RESUMEN
Microstructured optical fibers containing microchannels and Bragg grating inscribed were internally functionalized with a peptide nucleic acid (PNA) probe specific for a gene tract of the genetically modified Roundup Ready soy. These fibers were used as an optofluidic device for the detection of DNA by measuring the shift in the wavelength of the reflected IR light. Enhancement of optical read-out was obtained using streptavidin coated gold-nanoparticles interacting with the genomic DNA captured in the fiber channels (0%, 0.1%, 1% and 10% RR-Soy), enabling to achieve statistically significant, label-free, and amplification-free detection of target DNA in low concentrations, low percentages, and very low sample volumes. Computer simulations of the fiber optics based on the finite element method (FEM) were consistent with the formation of a layer of organic material with an average thickness of 39 nm for the highest percentage (10% RR soy) analysed.
Asunto(s)
ADN/análisis , ADN/genética , Tecnología de Fibra Óptica/instrumentación , Técnicas Analíticas Microfluídicas/instrumentación , Ácidos Nucleicos de Péptidos/genética , Refractometría/instrumentación , Técnicas Biosensibles/instrumentación , Mapeo Cromosómico/instrumentación , ADN/química , Diseño de Equipo , Análisis de Falla de Equipo , Miniaturización , Técnicas de Amplificación de Ácido Nucleico , Dispositivos Ópticos , Ácidos Nucleicos de Péptidos/químicaRESUMEN
We report a novel approach to genotyping single nucleotide polymorphisms (SNPs) using molecular beacons in conjunction with a suspended core optical fiber (SCF). Target DNA sequences corresponding to the wild- or mutant-type have been accurately recognized by immobilizing two different molecular beacons on the core of a SCF. The two molecular beacons differ by one base in the loop-probe and utilize different fluorescent indicators. Single-color fluorescence enhancement was obtained when the immobilized SCFs were filled with a solution containing either wild-type or mutant-type sequence (homozygous sample), while filling the immobilized SCF with solution containing both wild- and mutant-type sequences resulted in dual-color fluorescence enhancement, indicating a heterozygous sample. The genotyping was realized amplification-free and with ultra low-volume for the required DNA solution (nano-liter). This is, to our knowledge, the first genotyping device based on the combination of optical fiber and molecular beacons.
Asunto(s)
ADN/genética , Genotipo , Técnicas de Genotipaje/métodos , Polimorfismo de Nucleótido Simple/genética , Fluorescencia , Fibras ÓpticasRESUMEN
We describe a novel sensing approach based on a functionalized microstructured optical fiber-Bragg grating for specific DNA target sequences detection. The inner surface of a microstructured fiber, where a Bragg grating was previously inscribed, has been functionalized by covalent linking of a peptide nucleic acid probe targeting a DNA sequence bearing a single point mutation implicated in cystic fibrosis (CF) disease. A solution of an oligonucleotide (ON) corresponding to a tract of the CF gene containing the mutated DNA has been infiltrated inside the fiber capillaries and allowed to hybridize to the fiber surface according to the Watson-Crick pairing. In order to achieve signal amplification, ON-functionalized gold nanoparticles were then infiltrated and used in a sandwich-like assay. Experimental measurements show a clear shift of the reflected high order mode of a Bragg grating for a 100 nM DNA solution, and fluorescence measurements have confirmed the successful hybridization. Several experiments have been carried out on the same fiber using the identical concentration, showing the same modulation trend, suggesting the possibility of the reuse of the sensor. Measurements have also been made using a 100 nM mismatched DNA solution, containing a single nucleotide mutation and corresponding to the wild-type gene, and the results demonstrate the high selectivity of the sensor.
Asunto(s)
Técnicas Biosensibles/instrumentación , Técnicas Biosensibles/métodos , ADN/análisis , Tecnología de Fibra Óptica/instrumentación , Ácidos Nucleicos de Péptidos/química , Fibrosis Quística/genética , ADN/química , ADN/genética , ADN/metabolismo , Oro/química , Humanos , Nanopartículas del Metal/química , Modelos Genéticos , Fibras Ópticas , Ácidos Nucleicos de Péptidos/metabolismo , Mutación Puntual , Análisis EspectralRESUMEN
We report on the surface characterization, functionalization, and application of stable water suspensions of novel surface active maghemite nanoparticles (SAMNs), characterized by a diameter of 11 ± 2 nm and possessing peculiar colloidal properties and surface interactions. These features permitted the acquisition of titration curves and aqueous UV-vis spectra and suggested a role played by surface under-coordinated iron atoms. This new class of nanoparticles was obtained through an easy, inexpensive, one-step, green procedure and functionalized with ligands of high biotechnological interest, such as biotin and avidin, by simple incubation in aqueous solution. Bound avidin was determined by measuring the disappearance of free avidin absorbance at 280 nm, as a function of increasing nanoparticle concentration, showing the presence of 10 ± 3 avidin molecules per nanoparticle. The biological activity of the SAMN@avidin complex was evaluated and the number of available biotin binding sites was determined, using biotinyl-fluorescein as a probe, showing that each bound avidin molecule is able to bind 2.8 ± 0.8 biotin molecules, confirming the maintenance of biological activity and excellent binding capacity of the SAMN@avidin complex. Furthermore a Langmuir isotherm model was used to describe the biomolecule specific monolayer adsorption onto the particle surface, and in the case of avidin, the maximum adsorption capacity was 100 ± 27 µg avidin/mg SAMN, whereas the binding constant is 45.18 µL µg(-1). The SAMN@avidin complex was characterized by UV-vis spectroscopy, quartz crystal microbalance, FTIR spectroscopy, and transmission electron microscopy. Finally, SAMN@avidin was applied for the large scale purification of recombinant biotinylated human sarco/endoplasmic reticulum Ca(2+)-ATPase (hSERCA-2a), expressed by Saccharomyces cerevisiae. The protein was magnetically purified, and about 500 µg of a 70% pure hSERCA-2a were recovered from 4 L of yeast culture, with a purification yield of 64%.
Asunto(s)
Avidina/química , Avidina/metabolismo , Biotina/metabolismo , Compuestos Férricos/química , Nanopartículas/química , Proteínas Recombinantes/aislamiento & purificación , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/aislamiento & purificación , Biotina/química , Humanos , Modelos Moleculares , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/química , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Propiedades de SuperficieRESUMEN
The SERCA pump, a membrane protein of about 110kDa, transports two Ca(2+) ions per ATP hydrolyzed from the cytoplasm to the lumen of the sarcoplasmic reticulum. In muscle cells, its ability to remove Ca(2+) from the cytosol induces relaxation. The transport mechanism employed by the enzyme from rabbit muscle has been extensively studied, and several crystal structures representing different conformational states are available. However, no structure of the pump from other sources is known. In this paper we describe the crystal structure of the bovine enzyme, crystallized in the E1 conformation and determined at 2.9Å resolution. The overall molecular model is very similar to that of the rabbit enzyme, as expected by the high amino acid sequence identity. Nevertheless, the bovine enzyme has reduced catalytic activity with respect to the rabbit enzyme. Subtle structural modifications, in particular in the region of the long loop that protrudes into the SR lumen connecting transmembrane α-helices M7 and M8, may explain the difference.