Marli L. Moraes

Immunosensor Based on Immobilization of Antigenic Peptide NS5A‑1 from HCV and Silk Fibroin in Nanostructured Films

The peptide NS5A-1 (PPLLESWKDPDYVPPWHG), derived from hepatitis C virus (HCV) NS5A protein, was immobilized into layer-by-layer (LbL) silk fibroin (SF) films. Deposition was monitored by UV-vis absorption measurements at each bilayer deposited. The interaction SF/peptide film induced secondary structure in NS5A-1 as indicated by fluorescence and circular dichroism (CD) measurements. Voltammetric sensor (SF/NS5A-1) properties were observed when the composite film was tested in the presence of anti-HCV. The peptide-silk fibroin interaction studied here showed new architectures for immunosensors based on antigenic peptides and SF as a suitable immobilization matrix.

Layer-by-layer assembly of functionalized reduced graphene oxide for direct electrochemistry and glucose detection

We report an electrochemical glucose biosensor made with layer-by-layer (LbL) films of functionalized reduced graphene oxide (rGO) and glucose oxidase (GOx). The LbL assembly using positively and negatively charged rGO multilayers represents a simple approach to develop enzymatic biosensors. The electron transport properties of graphene were combined with the specificity provided by the enzyme. rGO was obtained and functionalized using chemical methods, being positively charged with poly(diallyldimethylammonium chloride) to form GPDDA, and negatively charged with poly(styrene sulfonate) to form GPSS. Stable aqueous dispersions of GPDDA and GPSS are easily obtained, enabling the growth of LbL films on various solid supports. The use of graphene in the immobilization of GOx promoted Direct Electron Transfer, which was evaluated by Cyclic Voltammetry. Amperometric measurements indicated a detection limit of 13.4μmol·L(-1) and sensitivity of 2.47μA·cm(-2)·mmol(-1)·L for glucose with the (GPDDA/GPSS)1/(GPDDA/GOx)2 architecture, whose thickness was 19.80±0.28nm, as determined by Surface Plasmon Resonance (SPR). The sensor may be useful for clinical analysis since glucose could be detected even in the presence of typical interfering agents and in real samples of a lactose-free milk and an electrolyte solution to prevent dehydration.

Lignin as immobilization matrix for HIV p17 peptide used in immunosensing

Immunosensors based on electrical impedance spectroscopy (EIS) are increasingly being used as a fast and potentially low cost method for clinical diagnostics. In this work we fabricated immunosensors by depositing layer-by-layer (LbL) films made with an antigenic peptide (p17-1) sequence (H2N-LSGGELDRWEKIRLRPGG-OH) and lignin on interdigitated gold electrodes, which could detect anti-p17 (HIV, human immune deficiency virus) antibodies (Ab) in phosphate buffered solutions (PBS). The molecular recognition interaction between the peptide (p17-1) and the specific Ab (anti-p17) yielded substantial changes in morphology of the with LbL films, with increased roughness according to atomic force microscopy data. This interaction is behind the high sensitivity of the immunosensor. Indeed, from the EIS results, we noted that the capacitance increased significantly with the specific Ab concentration, before getting close to saturation of available peptide sites at high concentrations. Concentrations of specific antibodies as low as 0.1 ng/mL could be detected and the immunosensors had their activity preserved for two months at least. The selectivity of the immunosensor was confirmed with two types of control experiments. First, no changes in impedance were observed when the lignin/peptide LbL immunosensor was immersed into a PBS solution containing the non-specific Ab (anti-HCV for Hepatitis C) antibodies. Furthermore, for sensing units made LbL films of lignin only, the electrical response was not affected by adding specific antibodies into the PBS buffer. The successful immunosensing for HIV with antigenic peptides in a lignin matrix is also relevant for valorization of lignin, which is an important biomass component in the sugar and ethanol industry, and brings the prospect for all-organic, biocompatible sensors if implantation is ever required.

Monoamine oxidase B layer-by-layer film fabrication and characterization toward dopamine detection

In this work nanostructured film composites of the monoamine oxidase B (MAO-B) enzyme, free or encapsulated in liposomes, were fabricated by the layer-by-layer (LbL) self-assembly technique, employing polyethylene imine (PEI) as polycation. Initially, the MAO-B enzyme was incorporated into liposomes in order to preserve its enzymatic structure ensuring their activity and catalytic stability. The LbL film growth was monitored by surface plasmon resonance (SPR) by gold resonance angle shift analysis after each bilayer deposition. Subsequently, the films were applied as amperometric biosensors for dopamine detection using Prussian Blue (PB) as the electron mediator. The biosensor fabricated by MAO-B incorporated into liposomes composed of DPPG:POPG in the ratio (1:4) (w/w) showed the best performance with a sensitivity of 0.86 (μA cm(-2))/(mmol L(-1)) and a detection limit of 0.33 mmol L(-1).

Silk fibroin organization induced by chitosan in layer-by-layer films: Application as a matrix in a biosensor.

In this paper, we show that chitosan may induce conformation changes in silk fibroin (SF) in layer-by-layer (LbL) films, which were used as matrix for immobilization of the enzyme phytase to detect phytic acid. Three chitosan (CH) samples possessing distinct molecular weights were used to build CH/SF LbL films, and a larger change in conformation from random coils to β-sheets for SF was observed for high molecular weight chitosan (CHH). The CHH/SF LbL films deposited onto interdigitated gold electrodes were coated with a layer of phytase, with which phytic acid could be detected down to 10-9M using impedance spectroscopy as the principle of detection and treating the data with a multidimensional projection technique. This high sensitivity may be ascribed to the suitability of the CHH/SF matrix, thus indicating that the molecular-level interactions between chitosan and SF may be exploited in other biosensors and biodevices.

Immunosensor for diagnosis of Alzheimer disease using amyloid-β 1-40 peptide and silk fibroin thin films.

Layer-by-Layer (LbL) films containing silk fibroin (SF) and the 40 aminoacid-long amyloid-β peptide (Aβ1-40) were prepared with the purpose of developing a new prototype of an electrochemical immunosensor. The film showed a satisfactory growth in quartz substrate and screen-printed carbon electrodes, as observed by UV-vis spectroscopy and cyclic voltammetric, respectively. The peptide immobilized in LbL films in junction with SF shows secondary structure induced, as shown by circular dichroism measurements, favoring the interaction SF/peptide LbL film with the specific antibody. Immunosensor showed a linear response in the presence of the antibody with concentrations from 0 to 10ngmL(-1) both analyzed by current changes in 0.3V and voltammogram area. This system can be applied as a new prototype for preliminary diagnosis of Alzheimers disease.

Electrical immunosensor made with antigenic peptide NS5A‑1 immobilized onto silk fibroin for diagnosing hepatitis C

Immunosensors based on impedance spectroscopy for diagnosing hepatitis C are reported where the sensing units were made with the antigenic peptide PPLLESWKDPDYVPPWHG (NS5A-1) derived from the NS5A protein of the hepatitis C virus (HCV) immobilized in layer-by-layer (LbL) films with silk fibroin (SF) and deposited on gold interdigitated electrodes. The electrical response of the sensing units varied upon immersion into solutions containing the antibody anti-HCV owing to the biomolecular recognition of NS5A-1. This was associated with morphological changes on the LbL films caused by adsorption of NS5A-1 and inferred from atomic force microscopy images. Buffer solutions with different anti-HCV concentrations down to 2 ng mL could be clearly distinguished by analyzing the impedance spectroscopy data with a multidimensional projection technique. The specificity toward anti-HCV antibodies was confirmed in control experiments where no significant changes in the electrical response were measured by exposing the sensing units to solutions containing an anti-human immunodeficiency virus (HIV) antibody. The high sensitivity and selectivity of the units made with LbL films demonstrate the feasibility of measuring electrical impedance as an immunosensing strategy to detect hepatitis C.

2 – SISTEMAS SUPRAMOLECULARES

RESUMO: Neste capitulo serao mostrados alguns conceitos basicos envolvidos nos sistemas supramoleculares, que sao montagens de subunidades quimicas unidas por interacoes covalentes ou intermoleculares. Entre esses casos, incluem-se as moleculas com varias estruturas em ciclos (macrociclos), e os sistemas dispersos de agregacao (micelas e lipossomos). Sera mostrado como os macrociclos podem ser usados para varias finalidades, incluindo processos de reconhecimento molecular, e tambem como micelas e vesiculas podem ser montadas a partir de substâncias anfifilicas, rendendo estruturas estaveis do ponto de vista termodinâmico e cinetico.