Nugaeva Natalia, Gfeller Karin Y, Backmann Natalija, Lang Hans Peter, Düggelin Marcel, Hegner Martin
Institute of Physics, University of Basel, Klingelbergstrasse 82, Switzerland.
Biosens Bioelectron. 2005 Dec 15;21(6):849-56. doi: 10.1016/j.bios.2005.02.004.
We demonstrate the use of micromechanical cantilever arrays for selective immobilization and fast quantitative detection of vital fungal spores. Micro-fabricated uncoated as well as gold-coated silicon cantilevers were functionalized with concanavalin A, fibronectin or immunoglobulin G. In our experiments two major morphological fungal forms were used--the mycelial form Aspergillus niger and the unicellular yeast form Saccharomyces cerevisiae, as models to explore a new method for growth detection of eukaryotic organisms using cantilever arrays. We exploited the specific biomolecular interactions of surface grafted proteins with the molecular structures on the fungal cell surface. It was found that these proteins have different affinities and efficiencies to bind the spores. Maximum spore immobilization, germination and mycelium growth was observed on the immunoglobulin G functionalized cantilever surfaces. We show that spore immobilization and germination of the mycelial fungus A. niger and yeast S. cerevisiae led to shifts in resonance frequency within a few hours as measured by dynamically operated cantilever arrays, whereas conventional techniques would require several days. The biosensor could detect the target fungi in a range of 10(3) - 10(6) CFUml(-1). The measured shift is proportional to the mass of single fungal spores and can be used to evaluate spore contamination levels. Applications lie in the field of medical and agricultural diagnostics, food- and water-quality monitoring.
我们展示了微机械悬臂阵列用于重要真菌孢子的选择性固定和快速定量检测。微加工的未涂层以及金涂层硅悬臂用伴刀豆球蛋白A、纤连蛋白或免疫球蛋白G进行功能化。在我们的实验中,使用了两种主要形态的真菌——丝状的黑曲霉和单细胞酵母形式的酿酒酵母,作为利用悬臂阵列探索真核生物生长检测新方法的模型。我们利用了表面接枝蛋白与真菌细胞表面分子结构之间的特定生物分子相互作用。发现这些蛋白结合孢子具有不同的亲和力和效率。在免疫球蛋白G功能化的悬臂表面观察到最大的孢子固定、萌发和菌丝生长。我们表明,黑曲霉和酿酒酵母的孢子固定和萌发会导致动态操作的悬臂阵列在几小时内测量到共振频率的变化,而传统技术则需要几天时间。该生物传感器能够检测10³ - 10⁶ CFUml⁻¹范围内的目标真菌。测量到的频率变化与单个真菌孢子的质量成正比,可用于评估孢子污染水平。其应用领域包括医学和农业诊断、食品和水质监测。