Effective antibodies immobilization and functionalized nanoparticles in a quartz-crystal microbalance-based immunosensor for the detection of parathion.

Bartolomeo Della Ventura,Massimo Pica Ciamarra,Riccardo Funari,Marco Iannaccone,Carlo Altucci,Sante Roperto,Rosanna Capparelli,Raffaele Velotta,Amitava Mukherjee

doi:10.1371/journal.pone.0171754

Bartolomeo Della Ventura, Massimo Pica Ciamarra + Show 7 more

Open Access

https://doi.org/10.1371/journal.pone.0171754

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Abstract

BackgroundBiosensor-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/FindingsThe 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/SignificanceThe 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.

Highlights

IntroductionThe detection of small molecules having a mass of a few hundreds of Dalton is of paramount importance in a variety of applications, since species like steroids, herbicides, pesticides, toxins and combustion products (e.g. polycyclic aromatic hydrocarbons) fall in this mass range and all of them are potentially harmful for human health [1,2,3]
The detection of small molecules having a mass of a few hundreds of Dalton is of paramount importance in a variety of applications, since species like steroids, herbicides, pesticides, toxins and combustion products fall in this mass range and all of them are potentially harmful for human health [1,2,3]
The small mass of the analyte is an issue for several transducers such as quartz-crystal microbalance (QCM) [4] and surface plasmon resonance (SPR) [5] both relying on a signal which would benefit from the increase of the effective interaction area as well as from ballasting the small molecule

Summary

Introduction

The detection of small molecules having a mass of a few hundreds of Dalton is of paramount importance in a variety of applications, since species like steroids, herbicides, pesticides, toxins and combustion products (e.g. polycyclic aromatic hydrocarbons) fall in this mass range and all of them are potentially harmful for human health [1,2,3]. The small mass of the analyte is an issue for several transducers such as quartz-crystal microbalance (QCM) [4] and surface plasmon resonance (SPR) [5] both relying on a signal which would benefit from the increase of the effective interaction area as well as from ballasting the small molecule. Limit of detection (LOD) of 20 ng/mL has been claimed in the detection of CRP (C-reactive protein) by a QCM immunosensors using secondary antibodies conjugated with Au-NPs of 20 nm in diameter[11]. In this case a complex procedure coupled more than one Au-NP on the same secondary antibody.

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