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Zinc oxide surface states can be utilized for ultra-specific detection of biomolecules. The major challenges in using ZnO for bio-sensing are attaining enhanced sensitivity and specificity. In this study, we explore the functionalization of zinc in ZnO through utilizing the thiol bond. The purpose of this study is to demonstrate that the ZnO based sensor is capable of achieving high specificity in presence of competitive surface binding through the thiol bond. The final goal is to design an ultra-specific biosensor to detect low occurring biomolecules. In this study, we have selected cortisol as a stress marker to demonstrate quantification and detection from synthetic sweat. In order to demonstrate ultra-specificity, we have used two competitive thiol based molecules binding to zinc, a linker Dithiobis succinimidyl propionate (DSP) and reducing agent of DSP, Dithiothreitol (DTT). Electrochemical impedance spectroscopy (EIS) is used to quantify the signal obtained through various ratiometric concentrations of DSP and DTT. To validate the EIS study results, inherent fluorescence studies are done by mapping changes in green emission spectrum of ZnO before and after linker functionalization. The optimal combination in terms of highest signal is identified to be of 25mM DTT and 50mM DSP. This is implemented in the experiments performed to calibrate the cortisol concentration in synthetic sweat. This study demonstrates the detection of cortisol antigen in synthetic sweat present within the physiological levels of 8 ng/mL to 140 ng/mL.
This study demonstrates the development of a zinc oxide (ZnO) based microelectrode sensor for the ultra-sensitive detection of protein biomarkers. Our research focuses on utilizing a materials-based approach to achieve this objective by utilizing ZnO as part of our biosensor for (1) improved surface binding to enhance sensitivity and (2) creating a nanotextured surface for enhanced output signal response. Nanotextured ZnO thin films were integrated onto printed circuit boards using RF magnetron sputter deposition. Films sputtered with and without the presence of oxygen were examined for possible differences in biosensor efficacy. These fabrication conditions not only dictate the number of oxygen vacancies within the film but also regulate the amount of zinc and oxygen terminated ends occurring on the material surface. The correlation between the surface terminations of the nanotextured ZnO to its performance as a biosensor was evaluated using two cross-linker molecules, dithiobis succinimidyl propionate and (3-aminopropyl)triethoxysilane, that maintain different binding chemistries to ZnO. Qualitative and quantitative assessment of cross-linker binding was accomplished using fluorescent microscopy and fluorescent intensity measurements. Electrical impedance spectroscopy (EIS) was used as the transduction mechanism for detection of the well-established cardiac biomarker, troponin-T. Utilizing EIS with a functionalized immunoassay on the ZnO surface, troponin-T was detected as low as 10 fg/mL using ZnO films sputtered without oxygen. This enhanced detection of the cardiac biomarker can be directly attributed to 1) oxygen vacancies within the metal oxide film, 2) the nanotexturing of the sensing site surface, and 3) the ability to bind a significant amount of cross-linker molecules for immobilizing capture antibodies.
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