Maria D.L. Oliveira

Carbohydrate–protein interactions and their biosensing applications

AbstractCarbohydrate recognition is clearly present throughout nature, playing a major role in the initial attachment of one biological entity to another. The important question is whether these prevalent interactions could provide a real suitable alternative to the use of antibodies or nucleic acid for detection and identification. Currently, examples of carbohydrates being employed in biological detection systems are limited. The challenges of using carbohydrate recognition for detection mainly come from the weak affinity of carbohydrate–protein interactions, the lack of versatile carbohydrate scaffolds with well-defined structures, and the less developed high-information-content, real-time, and label-free assay technology. In this review, we focus on discussing the characteristics of carbohydrate–protein interactions in nature and the methods for carbohydrate immobilization based on surface coupling chemistry in terms of their general applicability for developing carbohydrate- and lectin-based label-free sensors. Furthermore, examples of innovative design of multivalent carbohydrate–protein interactions for sensor applications are given. We limit our review to show the feasibility of carbohydrate and lectin as recognition elements for label-free sensor development in several representative cases to formulate a flexible platform for their use as recognition elements for real-world biosensor applications. FigureMultivalent protein–carbohydrate interactions at the cell surface (left) and development of a biosensor using carbohydrates (right)

Purification of a lectin from Eugenia uniflora L. seeds and its potential antibacterial activity

Aims:  The aim of this work was to analyse the antimicrobial properties of a purified lectin from Eugenia uniflora L. seeds.

Concanavalin A and polyvinyl butyral use as a potential dengue electrochemical biosensor.

Immobilization of concanavalin A on gold electrode by means of gold nanoparticles and polyvinyl butyral was carried out and investigated by cyclic voltammetry and electrochemical impedance spectroscopy. The system was tested with sera from patients infected by dengue fever (DF) and dengue hemorrhagic fever (DHF). Electrochemical impedance spectroscopy (in the frequency range from 100mHz to 100KHz), and cyclic voltammetry (from -0.2 to 0.7V vs. Ag/AgCl), was performed in phosphate buffer solution containing 10mM K(3)[Fe(CN)(6)]/K(4)[Fe(CN)(6)] (1:1) mixture as a redox probe. As biomolecules accumulated on the electrode surface the voltammetric response changed from a clear diffusional to an irreversible behavior. Impedance spectroscopy showed a clear increase of the electron-transfer resistance when the sensor is exposed to contaminated sera (DF or DHF) as compared to exposure to uncontaminated serum (NDF). The results were analyzed through an equivalent circuit and values of charge transfer resistance and capacitance were obtained. Variations in charge transfer resistance were used to distinguish the sensor response for the different sera investigated (DF, DHF and NDF). Alternatively, a three-dimensional graph gave the best response for differentiation of all three blood sera. The distinctive patterns of impedimetric responses observed were ascribed to different glycoprotein patterns in the sera investigated. Therefore, the lectin immobilization on electrode surface with gold nanoparticles and polyvinyl butyral, combined with the three-dimensional impedance analysis introduced herein are valuable tools in the development of a biosensor for immunological response to diseases.

Electrochemical evaluation of lectin–sugar interaction on gold electrode modified with colloidal gold and polyvinyl butyral

In this work, ConA and CramoLL lectins were immobilized on gold nanoparticles (AuNp) with polyvinyl butyral (PVB), and adsorbed on the surface of gold (Au) electrodes. Electrochemical impedance spectroscopy (EIS), in the frequency range from 100mHz to 100KHz, and cyclic voltammetry (CV), from -0.2 to 0.7V, were performed on these electrodes, in phosphate buffer (PBS) solution containing 10mM K(3)[Fe(CN)(6)]/K(4)[Fe(CN)(6)] (1:1) mixture as a redox probe. EIS and CV measurements showed that redox probe reactions on the modified Au electrodes were partially blocked due to the adsorption of AuNp-ConA-PVB and AuNp-CramoLL-PVB. SEM images showed the presence of aggregates of AuNp-ConA on PVB spherules in a tridimensional structure on the surface of the Au electrode. Bovine serum albumin (BSA) was adsorbed on the AuNp-Lectin-PVB modified electrode in order to block the remaining free gold sites. Both EIS and CV techniques yielded results that confirm positive responses of the lectins to ovalbumin agglutination. These results indicate an improvement of the sensitivity for detection of sugars that can be applicable to construction of a biosensor sensitive to glycoproteins in solution.

Impedimetric biosensor based on self-assembled hybrid cystein-gold nanoparticles and CramoLL lectin for bacterial lipopolysaccharide recognition

We report the development of a new selective and specific electrochemical biosensor for bacterial lipolysaccharide (LPS). An electrode interface was constructed using a l-cysteine-gold nanoparticle (AuNpCys) composite to be immobilized by electrostatic interaction in the network of a poly(vinyl chloride-vinyl acetate maleic acid) (PVM) layer on a gold bare electrode. The impedimetric biosensor is fabricated by self-assembled CramoLL lectin on the PVM-AuNpCys-modified gold electrode through electrostatic interaction. CramoLL is used as the recognition interface. AFM images showed that LPS was specifically recognized on the PVM-AuNpCys-CramoLL system surface. The measurements of cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) showed that the electrochemical response of a redox probe system (K(4)[Fe(CN)(6)](4-)/K(3)[Fe(CN)(6)](3-)) were blocked, due to the procedures of modified electrode with PVM-AuNpCys-CramoLL. In the majority of the experiments the lectin retained its activity as observed through its interaction with LPS from Escherichia coli, Serratia marcescens, Salmonella enterica and Klebsiella pneumoniae. The results are expressed in terms of the charge transfer resistance and current peak anodic using the EIS and CV techniques for the development of a biosensor for contamination by endotoxins. A new type of sensor for selective discrimination of LPS types with a high sensitivity has been obtained.

An impedimetric biosensor for detection of dengue serotype at picomolar concentration based on gold nanoparticles-polyaniline hybrid composites.

In this work, we describe the preparation and characterization of a novel gold nanoparticles-polyaniline hybrid composite (AuNpPANI) with SH-terminal groups that, due to its ability of immobilizing dengue serotype-specific primers 1, 2 and 3 (ST1, ST2 and ST3), can be used for the development of biosensors. Electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) were performed. CV and EIS results demonstrated that the AuNpPANI can immobilize ST1, ST2 and ST3, forming AuNpPANI-ST complexes. Well-defined cyclic voltammograms characteristic of a diffusion-limited redox process were observed both for the bare gold electrode and after these electrodes have been modified by the adsorption of AuNpPANI or AuNpPANI-ST. The AuNpPANI-ST(1-3) systems were able to recognize the dengue serotype of different patients at picomolar concentrations. Even when small volumes and low concentrations of the analyte were used, the CV and EIS results showed unequivocal evidence of an existing interaction between dengue serotype-specific primers and their complementary genomic DNA targets.

Impedimetric sensor of bacterial toxins based on mixed (Concanavalin A)/polyaniline films

In this paper, we report the use of Concanavalin A (ConA) and electrosynthesized polyaniline (PANI) thin films for the development of a new electrochemical sensor that allows the specific detection of two bacterial toxins: lipopolysaccharide (LPS) from Escherichia coli and lipoteichoic acid from Staphylococcus aureus. The impedimetric sensor is fabricated by using glutaraldehyde to self-assemble ConA lectin on PANI-modified steel electrodes through covalent binding. ConA acts as a recognition element for bacterial toxins. Electrical impedance spectroscopy (EIS) and scanning electron microscope (SEM) were applied to characterize the assembly process on the modified electrode. The EIS measurements revealed that the resistance charge transfer (RCT) of the electrode/electrolyte interface increases considerably after the ConA lectin interacts with specific carbohydrate moieties present in the molecule of the bacterial toxin. Our results showed that the ConA lectin retained its activity after immobilization on the PANI surface and also the existence of electrochemical impedance response of the bioelectrode which is linear to the extent of the lectin-toxin interaction, with maximum values of RCT for E. coli (14.40 kΩ), and S. aureus (17.80 kΩ). We have observed that electrosynthesized PANI is an excellent support layer for the covalent binding of lectins on the electrode surface. Thus, the recognition system provides an appropriate biomimetic interface for detection of specific constituents of gram-positive and gram-negative bacteria.

Diagnosis of dengue infection using a modified gold electrode with hybrid organic–inorganic nanocomposite and Bauhinia monandra lectin

A sensitive and selective biosensor for dengue serotyping was successfully developed. The biosensor uses a novel gold nanoparticles-polyaniline hybrid composite (AuNpPANI) for the immobilization of Bauhinia monandra lectin (BmoLL). The nanocomposite was applied to a bare gold electrode surface by chemical adsorption, and BmoLL was subsequently electrostatically adsorbed to the nanocomposite-modified surface. Atomic force microscopy (AFM), cyclic voltammetry (CV) and electrochemical impedance (EI) techniques were applied to evaluate the immobilization of BmoLL on AuNpPANI. The AFM images for AuNpPANI-BmoLL-DEN systems indicate a homogenous, compact and dense film of the conjugate. In the EI analyses, an obvious difference of the electron transfer resistance between the AuNpPANI-modified electrode and the bare gold electrode was observed. Among three dengue serotypes studied, dengue serotype 2 (DEN2) has higher values for R(CT), and lower values for both n and Q. These are indications of a larger blocking effect and smaller capacitive dispersion, resulting from the higher agglutination of glycoproteins from the DEN2 sera. The selective BmoLL recognition for various dengue serotypes may be attributed to different patterns of glycoproteins in the sera produced by the glycoprotein immunoresponse from patients infected by the dengue virus.

Optical and dielectric sensors based on antimicrobial peptides for microorganism diagnosis

Antimicrobial peptides (AMPs) are natural compounds isolated from a wide variety of organisms that include microorganisms, insects, amphibians, plants, and humans. These biomolecules are considered as part of the innate immune system and are known as natural antibiotics, presenting a broad spectrum of activities against bacteria, fungi, and/or viruses. Technological innovations have enabled AMPs to be utilized for the development of novel biodetection devices. Advances in nanotechnology, such as the synthesis of nanocomposites, nanoparticles, and nanotubes have permitted the development of nanostructured platforms with biocompatibility and greater surface areas for the immobilization of biocomponents, arising as additional tools for obtaining more efficient biosensors. Diverse AMPs have been used as biological recognition elements for obtaining biosensors with more specificity and lower detection limits, whose analytical response can be evaluated through electrochemical impedance and fluorescence spectroscopies. AMP-based biosensors have shown potential for applications such as supplementary tools for conventional diagnosis methods of microorganisms. In this review, conventional methods for microorganism diagnosis as well new strategies using AMPs for the development of impedimetric and fluorescent biosensors are highlighted. AMP-based biosensors show promise as methods for diagnosing infections and bacterial contaminations as well as applications in quality control for clinical analyses and microbiological laboratories.

Biosensor based on lectin and lipid membranes for detection of serum glycoproteins in infected patients with dengue

In this work, we developed a biosystem based on Concanavalin A (ConA) and lipid membranes to recognize glycoproteins from the serum of patients contaminated with dengue serotypes 1, 2 and 3 (DENV1, DENV2 and DENV3). The modified gold electrode was characterized using cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and atomic force microscopy. Morphological analyses of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), DPPC-ConA, DPPC-ConA-DENV1, DPPC-ConA-DENV2 and DPPC-ConA-DENV3 revealed the existence of a non-uniform covering and large globules. EIS and CV measurements have shown that redox probe reactions on the modified gold electrodes were partially blocked due to the adsorption of lipid-ConA system and reveal the interaction response of the immobilized ConA to the presence of glycoproteins of dengue serum. The biosystem exhibited a wide linear response to different concentrations of sera of dengue serotypes 1, 2 and 3. A higher impedimetric response to glycoproteins present in dengue serotype 3 was observed. Our results demonstrate the applicability of lectin and lipid membranes to the development of biosensors for dengue infections.