Silk: A bio-derived coating for optical fiber sensing applications

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Highlights

  • This work demonstrates the design and development of a silk coated micro structured fiber sensor.

  • The novel silk-coated fibre sensor was obtained through a simple and quick dip-coating procedure.

  • The bio-derived silk coating is optically transparent, biocompatible and non-immunogenic.

  • The silk coated fiber sensor is investigated in a mouse in vivo for real time pH sensing.

  • The work holds exciting potential to enable a wide range of in vivo biosensing applications.

Abstract

Optical fiber chemical sensing is generally achieved through attachment of sensor molecules to the fiber, a process that involves chemicals that are not biologically compatible or are limited to thin monolayer coatings. To address these limitations and enable in-vivo biosensing, we report here, for the first time, silk fibroin coating of optical fibers for encapsulating fluorescent sensor molecules. Silica exposed core fiber (ECF) samples were coated with a thin layer of silk – a naturally derived biopolymer composed entirely of proteins and amino acids. The silk was doped with the fluorophore 5,6-carboxynapthofluorescein (CNF), which allows optical measurement of pH by a robust ratiometric fluorescence method. The fluorescent signal from the doped-silk layer is coupled into the core of the ECF, enabling remote measurement of pH along the length of the fiber. We have demonstrated real time in vivo pH sensing measurements in a mouse model of hypoxia. Our results showed a continuous drop in the subcutaneous pH in the mouse lumbar area as hypoxia developed. The work explores, for the first time, the potential of a natural silk protein coating to perform fiber sensing inside the body.

Introduction

Biosensing is globally employed in health sciences and biomedical research for disease detection and monitoring of physiological changes [1]. Fluorescence-based biosensing can offer sensitive measurement of physiological parameters such as temperature [2], pH [3], pressure [4], levels of insulin and glucose [5], oxygen and concentration of ions in biological specimens [6]. Through measurement of the fluorescence intensity or wavelength, a biosensor can track changes in the biological system under examination, some of which may reach levels considered abnormal or pathological. The biosensors are usually tested in cells and tissues ex vivo, or in collected biological fluids such as blood or urine [7]. In vivo sensing is required for applications such as early diagnosis of cancers without surgical procedures, [7,8] and real-time health monitoring such as glucose tracking in diabetes [5] and pH sensing in the case of pulmonary exacerbation [8].

However, application of the biosensors in in vivo conditions remains a significant challenge due to limitations in detection techniques that are unable to precisely measure at the targeted area of interest deep inside the body [8]. To address this, optical fiber based sensors [5,9,10], which are thin and flexible enough to reach previously difficult-to-access regions, have found extensive use in the areas of early disease sensing and health monitoring [1,11]. These optical fiber technologies in combination with fluorescence biosensors enable tracking of physiological parameters at positions remote from the excitation source and detection equipment [1,6,12]. The optical fiber technology has thus emerged as a powerful and reliable tool, allowing the development of new techniques for biological sensing and detection [10].

For optical fiber-based biosensing, it is necessary that the sensor molecules are immobilised onto the glass surface of the fiber. Two classes of immobilisation are typically employed, (a) attachment via surface functionalisation or (b) physical encapsulation. The surface functionalisation method involves binding chemicals to the glass surface to provide appropriate functional groups for attachment of the sensor molecules. For example, silanes are covalently attached to the silica glass surface, which can subsequently be attached to biochemicals such as antibodies [13,14]. Alternatively, polyelectrolytes can be attached to the silica glass surface through electrostatic interaction with the electronegative glass surface to provide appropriate functional groups for subsequent binding [15,16]. Both processes produce a thin typically less than 10 nm coating, which is more appropriate for binding and sensing larger biomolecules and also provides no physical robustness for the bare optical fiber. For smaller chemicals of physiological interest, such as pH, oxygen, and metal ions, it is more appropriate to encapsulate the sensor molecule within a thicker, semi-permeable coating. For example, pH indicators can be embedded within acrylamide polymer coated onto the tip of an optical fiber, which has been applied to cancer margin detection [3]. However, for long duration in vivo measurements there is an ongoing need to develop biocompatible, optical grade coatings for encapsulating fluorescent chemical sensors, which can be coated at ambient temperature and pressure conditions, and homogenously cover the optical fiber surface.

A biocompatible, optical grade coating is hence needed that encapsulates the fluorescent biosensors, can be coated at ambient temperature and pressure conditions, and homogenously covers the optical fiber surface for insitu or invivo fluorescence-based optical fiber sensing. Silk obtained from caterpillars is an attractive biopolymer for biophotonic applications [17,18]. Silk fiber has been used in medicine for centuries as a suture. However, the unprocessed fiber is opaque and cannot be used in optical applications. Recently, a regenerated form of silk has been obtained through degumming the cocoons, dissolving the fibers and dialyzing in deionized water at room temperature to yield a transparent solution of pure silk fibroin in water [19]. The resulting liquid silk protein self assembles into various structures through application of physical stimuli [19]. The resulting structures exhibit biocompatibility, tunable biodegradability, disintegration into simple proteins, and in-vivo non-immunogenicity [17] and have been used as a biomaterial for enhancing optical emission of nanoparticles [17], tissue engineering, drug release [20], and other biomedical imaging and sensing applications [18]. Moreover, the elastic modulus of silk structures matches that of biological tissues, which reduces the likelihood of trauma to surrounding soft tissue due to either fiber motion or natural body motions [21,22]. Finally silk can be doped with a wide range of chemical indicators, fluorescent agents [23] and molecules [20] making it an excellent underlying substance for fabricating biomedical materials and biosensing devices.

Traditional solid optical fibers can only provide a single sensing point at the tip of the fiber [3,24]. To demonstrate the capability of utilizing silk coating to enable a sensing platform along the length of a fiber, we employed exposed core fiber (ECF) in this project. ECF is a class of specialty optical fiber where a micron-scale suspended core is partially exposed to the external environment, allowing the analyte to directly access the core [16]. Through interactions with the evanescent field, ECFs provide a platform for biochemical sensing integrated along the fiber length with the potential for high-sensitivity real-time measurements [25,26].

To demonstrate in vivo sensing with silk coated ECF probes, we have used real time pH sensing as an example . Sensing the pH in vivo enables the monitoring of the acidification levels in the body associated with pathological conditions [27]. The extracellular pH in normal tissue is known to be relatively basic [28]. Slight variations of pH provide an exploitable avenue for tracking of abnormalities, monitoring of physiological function and the treatment of disease. Perturbations in the levels of oxygen (O2) and carbon dioxide (CO2) may cause changes in the subcutaneous tissue pH. Increase in the levels of CO2 can increase acidity, which decreases the tissue pH.

This work reports a unique hybrid silk-coated fiber that combines the biocompatibility and optical transparency of silk with the real time remote sensing capability of microstructured ECF. Silk was functionalized with 5,6-carboxynapthofluorescein (CNF) – a ratiometric fluorescent pH sensor, where the dual emission profile is a result of the acid and base forms of CNF having two different fluorescence emission maxima. The first emission maximum is centred around 560 nm and the other one around 680 nm. The ratio of these two emission maxima change with pH and determines the acidity or basicity of the local environment efficiently within a biologically-relevant pH window of 6.5–8.0 [34]. The first emission peak mainly increases with the increase in acidity, while the 2nd emission peak rises when the environment is predominantly basic.

A change in pH leads to changes in dual emission peaks of the ratiometric fluorophore CNF. Ratiometric approach removes the intensity dependence which typically restricts the precision of intensity-based fluorophore measurements [3]. CNF silk solution was used to functionalize coating along the length of the ECF via a simple and quick dip-coating procedure. The thin-coated film (∼200 nm) of silk-CNF created a sensor capable of detecting events of interest along the ECF. The fabrication and characterization of a silk-CNF coated ECF was performed and successfully employed for real time in-vivo pH sensing.

Section 3.1 illustrates the synthesis and optical characterization of silk and CNF via in-solution spectroscopy. Section 3.2 performs a numerical simulation to investigate the optimum silk coating thickness required for effective light guidance through the ECF. Section 3.3 explores the fabrication of the silk-CNF coated ECF probes and thickness monitoring through scanning electron microscopy (SEM). The silk-CNF coated ECF probes are calibrated in a set of known pH media as presented in Section 3.4. Finally, in Section 3.5 we demonstrate real time, pH sensing measurements in a mouse model, which shows the potential of the silk coated ECF probes for remote biosensing applications.

Section snippets

Silk fibroin extraction

The silk fibroin solution was obtained by boiling and de-gumming Bombyx mori cocoons in an alkaline solution of 0.02 M sodium carbonate for 30 min. The silk fibroin hence obtained was then rinsed thoroughly with water and dissolved in 9.3 M LiBr aqueous solution. The solution was then dialyzed in water using a dialysis cassette with a molecular cut off weight of 3500 Da for 48 h. After dialysis, the fibroin solution was transferred into a centrifuge tube. Finally, the dialyzed solution was

Characterisation of silk CNF mixture

CNF is a sensitive dual-emission ratiometric dye, which fluoresces in the visible to near infrared range when excited in the blue spectral region (450−480 nm). As reported, the emission spectrum of the dye consists of two peaks, the first at approximately 560 nm and a second at 680 nm, with an intensity ratio dependent on the pH of the environment surrounding the fluorophore [3]. As reported in the literature, the CNF is able to detect pH values in the biologically relevant window of 6.5–8.0 [29

Conclusions

This work demonstrates the development of a bio-derived silk coating for fiber sensing applications. The performance of the fiber probe was investigated in an in vivo mouse model for real time pH sensing. A ratiometric fluorescent dye CNF was encapsulated in liquid silk and then coated on ECF probes. Numerical modeling indicated that a silk-CNF coating of thickness less or equal to 200 nm successfully guides the dye’s fluorescence through the ECF core. The structural and surface analysis of the

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

The authors acknowledge support from the ARC Centre of Excellence for Nanoscale Biophotonics (CE14010003) and a Premier's Research and Industry Fund grant provided by the South Australian Government Department for Industry and Skills. The authors like to acknowledge the RMIT Microscopic and Microanalysis Facility (RMMF) for SEM analysis and the RMIT node of the Australian Research Council Centre of Excellence for Nanoscale BioPhotonics (CNBP) for in solution spectroscopy data. The authors also

Dr Asma Khalid is a Vice Chancellor Postdoctoral Fellow at RMIT University. She received her PhD from the University of Melbourne in 2016 and worked as an exchange researcher at the Silk Lab, Tufts University in 2014. Her work focuses on exploring silk for designing and characterising hybrid optical structures that are implantable or injectable in the body. These optically transparent, biocompatible, tunably degradable silk-based materials have wide applications in biosensing, bioimaging and

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    Dr Asma Khalid is a Vice Chancellor Postdoctoral Fellow at RMIT University. She received her PhD from the University of Melbourne in 2016 and worked as an exchange researcher at the Silk Lab, Tufts University in 2014. Her work focuses on exploring silk for designing and characterising hybrid optical structures that are implantable or injectable in the body. These optically transparent, biocompatible, tunably degradable silk-based materials have wide applications in biosensing, bioimaging and drug release.

    Lu Peng achieved her M.S. from South China Normal University, China, in 2017. She is awarded an Adelaide University China Fee Scholarships (AUCFS) and is studying as a PhD student in the University of Adelaide since 2017. Her research interests are fiber interferometry sensors and plasmonic sensors for biochemical applications.

    Azim Arman is a PhD candidate in the Faculty of Health and Medical Sciences, University of Adelaide, Australia. His research interests focus on preclinical assessment of hypernociception.

    Dr Stephen C. Warren-Smith completed his PhD in 2011 at the University of Adelaide, Australia, on the topic of microstructured optical fiber chemical sensing. He was then employed from 2011 to 2014 as an Australian Research Council (ARC) Super Science Fellow at the Institute for Photonics and Advanced Sensing and the School of Chemistry and Physics at the University of Adelaide, working on optical fiber biosensing for women’s health applications. In 2015 and 2016 he worked as a European Union Marie Curie International Incoming Fellow at the Leibniz Institute of Photonic Technology, Jena, Germany, on a project investigating the micro/nano-structuring of optical fibers for sensing. Since October 2016 he is with the University of Adelaide as a Ramsay Fellow, developing microstructured optical fibers for sensing and imaging applications.

    Dr Erik Schartner is a postdoctoral researcher at the Institute for Photonics and Advanced Sensing and the ARC Centre of Excellence for Nanoscale Biophotonics at the University of Adelaide. His research focuses on optical fibre fabrication, primarily looking at sensing using these fibres within biological or industrial applications.

    Dr Georgina M. Sylvia is a postdoctoral researcher in the School of Physical Sciences, University of Adelaide. Her research focuses on the development and optimisation of biophotonic devices for medical and industrial applications.

    Prof Mark R Hutchinson BSc(Hons), PhD(Med) is the Director of the Australian Research Council Centre of Excellence for Nanoscale BioPhotonics, and ARC Future Fellow and a Professor in the Adelaide Medical School at the University of Adelaide. His research focus is on understanding the neuroimmune synapse through imaging and sensing it in a dynamic behaving environment and how this functional multicellular unit is involve in controlling brain behaviour.

    Prof Heike Ebendorff-Heidepriem is Research Professor in Photonics Materials and fibres at the University of Adelaide. She is Senior Investigator of the ARC Centre of Excellence for Nanoscale BioPhotonics (CNBP) and Deputy Director of the Institute for Photonics and Advanced Sensing of the University of Adelaide. Her research focuses on the development of novel optical glasses, specialty optical fibres, surface functionalization and sensing approaches.

    Prof Robert A. McLaughlin is the Chair of Biophotonics and a Professor at Adelaide Medical School, University of Adelaide. He is also co-founder of the company Miniprobes Pty Ltd. His research focuses on the design of optical fibre probes for biomedical applications and the development of intelligent algorithms for automated quantification.

    Prof Gibson was awarded his PhD from La Trobe University in 2004. From 2005-09, he was a Photonics Development Engineer at Quantum Communications Victoria (QCV) where he and colleagues designed and developed Australia’s first commercial quantum security product (QCV SPS 1.01). In 2011 he was awarded an Australian Research Council (ARC) Future Fellowship on Hybrid Diamond Materials for Next Generation Sensing, Biodiagnostic and Quantum Devices. Currently, Prof Gibson is a Deputy Director and RMIT Node Leader of the ARC Centre of Excellence for Nanoscale BioPhotonics. And he has wide-ranging research interests in the areas of fluorescent nanoprobes, biophotonics, hybrid integration and confocal microscopy.

    Dr Jiawen Li received her PhD degree in Biomedical Engineering from University of California Irvine in 2015. Her PhD work focused on developing optical coherence tomography (OCT) probes and multimodal optical-ultrasonic imaging catheters for in vivo applications. She joined the University of Adelaide as a Lecturer in 2016. She is currently supported by National Heart Foundation of Australia to develop in vivo intravascular fluorescence+OCT imaging catheters. Her research interests include OCT, fibre sensing, multimodality imaging, and ultra-thin endoscopes.

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