Building of a flexible microfluidic plasmo-nanomechanical biosensor for live cell analysis

https://doi.org/10.1016/j.snb.2019.04.038Get rights and content

Highlights

  • A methodology to build a microfluidic plasmo-nanomechanical biosensor capable of detecting cellular interactionsis presented.

  • This strategy offers control of each phase of the fabrication protocol without need of expensive clean room facilities.

  • The biosensor fosters cellular proliferation, opening the possibility of quantifying cellular adhesion forces.

Abstract

Biosensor devices can constitute an advanced tool for monitoring and study complex dynamic biological processes, as for example cellular adhesion. Cellular adhesion is a multipart process with crucial implications in physiology (i.e. immune response, tissue nature, architecture maintenance, or behaviour and expansion of tumor cells). This work focuses on offering a controlled methodology in order to fabricate a flexible plasmo-nanomechanical biosensor placed within a microfluidic channel as a new tool for future cell adhesion studies. We designed, fabricated, and optically and mechanically characterized this novel optical biosensor. As a proof-of-concept of its functionality, the biosensor was employed to observe fibroblasts adhesion in a cell culture. The device is configured by an hexagonal array of flexible rigid/soft polymeric nanopillars capped with plasmonic gold nanodisks integrated inside a microfluidic channel. The fabrication employs low-cost and large-scale replica molding techniques using two different polymers materials (EPOTECK OG142 and 310 M). By using those materials the spring constant of the polymer nanopillars (k) can be fabricated from 1.19E-02 [N/m] to 5.35E+00 [N/m] indicating different mechanical sensitivities to shear stress. Therefore, the biosensor has the feasibility to mimic soft and rigid tissues important for the description of cellular nanoscale behaviours. The biosensor exhibits a suitable bulk sensitivity of 164 nm or 206 nm/refractive index unit respectively, depending on the base material. The range of calculated forces goes from ≈1.98 nN to ≈.942 μN. This supports that the plasmo-nanomechanical biosensors could be employed as novel tool to study living cells behavior.

Introduction

Cell adhesion is an active process that cells undergo when they first interact with a substrate or with another cell. These interactions can last few seconds as in immunological signalling or can result enduring as in tissue formation [1]. It is a crucial physiological event which impacts cellular behavior, tissue formation, architecture, immune response and tumor cell spreading [2]. Although the nature of the static bonding process of a cell to a substrate is complex, numerous studies identify three main stages [1,3,4]: cell attachment, cell body flattening, spreading, and conformation of focal adhesion sites between the cell and the substrate product of actin cytoskeleton reorganization [4]. In other words, cells not only stick to the substrate, they form complex linkages between the extracellular matrix (ECM) and the intracellular actin cytoskeleton that provide a physical pathway to establish a bidirectional communication (integrin-ECM linkage). Failure in cell adherence will end in a compromised cell growth, proliferation, and even differentiation. Hence, it is paramount to develop controlled methodologies to create high-sensitive and non-invasive biosensing platforms capable of monitoring in real-time the cell attachment process.

The production of a biosensor with the previous characteristics is a technological challenge. Its design and fabrication requires the consideration of several aspects including the properties of the substrate, the nanostructuring of the sensor, the in-situ surface biofunctionalization to promote cell adhesion, and the non-invasive and real-time monitoring of the process. Regarding the properties of the substrate, the rigidity, the dimensions and topography of the ECM are important parameters that elucidate cell fate. Besides biochemical cues [5,6], cell phenotype and function can be regulated by biophysical cues of the substrate since the ECM presents a nanoscale topography and contains nanometer-sized proteins including collagen, fibronectin, and vitronectin [3].

On the one hand, stiff substrates can stimulate cell adhesion [7], promote cell spreading and proliferation [8], facilitate cell differentiation [9] and slow down the cell migration [10]. On the other hand, topographical cues can impact on the cell shape, regulate cell proliferation and facilitate stem cell differentiation [11]. For example, anisotropic arrays of polydimethylsiloxane (PDMS) micropillars promoted the elongation of epithelial cells along the axis of the stiffest direction, but this configuration reduced cell proliferation [12]. Meanwhile, cylindrical arrays of PDMS micropillars showed no preferable alignment of epithelial cells [13]. In another approach, differentiation of human Mesenchymal Stem Cells (hMSCs) was biased favouring osteogenesis on stiff arrays of PDMS micropillars with varying stiffness [14].

Although several attempts have been made to study the interplay between the cell behaviour, the rigidity and topography of the substrate, the interwoven effects of both biophysical cues is still a field of research for tissue engineering and regenerative medicine. Besides the material rigidity and topography, biocompatibility plays also an important role for fabricating a substrate for cell culture. To enhance cell attachment, there are numerous biofunctionalization procedures for substrate preparation [6]. In the case of nanopatterned surfaces, a common strategy consists of immersing the substrate in a fibronectin solution, letting fibronectin to be absorbed over the entire surface [15]. In other approaches, proteins such as collagen and vitronectin are covalently linked by microprinting onto the substrate surface [16]. Positively charged bioactive coatings can also be used to promote the initial attachment process by electrostatic interaction since the cellular membrane of some cells has a negative surface charge [17]. Subsequently, cells are seeded into the pretreated surface by pipetting or using a polymer-based fluidic system. For instance, Yang et al. used a flexi-perm silicon chamber for cell culturing and a gravity-based perfusion system to control the surrounding solutions to the targeted cells [18]. Although these processes are effective for substrate pretreatment, there is a lack of compatibility in the substrate dimensions and the volume of the solutions employed. It is not effective to integrate a nanopatterned surface within fluidic wells of macroscopic dimensions requiring solution in the order of milliliters.

In regard to the real-time monitoring tools, Localized Surface Plasmon Resonance (LSPR) is a highly sensitive and cost-effective optical technique for label-free biosensing, widely demonstrated in the literature, for example, for the detection of gluten peptides or tumor-associated autoantibodies [19,20]. Owing to the high sensitivity of LSPR to local refractive index changes near the metal surface, the LSPR change can be used to study the cell behaviour and, thereby, to study the cell integrin-ECM linkage. On other hand, plasmonic nanostructures can be fabricated over polymer nanostructures to provide them height and flexibility. There are two approaches that have employed SPR-based sensors for studying cell adhesion, spreading and contractility. Wang et al. mapped a single-cell-substrate interaction by SPR microscopy when introducing osmotic pressure changes into the cell environment. Yang et al. proposed an alternative approach named Long Range Surface Plasmon Resonance (LRSPR) to monitor the vertical displacement of the cell membrane of 3T3 fibroblasts and cancer cells associated with the cell micromotion mechanism [21]. While these approaches are suitable for studying cell behaviour, fabricating a clever biosensor combining both mechanical and optical properties may provide an efficient and low-cost detection platform capable of monitoring the cell-binding process when studying the interwoven effects of stiffness and topography of the nanosubstrate.

In our approach, we developed a plasmo-nanomechanical biosensor for detecting the adhesion and spreading of live fibroblasts both by changes of the refractive index in the environment as well as interactions between gold plasmonic nanodisks placed on flexible polymer nanopillars. The biosensor consists of a hexagonal array of closely spaced, vertical, elastomeric nanopillars capped with gold nanodisks embedded in a microfluidic channel. The fabrication of the biosensor is based on a replica molding technique that we reported previously [22,23]. The elastomeric nanopillars are designed to mimic soft or rigid tissues using two new polymer materials (EPOTECK OG142 and 310 M). The biosensor combines the mechanical flexibility of polymer nanopillars with the optical properties of plasmonic gold nanodisks that exhibit a LSPR response. LSPR is advantageous for studying cell adhesion and spreading owing to its low penetration depth (30–50 nm) compared to other SPR configurations (SPR 100–200 nm, and LRSPR 500–1000 nm) [21]. Finally, the optical detection does not require significant computational processing.

Herein, we underpin the following contributions: i) polymer nanopillars can support gold plasmonic nanodisks to detect not only changes in the refractive index of the medium but also the deflection of the flexible nanopillars by a shear force, ii) the nanopillar deflection is not limited to only one direction, iii) the spring constant of the nanopillars can be tuned changing their geometric dimensions and structural material to mimic the rigidity of different tissues, iv) the array of polymer nanopillars can be fabricated using biocompatible materials facilitating their integration within a microfluidic channel for reducing the biosensor dimensions and ensuring at the same time the use of small volumes of reagents, and v) by using an integrated microfluidic system, the biosensor can be easily functionalized to promote cell adhesion, if required. Our work faces the technological challenge of reproducing an adequate environment for cell interaction in an aim to mechanically imitate a physiological matrix resembling differential tissues in the human body. This, in order to study the existing cellular interactions at nanoscale while optimizing the available technological resources.

Section snippets

Fabrication of the biosensor

The plasmo-nanomechanical biosensor design consists of an hexagonal array of closely spaced, vertical, elastomeric nanopillars capped with plasmonic gold nanodisks embedded in a microfluidic channel. The fabrication of the biosensor includes the following steps: 1) fabrication of a Si master mold, 2) production of a patterned PDMS stamp from the master mold, 3) generation of a polymer replica of the original template, 4) evaporation of plasmonic gold nanodisks on the polymer nanopillars by

Characterization of the plasmo-nanomechanical biosensor

PDMS molds of nanoholes were fabricated from silicon masters molds according to the controlled procedure described in [23]. Fig. 3a shows a SEM image of a PDMS mold with a surface where a gold layer was deposited. The PDMS pattern (thickness 1 mm) contains the hexagonal array of nanoholes whose depths correspond to the heights of the SiNPs. Differences observed in the diameter of replicated nanopillars, and the presence of defects in the replicated mold can be explained by fabrication

Conclusions

We have described a controlled methodology to fabricate a novel plasmo-nanomechanical biosensor within a microfluidic channel for studying cell contractile forces. By combining our fabrication method with the mechanical properties (e.g., viscosity and Young’s Modulus) of EPOTECK-OG142-87 and 310 M polymers, we were able to modify the substrate rigidity along with its nanoscale topography. The use of these polymers allowed us to fabricate vertically aligned nanopillars tuning their spring

Author´s contributions

V. Solis-Tinoco was responsible for the nanofabrication and characterization of the sensors. Performed the optical simulations, cellular experiments on the sensors and SEM characterizations. Prepared the published work, specifically wrote the initial draft. S. Marquez contributed in the design of the bonding process of the sensors, literature review and writing. T. Quesada-Lopez and F. Villarroya contributed in the realization of the cellular experiments on the sensors. Provided the reagents,

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

V. Solis-Tinoco, S. Marquez, and T. Quesada-López acknowledge financial support from ¨Programa becas en el extranjero¨ from National Council for Science and Technology (CONACyT-Mexico). The ICN2 is funded by the CERCA programme/ Generalitat de Catalunya. The ICN2 is supported by the Severo Ochoa programme of the Spanish Ministry of Science, Innovation and Universities (Grant No. SEV-2017-0706).

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