MADRID Rossana Elena
congresos y reuniones científicas
Lectin-modified surfaces for the real-time determination of Bone Alkaline Phosphatase by Surface Plasmon Resonance (SPR) Spectroscopy
L. D. SAPPIA; PICCININI, E.; SANTILLI, N.; MARMISOLÉ, W.; MADRID, R.E.; AZZARONI, O.
Workshop; 1st Argentine-German Workshop on Nanotechnology and Nanobiosensors; 2017
The biological interaction of lectins and carbohydratesrules many recognitions processes found in living organisms . Theseinteractions can be exploited to develop highly stable lectin andglycoprotein biointerfaces [2,3]. Inthis work, we constructed carbohydrate layers on gold substrates for theimmobilization of the lectin wheat germ agglutinin (WGA). The WGA-modifiedsurfaces were used as a recognition element of glycosylated proteins present inhuman serum. The lectin-glycoprotein recognition event was evaluated byResonance Plasmon Spectroscopy (SPR), which has a resolution in the sub-nanometricscale .The glycosylation of aminated SPR gold substrates was doneas described before . Briefly, the substrates were aminated by theincubation in a cysteamine solution. Using the divinyl sulfone (DVS) chemistry,N-acetyl glucosamine (GlcNAc) is covalently attached to the surface (see Fig.1). Then, WGA was adsorbed to the glycosylated substrates, event that showedconsiderable changes in the angle of minimum reflectivity (θ min). Therobustness of the WGA attachment was evaluated exposing the substrate to a 100mM GlcNAc solution. These results are in agreement with the literature [6, 7]. The WGA-functionalizedsubstrates were exposed to increasing concentrations of human sera, from 1:500to 1:10 diluted in buffer (1-7 in Fig. 1). One of the serum was classified as?normal? (yellow line), while the other one was classified as pathological(blue line) due to their levels of an isoform of alkaline phosphatase (BALP) produced by bone cellsactivity. The SPR results are consistent with the differences in BALP activitymeasured by a reference method, that is based on the co- precipitation of thelectin WGA in solution with the ALP isoform and measuring the activity beforeand after this binding . From these results, it could be inferred that areal-time BALP determination from human serum can be developed and the methodmay be used to study diseases related to bone tissue disorders.