Toward Preserving the Structure of the Antigenic Peptide p17-1 from the HIV-1 p17 Protein in Nanostructured Films

Abstract

Antigenic peptides may be immobilized in nanostructured films in order to build highly specific immunosensors and other devices that require molecular recognition, with no need to use complex molecules. A major challenge for such endeavors, however, is to preserve the secondary structure of the peptides after immobilization. In this study, we show that the peptide p17-1 (LSGGELDRWEKIRLRPGG), derived from the HIV-1 p17 protein, may be immobilized in Layer-by-Layer (LbL) films made with polyelectrolytes. Its structure was preserved only if incorporated into phospholipid liposomes, according to fluorescence and circular dichroism (CD) spectroscopy. The lack of secondary structure for the peptide in the LbL film may be associated with the film-forming procedure in which p17-1 was adsorbed from an aqueous solution, where it does not form alpha helices. The importance of structure preservation was clear in the attempts to produce electrochemical immunosensors with the p17-1 peptide without being protected in liposomes in an LbL film. There was no detectable influence of the presence of anti-p17 antibodies, though some molecular interaction could be inferred from the voltammograms. In contrast, for p17-1 incorporated in liposomes electrochemical immunosensors could be obtained with the voltamogramms showing strong molecular recognition with the antibodies. These results indicated that phospholipids serve as a suitable matrix for immobilization of peptides, and confirmed the importance of structure preservation in electrochemical immunosensors.

Immunosensor for HIV-1 Diagnostics Based on Immobilization of the Antigenic Peptide p24-3 Into Liposomes

“…5 Therefore, all the LbL films reported here were obtained with p24-3 encapsulated into DPPG liposomes. Figure 2 displays maximum at 280 nm as a function of number of bilayers of PEI/p24-3-containing liposomes, indicating that film growth appears exponential for the first few bilayers, as in many LbL films made with biomolecules.…”

“…Scheme 1 shows the procedures for preparing liposomes and immobilizing them in LbL films, consisting in the following steps: (a) incorporation of p24-3 into DPPG liposomes was performed using the method described by Petri et al 5 (b) The DPPG liposomes containing p24-3 were immobilized in LbL films alternated with PEI layers onto a solid substrate. The latter was initially immersed in PEI solution for 3 min, while adsorption of liposomes was performed during 10 min, which was determined as optimized time from an adsorption kinetics study.…”

“…The latter was initially immersed in PEI solution for 3 min, while adsorption of liposomes was performed during 10 min, which was determined as optimized time from an adsorption kinetics study. 5 After each deposition step, the films were rinsed with Milli-Q water.…”

“…13 The use of antigenic peptides in immunosensors may be exploited in conjunction with platforms using nanostructured films and electrochemistry or impedance spectroscopy as the principle of detection. 5 [15][16][17][18][19]. In this paper, we produced an immunosensor for HIV-1 diagnostics that exploits the biorecognition between the antibody antip24 and the antigenic peptide p24-3 (AMATLRAE-QASQEVKNWMTE TLLVQNA) that corresponds to the residues 171-194 from the HIV-1 p24 protein.…”

Amperometric Detection of Lactose Using β-Galactosidase Immobilized in Layer-by-Layer Films

“…For fluorescence and optical absorption measurements, 2-bilayer (PEI/PVS) 2 LbL films were used as a cushion to reduce the influence of substrate morphology on film growth, 26 while the amperometric measurements were made with a 1-bilayer cushion of (PEI/PVS) 1 . LbL films were assembled by dipping the substrate in PEI and β-Gal solutions, both for 3 min, interspersing the immersion in sodium acetate buffer during 30 s for washing.…”