RESUMEN
Three-dimensional interdigitated electrodes (IDEs) have been investigated as sensing elements for biosensors. Electric field and current density were simulated in the vicinity of these electrodes as a function of the electrode width, gap, and height to determine the optimum geometry. Both the height and the gap between the electrodes were found to have significant effect on the magnitude and distribution of the electric field and current density near the electrode surface, while the width of the electrodes was found to have a smaller effect on field strength and current density. IDEs were fabricated based on these simulations and their performance tested by detecting C-reactive protein (CRP), a stress-related protein and an important biomarker for inflammation, cardiovascular disease risk indicator, and postsurgical recuperation. CRP-specific antibodies were immobilized on the electrode surface and the formation of an immunocomplex (IC) with CRP was monitored. Electrochemical impedance spectroscopy (EIS) was employed as the detection technique. EIS data at various concentrations (1 pg/mL to 10 microg/mL) of CRP spiked in buffer or diluted human serum was collected and fitted into an equivalent electrical circuit model. Change in resistance was found to be the parameter most sensitive to change in CRP concentration. The sensor response was linear from 0.1 ng/mL to 1 microg/mL in both buffer and 5% human serum samples. The CRP samples were validated using a commercially available ELISA for CRP detection. Hence, the viability of IDEs and EIS for the detection of serum biomarkers was established without using labeled or probe molecules.
Asunto(s)
Técnicas Biosensibles/métodos , Nanoestructuras/química , Técnicas Biosensibles/instrumentación , Proteína C-Reactiva/análisis , Impedancia Eléctrica , Electroquímica , Ensayo de Inmunoadsorción Enzimática , Oro/química , Humanos , Microelectrodos , Tamaño de la Partícula , Propiedades de SuperficieRESUMEN
Haptens are low-molecular-weight compounds that are usually nonimmunogenic in nature. These compounds are, in general, conjugated with carrier proteins to elicit an immune response for antibody production. In this work, we report the effect of multiple hapten loading on carrier protein after conjugation by monitoring the structural and immunogenic properties of the protein. Biochemical conjugation of carboxylated hapten (atrazine derivative) to bovine serum albumin via epsilon-amino groups of lysine residues was monitored by the intrinsic fluorescence intensity of tryptophan residues of protein. A significant blue shift of emission maxima confirmed the conformational changes with increasing molar ratio of hapten:protein. Circular dichroism spectroscopy suggested a decreasing trend for alpha-helical and increased formation of beta-sheet structures in hapten-loaded protein. A further insight was sought by using molecular modeling methods for understanding of structural changes in the native protein post-hapten conjugation. A sequential approach for hapten loading on the carrier confirmed that initial binding could affect the possible binding sites for subsequent incorporation of hapten molecules. These changes play a major role in the immunogenic response of hapten-carrier conjugate. The approach taken to develop this model is promising, and can be generalized for studies with other protein-hapten combinations.
Asunto(s)
Proteínas Portadoras/química , Haptenos/química , Secuencia de Aminoácidos , Animales , Atrazina/análogos & derivados , Atrazina/química , Bovinos , Dicroismo Circular , Epítopos/química , Epítopos/genética , Lisina/química , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Estructura Secundaria de Proteína , Albúmina Sérica Bovina/química , Albúmina Sérica Bovina/genéticaRESUMEN
This paper proposes an alternative solution to multimetal deposition (MMD) for the development of latent fingermarks on non-porous and porous surfaces. MMD offers a good sensitivity, however it is very time-consuming and requires many reagents to be carried out. Single-metal deposition (SMD) replaces the silver enhancement of the gold colloids by a gold enhancement procedure. This reduces the number of baths by one as well as the number of reagents and their cost, utilizes reagents with a longer shelf life, and most importantly reduces the labor-intensity of the procedure. It offers quasi-identical results to MMD and thus makes a very attractive alternative.
Asunto(s)
Dermatoglifia , Oro Coloide , Compuestos de Oro , Hidroxilamina , Costos y Análisis de Costo , Humanos , Procesamiento de Imagen Asistido por Computador , Fotograbar , Propiedades de SuperficieRESUMEN
Development of immunobiosensor detector surfaces involves the immobilization of active antibodies on the capture surface without any significant loss of antigen binding activity. An atomic force microscope (AFM) was used to directly evaluate specific interactions between pesticides and antibodies on a biosensor surface. Oriented immobilization of antibodies against two herbicide molecules 2,4-dichlorophenoxyacetic acid (2,4-D) and atrazine, on gold, was carried out to create the active immunobiosensor surfaces. The adhesive forces between immobilized antibodies and their respective antigens were measured by force spectroscopy using hapten-carrier protein functionalized AFM cantilevers. Relative functional affinity (avidity) measurements of the antibodies carried out prior to immobilization, well correlated with subsequent AFM force measurement observations. Analysis showed that immobilization had not compromised the reactivity of the surface immobilized antibody molecules for antigen nor was there any change in their relative quality with respect to each other. The utility of the immunoreactive surface was further confirmed using a Surface Plasmon Resonance (SPR) based detection system. Our study indicates that AFM can be utilized as a convenient immunobiosensing tool for confirming the presence and also assessing the strength of antibody-hapten interactions on biosensor surfaces under development.