RESUMEN
Mass spectrometry (MS) based methodology offers simple, fast and sensitive diagnosis. While it has become the predominate approach in biomolecular analysis, it has not been suitable for analyzing nucleic acid due to its low ionization efficiency. We report herein on a DNA assay based on monolayer-barcoded nanoparticles that were encoded with reporter mass molecules, which act as surrogate molecules for the matrix-assisted laser desorption/ionization time-of-flight MS (MALDI-TOF MS) identification of target DNA through mass spectrometry in combination with magnetic microprobes. This assay demonstrated high MS sensitivity, with the ability to detect target DNA at femtomolar (10(-15) M) levels. This inaugural effort using combined techniques is significant because it showed an extraordinary analytical capability for differentiating the single nucleotide polymorphism (SNP), which comprises the most abundant source of genetic variation in the human genome. We also report herein the feasibility of MS detection of two target DNAs that have the same mass but different nucleotide base composition, which classic MS methodology is inherently unable to differentiate.
Asunto(s)
ADN/análisis , Magnetismo , Nanopartículas del Metal/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Secuencia de Bases , ADN/química , ADN/genética , Sondas de ADN/química , Sondas de ADN/genética , Oro/química , Modelos Químicos , Peso Molecular , Polimorfismo de Nucleótido Simple , Reproducibilidad de los ResultadosRESUMEN
A general method is described to analyze pathogen DNAs in homogenous solution by a novel Bio-MassCode probe, using a small organic (namely disulfide) "reporter mass" that self-assembled in oligonucleotide-modified gold nanoparticles. With conventional MALDI TOF MS, the assay simultaneously detected DNAs for HIV, HBV, HCV, and TP with extraordinarily high sensitivity in the low attomolar (10(-18) M) range without any other amplification.
Asunto(s)
ADN Viral/análisis , ARN Viral/análisis , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Oro/química , VIH/genética , Hepacivirus/genética , Virus de la Hepatitis B/genética , Nanopartículas del Metal/química , Oligonucleótidos/químicaRESUMEN
Single nucleotide polymorphisms (SNPs) represent the most abundant source of genetic variation in the human genome, and they can be linked to genetic susceptibilities or varied pharmaceutical responses. Established SNP detection techniques are mainly PCR-based, which means that they involve complex, labor-intensive procedures, are easy contaminated, and can give false-positive results. Therefore, we have developed a simple and rapid MS-based disulfide barcode methodology that relies on magnifying the signal from a dual-modified gold nanoparticle. This approach permits direct SNP genotyping of total human genomic DNA without the need for primer-mediated enzymatic amplification. Disulfides that are attached to the gold nanoparticle serve as a "barcode" that allows different sequences to be discerned using MS detection. Specificity is based on two sequential oligonucleotide hybridizations, which include two steps: the first is the capture of the target by gene-specific probes immobilized onto magnetic beads; the second is the recognition of gold nanoparticles functionalized with allele-specific oligonucleotides. The sensitivity of this new method reaches down to the 0.1 fM range, thus approaching that of PCR. The feasability of this SNP identification methodology based on an MS-based disulfide barcode assay was demonstrated by applying it to genomic DNA samples representing all possible genotypes of the SNPs G2677T and C3435T in the human MDR1 gene. Due to its great advantage--the ability to perform SNP typing without the use of PCR--the assay was found to be simple, rapid and robust, and so may be highly suited to routine clinical detection as well as basic medical research.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Sondas de ADN/genética , Polimorfismo de Nucleótido Simple , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Espectrometría de Masas en Tándem/métodos , Subfamilia B de Transportador de Casetes de Unión a ATP , Sondas de ADN/química , Oro/química , Humanos , Nanopartículas del Metal/químicaRESUMEN
In the crystal structure of the title compound, C(26)H(33)N(3)O(4), there are two crystallographically independent mol-ecules. The two cyclohexane rings are trans-fused; the ring neighboring the phenyl group is in a half-chair conformation and the other is in a chair conformation. The two nitro groups and the benzene ring of the dinitro-phenyl group are almost coplanar. Intra-molecular N-Hâ¯O hydrogen bonds and weak inter-molecular C-Hâ¯O hydrogen bonds are observed.