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Investigating the Structure of Collagen using Surface-enhanced Raman Spectroscopy and X-ray Scattering

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posted on 2023-01-12, 03:36 authored by Yi Zhang

Collagen is the most abundant protein in mammals, comprising a variety of tissues including skin, tendon, and pericardium where it exhibits a hierarchical structure at molecular and fibrillar levels. The structure of collagen in native tissues can be modified by chemical processing which imparts desired properties to collagenous materials ranging from bioprostheses to leathers. However, conventional processing technologies involve intense chemical use and are under much scrutiny due to their undesirable environmental and health impacts.

Herein, investigations of three interrelated research questions were undertaken to provide an insightful mechanistic understanding of the binding and crosslinking of collagen with metallic species and organic crosslinkers. This work facilitated the design of chemically benign and efficient processing technologies to circumvent the environmental and health-related concerns existing with the current processing technologies. Surface-enhanced Raman spectroscopy (SERS) and X-ray scattering (in particular, small-angle X-ray scattering (SAXS)) were used during the investigations: SERS probed side chain interactions with metallic species, whereas SAXS provided information on the intermolecular packing of collagen.

The binding of chromium(III) (Cr(III)) to collagen in bovine skin was investigated using a combination of SAXS and SERS, focussing on the competition between covalent and electrostatic binding, as regulated by the anions in the system. Two counterions of Cr(III) were assessed: sulfate (SO42−) and chloride (Cl−). It was found that both covalent and electrostatic binding mechanisms occur concurrently in the collagen matrix under the conditions investigated. Higher kosmotropicity of the anion (e.g., sulfate (SO42−)) and weaker acidity (specifically, at pH = 4.5) facilitated the covalent binding of Cr(III) with side chain carboxylate (–COO−) groups (Cr–OOC) in collagen. In contrast, when a more chaotropic anion (e.g., chloride (Cl−)) was present or at stronger acidity (pH = 2.5), the electrostatic binding mechanism between Cr(H2O)63+ complex ions and side chain carboxylate (–COO−) groups dominate. The findings from this study highlight that the conventional high Cr use to exhaust covalent binding sites (–COO−) in collagen during the processing of skin into leather, is unnecessary. It also supports a potential correlation between the electrostatically bound Cr species and the health concerns related to Cr leaching and allergic contact dermatitis. Both re-emphasise the urgency to replace excess chromium salts in collagen treatments with benign chemicals.

Following the SERS measurements on collagen fibres in skins, the SERS measurements of collagen molecules in solution using citrate-reduced silver nanoparticles (AgNPs) led to the revealing of coadsorption phenomena on the surface of the AgNPs. When chloride ions (Cl−) were introduced to the AgNPs, citrate (Cit) species desorbed and were replaced by its thermal decomposition product, acetoacetate (AAc), which was produced during the synthesis of AgNPs. Protonated AAc was readily decomposed when acidified, giving a SERS spectrum of only the silver-halide bond and water peaks. The use of bromide (Br−) and iodide (I−) led to the displacement of AAc from the surface of AgNPs, but allowed the surface to adsorb and reveal the residual anionic surfactant (AS) in water. The residual was proposed to be a polyoxyethylene alkyl ether carboxylate. A mechanism was proposed to explain the variations in binding modes and affinities of the coadsorbed species based on the surface coverage of AgNPs by the strong ligands introduced, such as halides. Instead of forming a complete coating layer, the Cl ligand layer has minor gaps that repel Cit anions but allows AAc to bind to the exposed silver (Ag) atoms. The Br and I ligands are bulkier and thus leave larger gaps that allow the AS to fit into and directly bind to exposed Ag atoms.

The phenomena were then explored for potential interference with the SERS signals of collagen. The introduction of acidic collagen into Cl or Br-coated AgNPs gave clear SERS signals corresponding to collagen, whereas a remarkable persistence of AS on the I ligand layer was found to lead to overlapping SERS signals of AS and collagen. The strong persistence could be due to the deeper gaps in the ligand layer due to the larger radius of I− ions, which stabilise the binding of AS on the surface. The findings from this study highlight the importance of recording the background spectra of halide-coated AgNPs (especially after aggregation with salts or proteins) to examine any interference and avoid potential misinterpretation in SERS bioanalysis.

The effects of organic covalent crosslinking on the structure of collagen were also investigated using SAXS to reveal how the structural changes confer the corresponding enzymatic and thermal stability on bovine pericardia. Knowing the cytotoxicity of glutaraldehyde (GA) and its propensity to promote the calcification of pericardia, GA was chosen to be a candidate alongside 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) as an alternative crosslinker. Concentrations from as low as 50 times dilution of the conventional usage were investigated. A mechanism was proposed to highlight the preferred formation of telopeptidyl-helical intermolecular linkages at low concentrations, which led to major changes in the D-period and the denaturation temperature (Td) of collagen. Full collagenase resistance was observed at low concentrations, showing the key role of telopeptidyl-helical linkages in stabilising collagen. Higher concentrations were less efficient, as the crosslinkers mainly create helical-helical linkages with side reactions forming monovalent binding and self-polymerising in GA and hydrolysis in EDC. An optimal set of conditions was postulated to maximise crosslinking efficiency. The optimised reaction pathway directs future research to devise alternative methods that use more benign chemistries and minimise the level of adverse effects.


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Date of Award



Te Herenga Waka—Victoria University of Wellington

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Degree Discipline


Degree Grantor

Te Herenga Waka—Victoria University of Wellington

Degree Level


Degree Name

Doctor of Philosophy

Victoria University of Wellington Unit

Macdiarmid Institute for Advanced Materials and Nanotechnology

ANZSRC Socio-Economic Outcome code

280105 Expanding knowledge in the chemical sciences; 241105 Skins, leather and leather products; 240699 Environmentally sustainable manufacturing activities not elsewhere classified

ANZSRC Type Of Activity code

2 Strategic basic research

Victoria University of Wellington Item Type

Awarded Doctoral Thesis



Victoria University of Wellington School

School of Chemical and Physical Sciences


Le Ru, Eric Prabakar, Sujay

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