Della Ventura Bartolomeo, Iannaccone Marco, Funari Riccardo, Pica Ciamarra Massimo, Altucci Carlo, Capparelli Rosanna, Roperto Sante, Velotta Raffaele
Department of Physics "Ettore Pancini", University of Naples "Federico II", Naples, Italy.
Department of Agriculture, University of Naples "Federico II", Portici, Italy.
PLoS One. 2017 Feb 9;12(2):e0171754. doi: 10.1371/journal.pone.0171754. eCollection 2017.
Biosensor-based detection provides a rapid and low-cost alternative to conventional analytical methods for revealing the presence of the contaminants in water as well as solid matrices. Although important to be detected, small analytes (few hundreds of Daltons) are an issue in biosensing since the signal they induce in the transducer, and specifically in a Quartz-Crystal Microbalance, is undetectable. A pesticide like parathion (M = 292 Da) is a typical example of contaminant for which a signal amplification procedure is desirable.
METHODS/FINDINGS: The ballasting of the analyte by gold nanoparticles has been already applied to heavy target as proteins or bacteria to improve the limit of detection. In this paper, we extend the application of such a method to small analytes by showing that once the working surface of a Quartz-Crystal Microbalance (QCM) has been properly functionalized, a limit of detection lower than 1 ppb is reached for parathion. The effective surface functionalization is achieved by immobilizing antibodies upright oriented on the QCM gold surface by a simple photochemical technique (Photonic Immobilization Technique, PIT) based on the UV irradiation of the antibodies, whereas a simple protocol provided by the manufacturer is applied to functionalize the gold nanoparticles. Thus, in a non-competitive approach, the small analyte is made detectable by weighing it down through a "sandwich protocol" with a second antibody tethered to heavy gold nanoparticles. The immunosensor has been proved to be effective against the parathion while showing no cross reaction when a mixture of compounds very similar to parathion is analyzed.
CONCLUSION/SIGNIFICANCE: The immunosensor described in this paper can be easily applied to any small molecule for which polyclonal antibodies are available since both the functionalization procedure of the QCM probe surface and gold nanoparticle can be applied to any IgG, thereby making our device of general application in terms of target analyte.
基于生物传感器的检测为揭示水和固体基质中污染物的存在提供了一种快速且低成本的替代传统分析方法。尽管小分析物（几百道尔顿）的检测很重要，但在生物传感中却是一个问题，因为它们在换能器中，特别是在石英晶体微天平中诱导的信号无法检测到。像对硫磷（分子量 = 292 道尔顿）这样的农药就是一种典型的污染物，对于它而言，信号放大程序是很有必要的。
方法/发现：通过金纳米颗粒对分析物进行压载已被应用于蛋白质或细菌等较重的目标物，以提高检测限。在本文中，我们将这种方法的应用扩展到小分析物，表明一旦石英晶体微天平（QCM）的工作表面经过适当功能化处理，对硫磷的检测限可低于 1 ppb。有效的表面功能化是通过基于抗体的紫外线照射的简单光化学技术（光子固定技术，PIT）将抗体垂直固定在 QCM 金表面来实现的，而制造商提供的简单方案则用于对金纳米颗粒进行功能化。因此，在非竞争性方法中，通过与连接到重金纳米颗粒上的第二抗体采用“夹心方案”使其增重，从而使小分析物能够被检测到。已证明该免疫传感器对硫磷有效，并且在分析与对硫磷非常相似的化合物混合物时没有交叉反应。
结论/意义：本文所述的免疫传感器可以很容易地应用于任何有可用多克隆抗体的小分子，因为 QCM 探针表面和金纳米颗粒的功能化程序都可以应用于任何 IgG，从而使我们的设备在目标分析物方面具有广泛的适用性。
