RESUMO
Conductometric monitoring of protein-protein and protein-sterol interactions is here proved feasible by coupling quartz crystal microbalance with dissipation monitoring (QCM_D) to nucleic acid programmable protein arrays (NAPPA). The conductance curves measured in NAPPA microarrays printed on quartz surface allowed the identification of binding events between the immobilized proteins and the query. NAPPA allows the immobilization on the quartz surface of a wide range of proteins and can be easily adapted to generate innumerous types of biosensors. Indeed multiple proteins on the same quartz crystal have been tested and envisaged proving the possibility of analyzing the same array for several distinct interactions. Two examples of NAPPA-based conductometer applications with clinical relevance are presented herein, the interaction between the transcription factors Jun and ATF2 and the interaction between Cytochrome P540scc and cholesterol.
Assuntos
Fator 2 Ativador da Transcrição/química , Técnicas Biossensoriais , Enzima de Clivagem da Cadeia Lateral do Colesterol/química , Proteínas Proto-Oncogênicas c-jun/química , Fator 2 Ativador da Transcrição/genética , Fator 2 Ativador da Transcrição/metabolismo , Anticorpos/química , Anticorpos/metabolismo , Colesterol/química , Colesterol/metabolismo , Enzima de Clivagem da Cadeia Lateral do Colesterol/genética , Enzima de Clivagem da Cadeia Lateral do Colesterol/metabolismo , Condutometria , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Proteínas Imobilizadas/química , Proteínas Imobilizadas/genética , Proteínas Imobilizadas/metabolismo , Análise Serial de Proteínas , Ligação Proteica , Mapeamento de Interação de Proteínas , Proteínas Proto-Oncogênicas c-jun/genética , Proteínas Proto-Oncogênicas c-jun/metabolismo , Técnicas de Microbalança de Cristal de Quartzo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Propriedades de SuperfícieRESUMO
This paper describes the optimal implementation of three newly conceived sensors for both health and environmental applications, utilizing a wide range of detection methods and complex nanocomposites. The first one is inorganic and based on matrices of calcium oxide, the second is based on protein arrays and a third one is based on Langmuir-Blodgett laccase multi-layers. Special attention was paid to detecting substances significant to the environment (such as carbon dioxide) and medicine (drug administration, cancer diagnosis and prognosis) by means of amperometric, quartz crystal microbalance with frequency (QCM_F) and quartz crystal microbalance with dissipation monitoring (QCM_D) technologies. The resulting three implemented nanosensors are described here along with proofs of principle and their corresponding applications.
Assuntos
Técnicas Biossensoriais , Dióxido de Carbono/isolamento & purificação , Nanocompostos/química , Nanotecnologia , Compostos de Cálcio/química , Monitoramento Ambiental , Humanos , Lacase/química , Óxidos/química , Técnicas de Microbalança de Cristal de QuartzoAssuntos
DNA Complementar/química , Análise Serial de Proteínas/métodos , Proteínas/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Sequência de Aminoácidos , DNA Complementar/metabolismo , Humanos , Dados de Sequência Molecular , Proteínas/genética , Proteína Supressora de Tumor p53/química , Proteína Supressora de Tumor p53/genéticaAssuntos
Fibroblastos/citologia , Fibroblastos/metabolismo , Proteoma/metabolismo , Animais , Células CHO , Diferenciação Celular/efeitos dos fármacos , Cromatina/efeitos dos fármacos , Cromatina/metabolismo , Cricetinae , Cricetulus , AMP Cíclico/farmacologia , Fibroblastos/efeitos dos fármacos , Cadeias Pesadas de Miosina/metabolismo , Miosina não Muscular Tipo IIB/metabolismoRESUMO
Chinese Hamster Ovary fibroblasts (CHO-K1) have shown different protein contents when undergoing differentiation by 3',5'-cyclic adenosine monophosphate (cAMP), which is known to induce reverse transformation (RT) from malignancy to fibroblast-like characteristics. The mass spectrometry (MS) investigation here reported about the behavior of CHO-K1 cells before and after exposure to cAMP reveals a change in the composition of nuclear proteins associated to an inhibition of the protein expression. Possible implications of this finding on the control of cell reverse transformation are discussed.
Assuntos
AMP Cíclico/farmacologia , Proteínas Nucleares/metabolismo , Animais , Células CHO , Cromatografia Líquida de Alta Pressão/métodos , Cricetinae , Cricetulus , Membrana Nuclear/metabolismo , Espectrometria de Massas por Ionização por Electrospray/métodosRESUMO
Human lymphocytes gene expression before and after PHA stimulation is monitored by DNASER technology, a novel bioinstrumentation entirely constructed in our laboratories as previously reported. The validity of the DNASER measurements is confirmed by standard fluorescence microscopy equipped with CCD. The human lymphocytes gene expression here experimentally probed using commercially available DNA microarrays such as Human Starter, appears compatible both with independent bioinformatic prediction and with existing experimental data, pointing to MYC as the key gene in the G0-G1 transition induced by PHA in resting lymphocytes. It does not escape our notice that in cell biology and cancer research DNASER technology based on microarray constructed with few leader genes identified from bioinformatics represents a meaningful cost-effective route alternative to massive frequently misleading molecular genomics.