Sonochemically Synthesized Zinc Oxide Nanoflakes Based Electrochemical Immunosensor for Ethyl Glucuronide (EtG) Detection
Authors: Alam Fahmida , Sinha Raju , Jalal Ahmed Hasnain , Manickam Pandiaraj , Vabbina Phani Kiran , Bhansali Shekhar , Pala Nezih 
Published: February 2020

We report on label free highly sensitive electrochemical immunosensor for monitoring alcohol consumption through the detection and quantification of ethyl glucuronide (EtG) -a metabolite of ethanol-based on two-dimensional (2D) zinc oxide (ZnO) nanoflakes (NFs), which were synthesized on flexible Au-coated polyethylene terephthalate (PET) substrate using simple one step sonochemical approach. Highly sensitive detection of EtG using cyclic voltammetry (CV) is achieved by immobilizing EtG antibody on the as-synthesized sensing electrodes of ZnO NFs.

Continuous monitoring of alcohol detection is imperative for the prevention of accidents, point of care monitoring, and personal safety. The cutting-edge technologies are mostly focused to detect alcohol from the breath (e.g. breathalyzer), which is not compatible for the real-time monitoring. The relationship between the blood and breath alcohol concentrations and the improvement of the detection techniques of alcohol biomarkers from the sweat render an opportunity for point of care monitoring. Among different biomarkers, Ethylglucuronide (EtG) has been found as a promising indicator of alcohol due toit’s prolonged existence in sweat (24 hours for one or two “drinks” or 2-4 days for binge drinking), which prevents relapsing.

PET has gained popularity as substrate for wearables because of its intrinsic elasticity, thermal stability, hydrophobicity, excellent dielectric properties, low coefficient of thermal expansion, structural resiliency against repeated bending forces and compatibility with roll-to-roll fabrication processes for low cost and scalable manufacturing.

ZnO, a semiconducting material with piezoelectricity,high catalytic efficiency, biocompatibility, chemical stability in physiological environments, low toxicity and a high isoelectric point (IEP) of about 9.5, has been proposed for biosensing applications. ZnO nanostructures (NSs) with different morphologies such as nanorods, nanowalls, nanobelts and quantum dots have been used as immobilizing matrix in fabrication of biosensors for the detection of physiologically relevant biomarkers such as cholesterol, galactose, glucose. Considering their promising properties and potential applications, many techniques have been developed to synthesize various ZnO NSs. In comparison to the more conventional approaches such as hydrothermal method, sonochemical synthesis method is not only significantly faster, inexpensive and performed at ambient conditions but also highly stable and reproducible with the advantages of more uniform size distribution, faster reaction time and a higher surface area, which are essential to design sensing platform withimproved performance. Compared to bulk materials, the 2D ZnO NFs provide unique sensing advantages with polarized (0001) plane orientation and high surface charge density, which could enhance EtG antibody loading and thus improve sensing performance. High isoelectric point allows immobilization of most biomolecules without any additional biding layer. This provides a direct, stable pathway for rapid electron transport when an analyte is immobilized on NFs and improves electron transfer rate greater than 1D ZnO nanorods, high isoelectric point,that results in change of the resistance.Taking advantage of these unique properties, we immobilizedEtG antibody on the synthesized ZnO NFs. When an analyte binds the antibody, there is a change in the current due to increase in the charge transfer resistance. The cyclic voltammetry (CV) measurements were taken in the potential range of -0.1V-0.8V at a scan rate of 50mVs-1. For the EtGconcentrationsof1ng/mL-100µg/mLusing PET/Au/ZnO NFs as the working electrode. The minimum detection limit was found to be much lower than 1ng/mL which covers the physiological range. The sensitivity of the sensor was found to be 18.48 mA/ng/mL/cm-2.