Immunosensor for the Diagnostics of Autoimmune Hemolytic Anemia (AIHA) Based on Immobilization of a Monoclonal Antibody on a Layer of Silk Fibroin

European Regional Development Fund (ERDF)The diagnostics of the autoimmune hemolytic anemia (AIHA), a rare disease caused by autoantibody-induced hemolysis, is still prone to false positives for it is based on visual observation in the so-called Direct Coombs test. In this study, we developed a specific IgG hemolysis immunosensor produced with layer-by-layer (LbL) films containing a monoclonal antibody against human immunoglobulin (mAbIMUG) deposited along with a layer of silk fibroin (SF) derived from Bombyx mori cocoons. Adsorption of mAbIMUG on a SF layer was confirmed by the fluorescence emission band at 326 nm. Immunosensors were prepared with LbL films deposited on interdigitated gold electrodes for impedance spectroscopy and on screen printed carbon electrodes for electrochemical measurements. When the SF/mAbIMUGLbL film was exposed to healthy red blood cells (RBCs), no cell binding was observed by the optical microscopy images. In addition, no major changes were observed in the signals of the square wave voltammogram and in the impedance spectra. In contrast, the electrochemical signal was significantly increased and the dielectric loss curve shifted for the sensing units containing RBCs with the antibody attached on the surface (“sick cells”). Furthermore, cell attachment was so strong that optical images still showed covered electrodes even after washing in PBS buffer. The detection with two distinct methods seems promising for an effective diagnosis of AIHA

Information visualization techniques for sensing and biosensing

The development of new methods and concepts to visualize massive amounts of data holds the promise to revolutionize the way scientific results are analyzed, especially when tasks such as classification and clustering are involved, as in the case of sensing and biosensing. In this paper we employ a suite of software tools, referred to as PEx-Sensors, through which projection techniques are used to analyze electrical impedance spectroscopy data in electronic tongues and related sensors. The possibility of treating high dimension datasets with PEx-Sensors is advantageous because the whole impedance vs. frequency curves obtained with various sensing units and for a variety of samples can be analyzed at once. It will be shown that non-linear projection techniques such as Sammon's Mapping or IDMAP provide higher distinction ability than linear methods for sensor arrays containing units capable of molecular recognition, apparently because these techniques are able to capture the cooperative response owing to specific interactions between the sensing unit material and the analyte. In addition to allowing for a higher sensitivity and selectivity, the use of PEx-Sensors permits the identification of the major contributors for the distinguishing ability of sensing units and of the optimized frequency range. The latter will be illustrated with sensing units made with layer-by-layer (LbL) films to detect phytic acid, whose capacitance data were visualized with Parallel Coordinates. Significantly, the implementation of PEx-Sensors was conceived so as to handle any type of sensor based on any type of principle of detection, representing therefore a generic platform for treating large amounts of data for sensors and biosensors.FAPESPCNPqCAPESINCT-INEOnBioNet Films & Sensor

Use of hemoglobin as alternative to peroxidases in cholesterol amperometric biosensors

Sophisticated molecular architectures can be produced with the layer-by-layer (LbL) method, which may combine distinct materials on the same film. In this study, we take advantage of this capability to produce cholesterol amperometric biosensors from LbL films containing hemoglobin (Hb) and cholesterol oxidase in addition to the polyelectrolytes poly(allylamine hydrochloride) (PAH) and poly(ethylene imine) (PEI). Following an optimization procedure, we found that an LbL film deposited onto ITO substrates, with the architecture ITO(PEI/Hb)5(PEI/COx)10, yielded a sensitivity of 93.4 μA μmol L-1 cm-2 for cholesterol incorporated into phospholipid liposomes, comparable to state-of-the-art biosensors. Hb acted as efficient electron mediator and did not suffer interference from phospholipids. Significantly, cholesterol could also be detected in real samples from chicken egg yolk, with no effects from potential interferents, including phospholipids. Taken together these results demonstrate the possible fabrication of low cost, easy-to-use cholesterol amperometric biosensors, whose sensitivity can be enhanced by further optimizing the molecular architectures of the LbL films. © 2012 Elsevier B.V

Phytase immobilization on modified electrodes for amperometric biosensing

Phytase (myo-inositol hexaphosphate phosphohydrolase) and phytic acid (myo-inositol hexaphosphate) play an important environmental role, in addition to being a health issue in food industry. Phytic acid is antinutritional due to its ability to chelate metal ions and may also react with proteins decreasing their bioavailability. In this work, we produced biosensors with phytase immobilized in Layer-by-Layer (LbL) films, which could detect phytic acid with a detection limit of 0.19 mmol L-1, which is sufficient to detect phytic acid in seeds of grains and vegetables. The biosensosrs consisted of LbL films containing up to eight bilayers of phytase alternated with poly(allylamine) hydrochloride (PAH) deposited onto an indium-tin oxide (ITO) substrate modified with Prussian Blue. Amperometric detection was conducted in an acetate buffer solution (at pH 5.5) at room temperature, with the biosensor response attributed to the formation of phosphate ions. In subsidiary experiments with the currents measured at 0.0 V (vs. SCE), we demonstrated the absence of effects from some interferents, pointing to a good selectivity of the biosensor. (c) 2007 Elsevier B.V. All rights reserved

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

Layer-by-Layer (LbL) films containing silk fibroin (SF) and the 40 aminoacid-long amyloid-beta peptide (A beta 1-40) 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 10 ng mL(-1) both analyzed by current changes in 0.3 V and voltammogram area. This system can be applied as a new prototype for preliminary diagnosis of Alzheimer's disease. (C) 2016 Elsevier B.V. All rights reserved.Univ Estadual Paulista Julio de Mesquita Filho UN, Inst Quim, Rua Prof Francisco Degni 55, BR-14800060 Araraquara, SP, BrazilUniv Fed Sao Paulo UNIFESP, Inst Ciencia & Tecnol, Rua Talim 330, BR-12231280 Sao Jose Dos Campos, SP, BrazilInstituto de Ciência e Tecnologia, Universidade Federal de São Paulo (UNIFESP), Rua Talim, 330, 12231-280 São José dos Campos, SP, BrazilWeb of Scienc

Immobilization of liposomes in nanostructured layer-by-layer films containing dendrimers

Artificial vesicles or liposomes composed of lipid bilayers have been widely exploited as building blocks for artificial membranes, in attempts to mimic membrane interaction with drugs and proteins and to investigate drug delivery processes. In this study we report on the immobilization of liposomes of 1,2-dipalmitoyi-sn-Glycero-3-[Phospho-rac-(1-glycerol)] (Sodium Salt) (DPPG) in layer-by-layer (LbL) films, alternated with poly (amidoamine) G4 (PAMAM) dendrimer layers. The average size of the liposomes in solution was 120 nm as determined by dynamic light scattering, with their spherical shape being inferred from scanning electron microscopy (SEM) in cast films. LbL films containing up to 20 PAMAM/DPPG bilayers were assembled onto glass and/or silicon wafer substrates. The growth of the multilayers was achieved by alternately immersing the substrates into the PAMAM and DPPG solutions for 5 and 10 min, respectively. The formation of PAMAM/DPPG liposome multilayers and its ability to interact with BSA were confirmed by Fourier transform infrared spectroscopy (FTIR). The structural features and film thickness were obtained using X-ray diffraction and surface plasmon resonance (SPR). (c) 2007 Elsevier B.V. All rights reserved

Silk fibroin-antigenic peptides-YVO4:Eu3+ nanostructured thin films as sensors for hepatitis C

Nanostructured films prepared by Layer-by-Layer technique and containing silk fibroin, antigenic peptide NS5A-1 derived from hepatitis C virus (HCV) NS5A protein and YVO4:Eu3+ luminescent nanoparticles, were utilized in sensing of hepatitis C. Detection system exploits the biorecognition between the antibody anti-HCV and the antigenic peptide NS5A-1 through changes in luminescence properties. Films deposition was monitored by UV-vis Absorption and Fluorescence Spectroscopy measurements at each bilayer deposited. The Eu3+ luminescence properties were evaluated in the presence of anti-HCV for optical detection of specific antibody and anti-HIV used as negative control. Significant changes in luminescence were observed in the presence of anti-HCV concentrations. A new immunosensor platform is proposed for optical detection of hepatitis C. (C) 2015 Elsevier B.V. All rights reserved.Brazilian funding agency CNPqBrazilian funding agency FAPESPNanobiotec-CAPES network (Brazil)Sao Paulo State Univ, Inst Chem, UNESP, BR-14801970 Araraquara, SP, BrazilUniv Fed Sao Paulo, Inst Sci & Technol, UNIFESP, BR-12231280 Sao Jose Dos Campos, BrazilUniv Fed Sao Paulo, Inst Sci & Technol, UNIFESP, BR-12231280 Sao Jose Dos Campos, BrazilWeb of Scienc

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

A direct, low-cost method to determine the concentration of lactose is an important goal with possible impact in various types of industry. in this study, a biosensor is reported that exploits the specific interaction between lactose and the enzyme beta-galactosidase (beta-Gal) normally employed to process lactose into glucose and galactose for lactose-intolerant people. the biosensor was made with beta-Gal immobilized in layer-by-layer (LbL) films with the polyelectrolyte poly(ethylene imine) (PEI) and poly(vinyl sufonate) (PVS) on an indium tin oxide (ITO) electrode modified with a layer of Prussian Blue (PB). With an ITO/PB/(PEI/PVS)(1)(PEI/beta-Gal)(30) architecture, lactose could be determined with an amperometric method with sensitivity of 0.31 mu A mmol(-1) cm(-2) and detection limit of 1.13 mmol L-1, which is sufficient for detecting lactose in milk and for clinical exams. Detection occurred via a cascade reaction involving glucose oxidase titrated as electrolytic solution in the electrochemical cell, while PB allowed for operation at 0.0 V versus saturated calomel electrode, thus avoiding effects from interfering species. Sum-frequency generation spectroscopy data for the interface between the LbL film and a buffer containing lactose indicated that beta-Gal lost order, which is the first demonstration of structural effects induced by the molecular recognition interaction with lactose.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)nBioNet network (Brazil)Univ Fed Sao Carlos, UFSCar, BR-18052780 Sao Carlos, SP, BrazilUniversidade Federal de São Paulo, Inst Ciencia & Tecnol, Unifesp, BR-12231280 Sao Jose Dos Campos, SP, BrazilUniv São Paulo, Sao Carlos Inst Phys, BR-13560970 Sao Carlos, SP, BrazilUniversidade Federal de São Paulo, Inst Ciencia & Tecnol, Unifesp, BR-12231280 Sao Jose Dos Campos, SP, BrazilFAPESP: 2012/16158-0Web of Scienc