RESUMO
In biochip development two issues are critical: stable and specific immobilization of the ligand and achievement of high signal-to-background ratio. In this work we have addressed these issues for the development of biochips, produced by sputtering multilayers of thin metal films, metal oxides, and metal nitrides (tens to hundreds of nanometers thick) onto glass wafers. Optimized surfaces have shown good results in genomic and proteomic experiments with biochips based on surface-enhanced fluorescence and absorption techniques.
Assuntos
Materiais Revestidos Biocompatíveis/química , Cristalização/métodos , Metais/química , Nanotecnologia/instrumentação , Nanotubos/química , Nanotubos/ultraestrutura , Análise de Sequência com Séries de Oligonucleotídeos/instrumentação , Análise Serial de Proteínas/instrumentação , Adsorção , Materiais Revestidos Biocompatíveis/síntese química , Desenho de Equipamento , Análise de Falha de Equipamento , Gases , Temperatura Alta , Teste de Materiais , Membranas Artificiais , Nanotecnologia/métodos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Análise Serial de Proteínas/métodos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Propriedades de SuperfícieRESUMO
The combination of phage display antibody arrays with a novel nanotransducer technique based on resonant nanoparticles in a nanosandwiched film enables the sensitive parallel screening of proteins. Using the resonance of nanoparticles with their induced mirror dipoles in a thin-film structure, limitations of fluorophores, such as unspecific background and nonvisibility to the eye, can be overcome, thereby leading to an optical signal significantly more sensitive than that of standard colloid techniques. The signal can be both directly observed as a color change of a microdot at the sensor surface and tuned throughout the visible range of the spectrum. Here we report the application of an optical chip using scFv-antibody-antigen interactions. Artificial scFv-antibodies against a variety of proteins, including yeast enzymes and bovine serum albumin (as a standard), were constructed via Phage Display. These scFv-antibodies were then coated onto metal nanoclusters and bound to their antigens that were arrayed as nanodroplets at the resonance layer of the chip. ScFv-Antibody-antigen interaction resulted in a visible array of microdots. Using resonance-enhanced absorption, the absorption signal of the spots was amplified by one to two orders of magnitude (compared to colloid-based techniques). For quantitative analysis, either an 8-micron scanner or a CCD camera (resolution 4 microns) was employed to gain direct-reflection spectra rather than unspecific scatter data (prone to dust and unspecific interaction). Our results demonstrate that this device enables high-throughput proteomics to overcome some limitations of fluorescence, enzyme labels, and colloid techniques.