Zucolotto Valtencir, Pinto Andressa P A, Tumolo Tathyana, Moraes Marli L, Baptista Maurício S, Riul Antonio, Araújo Ana Paula U, Oliveira Osvaldo N
Instituto de Física de São Carlos, USP, CP 369, 13560-970 So Carlos, SP, Brazil.
Biosens Bioelectron. 2006 Jan 15;21(7):1320-6. doi: 10.1016/j.bios.2005.06.001. Epub 2005 Jul 27.
The detection of aromatic compounds from pesticides and industrial wastewater has become of great interest, since these compounds withstand chemical oxidation and biological degradation, accumulating in the environment. In this work, a highly sensitive biosensor for detecting catechol was obtained with the immobilization of Cl-catechol 1,2-dioxygenase (CCD) in nanostructured films. CCD layers were alternated with poly(amidoamine) generation 4 (PAMAM G4) dendrimer using the electrostatic layer-by-layer (LbL) technique. Circular dichroism (CD) measurements indicated that the immobilized CCD preserved the same conformation as in solution. The thickness of the very first CCD layers in the LbL films was estimated at ca. 3.6 nm, as revealed by surface plasmon resonance (SPR). PAMAM/CCD 10-bilayer films were employed in detecting diluted catechol solutions using either an optical or electrical approach. Due to the mild immobilization conditions employed, especially regarding the pH and ionic strength of the dipping solutions, CCD remained active in the films for periods longer than 3 weeks. The optical detection comprised absorption experiments in which the formation of cis-cis muconic acid, resulting from the reaction between CCD and catechol, was monitored by measuring the absorbance at 260 nm after film immersion in catechol solutions. The electrical detection was carried out using LbL films deposited onto gold-interdigitated electrodes immersed in aqueous solutions at different catechol concentrations. Using impedance spectroscopy in a broad frequency range (1Hz-1kHz), we could detect catechol in solutions at concentrations as low as 10(-10) M.
由于农药和工业废水中的芳香族化合物具有抗化学氧化和生物降解的特性，并在环境中不断累积，因此对其进行检测备受关注。在本研究中，通过将氯代儿茶酚1,2-双加氧酶（CCD）固定在纳米结构薄膜中，制备了一种用于检测儿茶酚的高灵敏度生物传感器。采用静电逐层（LbL）技术，将CCD层与第4代聚（酰胺胺）（PAMAM G4）树枝状大分子交替沉积。圆二色性（CD）测量表明，固定化的CCD保持了与溶液中相同的构象。表面等离子体共振（SPR）显示，LbL薄膜中最初几层CCD的厚度估计约为3.6 nm。采用光学或电学方法，利用PAMAM/CCD 10层双层膜检测稀释的儿茶酚溶液。由于采用了温和的固定化条件，特别是关于浸渍溶液的pH值和离子强度，CCD在薄膜中保持活性超过3周。光学检测包括吸收实验，通过测量薄膜浸入儿茶酚溶液后在260 nm处的吸光度，监测CCD与儿茶酚反应生成的顺-顺粘康酸的形成。电学检测是在不同儿茶酚浓度的水溶液中，使用沉积在金叉指电极上的LbL薄膜进行的。通过在宽频率范围（1Hz - 1kHz）内使用阻抗谱，我们能够检测到浓度低至10^(-10) M的溶液中的儿茶酚。
