A graphene oxide-based FRET platform for sensing xenogeneic

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A graphene oxide-based FRET platform for sensing xenogeneic collagen co-assembly Benmei Wei, Zhai Zhongwei, Wang Haibo, Zhang Juntao, Xu Chengzhi, Xu Yuling, Lang He, and Xie Dong J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b02554 • Publication Date (Web): 25 Jul 2018 Downloaded from http://pubs.acs.org on August 1, 2018

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Journal of Agricultural and Food Chemistry

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A graphene oxide-based FRET platform for sensing xenogeneic

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collagen co-assembly

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Benmei Wei , Zhongwei Zhai , Haibo Wang* , Juntao Zhang , Chengzhi Xu ,

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Yuling Xu , Lang He , and Dong Xie

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†School of Chemical and Environmental Engineering, Wuhan Polytechnic University, Wuhan, 430023, P. R. China *Corresponding author: [email protected]

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Benmei Wei et al. Page 1

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ABSTRACT

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Xenogeneic collagen co-assembly (XCCA) offers a new view for the design and performance

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regulation of novel collagen-based biomaterials. But there is still a lack of accurate and

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sensitive method for monitoring XCCA. In this study, a simple and efficient graphene oxide

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(GO)-based fluorescence resonance energy transfer (FRET) platform has been developed to

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sense XCCA. We first designed a fluorescein isothiocyanate (FITC)-labeled porcine skin

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collagen (PSC) that adsorbed on the GO surface and effectively quenched its fluorescence.

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Upon addition of grass carp skin collagen (GCSC), the XCCA between PSC and GCSC

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resulted in desorption of FITC-PSC from GO surface, thus caused an increase of fluorescence

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signal. Under the optimal conditions, the fluorescence signal linearly increased as the increase

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of the GCSC concentration in the ranges of 50-1000 µg/mL, with a sensitivity of 22 µg/mL

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(S/N=3). Furthermore, the developed strategy also exhibited excellent specificity and

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anti-interference ability. More interestingly, the thermal stability of collagen fibrils formed by

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XCCA is linearly related to the GCSC concentration. These results open a facile, effective and

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sensitive approach for sensing XCCA, and provide a new strategy for arbitrarily regulating

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the thermal stability of collagen fibrils.

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KEY WORDS: Collagen; Xenogeneic co-assembly; Graphene oxide; Thermal stability

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INTRODUCTION

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Collagens, the major structural component of extracellular matrix and connective tissues,

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are polymeric proteins broadly utilized in food engineering, regenerative medicine and

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biomaterial fields.1-4 It is known that self-assembly is a very important molecular behavior of

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collagen.5-7 In vivo, collagen with triple helical conformation can form a fibrous network by

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orderly arrangement, and offer a structural platform for cell growth, adhesion and migration.

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In vitro, collagen molecules can also self-assembly to produce a fibril under suitable

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conditions.8-10 Moreover, the formed collagen fibril in vitro exhibited the similar structure,

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mechanical and biological properties to those of assembled fibril in vivo. In the past few

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decades, the self-assembled products have been effectively used as food thickening agents,

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3D scaffold and meat tenderizers.11-13 More in-depth studies showed that there are not only a

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large number of fibrils fabricated by self-assembly of single collagen in living body, but also

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hybrid fibrils constructed by co-assembly of different types of collagen. For example,

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collagen type I and III hybrid fibrils have been found in the normal rat liver and collagen type

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II and III hybrid fibrils were observed in human articular cartilage.14-15 Moreover, these hybrid

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fibrils play a pivotal role for the regulation of extracellular matrix and the provision of special

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mechanics and biological properties of tissues.16-17 Inspired by these observations, scientists

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also tried to fabricate hybrid fibrils through the co-assembly of two collagens from different

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species, and further analyzed their physical and biological properties. For example, Nomura

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and other groups explored that the co-assembled fibrils of type I fish-sourced collagen and

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type I porcine collagen had a completely different thermal stability than that of assembled

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products of single collagen.18-19 Recently, a similar study on our group showed that the




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co-assembled fibrils exhibited totally different viscoelastic and cell proliferation properties.20

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These studies showed that the fibrils formed by xenogeneic collagen co-assembly (XCCA)

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displayed new physical and biological properties, which offer a new approach to design and

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regulate the performances of novel collagen-based biomaterials. However, there is still a lack

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of accurate and sensitive methods for monitoring XCCA, which will provide theoretical

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guidance for the design of collagen fibrils with different properties. Collagen fibrils have been

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broadly used in food, medicine and biomaterial fields. Furthermore, different utilizations have

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distinct demands for the properties of assembled products. Based on sensitive monitoring of

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XCCA, hybrid collagen fibrils with different performances can be specifically fabricated by

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simply varying the collagen concentration, and thereby meet the demand for collagen-based

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biomaterials in different application fields. Commonly, XCCA is monitored by turbidity

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measurement, which is a simple and intuitive method based on light transmittance.18-20

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Furthermore, it can only provides rough information about collagen aggregation, cannot

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directly reflect the occurrence of XCCA, also exhibit poor sensitivity. It remains a challenge

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to fabricate an accurate and sensitive method for sensing XCCA.

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Fluorescence resonance energy transfer (FRET) is an efficient method to explore

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molecular interactions because of its simplicity, sensitivity and reproducibility.21-22 Graphene

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oxide (GO) is a single layer two-dimensional nanomaterial possessing high water

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dispersibility, excellent biocompatibility and large surface area.23-24 It is worth noting that GO

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has proven to be an excellent quencher for a series of fluorophores due to non-radioactive

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electronic excitation energy transfer between the fluorophore and GO. Meanwhile, the large

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adsorption capacity of GO enables it as an excellent candidate acceptor for FRET



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biosensors.25-26 In the past few years, GO-based FRET strategies have been widely used in the

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analysis of DNA, proteins and metal ions because of its high sensitivity and selectivity.27-29

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Recently, Xiao et al have utilized this strategy to sense collagen-like peptide triple helix and

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unfolded collagen fragments.30-31 These works provided meritorious informations to

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comprehend biologic functions of the target collagen fragments. However, they are limited to

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unstructured collagen mimic peptide till present. The co-assembly of structured natural

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collagens has not yet been investigated by the similar sensing platform.

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Herein, we developed for the first time a simple GO-based FRET platform for sensing

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XCCA using porcine skin collagen (PSC) and grass carp skin collagen (GCSC) as model

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molecules. As shown in Scheme 1, the fluorescein isothiocyanate (FITC)-labeled PSC was

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adsorbed on GO surface by π-π and hydrophobic interactions. At this time, the distance

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between GO and FITC is relatively close. Thus, FRET occurred and caused the quenching of

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FITC fluorescence. Interestingly, the XCCA between GCSC and FITC-PSC induce the

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detachment of FITC-PSC from GO surface, resulting in the recovery of FITC fluorescence.

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We have proved that the strategy can be successfully applied to sense sensitively and

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effectively XCCA. Moreover, the developed assay also exhibited excellent specificity and

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anti-interference ability. Based on this result, we further achieved the arbitrarily regulation of

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thermal stability of co-assembled products by simply varying the GCSC concentration.

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MATERIALS AND METHODS

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Materials. Porcine and grass carp skins were purchased from a supermarket in Wuhan, Hubei

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Province, China. The skins were rinsed with chilled water and then stored at -25 °C until used.

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Collagens from porcine and grass carp skins were prepared according to previous studies.32-33




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Briefly, the non-collagenous proteins were firstly removed by soaking and stirring porcine

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skins in 0.1 M NaOH for 1 day. Then, the skins were rinsed with cold water and soaked with

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10% n-butyl alcohol to remove fat. Subsequently, the defatted skins were immersed in 0.5 M

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ethylic acid (m/v=1/20) and centrifuged at 20,000×g for 1 h. After that, 0.5 M ethylic acid

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containing 1.0% pepsin was used to re-suspend the obtained precipitate. The suspension was

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then centrifuged at 8000×g for 1 h and salted out using sodium chloride. The solution was

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further centrifuged at 20000×g for 0.5 h and the obtained precipitate was re-dissolved in 0.5

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M ethylic acid. The dissolved sample was dialyzed with 0.1 M ethylic acid and cold water

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respectively. Finally, PSC was received by freeze-drying. Similarly, GCSC and other

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collagens were also obtained by the similar way. All operations were carried out below 15 °C.

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GO was purchased from XFNANO Materials Tech. Co., Ltd., Nanjing, Jiangsu Province,

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China. All other chemicals obtained from commercial sources were of analytical grade and

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used without further purification. All solutions were prepared with ultrapure water (resistivity

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= 18.2 MΩ·cm) from a Millipore system.

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Instruments. All fluorescence measurements were performed on a fluorescence

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spectrophotometer (Varian, Cary Eclipse, USA). The turbidity analysis of collagen assembly

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was performed by a UV-Vis spectrophotometer (UV-2000, Unico, Shanghai, China). The

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thermal stability of collagen fibrils was measured by differential scanning calorimetry

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(DSC-Q10, TA Instruments, New Castle, Delaware, USA). The triple-helix conformation of

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collagen was examined by circular dichroism (CD) spectroscopy (JACSO J-1500, Hachioji,

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Tokyo, Japan). Amino acid (AA) composition analysis of PSC and GCSC was carried out by a

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AA analyzer (Hitachi 835-50; Hitachi Limited Co., Tokyo, Japan).



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Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of PSC

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and GCSC. SDS-PAGE analysis of PSC and GCSC were carried out by following the

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previous work.34 4% stacking gel and 7.5% resolving gel was firstly mixed to prepare

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polyacrylamide gel. Meanwhile, two collagen samples were dissolved in 0.1 M ethylic acid to

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4 mg/mL respectively. Subsequently, the solutions were diluted with 0.5 M Tris-HCl buffer

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(pH 6.8, containing 5% SDS and 20% glycerol, v/v=1:2). After electrophoresis, the gels were

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stained with Coomassie Blue R 250 for 0.5 h and rinsed with a methanol/ethylic acid mixture

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(v/v=2:1) for 1 day. The molecular weights of PSC and GCSC were evaluated using protein

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makers as a reference.

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CD spectroscopy measurement and AA composition analysis of PSC and GCSC. For CD

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spectroscopy measurement, collagen samples were firstly dissolved in 0.1 M ethylic acid to 2

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mg/mL. Then, the solution was diluted with distilled water (v/v=1:9). After that, the diluted

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solution was placed in a quartz cell of the CD spectrometer with a path light 10 mm at 0.2

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intervals. The CD spectra of two collagens in the range of 190-250 nm were collected under

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the condition of with 1 nm bandwidth and nitrogen atmosphere. AA composition of PSC and

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GCSC were determined by following the previous study.35 Collagen samples were firstly

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hydrolyzed with 6 M hydrochloric acid containing 2% (v/v) carbolic acid at 105°C for 1 day.

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Subsequently, 3.0 M NaOH was used to neutralize this solution. The AA composition of

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collagen samples was obtained by the AA analyzer.

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Preparation of FITC-PSC and GCSC solutions. PSC was dissolved in 0.1 M ethylic acid to

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2 mg/mL and dialyzed with PBS buffer (200 mM, pH 7.40). FITC was dissolved at 0.5

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mg/mL in PBS. PSC and FITC solutions were mixed (v/v=100:1) and conjugated at 4 °C until




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the reaction was complete. After that, excess FITC was eliminated by dialyzing the solution

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with PBS. To ensure that excess FITC was completely removed, the fluorescence intensity of

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FITC in the dialysis buffer after exchange was continuously monitored. In addition, 2 mg/mL

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GCSC solution was also obtained by dissolving it in 0.1 M ethylic acid and dialyzing against

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PBS buffer. Moreover, different concentrations of collagen solutions were prepared by

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diluting them with PBS.

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Fluorescence monitoring the XCCA of FITC-PSC and GCSC. FITC-PSC (2 mg/mL, 2mL)

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was mixed with GO (2 mg/mL, 40 µL) prior to the addition of GCSC. Subsequently, different

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concentrations of GCSC (0-1000 µg/mL, 2 mL) was added to the GO/FITC-PSC solution and

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incubated at 30°C for 45 min. The fluorescence of the mixture of GO/FITC-PSC/GCSC was

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monitored at 517 nm.

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Turbidity analysis of XCCA. The turbidity analysis of XCCA was performed by following

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the previous report.36 The mixture of FITC-PSC (2 mg/mL, 2 mL), GO (2 mg/mL, 40 µL) and

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GCSC (1 mg/mL, 2 mL) were incubated at 30 °C and continuously monitor the absorbance at

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310 nm up to 2 h.

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Thermal stability measurement of collagen fibrils. The mixture of PSC (2 mg/mL, 2 mL)

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and different concentration of GCSC (2 mL) were incubated at 30 °C for 2 h. Subsequently,

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the incubated solution was centrifuged at 10000×g for 5 min and washed with distilled water.

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Then, the precipitate was freeze-dried and swollen with distilled water for 1 day in a capped

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aluminum cell. The DSC curve was obtained by scanning at 2 °C/min over the range from 30

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to 60 °C and using distilled water as a reference substance. The maximum transition

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temperature (Tm) was assessed from the maximum peak of the DSC curve. Meanwhile, the



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thermal stability of PSC fibrils, GCSC fibrils and their mixture were also measured in the

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same way.

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RESULTS AND DISCUSSION

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SDS-PAGE analysis of PSC and GCSC. The obtained PSC and GCSC were characterized

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by SDS-PAGE (Figure S1). It can be seen that 2 α-chains (α1 and α2) were the major

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components of PSC and GCSC. Furthermore, we also found obvious β-chains band and

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obscure γ-chains band for both collagens, indicating that two collagens were confirmed as

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type I collagen.37

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CD spectroscopy measurement and AA composition analysis of PSC and GCSC. Native

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collagen shows a characteristic CD spectrum with a maximal positive peak at ~220 nm and a

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minimum negative peak at ~197 nm.33 The spectra of PSC and GCSC showed a positive peak

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at 222 nm and a negative peak at 197 nm respectively (Figure S2), which was consistent with

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the previous report.33 The AA composition analysis of PSC and GCSC showed that the most

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abundant AA in two collagens was glycine (about one third). Furthermore, two collagens were

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rich in proline (Pro), alanine (Ala) and hydroxyproline (Hyp), which were accorded with AA

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composition of collagen. However, two collagens showed a subtle difference in AA

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composition. Total content of Pro and Hyp in GCSC was lower than that of PSC. In addition,

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the non-polar AA content for PSC was much higher than that of GCSC.

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The fluorescence quenching of FITC-PSC by GO. The preparation of FITC-PSC was first

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confirmed by fluorescence method. PSC had no detectable fluorescence signal at 517 nm.

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However, an apparent fluorescence signal was produced after PSC was modified with FITC,

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suggesting that PSC was labeled successfully with FITC (Figure S3). In addition, we further




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evaluated the fluorescence quenching ability of GO towards FITC-PSC. The fluorescence

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signals were tested for FITC-PSC in the presence of various concentration of GO.

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Fluorescence intensity of FITC-PSC was gradually decreased when the concentration of GO

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was increased (Figure 1A), and the quenching efficiency monotonically increased (Figure 1B).

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When the concentration of GO exceeded 40 µg/mL, the quenching efficiency was estimated

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about 80% and remains almost unchanged. Therefore, 40 µg/mL GO was selected for the

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subsequent experiments.

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Fluorescence monitoring the XCCA of FITC-PSC with GCSC. The fluorescence signal

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was measured for the GO/FITC-PSC complex in the presence of GCSC. The fluorescence of

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FITC-PSC was effectively quenched by GO in the absence of GCSC, while the introduction

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of GCSC significantly enhanced the fluorescence, indicating that there are strong interactions

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between GCSC and FITC-PSC, and resulted in the detachment of FITC-PSC from GO surface

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(Figure 2A). In order to confirm the interaction of GCSC and FITC-PSC originated from

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XCCA, we further perform the control experiments under the same conditions. We only

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observed weak changes in fluorescence intensity by directly mixing GCSC and FITC-PSC

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without incubation or incubating the mixture at 4 °C for 45 min (Figure 2B b-c). When the

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incubation temperature is 30 °C, GCSC can perform self-assembly, whereas PSC cannot

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achieve this process because the self-assembly threshold temperature of PSC is higher than

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incubated temperature.20,33 However, PSC can serve as a nucleation site for GCSC assembly,

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resulting in the occurrence of XCCA between GCSC and PSC.38 Therefore, the co-assembly

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of GCSC and PSC lead to the detachment of FITC-PSC from GO surface and recovered its

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fluorescence. Lack of incubation or low temperature incubation does not lead to the



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occurrence of XCCA, resulting in weaker fluorescence changes. At the same time, the

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introduction of unassembled other proteins, such as BSA and HSA, did not induce any

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significant increase in fluorescence intensity under the same conditions (Figure 2B d-e).

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These results suggested that XCCA is the basis of the fluorescence restoration. Previous

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studies have shown that hydrophobic interaction is the prime driving force for the

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self-assembly of collagen.39 GCSC may assemble with itself or with PSC under the condition

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of 30 °C. On the basis of the results of AA composition analysis, the non-polar AA content for

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PSC was much higher than that of GCSC. That is to say, PSC has more hydrophobic groups

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than GCSC. Therefore, PSC-GCSC hydrophobic interaction is stronger than GCSC-GCSC

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interaction. Namely, GCSC is easier to co-assemble with PSC than the self-assembly of

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GCSC.

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In order to evaluate the specificity of this strategy, we also investigated the fluorescence

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intensity change by introducing other natural collagens into GO/FITC-PSC complex under the

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same conditions. The introduction of bullfrog skin collagen (BSC) and carp skin collagen

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(CSC) has resulted in significant fluorescence changes, while the introduction of PSC and

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bovine tendon collagen (BTC) only lead to weaker fluorescence changes (Figure 3). BSC and

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CSC can carry out self-assembly at 30 °C because the incubation temperature reach their

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assembly threshold temperature,20,33 resulting in the occurrence of XCCA and significant

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fluorescence changes. The assembled threshold temperatures of PSC and BTC are higher than

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30 °C,20,40 namely, this two collagens could not perform self-assembly. Obviously, FITC-PSC

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could not carry out co-assemble with them under the experimental conditions, resulting in

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weaker fluorescence changes. These results proven that this strategy exhibited excellent




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specificity for XCCA. In addition, we also investigated the anti-interference ability of this

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assay by performing the experiment in 10% blood serum. Compared with buffer solution,

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almost the same fluorescence curve was observed in blood serum (Figure S4). This result

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indicated that the assay has a good anti-interference ability.

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Turbidity analysis of XCCA. We observed the XCCA of FITC-PSC and GCSC by a

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turbidity assay. A significant turbidity variation at 310 nm was usually produced after collagen

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assembly. Moreover, the equilibrium turbidity corresponds to the amount of assembled

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fibril.41 The turbidity of FITC-PSC and GO mixture was almost unchanged at 30 °C, while

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significant change in turbidity was observed at 35 °C (Figure S5A), which confirmed that

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PSC could not achieve self-assembly at 30 °C. In addition, we also investigated the effect of

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GO on XCCA. In the presence of GO, the assembly rate of XCCA decreased (Figure S5B),

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which may attributed to the hydrophobic interaction between GO and collagen. However, the

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introduction of GO does not lead to the disappearance of XCCA. Furthermore, the

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equilibrium turbidity of FITC-PSC/GO/GCSC mixture was higher than that of GCSC/GO

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mixture (Figure S5C). This result showed that the turbidity variation of FITC-PSC/GO/GCSC

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mixture did not only originate from GCSC self-assembly, but also PSC participated in the

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assembly. This result further confirmed that the XCCA of PSC and GCSC occurred.

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Thermal stability analysis of collagen fibrils. The results of fluorescence and turbidity

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methods confirmed the occurrence of XCCA, but these results may be caused by two

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possibilities: (i) a sole PSC/GCSC xenogeneic co-assembly; (ii) both PSC/GCSC xenogeneic

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co-assembly and GCSC self-assembly. In order to confirm the above hypothesis, we further

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investigated the thermal stability of self-assembled products by DSC (Figure S6). The DSC



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curves of the GCSC fibril and PSC fibril showed the maximum transition temperature (Tm)

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about 38.85 and 51.00 °C respectively. Previous reports have proven that the thermal stability

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of collagen is correlated with the total content of Pro and Hyp.20 The total content of Pro and

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Hyp in PSC was higher than that of GCSC (Table S1), resulting in a higher Tm. Meanwhile,

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the mixture of GCSC fibril and PSC fibril exhibited two independent endothermic peaks,

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which correspond to GCSC fibril (~38.64 °C) and PSC fibril (~51.35 °C) respectively.

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Furthermore, the xenogeneic co-assembled product exhibited only one endothermic peak

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around 48.22 °C, and the endothermic peak of GCSC fibril was not observed. These results

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showed that the fibril formed by XCCA is a new kind of collagen fibril rather than a mixture

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of multiple fibrils. That is to say, the results of fluorescence and turbidity methods are only

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caused by a sole PSC/GCSC xenogeneic co-assembly.

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Sensitivity of GO-based FRET platform. To further test if the GO-based FRET platform

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could serve as a quantitative assay for XCCA, the fluorescence intensity was measured for

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GO/FITC-PSC complex in the presence of different concentrations of GCSC. When the

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concentration of GCSC was increased, the fluorescence intensity of the GO/FITC-PSC

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complex was gradually increased (Figure 4A). Moreover, a linear relationship between the

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change of the fluorescence intensity and the logarithm of GCSC concentration from 50 µg/mL

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to 1000 µg/mL was observed (R2=0.9452) (Figure 4B). The GO-based FRET platform

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allowed the accurate and sensitive sense of XCCA at a concentration of GCSC as low as 22.0

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µg/mL (S/N=3). Current characterization of XCCA is basically limited to a turbidity method,

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which requires samples with much higher concentrations at mg/mL level (0.25-0.50

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mg/mL).18-20 In contrast, the sensitivity of the proposed method is superior than that of




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previously reported strategies.

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Regulation of thermal stability of hybrid fibrils formed by XCCA. According to the

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results of DSC, the obtained fibrils from XCCA exhibited different thermal stability with

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syngeneic collagen fibrils. Furthermore, the assembly degree of xenogeneic collagens was

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closely related to the concentration of GCSC. Thus, we attempted to investigate the

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correlation between GCSC concentrations with the thermal stability of hybrid collagen fibrils.

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All of the xenogeneic co-assembled product exhibited only one endothermic peak, and the Tm

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values of all hybrid collagen fibrils showed an intermediate range between PSC and GCSC

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fibrils, indicating that the change of collagen concentration did not affect the occurrence of

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XCCA. When the concentration of GCSC was increased, the Tm of hybrid collagen fibril was

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gradually increased (Figure 5A). When the mixture of PSC and GCSC was incubated at 30 °C,

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GCSC can thoroughly self-assembly regardless of its concentration because the incubation

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temperature is matched with its self-assembly threshold temperature, whereas PSC can not

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perform this process alone at this temperature. The greater the concentration of GCSC, the

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stronger the fluorescence signal (Figure 4), indicating that more PSC was involved in XCCA.

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That is to say, the proportion of PSC in hybrid collagen fibril was gradually increased.

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Moreover, the thermal stability of PSC fibril was stronger than that of GCSC fibril (Figure

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S6). Therefore, the thermal stability of hybrid collagen fibrils was increased by the addition of

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GCSC concentration. More importantly, the Tm values were linear with GCSC concentration

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from 50 µg/mL to 1000 µg/mL (R2= 0.9884) (Figure 5B). This result showed that the thermal

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stability of hybrid collagen fibrils can be arbitrarily regulated by simply changing the

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concentration of collagen. Thermal stability is a key indicator for the collagen-based



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biomedical materials.42 Furthermore, different application fields of collagen assembled

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products have differentiated demands for its thermal stability. For example, the slow-release

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time can be regulated by adjusting the thermal stability of collagen-based materials for

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targeted drug release.43-44 The good thermal stability of collagen products is helpful for

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reducing the technical difficulty of its processing and storage, and prolonging the timeliness

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of its function.45-46 In addition, collagen has been successfully applied as a food thickening.

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Meanwhile, thickeners for different food have different demands for thermal stability.47 The

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result of this work provided a new strategy for the regulation of the thermal stability of

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collagen assembled products, which can offer guidance to users in different fields of collagen.

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In summary, a novel GO based FRET platform for sensing XCCA was presented. The

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FITC-PSC was adsorbed onto GO surface, resulting in the fluorescence quenching due to

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FRET between GO and FITC. In the presence of another collagen, the co-assembly of two

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collagens lead to the detachment of FITC-PSC from GO surface. Therefore, the final system

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retained strong fluorescence signal. Based on this principle, the XCCA can be monitored

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simply and efficiently. Meanwhile, the developed strategy exhibited good sensitivity,

324

excellent specificity and anti-interference ability. Furthermore, we achieved the arbitrary

325

regulation of the thermal stability of hybrid collagen fibrils according to the result of this

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strategy.

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ASSOCIATED CONTENT

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Supporting Information

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SDS-PAGE analysis (Figure S1), CD (Figure S1) spectroscopy and AA composition (Table S1)

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of PSC and GCSC. Confirmation of the preparation of FITC-PSC (Figure S3).




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Anti-interference ability test of this assay (Figure S4). Turbidity analysis of xenogeneic

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collagen co-assembly (Figure S5). Thermal stability measurement of GCSC fibrils, PSC

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fibrils, hybrid fibrils and the mixture of GCSC and PSC fibrils (Figure S6). This material

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available free of charge via the Internet at http://pubs.acs.org.

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FUNDING SOURCES

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This research was supported by the National Natural Science Foundation of China (No.

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21676208, 21706201), Hubei Provincial Nature Science Foundation of China (No.

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2017CFB507, 2018CFA030, 2016CFB299), Foundation of Hubei Educational Commission

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(No. D20161703), Science and Technology Project of Wuhan City, China (No.

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2016020101010082) and Wuhan Morning Light Plan of Youth Science and Technology (No.

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2017050304010326).

342 343 344 345 346 347 348 349 350 351 352



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SCHEME

Scheme 1 Schematic illustration of GO-based FRET platform for sensing xenogeneic collagen co-assembly.




FIGURES

Figure 1 (A) The fluorescence quenching of FITC-PSC by various concentrations of GO. (B) The quenching efficiency monitored as a function of GO concentration.



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Figure 2 (A) Xenogeneic co-assembly of PSC and GCSC was monitored by fluorescence assay. (B) Control experiments under different conditions: (a) GCSC + GO/FITC-PSC, 30 °C, 45 min; (b) GCSC + GO/FITC-PSC, direct mixing; (c) GCSC + GO/FITC-PSC, 4 °C, 45 min; (d) BSA + GO/FITC-PSC, 30 °C, 45 min; (e) HSA + GO/FITC-PSC, 30 °C, 45 min.




Figure 3 Specificity of the developed strategy was shown for XCCA by incubating the mixture FITC-PSC and multiple collagen samples including GCSC, BSC, CSC, PSC and BTC.



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Figure 4 (A) The fluorescence signals were measured for the GO/FITC-PSC complex in the presence of different concentrations of GCSC (0, 50, 100, 200, 500, and 1000 µg/mL). (B) The linear relationship between the change of the fluorescence intensity and the logarithm of GCSC concentration from 50 µg/mL to 1000 µg/mL.




Figure 5 (A) DSC curves of hybrid collagen fibrils in the presence of various concentrations of GCSC (a 50 µg/mL, b 100 µg/mL, c 500 µg/mL, d 1000 µg/mL). (B) The linear relationship between the Tm values and GCSC concentration from 50 µg/mL to 1000 µg/mL.



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GRAPHIC FOR TABLE OF CONTENTS



A graphene oxide-based FRET platform for sensing xenogeneic

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