Microfluidic Cell Microarray Platform for High Throughput Analysis of

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Microfluidic cell microarray platform for high throughput analysis of particle-cell interactions Ziqiu Tong, Gayathri Rajeev, Keying Guo, Angela Ivask, Scott McCormick, Enzo Lombi, Craig Priest, and Nicolas H. Voelcker Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b03079 • Publication Date (Web): 02 Mar 2018 Downloaded from http://pubs.acs.org on March 3, 2018

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Analytical Chemistry

Microfluidic cell microarray platform for high throughput analysis of particle-cell interactions Ziqiu Tong1,2, Gayathri Rajeev2, Keying Guo1,2, Angela Ivask2,#, Scott McCormick2, Enzo Lombi2, Craig Priest2,*, and Nicolas H. Voelcker1-5,*

1. Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville, Victoria, Australia 2. Future Industries Institute, University of South Australia, Mawson Lakes, 5095, South Australia, Australia 3. Commonwealth Scientific and Industrial Research Organisation (CSIRO), Clayton, VIC, Australia 4. Melbourne Centre for Nanofabrication, Victorian Node of the Australian National Fabrication Facility, Clayton, VIC, Australia 5. Monash Institute of Medical Engineering, Monash University, Clayton, Victoria, Australia # Current address: Laboratory of Environmental Toxicology, National Institute of Chemical Physics and Biophysics, Akadeemia tee 23, 12618, Tallinn, Estonia

* Correspondence to: Email: [email protected]; [email protected]

Keywords: microfluidics, cell microarray, high throughput screening, nanotoxicity, particle-cell interactions

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Abstract With the advances in nanotechnology, particles with various size, shape, surface chemistry and composition can be easily produced. Nano- and microparticles have been extensively explored in many industrial and clinical applications. Ensuring that the particles themselves are not possessing any toxic effects to the biological system is of paramount importance. This paper describes a proof of concept method in which a microfluidic system is used in conjunction with a cell microarray technique aiming to streamline the analysis of particle-cell interaction in a high throughput manner. Polymeric microparticles, with different particle surface functionalities, were firstly used to investigate the efficiency of particle-cell adhesion under dynamic flow. Silver nanoparticles (AgNPs,10 nm in diameter) perfused at different concentrations (0 to 20 µg/ml) in parallel streams over the cells in the microchannel exhibited higher toxicity compared to the static culture in the 96 well plate format. This developed microfluidic system can be easily scaled up to accommodate larger number of microchannels for high throughput analysis of potential toxicity of a wide range of particles in a single experiment.


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1. Introduction The earliest studies using particles for biological assays and other applications were reported in the 1950’s.1 Latex microspheres and gold nanoparticles were probably the most commonly explored early examples. With the recent advances in nanotechnology, particles of various size, shape and composition can be easily fabricated, and their applications have hence been drastically expanded.2 Especially important for their applications in diverse areas of biology and medicine, micro- and nanoparticles are considered suitable materials due to their unique features, such as a large surface to volume ratio and the feasibility to tune their size and surface chemistry (e.g., hydrophobicity).3 For instance, microparticles of size comparable to that of capillaries have been used to occlude blood vessels in treatments of vascular anomalies or hemoptysis (severe bleeding)4,5 and to combat the growth and development of solid tumors by blocking the nutrient supply.6,7. Microparticles with magnetic properties have also been routinely used for various in vitro assays, such as white blood cell isolation, immunoassays, and protein purifications.8-10 Submicron particles are even more suitable for in vivo applications due to their improved bioavailability, increased residence time in the body and ability to translocate into subcellular compartments.11 For example, lipid and polymeric nanoparticles have been extensively investigated to deliver DNA/RNA to cells as gene delivery systems.12 Their reduced immune response and toxicity have made them more attractive choices as delivery vehicles than their viral vector counterparts.12 Moreover, nanoparticles have been widely explored as drug delivery carriers as they are able to transport high concentrations of therapeutic agents, while protecting them from degradation by the harsh


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biological conditions.13 Furthermore, formulated nanoparticles are considered one of the most promising and versatile drug delivery platform into normally inaccessible areas, such as the brain.14 Microparticles and submicron particles indeed possess great opportunities as drug delivery vehicles and other important applications in biomedicine, yet their interactions with cells still remain to be carefully characterized and dependent on particle size, shape, composition and surface chemistry. In particular, it is important to assess their potential inherent toxicity to cells.15 Traditionally, monitoring particle-cell interactions has been routinely carried out in flask or well-plate formats where cells are grown at the bottom of a culture plate and incubated with testing particle suspensions.16-18 The particles are assumed to diffuse freely in the medium and having the same concentration on the cell surface as in the bulk suspension. However, the reality is that some particles tend to aggregate and change their physical characteristics when they are suspended in a culture medium.19 Furthermore, large and heavy particles tend to settle down to the bottom of the culture plate due to gravitational force causing inhomogeneous distribution of particles.20 Hence, the actual concentration that cells are experiencing can differ greatly from the initial concentration. To tackle this sedimentation effect, recent efforts have focused in designing experiments of an inverted cell culture configuration where cells are grown on a glass cover slide and inverted into cell growing media conditioned with nanoparticles.21,22 However, with this inverted configuration, cells experience an opposite trend where sedimentation reduces the concentration of particles at the cell interface. Microfluidic systems are becoming a popular alternative platform for cell culture due to the small quantity of reagents required (nanoliter to microliter range) and high-level of


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manipulation. They have been utilized in applications such as in cell-based assays and for drug screening purposes.23 Microfluidic devices perfuse fluids continuously and are able to create a uniform particle concentration profile.20 Moreover, the controlled perfusion mimics more closely in vivo physiological flow condition.23 As such, it has been reported that fluid flow can induce various processes which are important in cell-particle interactions, such as tissue biodistribution and cellular uptake through stress forces at the cell surface-particle level.24,25 Nevertheless, reports of using microfluidic systems for the study of particle-cell interactions (especially nanoparticles) under flow condition have been very limited so far20,24, and usually restricted to test single nanoparticle suspensions. Herein, we have demonstrated using a crossed flow microfluidic platform configuration recently developed in our group26, in conjunction with a cell microarray for assessing particle-cell interactions in a high throughput and accurate manner. Polymeric microparticles with different surface characteristics were simultaneously perfused onto the immobilized cell microarray. AgNPs at different concentrations were also used to validate this system for evaluating nanoparticle-induced toxicity under flow condition. Standard well plate static control experiments were carried out for comparison. This proof of concept using crossed flow microfluidic configuration platform for assessing particle-cell interaction can provide a faster readout, offers time-resolved monitoring, and retains high magnification imaging capability.

2. Results and Discussion 2.1 Design and principle of the microfluidic system


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A microfluidic chip platform, in conjunction with a cell microarray aiming for high throughput delivery of multiple cell treatments, especially designed for particle suspensions was developed. The microfluidic channel network was fabricated in polydimethylsiloxane (PDMS), and via a protected plasma bonding process adhered to glass cover slide pre-patterned with antibody microarray26 (Figure 1, Steps 1 and 2). A pressure pump was used for manipulation of fluid inside the microfluidic chip for easy automation, fast and stable flow controls. The sequence of operation is the following: delivery of cells, crossed-flow delivery of particles, and then performing viability assay (Figure 1). This is achieved through sequential activation of flows in Inlets 1 and Inlets 2 as shown in Figure 1 (Steps 3-5). Inlets 1 containing a cell suspension were activated together with combined Outlet 1 to introduce the cell population inside the chip and to generate a cell microarray while Inlets 2 and Outlet 2 were closed (Figure 1, Step 3). Subsequently, Inlets 2 containing cell treatments (e.g., particle suspension) and Outlet 2 were then switched on while Inlets 1 and Outlet 1 were switched off (Figure 1, Step 4). Lastly, Inlets 1 with new solutions (e.g., cell detection assay reagents, or washing buffer) and Outlet 1 were re-activated (Figure 1, Step 5). Each inlet microchannel is 350 µm wide and 125 µm high. The final assembled microfluidic chip on glass cover slide with food coloring dye for visualization is shown in Figure 1. One important element for generation of a stable cell microarray is the strong anchorage of cell capturing molecules (such as antibody in our case). We applied a surface modification that allows covalent biomolecule immobilization onto a glass cover slide surface through a silanization process27 (Figure 2). Amine – epoxy chemistry was further applied and to covalently link antibody biomolecules onto the silanized surface27, thus we


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generated a 5 x 5 array of anti-CD20 antibody spots (a total of 25 spots, each 350 µm in diameter) via a microarrayer. B-lymphocytes (HR1K cells), expressing CD20 cell surface antigen, bind preferentially onto the anti-CD20 antibody patterned spots than to the BSA passivated regions (Figure 2). We first used two fluorescent cell dyes, CellTracker Red (CTR) and CellTracker Green (CTG) to demonstrate the laminar flow profile over a cell microarray on chip. To this end, HR1K cell microarray containing 25 clusters (5 x 5 array) was generated as previously described (Figure 3a). Thereafter, Inlets 1 containing CTR with 5 serial dilutions in each inlet channel, were simultaneously perfused into the chip, where each dilution was positioned onto a column of the cell microarray (Figure 3 a and b). Similarly, Inlets 2 containing 5 serial dilutions of CTG were then activated and perfused perpendicularly to the CTR flow direction (i.e., each dilution was positioned over each row of the cell microarray) (Figure 3a and 3c). As expected, cell clusters that are receiving lower concentrations of CTR show decreased red fluorescence intensities (Figure 3b) and similarly decreased green fluorescence signals for the cell clusters receiving lower concentrations of CTG (Figure 3c). Moreover, by merging the two fluorescence channels together, we can observe that individual cell clusters have different color code information (from bright yellow color to dark) resulting from combinations of different intensities of red and green fluorescence (Figure 3d). Furthermore, by using the ImageJ software, we obtained the relative fluorescence intensity values for equal analysis areas. The results show that the variation in fluorescence intensity among cell clusters receiving the same treatment is very small (Figure 3e). This small variation also indicates that the distance of each cell cluster to the treatment inlet does not influence the cell uptake of the two dyes.


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In other words, the cell clusters closer to the treatment source exhibited the same behavior to the cell clusters further away from the inlet. This indicates that under constant fluid flow the concentration of the cell dye is maintained constant throughout the channel length (i.e., dye depletion by the upstream cell clusters becomes negligible when new dye molecules are constantly being replenished from the inlet source to the downstream cell clusters). However, the experiment based on serial dilutions of the same reagent does not convincingly prove that there are no inter-diffusion processes occurring between adjacent microfluidic streams for the length (~2.75 mm) of the cell microarray (Figure 1). In fact, a fluid stream inside a microfluidic system generally conforms to laminar flow profile meaning that two parallel streams can mix only through molecular diffusion.28 In order to confirm that inter-diffusion did not occur over the length of the cell microarray), we chose 5 distinct cellular treatment reagents: CTR and CTG mixture, Hoechst 33342, CTR, CTG, and phosphate buffered saline (PBS, no dye control). When these reagents were flown over the cell microarray, each column was observed to emit different fluorescence colors: yellow, blue, red, green or no color (dark), which corresponds to the treatments of mixture of CTR and CTG, Hoechst 33342, CTR, CTG, and PBS buffer, respectively (Figure 4a and 4b). To note, a scattered blue signal was observed in Figure 4a, which might be caused by some auto-fluorescent microscopic objects (such as dust particles) inside the PDMS chip. Nonetheless, since cell clusters from columns 1 and 3 did not show populations of blue cells on the spots, it was evident that cross contamination from column 2 (blue dye channel) did not occur.


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2.2 Delivery of polymeric particles with different surface modifications After proving that we were able to simultaneously deliver multiple solutions (5) and no detectable cross contamination was introduced into the neighboring streams using the current design setup, we investigated the ability of the microfluidic platform to analyze particle delivery to the immobilized cells. As test particles, we used polymeric microparticles that have been extensively explored in biological applications because of their well-defined physical properties (such as monodispersed size) and ease to conjugate bioactive molecules onto their surface.5 The polystyrene particles (Dynabeads, 2.8 µm diameter) used in our experiments have terminating carboxyl groups (-COOH) and we performed EDC-NHS chemistry to graft amine reactive NHS ester that allowed proteins and antibodies to bind onto the particle surface via stable amide bond linkages (Suppl. Figure 1). Particles were conjugated with human IgG (control antibody) and different concentrations (1,10 and 100 µg/ml) of anti-CD20 antibody, and were perfused over each column of the cell microarray (for a total of 5 cell clusters per particle type). Non-functionalized particles were used in parallel to assess the non-specific binding events. Within one minute perfusion, particles already started to bind to the immobilized cell surfaces (Figure 5a and 5b). Average number of bound particles per cell was quantified for each particle type. Among the particles functionalized with IgG, those decorated with anti-CD20 antibody showed significantly more bindings to HR1K cell clusters (Figure 5c). Moreover, particles functionalized with 1 µg/ml of anti-CD20 antibody had significantly less cellular binding than particles with either 10 µg/ml or 100 µg/ml conjugated antibody. The latter conditions resulted in similar cellular binding activities suggesting that the particle surfaces may have


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already reached saturation at concentration of 10 µg/ml. The non-functionalized particles also showed some binding affinity to immobilized cells probably as a result of non-specific binding of the active carboxyl groups which are not quenched (Figure 5c). To further prove that the anti-CD20 antibody-coated particles binding to HR1K cell surface was indeed through specific antibody-cell surface antigen interaction and not through other non-specific binding, we performed an additional antibody pre-blocking step prior to perfusion of the antibody conjugated particles. To this end, after the HR1K cell microarray was generated on chip, we selected cell clusters in columns 1 and 4 (as repeating conditions) to receive solutions containing anti-CD20 antibody (prior to particle perfusion), while the other three columns (2, 3, and 5) of cell clusters were perfused with PBS solutions. As HR1K cell surface expresses CD20 molecules, perfusion of anti-CD20 antibody should allow the solution phase antibody to bind to the exposed CD20 on the cell surfaces, and prevent further binding of particles conjugated with anti-CD20 antibody. As expected, anti-CD20 antibody decorated particles did not bind to the surface of cells in columns 1 and 4 where cell surface was pre-blocked with antibody (Figure 6a). In contrast, anti-CD20-functionalized particles perfused over cell clusters in columns 2 and 5 (as repeating conditions), were observed to bind to the cells (Figure 6b). Cell clusters in column 3 were delivered with non-specific IgG coated particles as negative control and were observed to bind minimally to the cells (Figure 6b). Using specific antibody as targeted delivery in vivo is particularly important in the case of micro- and nanoparticles for drug delivery since the antibody can guide the delivery of the particle-drug complex and to release the drug locally and more effectively.29


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2.3 Integration of cell viability assay into microfluidic chip after imposed cell treatments To test whether certain particles (e.g., nanoparticles) are exhibiting toxic effects toward biological systems, a cell cytotoxicity assay is commonly performed after incubating cells with particles for some pre-defined time. We would like to test if we were able to integrate such detection method into the crossed flow microfluidic system after the particles have been delivered onto the cell surfaces. However, the particles (Dynabeads) that we have used in our pilot study experiments are biocompatible and have been used in many biological assays, such as cell isolation and protein purification, and hence, will not likely to induce cell toxicity.30 Therefore, we chose instead solutions of different tonicity which are known to produce cell killing effects as model treatments31, and to assess the viability of the treated cells using fluorescent indicator stains. The model solution of different tonicity ranged from DI water (very low osmotic concentration, thus resulting in cell swelling, apoptosis31,32 and bursting) to PBS (having isotonic ionic strength and non-toxic to cells). We prepared five mixtures of varying volume ratio (ø) of DI water to PBS and perfused the solutions over the cell clusters for a one-minute treatment. By comparing the micrographs before and after the treatments, it is noticeable to observe that the first column that received pure DI water solution (ø = 1) had significantly fewer cells remaining on the spots; as DI water treatment has caused cell to lyse and cell debris were washed away by the flowing liquid stream (Figure 7 a and b). Furthermore, cell viability staining reagents were immediately introduced into the chip resulting in a red fluorescence signal indicating cell death, and green signal indicating healthy cells. Cell treatments containing higher water content showed more toxicity (indicated by higher number of red cells per spot) (Figure 7c-d). By using 5 solutions of different tonicity, a plot of percentage of cell


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viability was generated against DI water volume ratio in PBS (ø) in resemblance to a response-dose dependent relationship (Figure 7e). And the equivalent “IC50” was estimated to be roughly around ø = 0.75, for a treatment of one minute experiment. This microfluidic system has the advantage that a precise control of short reaction times is possible since switching from treatment solutions to washing buffer is almost instantaneous. In comparison, treatments to cells in suspension in Eppendorf tube or in well plate format require the extra time to spin down the cells (~3 - 5 mins), which can significantly contribute to experimental error. Furthermore, centrifugation steps may not be able to recover all the cells in the suspension, especially in the case of cells with damaged cell membranes. Using the 96 well plate format, we observed approximately 50% of cell loss after 3 washes (Suppl. Figure 2).

2.4 AgNPs induce higher cell toxicity in dynamic flow condition than in static condition AgNPs are known for their antimicrobial properties and have been utilized in many consumer and health care products.33,34 However, numerous reports have also indicated that they are toxic towards a number of mammalian cell lines.35-37 We have thus chosen AgNPs for testing in our microfluidic system. Immobilized HR1K cells exhibited dosagedependent cytotoxicity under dynamic flow conditions for AgNP concentrations at 0, 0.5, 5, 10 and 20 µg/ml for 6 h particle exposure (Figure 8a). Interestingly, under static conditions in the conventional 96 well plate format, hardly any cytotoxicity was induced at the same AgNP concentrations (Figure 8a). For example, with 20 µg/ml of AgNP treatment, less than 30% of cells were viable after 6 h particle exposure under flow, whereas greater than 80% of cell viability was observed under static condition (Figure 8b).


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Only when AgNP concentration was increased to 50 µg/ml or the exposure time was extended to 24 h, did we observe noticeable cytotoxic effect using static conditions (Suppl. Figure 3). The flow exposure condition eradicates the issue of particle precipitation which causes nonhomogeneous particle distribution, an acknowledged challenge under static conditions.20 Such particle precipitation may explain the higher cell viability observed in well plates particularly since HR1K cells are suspension cells. Furthermore, we have observed HR1K cells tend to grow as large cell spheroids with neighboring cells in the well plates38 (Suppl. Figure 3c), which might have shielded the inner core cells from exposure to AgNPs. The ability to expose individual cells to a uniform concentration of AgNPs continuously is an advantage of our microfluidic system, which may translate to more accurate nanotoxicity measurements. And the earlier onset of cytotoxicity triggered by AgNPs under dynamic flow condition (< 6 h) could be an important characteristic (i.e., faster readout) in nanotoxicity screening assays. Note that the shear stress induced by the fluid flow had minor or no toxicity towards the immobilized cells (the control streams contained no AgNPs). Alternative to compare nanotoxicity results based on particle concentrations, the total number of particles present in testing conditions can also be an interesting parameter to report, especially for particles with different sizes as the total number of particles scales strongly with particle radius (Supplementary Equation 1). However, for particles of the same size (e.g., 10 nm in this report), the total number of nanoparticles in the solution is directly proportional to particle concentrations (Supplementary Table 1). Due to the complex behavior of nanomaterials in a solution (e.g., sedimentation, Brownian motion effect, particle aggregation, and protein corona effect) and their unique interactions with


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biological system under different testing conditions (e.g., static and flow), comparison of obtained toxicity results should be exercised with extreme care as a slight perturbation in testing protocol can product quite different results. As a proof of concept for using our microfluidic flow system for delivering nanoparticles onto the cell microarray, AgNPs with 10 nm in diameter were used at different particle concentrations as to obtain an “IC50” equivalent of particle toxicity. However, the exact mechanism of particle induced toxicity under flow through system, the potential molecular signaling among cell clusters, physical property of nanoparticles tested (e.g. size and shape), and the effect of physical parameters of the device (e.g., flow rate and flow duration) should require further extensive studies.

High light transmittance of the microfluidic chip (PDMS on thin glass cover slide) makes our device extremely suitable for microscopy analysis. For instance, under 60x magnification, the morphology of single HR1K cells exposed to AgNPs (round, condensed nuclei, translucent membrane) and control cells (rough cell membrane with protrusions) can be distinguished (Figure 8c). Using conventional 96 well plates, the maximum magnification for microscopy imaging is limited to 20x due to the thick plastic plate. Immunostaining for F-actin filament of the immobilized cells could also be performed and imaged on chip (Suppl. Figure 4). Furthermore, this microfluidic chip system is compatible with automation and live cell time-resolved monitoring of cells exposed to different concentrations of nanoparticles (see Suppl. Videos).

3. Conclusions


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With continuous innovations in nanotechnology, novel and more sophisticated particles are being developed for a range of applications in biological and medical fields. To truly harness their potentials as tools to probe biological systems, we still need to better understand their interactions with biological entities and to fully characterize their potential toxicities. Especially in the field of drug delivery, monitoring particle interactions with targeted cells under physiological flow conditions in vitro and in high throughput screening can fast track the discovery of potential drug carriers. Here we have demonstrated as a proof of concept utilizing a microfluidic device system to deliver particle suspensions onto a microarray of cell clusters using a crossed-flow model that we have previously developed.26 We firstly used polymeric microparticles with various surface modifications to simultaneously perfused them onto cell surface under flow. By using antibody preblocking treatment, we have demonstrated that the binding of particles to cell surface were through “targeted” antibody and antigen interaction. For proof of principle, we have extended our experiments to use AgNPs which are known to be cytotoxic. We observed higher cell toxicity under shear flow in comparison to static conditions, and this result is in accordance with other literature reports.39,40 This unique combination of cell microarray technique with microfluidic system could indeed increase the experimental accuracy and degree of manipulation, especially for the application in nanotoxicity screening. Choosing a cell microarray over a continuous monolayer cell surface has the following key advantages: with predefined area of cell spots (350 µm in diameter), more precise and consistent cell quantification can be achieved. Furthermore, with distinct spatial distribution of cell array spots (5 rows x 5 columns), cell clusters that have been subjected to particular nanoparticle treatments can


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be easily distinguished. On the other hand, using a continuous cell monolayer surface, a subsequent calculation/measurement step is necessary in order to determine the exact locations of cell subpopulation of interest, which is more tedious and prom to introduce errors. Spotting technique is also a simple method to introduce multiple biomolecules onto a surface, hence multiple cell lines.26 Further development to incorporate multiple cell lines into a single screening platform would be desirable to increase throughput. In our ongoing efforts, we will scale up this microfluidic setup to be able to screen a larger set of nanoparticles, and assess the effects contributed by particle size, shape, surface chemistry, flow rate, flow duration and so on. Our cross flow microfluidic configuration platform can provide a faster readout, is cost effective, offers time-resolved monitoring, and retains high magnification imaging capability. It could potentially become a useful tool for high throughput screening of particle-cell interactions in vitro under physiological condition and eventually lead to the reduction of animal model experiments.

4. Experimental Section Cell culture: HR1K cell line (acute B-cell leukemia, ATCC), was cultured in RPMI 1640 medium (Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS), 100 units/mL penicillin and 100 μg/mL streptomycin (Invitrogen), 4.5 g/L glucose, 1x Glutamax (Thermo Fisher), and 1 mM sodium-pyruvate. The cell line was maintained at 37 oC in a humidified 5% CO2 incubator. Device fabrication: Microfluidic chip with crossed 5 x 5 inlets and two outlets was fabricated via photolithography and soft-lithography as previously reported

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. In brief,

SU8 50 photoresist (MicroChem) was spun on a cleaned and dehydrated 3” silicon wafer


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for achieving a height of 125 µm. A laser writer (Cloe Dilase 650) with beam size of 10 µm was used to create patterns on SU8 coated wafer. SU8 developer (MicroChem) was used to dissolve away uncross-linked SU8 50. Vapor deposition of hexamethyldisilazane (Sigma) on patterned silicon wafer was performed to passivate the surface. Polydimethylsiloxane (PDMS) was mixed at 10:1 ratio (monomer to catalyst) and poured onto the passivated silicon wafer surface and cured inside an hour for 2 h at 80 oC. The cured PDMS mold was then carefully detached from the SU8 50 patterned silicon master and the inlets and outlets holes were cut out using Haris Uni-Core biopsy punches (Ted Pella). The final PDMS chip was cleaned using ethanol and then water before its subsequent usage. Generation of antibody array spots: Thin glass cover slides (24 mm x 60 mm, MenzelGlaser) were treated with 10% v/v (3-glycidylxoypropyl)-trimethoxysilane (GPTMS) in anhydrous toluene to enhance protein adsorption. Functional silanes, such as GPTMS, are bifunctional coupling agents that can bind to a substrate and leave the reactive organic component to couple to antibodies, proteins, or any other biomolecule possessing appropriate chemical groups for reaction.27 More specifically, the inorganic hydroxyl terminating groups (-OH) on a glass substrate initiated covalent bonds with organosilane of GPTMS via a siloxane bond. The reactive epoxy groups of GPTMS were then exposed to biomolecules, such as antibodies or proteins having amine groups (-NH2) to initiate another coupling reaction, resulting in a secondary amine bond. An array of 5 x 5 antihuman CD20 antibodies (provided by Dr. Peter Macardle) of circular spot was generated on GPTMS treated glass substrate using Xactll™ Compact Microarray System equipped with 350 µm diameter pin head. The printed protein array slide was then stored in a


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humidified petri dish at 4 oC overnight. It was washed with copious amount of Dulbecco’s phosphate buffered saline (PBS, Sigma) and then blocked with 5% bovine serum albumin (BSA, Sigma) overnight at 4

o

C. The antibody deposited glass slide was then

subsequently bonded to a PDMS counterpart by oxygen plasma treatment (Harrick Plasma). The antibody printed area was protected by the use of a hollowed-out PDMS piece26 preventing protein degradation by the plasma activation. Microparticle conjugation: Carboxyl terminated magnetic polystyrene particles (2.8 µm diameter, Dynabeads M-270 carboxylic acid, Invitrogen) were purchased from Thermo Fisher scientific, Australia. Microparticle stock suspension was initially prepared by diluting 10 µl of particle suspension in 990 µl of PBS buffer (Sigma Aldrich), yielding a stock concentration of 6 X 107 particles per mL. Particles were decorated with antibodies via EDC-NHS chemistry. In brief, particles were washed twice with cold 2-(Nmorpholino)ethanesulfonic acid (MES) buffer (pH 5) by adding equal amount of particle stock suspension (100 µl). Each washing step consists of mixing for 5 min in 100 µl cold MES buffer and placing the Eppendorf tube containing particles on a magnet for 5 min and removing the supernatant. After washing the particles, carboxyl-terminals were activated to form NHS ester-terminated surface by reacting with a mixture of 50 µl of 50 mg/ml of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC, Fluke) and 50 µl of 50 mg/ml of N-hydroxysuccinimide (NHS, Sigma) in cold MES buffer. This reaction was conducted under mixing in an orbital shaker for 30 min at room temperature. After this reaction, Eppendorf tube was placed on the magnet for 5 min to remove supernatant. Activated particles were then incubated with control human IgG (Sigma), and anti-CD20 antibody at three different concentrations (1 µg/ml, 10 µg/ml, and 100 µg/ml) for 1 h at


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room temperature on an orbital shaker. Antibody decorated particles were then washed 4 times in PBS containing 0.01% Tween 20 by the same washing step used previously to remove non-specifically bound antibodies. Antibody modified particles were then reacted with 100 µl of 0.1 M Ethanolamine (Sigma) in PBS solution for 1 h to block any uncovered surface on the particles by reacting with the remaining active ester groups on particle surface. Modified particles were stored at 4 °C until further use. Dual treatment with CellTracker Red (CTR) and CellTracker Green (CTG): The assembled microfluidic device was placed inside a stage top incubator on a EVOS FL Auto Imager microscope (Thermo Fisher) with humidity, temperature, and CO2 controls. HR1K cells (1x107cells/ml) were perfused into the antibody patterned area and incubated for 10 to 15 min for cells to bind to underlying anti-CD20 antibody. PBS was then perfused into the chip to wash away non-specifically bound cells. A matrix of 25 circular clusters (5 columns x 5 rows) of HR1K cells were generated. Five parallel laminar flowing streams containing different dilutions of CTR (10 µM, 5 µM, 1 µM, 0.1 µM, and 0.01 µM) were perfused over cell clusters for 5 min. Each treatment solution was precisely in contact with each column of the cell array. The cell microarray was then rinsed with PBS solution by quickly switching to the pumping streams from the perpendicular inlets. Similarly, five different concentrations of CTG (10 µM, 5 µM, 1 µM, 0.1 µM, and 0.01 µM) were perfused orthogonally over the cell clusters for 5 min. The reaction chamber was once again washed with PBS solution. Fluorescence images were then acquired using EVOS monochrome and color cameras. Fluorescence intensity measurement of individual cell cluster with same analysis area were characterized using ImageJ software and were plotted via GraphPad Prism version 7, GraphPad Software, La Jolla.


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Simultaneous cell dye treatments (5 solutions) to cell clusters: Parallel streams containing different cell staining reagents, CellTracker Red (CTR, 5 µM, Life Technology), CellTracker Green (CTG, 5 µM, Life Technology), Hoechst 33342 (10 µg/ml, Sigma), equal ratio of CTR and CTG (both at 5 µM), and control PBS streams were perfused simultaneously onto the cell microarray. After 5 min of dye perfusion, the reaction area was washed and equilibrated with PBS buffer. Fluorescence microscopy images were acquired using color and monochrome cameras of EVOS microscope, and were further processed via ImageJ software. Particle perfusion (Dynabead microparticles and AgNPs) to bound HR1K cell microarray: A HR1K cell microarray was generated as described in previous session. For perfusion of Dynabead microparticles, parallel streams containing particle suspensions with equal concentrations of: unmodified particles, control IgG modified particles, and particles pre-incubated with anti-CD20 antibody of different concentrations (1 µg/ml, 10 µg/ml, and 100 µg/ml) were perfused over the cell clusters for 1 min. In selected experiments, an anti-CD20 antibody (1 µg/ml) preblocking step was performed. Perpendicular streams of PBS buffer were activated to wash off non-specifically bound particles. The extent of particles bound to cells were imaged via EVOS system and were counted using ImageJ software. For perfusion of polyvinylpyrrolidone (PVP) coated AgNPs (10 nm, nanoComposix, USA), parallel streams of AgNPs at five different concentrations (0, 0.5, 5, 10, and 20 µg/ml) were perfused over cell clusters at 1 µL/min for 6 h before switching to cell staining reagent containing propidium iodide (PI, 2.5 µg/ml, Sigma) and Hoechst 33342 (2.5 µg/ml). In selected experiments, Phalloidin-TRITC solution (100 μg/ml, Sigma) was perfused into the chip for staining for F-actin. Dead cells


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(positive staining by PI) were counted using automized cell counter ImageJ software against the total cell number (positive staining by Hoechst 33342) to assess cell viability. Static control experiments were also carried out using 96 well plate format at different AgNP concentrations (0, 0.5, 5, 10, 20 and 50 µg/ml) and incubated for 6 h and 24 h. For assessing the percentage of cell loss during the washing steps, the 96 well plate was subjected to centrifugation at 200 rcf for 5 min, and the supernatant discarded. PBS was added to resuspend cells and this process was repeated thrice. Cells were counted via ImageJ after each wash. Statistical analyses were performed using GraphPad Prism software. Cell viability assessment with solutions of different tonicity: Streams containing different water to PBS volume ratio (ø= Vwater : VPBS = 1, 0.75, 0.5, 0.25, 0) were perfused over the cell clusters for 1 min. A mixture of propidium iodide (PI, 10 µg/ml, Sigma) and fluorescein diacetate (FDA, 5 µg/ml, Sigma) staining dead and live cells, respectively, were perfused into the reaction area. The cells were then washed with PBS solution before subjected to fluorescence imaging. Percentage of cell viability is calculated by counting the number of green cells (live cells) in each cell cluster after the treatments averaged by the total number of cells in that corresponding cluster prior to the treatments. Dose-response plot was generated using GraphPad Prism. Scanning Electronic Microscopy (SEM) imaging of microparticles binding to cells: Cells were captured onto anti-CD20 antibody patterned glass slide as previously described. A suspension of anti-CD20 antibody conjugated beads was incubated over the cell cluster for 15 min. The unbound beads were washed away by gentle rinse in PBS. Formaldehyde solution (4%, Sigma) was used as fixing agent and was incubated over


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the bound cell for half an hour. The sample was then dehydrated by incubation in sequential ethanol dilutions of 50%, 70%, and 100% for 15 min in each drying process. The dried sample was then subjected to SEM imaging.

Acknowledgements This work was supported by an Australian Research Council Discovery Grant (DP150101774) and part by South Australian PRIF project "International Cluster of Nanosafety". This work was performed (in part) at the South Australian and Victorian nodes of the Australian National Fabrication Facility under the National Collaborative Research Infrastructure Strategy to provide nano and microfabrication facilities for Australia’s researchers. The authors would like to thank Dr. Nicholas Welch from CSIRO for his help preparing antibody protein microarrays.

References (1) Singer, J. M.; Plotz, C. M. The American Journal of Medicine 1956, 21, 888-892. (2) Hermanson, G. T. In Bioconjugate Techniques (Second Edition); Academic Press: New York, 2008, pp 582-626. (3) Singh, S. K.; Kulkarni, P. P.; Dash, D. In Bio-Nanotechnology; Blackwell Publishing Ltd., 2013, pp 132. (4) Laurent, A. Techniques in Vascular & Interventional Radiology 2007, 10, 248-256. (5) Saralidze, K.; Koole, L. H.; Knetsch, M. L. W. Materials 2010, 3, 3537. (6) Kettenbach, J.; Stadler, A.; Katzler, I. v.; Schernthaner, R.; Blum, M.; Lammer, J.; Rand, T. CardioVascular and Interventional Radiology 2008, 31, 468-476. (7) Cavalieri, F.; Chiessi, E.; Villa, R.; Viganò, L.; Zaffaroni, N.; Telling, M. F.; Paradossi, G. Biomacromolecules 2008, 9, 1967-1973. (8) Neurauter, A. A.; Bonyhadi, M.; Lien, E.; Nøkleby, L.; Ruud, E.; Camacho, S.; Aarvak, T. In Cell Separation: Fundamentals, Analytical and Preparative Methods, Kumar, A.; Galaev, I. Y.; Mattiasson, B., Eds.; Springer Berlin Heidelberg: Berlin, Heidelberg, 2007, pp 41-73. (9) Hubbard, N.; Hagin, D.; Sommer, K.; Song, Y.; Khan, I.; Clough, C.; Ochs, H. D.; Rawlings, D. J.; Scharenberg, A. M.; Torgerson, T. R. Blood 2016. (10) Plouffe, B. D.; Murthy, S. K.; Lewis, L. H. Reports on progress in physics. Physical Society (Great Britain) 2015, 78, 016601-016601. (11) Jain, K. K. Drug discovery today 2005, 10, 1435-1442.


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(12) Levy, O.; Han, E.; Ngai, J.; Anandakumaran, P.; Tong, Z.; Ng, K. S.; Karp, J. M. In Micro- and Nanoengineering of the Cell Surface; William Andrew Publishing: Oxford, 2014, pp 253-279. (13) Cristiana, S. O. P.; Ricardo Pires das, N.; Lino, S. F. Nanotechnology 2011, 22, 494002. (14) Saraiva, C.; Praça, C.; Ferreira, R.; Santos, T.; Ferreira, L.; Bernardino, L. Journal of Controlled Release 2016, 235, 34-47. (15) Nel, A.; Xia, T.; Madler, L.; Li, N. Science (New York, N.Y.) 2006, 311, 622-627. (16) Burke, T. J.; Loniello, K. R.; Beebe, J. A.; Ervin, K. M. Combinatorial Chemistry & High Throughput Screening 2003, 6, 183-194. (17) Jan, E.; Byrne, S. J.; Cuddihy, M.; Davies, A. M.; Volkov, Y.; Gun’ko, Y. K.; Kotov, N. A. ACS Nano 2008, 2, 928-938. (18) Jones, C. F.; Grainger, D. W. Advanced Drug Delivery Reviews 2009, 61, 438-456. (19) Sager, T. M.; Porter, D. W.; Robinson, V. A.; Lindsley, W. G.; Schwegler-Berry, D. E.; Castranova, V. Nanotoxicology 2007, 1, 118-129. (20) Mahto, S. K.; Yoon, T. H.; Rhee, S. W. Biomicrofluidics 2010, 4, 034111. (21) Agarwal, R.; Singh, V.; Jurney, P.; Shi, L.; Sreenivasan, S. V.; Roy, K. Proceedings of the National Academy of Sciences 2013, 110, 17247-17252. (22) Cho, E. C.; Zhang, Q.; Xia, Y. Nature nanotechnology 2011, 6, 385-391. (23) Giridharan, V.; Yun, Y.; Hajdu, P.; Conforti, L.; Collins, B.; Jang, Y.; Sankar, J. Journal of Nanomaterials 2012, 2012, 14. (24) Kang, T.; Park, C.; Choi, J.-S.; Cui, J.-H.; Lee, B.-J. Journal of Drug Delivery Science and Technology 2016, 31, 130-136. (25) Henriksen-Lacey, M.; Carregal-Romero, S.; Liz-Marzán, L. M. Bioconjugate Chemistry 2017, 28, 212-221. (26) Tong, Z.; Ivask, A.; Guo, K.; McCormick, S.; Lombi, E.; Priest, C.; Voelcker, N. H. Lab on a Chip 2017, 17, 501-510. (27) Hermanson, G. T. In Bioconjugate Techniques (Second Edition); Academic Press: New York, 2008, pp 562-581. (28) Kamholz, A. E.; Weigl, B. H.; Finlayson, B. A.; Yager, P. Analytical Chemistry 1999, 71, 5340-5347. (29) Sun, T.; Zhang, Y. S.; Pang, B.; Hyun, D. C.; Yang, M.; Xia, Y. Angewandte Chemie International Edition 2014, 53, 12320-12364. (30) Tiwari, A.; Punshon, G.; Kidane, A.; Hamilton, G.; Seifalian, A. M. Cell biology and toxicology 2003, 19, 265-272. (31) Selzner, N.; Selzner, M.; Graf, R.; Ungethuem, U.; Fitz, J. G.; Clavien, P. A. Cell death and differentiation 2004, 11 Suppl 2, S172-180. (32) Collins, S. The Laryngoscope 1993, 103, 825-827. (33) Vance, M. E.; Kuiken, T.; Vejerano, E. P.; McGinnis, S. P.; Hochella, M. F., Jr.; Rejeski, D.; Hull, M. S. Beilstein Journal of Nanotechnology 2015, 6, 1769-1780. (34) Nowack, B.; Krug, H.; Height, M. 120 Years of Nanosilver History: Implications for Policy Makers, 2011; Vol. 45. (35) Nguyen, K. C.; Seligy, V. L.; Massarsky, A.; Moon, T. W.; Rippstein, P.; Tan, J.; Tayabali, A. F. Journal of Physics: Conference Series 2013, 429, 012025. (36) Richter, L.; Charwat, V.; Jungreuthmayer, C.; Bellutti, F.; Brueckl, H.; Ertl, P. Lab on a Chip 2011, 11, 2551-2560. (37) Vecchio, G.; Fenech, M.; Pompa, P. P.; Voelcker, N. H. Small 2014, 10, 2721-2734. (38) Wang'ondu, R.; Teal, S.; Park, R.; Heston, L.; Delecluse, H.; Miller, G. PLoS One 2015, 10, e0126088. (39) Park, M. S. Y., Tae Hyun Bulletin of the Korean Chemical Society 2014, 35, 123-128. (40) Kim, D.; Lin, Y.-S.; Haynes, C. L. Analytical Chemistry 2011, 83, 8377-8382.


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Figure 1. Workflow of experimental procedure. Antibody was first deposited onto a silane functionalized glass cover slide. Second, the glass slide and PDMS chip was covalently bonded together via a plasma activation. Third, Inlets 1 and Outlet 1 were switched on for the flow of cell suspension into the chip for cell immobilization. Forth step, Inlets 2 and Outlet 2 were activated while Inlets 1 and Outlet 1 were turned off to introduce cell treatment streams (e.g., particle containing solutions) over the immobilized cell microarray. Lastly, Inlets 1 and Outlet 1 were reactivated for perfusion of additional reagents, such as washing buffer, cell staining dyes, or cell viability assay compounds. A micrograph of assembled microfluidic chip on glass cover slide is shown with food coloring dye indicating the interconnected channel network.

Figure 2. Glass slide silanisation for attachment of antibody and cell capture. Cleaned glass cover slide was first treated with epoxy silane to graft epoxy groups onto the glass slide surface. Anti-CD20 antibody was then covalently attached to the glass slide, while BSA was used for blocking of the non-antibody spotted regions. HR1K cells were then captured by patterned anti-CD20 antibody.


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Figure 3. Dual cell treatments in crossed flow configuration microfluidic chip. Bright field image of HR1K cell microarray (a). Fluorescence images of cell clusters treated serially with 5 dilutions of CTR (b) and 5 dilutions of CTG (c). Merged fluorescence channels after cell microarray was treated with two cell dyes (d). Fluorescence intensity plot of CTR and CTG at different dilutions (e). Each scattered data point in (e) represents an averaged fluorescent intensity value of each cell cluster, i.e., 5 data points per dye concentration were acquired. Scale bars: (a-d) 300 µm.

Figure 4. Simultaneous delivery of 5 different treatments via laminar flow streams. Inlets containing five solutions of cell treatment dyes (mixture of CTR and CTG, Hoechst 33342, CTR, CTG, and PBS) were perfused over a cell microarray and fluorescence microscopy


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images were taken under a 4x objective (a). Higher magnification (20x) of representative fluorescent micrographs of stained cell clusters from each condition (b). Scale bars: (a) 200 µm and (b) 50 µm.

Figure 5. Microparticle binding to cell microarray. Microparticles with different surface modifications: unmodified, conjugated with control IgG, conjugated with anti-CD 20 antibody at 1 µg/ml, 10 µg/ml, and 100 µg/ml were perfused over the HR1K cell clusters (a). SEM image of microparticles attached to the cell surface (b). Average number of particles per cell was quantified for different surface modifications of microparticles. Data points represent mean ± SE with N = 3. Column plot and statistical analysis were generated using GraphPad Prism (c). Unpaired Student’s t tests were performed in comparison with IgG control. *p < 0.05 and ***p < 0.0001 denote significantly different, and ns (p > 0.05) denotes not significantly different. Scale bars: (a) 50 µm, and (b) 2 µm.


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Figure 6. Inhibition of microparticle binding by antibody pre-blocking. Representative micrographs of microparticles conjugated with control human IgG or anti-CD 20 antibody perfused over cell surfaces with or without pre-blocking with anti-CD20 antibody (a). Average number of particles per cell was quantified for each condition (b). Data points represent mean ± SE, with N = 3. Column plot and statistical analysis were generated using GraphPad Prism (b). Unpaired Student’s t tests were performed for each pair testing. ***p < 0.0001 denotes significantly different, and ns (p > 0.05) denotes not significantly different. Scale bars: (a) 50 µm.


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Figure 7. Cell viability on chip assay using 5 treatments of solutions of different tonicity. Food coloring dye was used to visualize the fluid flow pattern over HR1K cell microarray (a). 5 different dilutions of DI water volume ratio in PBS (ø = Vwater : VPBS) were perfused over the cell microarray (b). Arrows indicating where the cells underwent membrane disintegration and were washed away by flowing fluids. Propidium iodide and fluorescein diacetate mixture were perfused inside the chip for distinguishing live and dead cells (c). Representative images of cell clusters after treatment with different tonicity solutions (d). Dose-response plot was generated using GraphPad Prism (e). Data points represent mean ± SE with N=3. Scale bars: (a-c) 300 µm, and (d) 10 µm.


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Figure 8. Cell viability on chip assay in response to AgNPs. (a) AgNPs with 5 different concentrations (0, 0.5, 5, 10 and 20 µg/ml) were used to determine nanoparticle induced cell cytotoxicity under shear flow condition and under static 96 well plate format. (b) Representative micrographs of HR1K cells, in bright field images and merged fluorescent images (stained with PI and Hoechst 33342) after 6 hour treatments at 0.5 µg/ml and 20 µg/ml particle concentration. (c) High magnification images of AgNP treated cells at 20 µg/ml (top) and control cells without AgNP treatment (bottom) for 6 h on chip. Bright field images (column 1), PI or FDA stained (column 2), Hoechst 33342 stained (column 3), and merged bright field and fluorescence channel images (column 4) showing the morphological differences of dead and live cells after treatments. Data points represent mean ± SE with N=3. Scale bars: (b) 200 µm, and (c) 20 µm.


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For TOC only A microfluidic system is used in conjunction with a cell microarray technique aiming to streamline the analysis of particle-cell interaction in a high throughput manner.


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Microfluidic Cell Microarray Platform for High Throughput Analysis of

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