Protein immobilization on functionalized fluorine-terminated nanocrystalline (NCD) films was studied by attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy using an immobilization protocol developed to specifically bind C-reactive protein (CRP). Using an ATR-FTIR spectroscopy method employing a force-controlled anvil-type configuration, three critical steps of the ex situ CRP immobilization were analyzed. First, the NCD surface was passivated by deposition of a copolymer layer consisting of polyethylene oxide and polypropylene oxide. Second, a synthetic modified polypeptide binder with high affinity to CRP was covalently attached to the polymeric film. Third, CRP dissolved in aqueous buffer in concentrations of 10–20 μg/mL was added on the functionalized NCD surface. Both the amide I and II bands, due to the polypeptide binder and CRP, were clearly observed in ATR-FTIR spectra. CRP amide I bands were extracted from difference spectra and yielded bands that agreed well with the reported amide I band of free (non-bonded) CRP in solution. Thus, our results show that CRP retains its secondary structure when it is attached to the polypeptide binders. Compared to previous IR studies of CRP in solution, about 200 times lower concentration was applied in the present study.
Infrared spectroscopy ATR-FTIR Nanocrystalline diamond CRP Protein binders Biosensor
This is a preview of subscription content, log in to check access.
Dr. Karin Fromell and Prof. Lars Baltzer (both at Uppsala University) are acknowledged for their help with binder and CRP samples. The work was supported financially by grants from Uppsala Berzelii Technology Centre for Neurodiagnostics and VR-project 621-2014-5959.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
Bahadir EB, Sezginturk MK. Applications of commercial biosensors in clinical, food, environmental, and biothreat/biowarfare analyses. Anal Biochem. 2015;478:107–20.CrossRefGoogle Scholar
Bhakta SA et al. Protein adsorption onto nanomaterials for the development of biosensors and analytical devices: a review. Anal Chim Acta. 2015;872:7–25.CrossRefGoogle Scholar
Verma N, Bhardwaj A. Biosensor technology for pesticides—a review. Appl Biochem Biotechnol. 2015;175(6):3093–119.CrossRefGoogle Scholar
Hartl A et al. Protein-modified nanocrystalline diamond thin films for biosensor applications. Nat Mater. 2004;3(10):736–42.CrossRefGoogle Scholar
Fromell K et al. Designed protein binders in combination with nanocrystalline diamond for use in high-sensitivity biosensors. Anal Bioanal Chem. 2012;404(6–7):1643–51.CrossRefGoogle Scholar
Devouge S et al. Surface functionalization of germanium ATR devices for use in FTIR-biosensors. J Colloid Interface Sci. 2009;332(2):408–15.CrossRefGoogle Scholar
Andersson PO et al. A novel ATR-FTIR approach for characterisation and identification of ex situ immobilised species. ChemPhysChem. 2007;8(5):712–22.CrossRefGoogle Scholar
Andersson PO et al. A novel ATR-FTIR method for functionalised surface characterisation. Surf Interface Anal. 2008;40(3–4):623–6.CrossRefGoogle Scholar
Bijnens N et al. Synthetic diamond films as a platform material for label-free protein sensors. Physica Status Solidi a-Applications and Materials Science. 2009;206(3):520–6.CrossRefGoogle Scholar
Quershi A et al. A novel interdigitated capacitor based biosensor for detection of cardiovascular risk marker. Biosensors & Bioelectronics. 2009;25(4):877–82.CrossRefGoogle Scholar
Dong A et al. Secondary structure of the pentraxin female protein in water determined by infrared-spectroscopy—effects of calcium and phosphorylcholine. Biochemistry. 1992;31(39):9364–70.CrossRefGoogle Scholar
Dong AC, Caughey WS, Duclos TW. Effects of calcium, magnesium, and phosphorylcholine on secondary structures of human c-reactive protein and serum amyloid-p component observed by infrared-spectroscopy. J Biol Chem. 1994;269(9):6424–30.Google Scholar
Heaton RJ, Raynes JG, Johnston DS. A study of the denaturation of human C-reactive protein in the presence of calcium ions and glycero-phosphorylcholine. Thermochim Acta. 1999;334(1–2):97–106.CrossRefGoogle Scholar
Bergholt MS, Hassing S. Quantification of C-reactive protein in human blood plasma using near-infrared Raman spectroscopy. Analyst. 2009;134(10):2123–7.CrossRefGoogle Scholar
Tegler LT et al. Powerful protein binders from designed polypeptides and small organic molecules—a general concept for protein recognition. Angewandte Chemie-International Edition. 2011;50(8):1823–7.CrossRefGoogle Scholar
Rigler P et al. Downscaling Fourier transform infrared spectroscopy to the micrometer and nanogram scale: secondary structure of serotonin and acetylcholine receptors. Biochemistry. 2003;42(47):14017–22.CrossRefGoogle Scholar
Wang XF et al. Diamonds are a spectroscopist’s best friend: thin-film diamond mid-infrared waveguides for advanced chemical sensors/biosensors. Anal Chem. 2014;86(16):8136–41.CrossRefGoogle Scholar