Síntese, caracterização e aplicação de Quantum Dots como nanosondas fluorescentes para aplicações bioanalíticas

Abstract

In the process of quantifying biomolecules, the traditional methods of determination have some limitations, such as high operational cost, complexity of analysis, need for specialized instruments, complexity of analysis, among other factors. These same mishaps are present for the detection of protamine (PT) and trypsin (TRIP), which are two biomolecules with great importance and application in the medicinal and pharmaceutical area, being essential to carry out monitoring in samples in vivo and in vitro in both. cases. Featuring high versatility and unique properties, such as high quantum yield, high stability, broad spectrum of absorption and emission and low toxicity compared to the organic fluorophores used, quantum dots (quantum dots, QD) are nanoparticles that have been explored and applied as markers or as photoluminescent probes of various components, such as metals, biomolecules, cells and tissues, where the analyte is usually detected due to the increase or attenuation of fluorescence during the interaction process. In order to determination of protamine, QDs of mercaptosuccinic acid functionalized CdTe were synthesized (pKa1 = 3.30 and pKa2 = 4.60). The nanoprobe were characterized by a direct relationship between synthesis time and QD size (2.61 – 3.04 nm). The QD-CdTe-MSA interacted with protamine (PT), a cationic protein, forming a bioconjugate, thus quenching the photoluminescence intensity and generating an ON-OFF system. The nanoprobe produced at a synthesis time of 1 h (QD-CdTe1) presented PT's best sensitivity in a succinate buffer (pH = 5). Under the optimized conditions, the proposed method presented a linear range of 0.05 - 0.5 mg L-1 (10 - 100 nM), LOD 0.01 mg L-1 (2 nM), and RSD ≤ 2.01% (n = 10). The interaction of the nanoprobe and PT led to aggregation due to a bioconjugate formation. The systems' hydrodynamic radius varied from 4.31 nm (QD-CdTe1) to 30.50 nm for the bioconjugate (QD-CdTe1-PT). The method was sensitive to variation in ionic strength and based on thermodynamic parameters; it was demonstrated that the interaction mechanism occurred preferentially through electrostatic forces. Finally, the method proved to be fast, sensitive, and viable for quantifying PT in drugs and synthetic urine samples with recoveries above 95%. The bioconjugate QD-CdTe6-PT was then explored for the quantification of trypsin (TP), an enzyme with specific cleavage in the amino acids lysine and arginine, through an ON-OFF-ON system. In this system, the size of the QD varied between 2.56 - 3.21 nm, where the bioconjugate QD-CdTe6-PT was the one that showed the best sensitivity compared to TP in ammonium buffer solution (75 mM, pH = 8.5). Under the optimized conditions, the proposed method had a linear range of 0.005 - 0.07 mg L-1 (0.22 - 3.08 nM), with an LOD of 0.0014 mg L-1 (0.06 nM) and RSD  1 , 81%. The interaction process between QD-CdTe6-PT and TP led to the release of the nanoparticle due to TP cleavage, with a variation in the hydrodynamic radius from 234 de 37.13 to 83.20  40.00, 30.90  21.10 and 23.42  12.43 nm (TP at 0.015, 0.03 and 0.05 mg L-1, respectively). Finally, the proposed method feasible for quantification of PT in plasma samples with recoveries above 91%.