Applied Materials Today
Volume 20, September 2020, 100726
Journal home page for Applied Materials Today

3D printing enables the rapid prototyping of modular microfluidic devices for particle conjugation

https://doi.org/10.1016/j.apmt.2020.100726Get rights and content

Highlights

  • Direct 3D printing allows quick and easy tailoring of design features for modular micromixers

  • 3D printed microfluidics dramatically reduces the turnover time for optimizing experiments from days to hours

  • 3D micromixers facilitate the production of micro and nanoparticles conjugated polymers and functional antibodies by mixing multiple reagents.

Abstract

Antibody micro/nano-particle conjugates have proven to be essential tools in many diagnostic and nanomedicine applications. However, their production with homogenous coating and in a continuous fashion remains a tedious, labor-intensive, and costly process. In this regard, 3D micromixer-based microfluidic devices offer significant advantages over existing methods, where manipulating the flow in three dimensions increases fluid contact area and surface disruption, facilitating efficient mixing. While conventional softlithography is capable of fabricating simple 2D micromixers, complications arise when processing 3D structures. In this paper, we report the direct fabrication of a 3D complex microchannel design using additive manufacturing for the continuous conjugation of antibodies onto particle surfaces. This method benefits from a reduction in cost and time (from days to hours), simplified fabrication process, and limited post-processing. The flexibility of direct 3D printing allows quick and easy tailoring of design features to facilitate the production of micro and nanoparticles conjugated with functional antibodies in a continuous mixing process. We demonstrate that the produced antibody-functionalized particles retain their functionality by a firm and specific interaction with antigen presenting cells. By connecting 3D printed micromixers across the conjugation process, we illustrate the role of 3D printed microchannels as modularized components. The 3D printing method we report enables a broad spectrum of researchers to produce complex microfluidic geometries within a short time frame.

Introduction

Antibody (Ab) micro/nano-particle conjugates have become an integral part of biosciences with applications across therapy, diagnostics, biosensing, and sorting [1], [2], [3]. The adsorption of functionalized groups onto gold nanoparticles (AuNPs) has proven effective for optical imaging techniques and in vivo and in vitro diagnostics [4], [5], [6]. Microparticle conjugates have also proven useful for cell targeting. Despite the many advantages and applications of micro/nanoparticle-Ab conjugates [7], [8], [9], their preparation remains a challenging issue, involving labor-intensive and time-consuming processes. Regularly involving multiple steps, these batch processes often suffer from varying levels of reproducibility (unwanted variations in size, adsorptivity, and functionality) and quality [10]. Batch scale production is also limited in terms of volume; often appropriate for fundamental studies, not amenable to commercial upscaling due to the lack of control in the mixing that results in batch-to-batch variations. Upscaling a batch scale protocol will aggravate the insufficient mixing and mass transfer issues at play, negatively impacting the physical and chemical characteristics of conjugates. Furthermore, the batch production lacks fast screening of products and optimization of synthesis conditions. As such, a continuous, automated approach that minimizes the chance of error and human interaction is an appealing concept.

Microfluidics is uniquely suited to address the issues associated with producing particle conjugates. In particular, continuous flow micromixers are widely studied for their precise control, elevated heat and mass transfer, high mixing efficiency, and increased number of particle interactions [11]. These systems operate at a steady-state condition, offering control over the introduction of reagents, incubation time, and mixing efficacy [12]. Microfluidic micromixer geometries may exist in either two or three dimensions and can be further classed as active or passive. Active micromixers rely upon the application of external forces, making them a less viable option for rapid prototyping and commercial translation owing to added complexity. By contrast, passive micromixers rely upon their geometry to stretch, fold, break, and split fluid flow to combine reagents. While the synthesis of various micro and nanoparticles via planar microfluidic platforms have been well explored [13], [14], [15], [16], [17], particle functionalization with biological moieties via micromixing is less investigated.

While planar passive micromixers have well-established methods of fabrication, they often fail to deliver high mixing efficiencies over short channel lengths and wide ranges of Reynolds number (Re) [18]. Additionally, many nanoparticle types readily diffuse into the matrix of polydimethylsiloxane (PDMS) (commonly used in 2D micromixer manufacturing) and are prone to particle precipitation, effectively fouling the microchannel and reduce the functionality of the device [13]. On the other hand, 3D micromixers offer significant advantages over planar designs as the addition of z-direction greatly impacts the generation of vortices. By manipulating the flow in a third dimension, there is an increase in fluid contact times, surface disruption, and a decrease in required channel length [19], resulting in significant improvements in mixing efficiency across a range of Re when compared to planar micromixers. Despite these advantages, the fabrication process for 3D passive micromixers by conventional photolithography and chemical etching processes remains a standing challenge due to the complexity in processing complicated structures, leading to an inability for direct industrial translation [20,21].

Additive manufacturing is a rapidly expanding field of fabrication methods, well suited to addressing these issues [22]. The emergence of 3D printers has realized the potential for the fabrication of complex microstructures [23]. 3D printing enables a reduction in the resources and skills required to manufacture microfluidic devices, micromixers included. One of the major advantages of direct 3D printing over other existing methods is the ability to modify the design quickly. Furthermore, there is no need for plasma treatments, lengthy heat curing, or use of adhesives. There exist several printing technologies, each with their own inherent limitations. Among them, multijet modelling (MJM) has been explored for the printing of internal and external features [24,25], but requires further post-processing and is not proper for extremely fine features in order micrometers. Fusion deposition modelling (FDM) provides low-cost production of microfluidic devices and sensors [26], at the cost of resolution. Many other printing methods often involve several labor-intensive steps, compounding fabrication time; as such, researchers are not afforded the flexibility to make design changes as their experiments may demand. The direct 3D printing of a three-dimensional micromixer that requires limited post-processing and allows rapid prototyping of designs for swift optimization has until now remained elusive.

In this work, we report the facile fabrication of 3D complex microchannel designs for the continuous and efficient mixing and production of Ab micro/nano-particle conjugates. The reported printing process enables us to fabricate complex internal microchannels with little required expertise in the microfabrication. We validate the flexibility and mixing capability of our design through numerical simulations. We demonstrate that the versatility of direct 3D printing allows quick and easy tailoring of design features to facilitate processes that require high mixing indexes (MI). Additionally, we demonstrate the adaptability of our mixing approach through the adsorption of functional antibodies onto a variety of particles, from microparticles to nanoparticles. Furthermore, the flexibility of direct 3D printing also enables the connection of modularized print parts to introduce new reagents and control mixing incubation time. With the rise of multiple lab-on-chip microfluidic technologies and considering how essential mixing is in many microfluidic procedures, the benefit of the quick and easy fabrication of micromixer modules that can be adjusted for integration with other microfluidic components is highly valuable (Fig. 1). Overall, the printing process we report here is versatile enough to be used for multiple conjugation processes; from particle synthesis to cell labeling, and even for the other microfluidic operations such as particle separation or 3D printed molds [27].

Section snippets

Materials

Printing was performed using a semi-clear proprietary resin (BV007A) purchased from Creative CADworks (Toronto, Canada). 70% ethanol and isopropanol (IPA) were used respectively for resin washing as provided. Carboxylated PS Microspheres (5.1 µm diameter) were purchased from Bangs Laboratories (Bangs Laboratories, Fishers, IN), while Anti-Epcam antibodies were supplied by BioLegend® (San Diego, USA). PC3 cells were used as supplied from cryopreservation and cultured with RPMI supplemented with

Results and discussion

Upscaling the fabrication of Ab micro/nano-particle conjugates remains a challenging issue due to batch production that involves labor-intensive and time-consuming processes. In this work, we have demonstrated the facile fabrication of 3D micromixers for the continuous and efficient mixing and production of Ab micro/nano-particle conjugates. The reported process based on 3D printing enables us to fabricate complex internal microchannels with little required expertise in microfabrication.

We

Conclusion

This study showcases the successful fabrication of directly 3D printed, modular microfluidic devices for the creation of consistent and continuous mixing of micro/nano-particles with biomolecules. AuNP-Ab and PS microsphere-Ab conjugates were produced within directly 3D printed micromixers to confirm their mixing capability. The printing process completed in a matter of hours, dramatically reduces the turnover time for optimizing experiments and is amenable to modularization. The flexibility

Author contributions

Conceptualization, S.V., S.R.B, O.S., M.E.W.; Funding acquisition, D.J.; Data curation, S.V. and O.S; Methodology, S.V., S.R.B., M.E.W., O.S.; Investigation, S.V. and S.R.B; Software, S.R.B; Resources, O.S, M.E.W., D.J.; Supervision, M.E.W, O.S.; Formal Analysis, S.V., S.R.B; Writing-original draft, S.V., S.R.B., O.S.; Writing-review and editing, S.V., S.R.B., O.S., M.E.W. All authors agree to the published version of the manuscript.

Associated content

Supporting information

The Supporting

Declaration of Competing Interests

The authors declare no competing financial interests.

Acknowledgment

M.E.W. would like to acknowledge the support of the Australian Research Council through Discovery Project Grants (DP170103704 and DP180103003) and the National Health and Medical Research Council through the Career Development Fellowship (APP1143377). O.S. acknowledges NHMRC-ARC dementia research development fellowship (APP1101258).

References (47)

  • I.H. El-Sayed et al.

    Surface plasmon resonance scattering and absorption of anti-EGFR antibody conjugated gold nanoparticles in cancer diagnostics: applications in oral cancer

    Nano Lett.

    (2005)
  • S. Manohar et al.

    Synthesis and bioconjugation of gold nanoparticles as potential molecular probes for light-based imaging techniques

    Int. J. Biomed. Imaging

    (2007)
  • K. Sokolov et al.

    Real-time vital optical imaging of precancer using anti-epidermal growth factor receptor antibodies conjugated to gold nanoparticles advances in brief real-time vital optical imaging of precancer using anti-epidermal growth factor receptor antibodies conj

    Cancer Res.

    (2003)
  • A. Kaur et al.

    Novel screening test for celiac disease using peptide functionalised gold nanoparticles

    World J. Gastroenterol.

    (2018)
  • N. Mustafaoglu et al.

    Site-specific conjugation of an antibody on a gold nanoparticle surface for one-step diagnosis of prostate specific antigen with dynamic light scattering

    Nanoscale

    (2017)
  • S. Kataria et al.

    Microsphere: a review kataria

    Int. J. Res. Pharm. Chem.

    (2011)
  • Gomez, L.; Sebastian, V.; Irusta, S.; Ibarra, A.; Arruebo, M.; Santamaria, J.Lab on a chip scaled-up production of...
  • Razavi Bazaz, S.; Mehrizi, A. A.; Ghorbani, S.; Vasilescu, S.; Asadnia, M.; Warkiani, M. E.A hybrid micromixer with...
  • J. Ma et al.

    Controllable synthesis of functional nanoparticles by microfluidic platforms for biomedical applications-a review

    Lab Chip

    (2017)
  • S. Duraiswamy et al.

    Droplet-based microfluidic synthesis of anisotropic metal nanocrystals

    Small

    (2009)
  • J. Boken et al.

    Critical reviews in analytical chemistry microfluidic synthesis of nanoparticles and their biosensing applications microfluidic synthesis of nanoparticles and their biosensing applications

    Crit. Rev. Anal. Chem.

    (2016)
  • L. Gomez et al.

    Scaled-up production of plasmonic nanoparticles using microfluidics: from metal precursors to functionalized and sterilized nanoparticles

    Lab Chip

    (2014)
  • Y. Zhang et al.

    Design and simulation of passive micromixers based on capillary

    Microfluid. Nanofluid.

    (2012)
  • Cited by (0)

    1

    These authors contributed equally.

    View full text