4c) with 2 shows effective labelling on the surface of NP’s with 2. Reconstructed images after processing 5000 image stacks reveal intense green emission at 500-525 nm (Excitation at 480 nm) on SiNP-2 indicating success of the labeling method described. Supporting Information Figure S6 & S7 shows TEM/SEM and STORM images of SiNP3 particles, respectively.


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Figure 4. a) TEM images (inset one single particle) of SiNP-2. b) SEM images of SiNP2 at 20,000x (left) & 100,000x (right) magnification, respectively. Super resolution image of SiNP-2 particles, c) STORM image of SiNP-2 particles indicated by red arrows (clusters of SiNP-2 particles in dotted boxes). d) Selected area of STORM reconstructed image of two SiNP-2 particles (Scale bar in red = 5 µm). To study the potential and biocompatibility of on-chip functionalized Si-NPs for bioimaging, SiNP-2 and/or SiNP-3 particles were incubated with MCF-7 breast cancer


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cell line for 24 h and assessed for cytotoxicity using MTT viability assay (Section S3). As can be seen from Figure S8, the modified particles exhibited low cytotoxicity at a moderate concentration range which indicates the biocompatibility of the labelled particles for cellular assays. Furthermore, to study the extent of cellular uptake of SiNP2 and SiNP-3, flow cytometry was conducted using a moderate concentration range of 25-50 µg mL-1 (Figure S9). In addition to this, the cell labelling and bioimaging capability of SiNP-2 was investigated using SHSY-5Y neuroblastoma cells. Figure 5a-d represent fluorescence images of cells treated with SiNP-2 particles for 24 h. The images indicate a uniform dispersion of particles along the cytoplasm (Figure 5c) whilst very little particles managing to enter the nucleus. Images obtained using SiNP-3 particles can be found in the Figure S10. Finally, the effect of SiNP-2 and SiNP-3 nanoparticles on cell growth was investigated by incubating particles with MCF-7 breast cancer cells and monitoring them under a time-lapse experiments using an imaging biostation. Movies S2 & S3 demonstrate the uptake of SiNP-2 and SiNP-3 particles by MCF-7 cells. It was observed that the particles had very little or no significant influence on cell growth, thereby indicating their potential for bioimaging studies. This further reiterates the


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potential of SAW nanomechanical mixing based surface functionalization approach for application in numerous cellular assays. Over the years, several continuous-flow microfluidic systems have been developed for the synthesis or conjugation of nanoparticles with precise control of reaction conditions, rapid mass and heat transfer, environmental friendliness and high scalability. With the ability to control various steps in nanoparticle conjugation, nanoparticles conjugated with higher uniformity index can be prepared within microfluidic platforms with enhanced performance in various applications. In this regard, the developed approach provides three critical improvements over current strategies for engineering nanoparticle surfaces that include: (i) SAW nanomechanical mixing provides a tunable conjugation system (via applied field) that can enable high efficiency immobilization, (ii) the synthesis of a well-dispersed nanoparticle solution with homogeneous immobilization of designated fluorescence tags across individual particles in a green-chemistry protocol, and (iii) a facile allenamide-thiol chemistry enabled synthesis of biocompatible nanoparticles with potential applications in bioimaging and drug delivery. This approach is not just limited


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to allenamide-thiol chemistry alone and can very well be tailored to enable different nanoparticle bioconjugation strategies on-chip.

Figure 5. Representative confocal microscopy images of neuroblastoma cells (SHSY-5Y cells) incubated with SiNP-2. Images were obtained from the (a) Bright field image, (b) DAPI channel, (b) FITC (exc. 490 & emi. 525 nm) and (d) represents overlay of (a), (b) & (c). (× 100 oil objective). Scale bar is 20 μm.


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CONCLUSIONS We introduced and validated a new heterogeneous surface labelling protocol using allenamide-thiol chemistry and demonstrated the protocol on silica nanoparticle surface. Direct coupling of free thiol groups on nanoparticle surface with allenamide modified molecules in aqueous conditions was performed in aqueous conditions. By using silica nanoparticles as model nanosubstrates, we presented a microfluidic surface labelling approach using a microfluidic device equipped with SAW actuators. Our custom designed nanomechanical mixing microchannel enables acoustic streaming mediated rapid surface labelling with higher efficiency. We also demonstrated SAW mediated efficient surface labeling inside microchannel using two allenamide handle appended organic fluorophores. The stability and biocompatibility of the nanoparticles labelled using the new protocol was examined. Modified nanoparticles using allenamide thiol chemistry inside the SAW device were then examined for application in in vitro fluorescence bioimaging in live cell cultures. This technique allows direct, safe and high efficiency surface labelling of nanoparticles in aqueous conditions with simple actuation of acoustic wave mixing that can precisely control the conjugation process. Further, the


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simplicity and rapid functionalization of nanoparticles can render several applications in bioconjugation and nanomedicine.

EXPERIMENTAL SECTION

Synthesis and characterization of allenamide handles; 1, 2, & 3 were synthesized and purified as per previous literature report.24 For the preparation of 1a, a modified procedure was employed. To a continuously stirring solution of 1 in methanol, Cys.Meth-SH (1:1 mole ratio) was added in PBS in room temperature (Supporting Information Scheme S1). The mixture was stopped after 40 mins after confirming completion of reaction using thin layer chromatography and purified using column chromatography. Details of characterization including 1H NMR,

13

NMR, ESI-MS, Figure S1 and Figure S2 are provided in

the Supporting Information Section S1. Synthesis of SiNP. For the synthesis of silica nanoparticles (SiNP’s) we followed Stöber process.27 In a typical reaction tetra ethyl orthosilicate (TEOS) was added to a constantly stirring solution of 80 mL MeOH and ammonia solution (20 mL, 1.8 M). The mixture was heated to 40 °C inside a water bath. Formation of turbid solution indicates


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formation of silica particles. The reaction was stopped after 2h, centrifuged and repeatedly washed with ethanol to remove unreacted starting materials and ammonia. The obtained white powder was dried and characterized further. Synthesis of SiNP-SH. 2 mL of (3-mercaptopropyl)trimethoxysilane (MPTMS) was then added to 1g SiNP dispersed in 20 mL of dry toluene under nitrogen atmosphere in a two neck round bottom flask. The reaction mixture was refluxed for 12 h. The reaction mixture was then filtered and washed with dry toluene and ethanol to obtain, SiNP-SH. Freshly obtained SiNP-SH powder was dried in vacuum and stored in dry cabinet for further use. SAW device fabrication. The device used for the mixing experiment consists of a SAW device was equipped with a pair of interdigital electrodes (IDT) fabricated on the surface of a piezoelectric substrate, a Y-shaped microfluidic channel for transporting the nanoparticle solution and the dyes. A 1280 rotated Y-cut X propagation lithium niobate (LN, LiNbO3) was used as the substrate due to its high electromechanical coupling coefficient (k2 = 5.6%) compared to other substrates used for SAW generation.28 A detailed process flow diagram is given in Figure S3. The fabricated SAW device


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consists of 25 pairs of single electrode transducers, of 200 nm gold on top of a 20 nm Cr layer for adhesion, with a pitch of 300 µm, finger width/gap of 75 µm and an IDT overlap aperture of 4 mm. Figure S4 shows image of an as prepared SAW before bonding of PDMS channel. The center frequency of the IDT for a wavelength of 300 µm and speed of sound on the substrate (3976 m/s) is calculated to be equal to 13.26 MHz. The fabricated device has a center frequency of 12.9 MHz, which was obtained by measuring the S11 (reflection spectra) using an E5061B ENA Series network analyzer. The required frequency of 12.9 MHz was generated from a Tektronix AFG3252 arbitrary function generator and amplified using a power amplifier (LZY-22+, Minicircuits) with a gain of 40 dB.

The microfluidic channels were made using polydimethylsiloxane

(PDMS). The channel was bonded to the piezoelectric substrate using oxygen plasma (Covance, Femto Science, Korea). The microfluidic channel had a width of 200 µm and a height of 100 µm across the main channel. Figure S5 shows photograph of a ready to use SAW device with PDMS channel embedded.

ASSOCIATED CONTENT


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Supporting Information. Details of characterization of organic precursors, nanoparticles, SAW substrate preparation process and details of bioimaging is provided.

AUTHOR INFORMATION

Corresponding Author *Prof. Dr. C. T. Lim: [email protected]

*Dr. S. Sreejith: [email protected] ; [email protected]

*Prof. Dr. Y. Zheng: [email protected]

Author Contributions SS conceived the idea. SS prepared and characterized nanoparticles and optimized labelling protocols. AA prepared and provided allenamide handles. RK prepared the SAW device and the microfluidic channel, including microfabrication process. RK, SS, YPS and RT conducted the experiments on SAW induced microfluidic mixing. VD and


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LZ conducted neuroblastoma cultures and imaging. TY, RV and SS performed live bioimaging and conducted super resolution microscopy imaging. SS, RK and CTL wrote the manuscript through contributions of all authors. YZ and CTL supervised the project. All authors have given approval to the final version of the manuscript.



SS, RK and AA contributed equally to this work.

ACKNOWLEDGMENT S.S. and C. T. L. acknowledge support from the Biomedical Institute for Global Health Research & Technology (BIGHEART), National University of Singapore.

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Nanomechanical Microfluidic Mixing and Rapid Labeling of Silica

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