Plasma cleaning for the study of biomaterials

Plasma cleaning to improve coating and adhesion in biomaterials research.

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The Henniker HPT-100 benchtop plasma system is being put to good use within the Department of Polymer and Biomaterials Science at the West Pomeranian University of Technology, Szczecin (ZUT) where Dr. Peter Sobolewski and his colleagues are working in the area of polymeric biomaterials.

Biomaterials are engineered materials intended to interact with biological systems, for example during the use of a medical device or implant. A growing interest in the fields of regenerative medicine and tissue engineering has led to a need to develop new biomaterials, especially polymers, suitable for use in soft tissue implants or as scaffolds for tissue regeneration. The synthesis and study of such polymeric biomaterials is the focus of Dr. Sobolewski and his colleagues in the Department of Polymer and Biomaterials Science, ZUT.

While end-use forms of biomaterials can take on many shapes and sizes—ranging from nanoparticles to nano- and micro-fibers, or macroscopic objects (sutures, bandages, implants)—it’s their surfaces that play a crucial role in determining their so-called ‘biocompatibility.’ This is essentially the ability of the material to perform its function while being tolerated by the tissue of the host—producing an appropriate host response. Thus, numerous techniques are used for studying the surfaces of biomaterials, as well as their interactions with proteins and cells.

One strategy to precisely study various polymer surface interactions and properties involves preparing carefully controlled thin films using spin coating. Depending on the study, an appropriate substrate, such as a sensor or glass coverslip is coated with a layer of polymer in the range of 100 to 300 nm by depositing the polymer in solution and rapidly rotating the substrate to spread the film evenly and evaporate the solvent. To obtain reliable data, the films must be pristine—free of defects, such as pinholes, or contaminants, such as dust. Of course, if the substrate is not pristine, neither will be the film. As a result, cleaning and preparing substrates for the spin coating procedure is an absolutely crucial step.

At present, Dr. Sobolewski and his colleagues have been focused on the study of a family of poly(butylene succinate-dilinoleic succinate) copolyesters (PBS-DLS), recently developed within their Department.[1,2] These copolymers are characterized by excellent tuneable elastomeric properties, biodegradability, and can be synthesized from bio-based monomers using non-toxic catalysts, including enzymes.

Recently, Dr. Sobolewski studied the interactions between these new polymers and two proteins that play a crucial role in wound healing and cell-material interactions: fibronectin and fibrinogen—components of the provisional matrix.[3] This study involved spin-coated thin films prepared on quartz crystal sensors for analysis using a quartz crystal microbalance (QCM). The results predict excellent cell-polymer interactions. However, the quartz substrates are not transparent making direct visualization of cell adhesion a problem.

This was the challenge faced by Master’s student Nina Kantor-Malujdy: preparing analogous thin films on a transparent substrate. Together with Dr. Sobolewski, she selected glass coverslips as suitable substrates and set out optimize the spin coating process. Unfortunately, the as-manufactured glass coverslips were contaminated with organic residues that made obtaining pristine coatings difficult. Ms. Kantor-Malujdy followed a previously established cleaning protocol involving sonication in isopropyl alcohol, then acetone, and finally drying using filtered compressed air. This protocol was time-consuming, low-throughput, and involved extensive handling and manipulation of each delicate coverslip—particularly during drying.

ZUT Case Study - Plasma Cleaning Biomaterials

Figure 1: Plasma cleaning coverslips using HPT-100. Master’s student Nina Kantor-Malujdy seen in the reflection.

At this point, the team decided to try plasma cleaning using their new Henniker HPT-100 instrument. In fact, Dr. Sobolewski reports that they obtained the protocol from an article Tweeted by @HennikerPlasma. The process parameters were: 30s, 100% Power, and 50 sccm airflow. The effects were immediately apparent, as the water contact angle dropped from ~80° to virtually zero, as compared to ~60° for the solvent cleaning method. Thus, not only were the organic contaminants removed, but the glass also became activated, having a high surface free energy.

Ms. Kantor-Malujdy confirmed that the effect could be leveraged in the next steps of her process, by monitoring the water contact angle over time: after 24 hours, it was still ~5°, meaning she had plenty of time for the spin coating stage of the process.

Importantly, laser scanning microscopy confirmed the glass was free of any contaminates but had not been etched: the roughness parameter Ra was slightly reduced from 11 nm for as-manufactured to 10 nm—both measurements virtually at the sensitivity limit of the technique.

“Using the Henniker plasma system significantly reduced the time of preparing samples, because cleaning the cover glasses was an integral part of the process and previously was laborious and took a substantial amount of time.” Master’s student Nina Kantor-Malujdy explains “Plasma cleaning yielded clean and dry coverslips in 30 s, without etching the glass or needless handling. The Henniker instrument was easy to operate with a user-friendly interface.”

With the substrate preparation sorted, Ms. Kantor-Malujdy could focus on the spin coating process. Unexpectedly, the protocol developed for quartz crystal sensors was not suitable for glass coverslips. Thus, before the team could proceed with their cell studies, she had to develop a new optimised spin coating protocol. The time saved on cleaning the glass coverslips turned out to be crucial! In the end, she needed to change solvents from dichloromethane to tetrahydrofuran, which is less volatile.

ZUT Case Study - Plasma Cleaning Biomaterials -2 ZUT Case Study - Plasma Cleaning Biomaterials -3

Figure 2: Left: Two types of coverslips spin coated with PBS-DLS 50:50 copolyester (1.5 wt.% solution in THF, 4000 rpm) Right: Laser scanning micrograph of a scratch test of the coating on round coverslip confirming ~100 nm thickness and Ra ~10 nm.

Ultimately, Ms. Kantor-Malujdy was able to reliably obtain PBS-DLS copolymer coatings ~100 nm thick with similar roughness to the substrate (Ra ~10 nm), thus replicating the polymer surfaces used in the QCM protein adsorption studies. The team was finally ready to proceed with the planned cell adhesion studies.

Just before the lab was shut down by the COVID-19 pandemic, they conducted the first experiment, using L929 mouse fibroblasts, a workhorse cell line in preliminary biomedical studies. The results were very encouraging: after 24 hours in culture they observed robust adhesion of cells to the experimental copolymers and similar morphology of the cells to tissue culture plastic control surfaces.

ZUT Case Study - Plasma Cleaning Biomaterials

Figure 3: L929 murine fibroblasts adhered to PBS-DLS 50:50 copolyester spin coated surface after 24 hours of culture.

“The results were very encouraging: after 24 hours in culture they observed robust adhesion of cells to the experimental copolymers and similar morphology of the cells to tissue culture plastic control surfaces.”

The next steps of their research—once the COVID-19 pandemic subsides enough to permit a return to the lab—will involve careful quantification and replication of the initial study, as well as live cell imaging, to monitor the adhesion in real-time. Of course, keeping cells alive and healthy during various experimental manipulations and live cell imaging can be challenging. As a result, it’s important that the methodology of preparing the substrates be as reliable and reproducible as possible. Thanks to their Henniker HPT-100 plasma system and the diligent work of Master’s student Nina Kantor-Malujdy these aspects have been streamlined and optimised.

“These coatings are a model of the surface of a device that may be an implant in the body. To determine the biological properties and thus optimal applications of these new copolyesters, we need to study whether cells will attach and proliferate on such surfaces.” Dr. Sobolewski explains “The complexity and variability of biological systems makes our studies challenging, so I was extremely happy that the process of cleaning and spin-coating the glass coverslips could become simple and routine. I’m grateful for the excellent work of Master’s student Nina Kantor-Malujdy.”

An image of our UK Manufactured Henniker Plasma Treatment Equipment - HPT-100

Figure 3: The Henniker HPT-100 Plasma System

“Regarding the plasma system, we needed a cost-effective plasma system that could be easily set-up and produce the right process for us. We were already thinking about cleaning samples but also wanted to use it for activating polymer surfaces. Finally, we’d like to move into the area of plasma polymerization. We chose the HPT-100 plasma system from Henniker Plasma, because of the flexibility of the system and the excellent support from Henniker Plasma and Polish representative Jacek Łatkowski of EDFelectronics.”
– Dr. Peter Sobolewski, @psobolewskiPhD

References
(1) Stepień, K.; Miles, C.; McClain, A.; Wiśniewska, E.; Sobolewski, P.; Kohn, J.; Puskas, J.; Wagner, H. D.; el Fray, M. Biocopolyesters of Poly(Butylene Succinate) Containing Long-Chain Biobased Glycol Synthesized with Heterogeneous Titanium Dioxide Catalyst. ACS Sustainable Chemistry & Engineering 2019, 7 (12), 10623–10632. https://doi.org/10.1021/acssuschemeng.9b01191.
(2) Sonseca, A.; el Fray, M. Enzymatic Synthesis of an Electrospinnable Poly(Butylene Succinate-Co-Dilinoleic Succinate) Thermoplastic Elastomer. RSC Adv. 2017, 7 (34), 21258–21267. https://doi.org/10.1039/C7RA02509B.
(3) Sobolewski, P.; Murthy, N. S.; Kohn, J.; el Fray, M. Adsorption of Fibrinogen and Fibronectin on Elastomeric Poly(Butylene Succinate) Copolyesters. Langmuir 2019, 35 (26), 8850–8859. https://doi.org/10.1021/acs.langmuir.9b01119.

 

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Oxygen Plasma Ashing for successful preparation of samples

Oxygen Plasma Ashing for successful preparation of samples by Stable Isotope Mass Spectrometry

Stable isotope ratio measurements, performed by mass spectrometry, can provide accurate information in a number of geoscience applications, including the reconstruction of past glacial/interglacial temperature variations for example.

One of the most common problems in stable isotopic measurements is the presence of hydrocarbon (e.g. bitumen) and adhering organic material. Effective removal of this hydrocarbon material needs to be accomplished by a method which in turn does not itself also alter the initial isotopic composition.

Oxygen plasma ashing can be employed for this purpose but care is needed to ensure that the initial isotopic composition is retained.

Recently published work from Imperial College London using the Henniker HPT-100 demonstrated the routine preparation of geological samples without altering the initial isotopic composition and provided the conditions and methods by which this should be undertaken.

Please find the abstract below;

Effects of Oxygen Plasma Ashing treatment on Carbonate Clumped Isotopes

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Enhanced photocatalytic hydrogen evolution from organic semiconductor heterojunction nanoparticles

Recently published research in Nature Materials, by groups working at King Abdullah University of Science and Technology (KAUST), Imperial College London and the Rutherford Appleton Laboratory details advances in the development of organic semiconductor photocatalysts which could be used in solar panels to harness more of the sun’s energy than was previously possible. The work demonstrates that incorporating a heterojunction between a donor polymer (PTB7-Th) and non-fullerene acceptor (EH-IDTBR) in organic nanoparticles (NPs) can result in hydrogen evolution photocatalysts with greatly enhanced photocatalytic activity. The Henniker HPT-100 plasma system was used to prepare nanoparticles prior to atomic force microscopy to confirm the core-shell morphology of the deposited nanoparticle layer.

Please find the abstract below;

Enhanced photocatalytic hydrogen evolution from organic semiconductor heterojunction nanoparticles

[All information courtesy of  Nature Materials (2020), Jan Kosco, Matthew Bidwell, Hyojung Cha, Tyler Martin, Calvyn T. Howells, Michael Sachs, Dalaver H. Anjum, Sandra Gonzalez Lopez, Lingyu Zou, Andrew Wadsworth, Weimin Zhang, Lisheng Zhang, James Tellam, Rachid Sougrat, Frédéric Laquai, Dean M. DeLongchamp, James R. Durrant & Iain McCulloch |Published:

 

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Photocatalysts formed from a single organic semiconductor typically suffer from inefficient intrinsic charge generation, which leads to low photocatalytic activities. We demonstrate that incorporating a heterojunction between a donor polymer (PTB7-Th) and non-fullerene acceptor (EH-IDTBR) in organic nanoparticles (NPs) can result in hydrogen evolution photocatalysts with greatly enhanced photocatalytic activity. Control of the nanomorphology of these NPs was achieved by varying the stabilizing surfactant employed during NP fabrication, converting it from a core-shell structure to an intermixed donor/acceptor blend and increasing H2 evolution by an order of magnitude. The resulting photocatalysts display an unprecedentedly high H2 evolution rate of over 60,000 µmol h−1 g−1 under 350 to 800 nm illumination, and external quantum efficiencies over 6% in the region of maximum solar photon flux.

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Investigating the nanoscale adhesion mechanisms between polymeric biomaterials and biological samples

Recently published research from scientists at Queen Mary University of London and GlaxoSmithKline demonstrates the complex nature of nanoscale adhesion mechanisms between polymeric biomaterials and biological samples.  Henniker’s HPT-200 plasma system is used in this work to provide the clean substrate surfaces (silicon and gold) onto which a variety of polymer brushes and self-assembled monolayers (SAMs) were synthesized.

Please find the abstract below;

The physico-chemistry of adhesions of protein resistant and weak polyelectrolyte brushes to cells and tissues

[All information courtesy of https://pubs.rsc.org/ Edward J. Cozens ab, Dexu Kong ab, Nima Roohpour c, and Julien E. Gautrot | Received 11th July 2019, Accepted 15th October 2019 | First published on 5th December 2019]

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Abstract

The non-specific adhesion of polymers and soft tissues is of great interest to the field of biomedical engineering, as it will shed light on some of the processes that regulate interactions between scaffolds, implants, and nanoparticles with surrounding tissues after implantation or delivery. In order to promote adhesion to soft tissues, a greater understanding of the relationship between polymer chemistry and nanoscale adhesion mechanisms is required.

In this work, we grew poly(dimethylaminoethyl methacrylate) (PDMAEMA), poly(acrylic acid) (PAA) and poly(oligo ethylene glycol methacrylate) (POEGMA) brushes from the surface of silica beads and investigated their adhesion to a variety of substrates via colloidal probe-based atomic force microscopy (AFM). We first characterized adhesion to a range of substrates with defined surface chemistry (self-assembled monolayers (SAMs) with a range of hydrophilicities, charge and hydrogen bonding), before studying the adhesion of brushes to epithelial cell monolayers (primary keratinocytes and HaCaT cells) and soft tissues (porcine epicardium and keratinized gingiva).

Adhesion assays to SAMs reveal the complex balance of interactions (electrostatic, van der Waals interactions and hydrogen bonding) regulating the adhesion of weak polyelectrolyte brushes. This resulted in particularly strong adhesion of PAA brushes to a wide range of surface chemistries. In turn, colloidal probe microscopy on cell monolayers highlighted the importance of the glycocalyx in regulating non-specific adhesions.

This was also reflected by the adhesive properties of soft tissues, in combination with their mechanical properties. Overall, this work clearly demonstrates the complex nature of interactions between polymeric biomaterials and biological samples and highlights the need for relatively elaborate models to predict these interactions.

TO READ THE FULL PAPER CLICK HERE

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New Henniker Plasma Spanish distribution partners Irida Iberica

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Irida Iberica – Advanced Solutions for Science.

Henniker Plasma announces new Spanish distribution partners, Irida Ibérica.

Irida are an established supplier of high-end measurement apparatus for research and development. Irida currently offers the latest and most innovative technology in thin film deposition and nanostructures lithography as well as a variety of products for surface chemical and physical characterization techniques.

With longstanding industry experience in surface and sample preparation & analysis, Henniker are confident that Irida will make an excellent distributor partner for expansion into the Spanish territory.

You can visit Irida at their free microscopy workshop on the 11th February in Madrid. ‘In-situ Electron Microscopy: Capabilities and Applications’

More details can be found by downloading the event guide here.

VISIT OUR DEDICATED SPANISH WEBSITE HERE

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UK Electronics and Photonics Innovation Centre (EPIC) invest in Henniker Plasma Technology

Press Release HPP0821 | 08/01/2019 | Henniker Plasma Collaboration News | For immediate release

UK Electronics & Photonics Innovation Centre EPIc invest in henniker Plasma Treatment Technology.

The EPIC Centre, built to serve the Electronics and Photonics industry, recently acquired an advanced plasma treatment system from UK based manufacturer Henniker Plasma.

The newly opened Centre, based in Torbay UK – the home of an established photonics and microelectronics cluster, aims to serve current and future users wishing to draw on its innovative facilities providing them with the opportunity to expand their capabilities through investment in modern technological advances.

Henniker plasma – EPIC partnerships

henniker-plasma-treatment-system  An image of our UK Manufactured Henniker Plasma Cleaning Equipment - HPT-100

Henniker Plasma, an established leading UK manufacturer of plasma treatment systems, were approached by EPIC to provide them with a plasma technology solution.

Wayne Loschi, EPIC Centre Director, explains why they felt the acquisition of a Henniker System would best serve their current users and in addition attract more tech businesses to the region;

“Historically Henniker has worked with numerous academic institutes and industrial companies to develop processes from inception to completion, making them the ideal candidate.”
Wayne Loschi, EPIC Centre Director.

Henniker’s successes as a provider of plasma technology are built around an exceptional body of knowledge and expertise in surface engineering for cleaning, activation and coating applications.

“Henniker welcomed the opportunity to provide the Centre with the HPT-200 plasma treatment system, hoping to widen not only EPIC’s capabilities but in turn increase awareness of the technology.”
Gill Bolton Marketing & Comms Manager, Henniker Plasma.

Plasma Technology explained

Plasma technology has been an important production tool for more than 30 years in the fabrication of microelectronic devices. Over this period, the technology has also permeated a much broader range of industries including automotive, medical device, textiles, and aerospace to name but a few.

Plasma treatments are used to alter the surface properties of a wide range of materials to make them easier to bond, glue, and paint. By treating parts, they both clean and activate the surface, improving their adhesion characteristics.

Today Henniker plasma technology is routinely used in academic institutes and industrial partners across the UK and is evolving into a global brand.

Future endeavours

Both parties look forward to the opportunities this collaboration will bring to the Electronics and Photonics UK Community.

EPIC-microelectronics-image

Read the full press release here

Contact: Henniker Plasma

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Gill Bolton – Marketing and Comms Manager
gbolton@plasmatreatment.co.uk
www.plasmatreatment.co.uk
info@plasmatreatment.co.uk
Tel: +44 (0)1925 830 771

Contact: EPIC Torbay Electronics & Photonics Innovation Centre

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Wayne Loschi – Centre Director
Wayne.Loschi@tda.uk.net
www.epic-centre.co.uk
epic@tda.uk.net
Tel: +44 (0)1803 714 714

Categorised in: News & Events

UK Electronics and Photonics Innovation Centre (EPIC) invest in Henniker Plasma Technology

The EPIC Centre, built to serve the Electronics and Photonics industry, recently acquired an advanced plasma treatment system from UK based manufacturer Henniker Plasma.

Download the press release