Caracterização e purificação da lactoferrina de bubalina (Bubalus bubalis), associação com quantum dots e sua determinação utilizando sensor a base de Au/PAMAM

Abstract

Lactoferrin (Lf), a protein present in milk, is a monomeric glycoprotein of around 80 kDa, with a single polypeptide chain of about 690 aminos acid residues and is an important component of the immune system, including bacteria, viruses, fungi, parasites. and also has anti-inflammatory and anti-cancer activities. In this way it is an important additive in medicines, foods and cosmetics, such as health products and infant formulas. The amino acid sequence relationships place it in the transferrin family. Fluorometric analyzes of bovine, caprine, and buffalo Lf reveal a difference between species. The molecule is folded into homologous terminal N and C lobes, each comprising two domains that include a local conserved iron bond. There are few analytical methodologies that can be applied for the determination of lactoferrin, among these are the spectroscopic and electrochemical methods. The latter stand out for presenting good sensitivity, selectivity, stability, low cost, in situ analysis capacity and the possibility of miniaturization. The objective of this work is to isolate, purify, characterize Lf buffalo, evaluate its interaction with quantum dots and determine its concentration using an Au/PAMAM-based electrochemical sensor. For this we will use purification methodologies adapted from the literature. Lf was isolated in the method proposed in this work and a greater amount of it was isolated from buffalo milk by Salting Out and after purification in a Sephacryl S-200 chromatographic column. The interaction between buffalo lactoferrin (bLf) and CdSeCdS quantum dots (QD) was investigated by spectrofluometry. The effect of CdSe-CdS on buffalo Lf conformation was also analyzed in which it was possible to correlate fluorescence with concentration by obtaining Stern - Volmer constant values. Fluorescence results showed that QDs effectively quenched lactoferrin fluorescence. The influence of QDs on protein functional alterations was observed through the analysis of bLf surface alterations and investigation of the binding forces between bLf and iron ions. These results indicated that the changes in the structure and function of bLf were due to the interaction between bLf and QDs. Lf also formed complex with Quercetin. With the electrochemical sensor formed by an Au electrode modified with 2nd generation PAMAM, it was possible to build an analytical curve between the peak current and the Lf concentration, with good linearity (R= 0.994) with a detection limit of 0.010 µg/L and a limit of 0.010 µg/L. quantification 0.03µg/L, as well as to determine the concentration of Lf in samples of bovine, buffalo and goat milk.