RESUMEN
In the development of colorimetric biosensors, the use of electrochromic mediators has been accepted and widely used during decades. The main drawback of these types of enzymatic substrates is the difficult recovery of the initial redox state of the molecule, which can be done electrochemically or by antioxidants addition, complicating the initially simple structure of the biosensor. those strategies are rarely followed Actually, being the disposable biosensor configuration the most extended for this detection mechanisms. Alternatively, we propose the first reported use of a diacid dithienylethene 1,2-bis(5-carboxy-2-methylthien-3-yl)cyclopentene (DTE) photoelectrochromic compound as a substrate of the horseradish peroxidase (HRP). The photoisomerization between the open (DTEo) and closed (DTEc) forms of the molecule and the respective shift in the redox potential allowed the light-induced enzymatic detection of glucose in the glucose oxidase [(GOx)]-HRP cascade system. This fast and easy control over the enzymatic substrate availability by light pulses permits a gradually consumption and the light-regeneration of the biosensor for a number of cycles. We consider the presented results transcendent in the development of reusable and light-controlled photonic biosensing systems.
Asunto(s)
Glucosa Oxidasa/metabolismo , Glucosa/química , Peroxidasa de Rábano Silvestre/metabolismo , Técnicas Biosensibles/métodos , Colorimetría/métodos , Glucosa Oxidasa/química , Peroxidasa de Rábano Silvestre/química , Oxidación-ReducciónRESUMEN
The inhibitor of DNA binding and cell differentiation 2 (Id2) is a helix-loop-helix (HLH) protein that acts as negative dominant regulator of basic-HLH transcription factors during development and in cancer. The structural properties of Id2 have been investigated so far by using synthetic or recombinant fragments reproducing single domains (N-terminus, HLH, C-terminus): the HLH domain tends to dimerize into a four-helix bundle, whereas the flanking regions are flexible. In this work, the intact protein was expressed in E. coli, solubilized from inclusion bodies with urea, purified and dissolved in water at pH~4. Under these conditions, Id2 was obtained with both cysteine residues disulfide-bonded to ß-mercaptoethanol that was present during the solubilization process. Moreover, it existed in a self-assembled state, in which the N-terminus remained highly flexible, while the HLH domain and, surprisingly, part of the C-terminus, which corresponds to the nuclear export signal (NES), both were involved in slowly tumbling, rigid structures. The protein oligomers also formed twisted fibrils that were several micrometers long and up to 80 nm thick. These results show that self-assembly decreases the backbone flexibility of those two protein regions (HLH and NES) that are important for interaction with basic-HLH transcription factors or for nucleocytoplasmic shuttling.
Asunto(s)
Escherichia coli/crecimiento & desarrollo , Proteína 2 Inhibidora de la Diferenciación/química , Proteína 2 Inhibidora de la Diferenciación/genética , Transporte Activo de Núcleo Celular , Dicroismo Circular , Escherichia coli/genética , Escherichia coli/metabolismo , Secuencias Hélice-Asa-Hélice , Humanos , Cuerpos de Inclusión/química , Cuerpos de Inclusión/metabolismo , Proteína 2 Inhibidora de la Diferenciación/metabolismo , Modelos Moleculares , Señales de Exportación Nuclear , Multimerización de Proteína , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Protein adsorption at nanostructured oxides strongly depends on the synthesis conditions and sample history of the material investigated. We measured the adsorption of bovine serum albumin (BSA) to commercial Aeroxide TiO2 P25 nanoparticles in aqueous dispersions. Significant changes in the adsorption capacity were induced by mild sample washing procedures and attributed to the structural modification of adsorbed water and surface hydroxyls. Motivated by the lack of information about the sample history of commercial TiO2 nanoparticle samples, we used vapor-phase-grown TiO2 nanoparticles, a well-established model system for adsorption and photocatalysis studies, and performed on this material for the first time a systematic and quantitative BSA adsorption study. After alternating vacuum and oxygen treatment of the nanoparticle powders at elevated temperatures for surface purification, we determined size distributions covering both the size of the individualized nanoparticles and nanoparticle agglomerates using transmission electron microscopy (TEM), X-ray diffraction (XRD), and dynamic light scattering (DLS) in an aqueous dispersion. Quantitative BSA adsorption measurements at different pH values and thus variable combinations of surface-charged proteins and TiO2 nanoparticles revealed a consistent picture: BSA adsorbs only at the outer agglomerate surfaces without penetrating the interior of the agglomerates. This process levels at coverages of single monolayers, which resist consecutive simple washing procedures. A detailed analysis of the protein-specific IR amide bands reveals that the adsorption-induced protein conformational change is associated with a decrease in the helical content. This study underlines that robust qualitative and quantitative statements about protein adsorption and corona formation require well-documented and controllable surface properties of the nanomaterials involved.
RESUMEN
BACKGROUND: Activity retention upon enzyme adsorption on inorganic nanostructures depends on different system parameters such as structure and composition of the support, composition of the medium as well as enzyme loading. Qualitative and quantitative characterization work, which aims at an elucidation of the microscopic details governing enzymatic activity, requires well-defined model systems. RESULTS: Vapor phase-grown and thermally processed anatase TiO2 nanoparticle powders were transformed into aqueous particle dispersions and characterized by dynamic light scattering and laser Doppler electrophoresis. Addition of ß-galactosidase (ß-gal) to these dispersions leads to complete enzyme adsorption and the generation of ß-gal/TiO2 heteroaggregates. For low enzyme loadings (~4% of the theoretical monolayer coverage) we observed a dramatic activity loss in enzymatic activity by a factor of 60-100 in comparison to that of the free enzyme in solution. Parallel ATR-IR-spectroscopic characterization of ß-gal/TiO2 heteroaggregates reveals an adsorption-induced decrease of the ß-sheet content and the formation of random structures leading to the deterioration of the active site. CONCLUSIONS: The study underlines that robust qualitative and quantitative statements about enzyme adsorption and activity retention require the use of model systems such as anatase TiO2 nanoparticle agglomerates featuring well-defined structural and compositional properties.
Asunto(s)
Nanopartículas/química , Titanio/química , beta-Galactosidasa/química , Adsorción , Activación Enzimática , Concentración de Iones de Hidrógeno , Microscopía Electrónica de Rastreo , Tamaño de la Partícula , Porosidad , Propiedades de Superficie , AguaRESUMEN
In biosensor development, silk fibroin is advantageous for providing transparent, flexible, chemically/mechanically stable, biocompatible, and sustainable substrates, where the biorecognition element remains functional for long time periods. These properties are employed here in the production of point-of-care biosensors for resource-limited regions, which are able to display glucose levels without the need for external instrumentation. These biosensors are produced by photopatterning silk films doped with the enzymes glucose oxidase and peroxidase and photoelectrochromic molecules from the dithienylethene family acting as colorimetric mediators of the enzymatic reaction. The photopatterning results from the photoisomerization of dithienylethene molecules in the silk film from its initial uncolored opened form to its pink closed one. The photoisomerization is dose-dependent, and colored patterns with increasing color intensities are obtained by increasing either the irradiation time or the light intensity. In the presence of glucose, the enzymatic cascade reaction is activated, and peroxidase selectively returns closed dithienylethene molecules to their initial uncolored state. Color disappearance in the silk film is proportional to glucose concentration and used to distinguish between hypoglycemic (below 4 mM), normoglycemic (4-6 mM), and hyperglycemic levels (above 6 mM) by visual inspection. After the measurement, the biosensor can be regenerated by irradiation with UV light, enabling up to five measurement cycles. The coupling of peroxidase activity to other oxidoreductases opens the possibility to produce long-life reusable smart biosensors for other analytes such as lactate, cholesterol, or ethanol.
Asunto(s)
Técnicas Biosensibles , Seda , Seda/química , Colorimetría/métodos , Peroxidasas , Técnicas Biosensibles/métodos , Peroxidasa , GlucosaRESUMEN
In optical biosensing, silk fibroin (SF) appears as a promising alternative where other materials, such as paper, find limitations. Besides its excellent optical properties and unmet capacity to stabilize biomacromolecules, SF in test strips exhibits additional functions, i.e. capillary pumping activity of 1.5 mm s-1, capacity to filter blood cells thanks to its small, but tuneable, porosity and enhanced biosensing sensitivity. The bulk functionalization of SF with the enzymes glucose oxidase and peroxidase and the mediator ABTS produces colourless and transparent SF films that respond to blood glucose increasing 2.5 times the sensitivity of conventional ABTS-based assays. This enhanced sensitivity results from the formation of SF-ABTS complexes, where SF becomes part of the bioassay. Additionally, SF films triple the durability of most stable cellulose-based sensors. Although demonstrated for glucose, SF microfluidic test strips may incorporate other optical bioassays, e.g. immunoassays, with the aim of transferring them from central laboratories to the place of patient's care.
Asunto(s)
Glucemia/análisis , Fibroínas , Nanoporos , Acción Capilar , HumanosRESUMEN
At the point of care (POC), on-side clinical testing allows fast biomarkers determination even in resource-limited environments. Current POC systems rely on tests selective to a single analyte or complex multiplexed systems with important portability and performance limitations. Hence, there is a need for handheld POC devices enabling the detection of multiple analytes with accuracy and simplicity. Here we present a reconfigurable smartphone-interfaced electrochemical Lab-on-a-Chip (LoC) with two working electrodes for dual analyte determination enabling biomarkers' selection in situ and on-demand. Biomarkers selection was achieved by the use of electrodepositable alginate hydrogels. Alginate membranes containing either glucose oxidase (GOx) or lactate oxidase (LOx) were selectively electrodeposited on the surface of each working electrode in around 4â¯min, completing sample measurement in less than 1â¯min. Glucose and lactate determination was performed simultaneously and without cross-talk in buffer, fetal bovine serum (FBS) and whole blood samples, the latter being possible by the size-exclusion filtration capacity of the hydrogels. At optimal conditions, glucose and lactate were determined in a wide linear range (0-12â¯mM and 0-5â¯mM, respectively) and with high sensitivities (0.24 and 0.54⯵Aâ¯cm-2 mM-1, respectively), which allowed monitoring of Type-1 diabetic patients with a simple dual analysis system. After the measurement, membranes were removed by disaggregation with the calcium-chelator phosphate buffer. At this point, new membranes could be electrodeposited, this time being selective to the same or another analyte. This conferred the system with on-demand biomarkers' selection capacity. The versatility and flexibility of the current architecture is expected to impact in POC analysis in applications ranging from homecare to sanitary emergencies.
Asunto(s)
Diabetes Mellitus Tipo 1/sangre , Dispositivos Laboratorio en un Chip , Teléfono Inteligente , Alginatos , Animales , Glucemia/análisis , Humanos , Hidrogel de Polietilenoglicol-Dimetacrilato , Ácido Láctico/sangre , Masculino , Ratones , Ratones Endogámicos C57BL , Sistemas de Atención de Punto , Distribución AleatoriaRESUMEN
The determination of ethanol intoxication in whole blood samples may open the opportunity for a precise and quick point-of-measurement in the ambit of medical emergency or law enforcement. In contrast with traditional techniques based on breath sampling, direct blood measurements present greater immunity to errors specially in case of unconscious or non-collaborative patients. In this context, a portable, sensitive and easy-to-use instrument is highly desirable. In the current work we present a smartphone-based µPotentiostat which combines a novel circuital technique for sensor readout digitalization with a reusable lab-on-a-chip (LoC) concept. Such system allows both chronoamperometric and cyclic voltammetry measurements with a reduced number of electronic components on a very compact PCB (38.5â¯× 22.5â¯mm2). Power, data-link and user interface are provided in combination with a standard smartphone, enabling cost-effectiveness and reconfigurability without sacrificing precision. The readout platform discussed in this work has been coupled to a LoC for point-of-care combining Pt electrodes microfabricated on silicon substrate for electrochemical measurement and a microfluidic structure of methacrylate for fluid management. Biosensing is enabled by in situ electrodeposition of a calcium alginate hydrogel containing horseradish peroxidase (HPR) and alcohol oxidase (AOx) for selective ethanol detection. Alginate membrane electrodeposition has been here optimized for rapid generation (2â¯min) and to retain the cellular fraction, thus allowing the measurement in whole blood samples. The µPotentiostat features a sensitivity of 36â¯nA/gâ¯L-1 to ethanol concentration in blood in the 0-1.25â¯g;L-1 range, with a limit of quantification (LoQ) of 4.5â¯nA, which is a suitable response for discerning the legal, illegal, severely illegal thresholds in a 40⯵L sample of blood.