Fluorescence And Electrochemical Dual-signal Sensing Based On Functionalized Glass Nanopores

In recent years,with the improvement of sensitivity and the diversification of functional methods,nanopore sensing technology has been developed rapidly.Without the need for covalently labelling the target analyte,the nanopore sensing techniques can detect a variety of analytes,including DNA,RNA,protein complexes,etc.Great progress has been made in the application of biotechnology.However,there are still limitations in nanopore sensing based on the change of ion current.Most of the previously reported assays rely on analyte-mediated ion current fluctuations,which are susceptible to the interference from non-specific adsorption.Therefore,how to correct the false-signal and improve the selectivity and accuracy of the detection method become the focus of this paper.To address the above challenges,we rationally designed terbium ion-functionalized carbon dots(CDs-Tb)and bovine serum albumin-protected gold nanoclusters(BSA-Au NCs)with fluorescent properties and recognition units.Through the electrostatic deposition,the fluorescent nanomaterials-modified glass nanopores were successfully prepared.Upon binding to the target molecule,the surface chemistry of fluorescent nanomaterial inside the nanopore changes,triggering fluorescence enhancement as well as ionic current response.Based on the above variations,we constructed a dual-signal sensing platform for fluorescence and ion current.Although the CDs-Tb film-modified glass nanopore can achieve dual-signal sensing detection,using Terbium ion as the sensing unit,it needs to be excited at short wavelengths,and there is strong Rayleigh scattering of green-emitting CDs-Tb at the nanopores,which is not conducive to nanopore imaging and quantification.Therefore,in the second part,we further selected BSA-Au NCs with long wavelength excitation and tried to modify the glass nanopore platform with BSA-Au NCs.The red-emitting BSA-Au NCs effectively attenuate the Rayleigh scattering,which overcomes the defects of the first part and improves the sensitivity of the sensor.The dual-signal output mode of fluorescence and ion current constructed based on functionalized glass nanopores enables the correction of false-signals and improves the selectivity and accuracy of the method.The full paper is divided into three parts and the detailed are as follows:Chapter 1: OverviewThis chapter firstly introduces the types of nanopores,which can be classified into biological nanopores and artificial nanopores according to the material sources.Then,we describe the preparation and functionalization of nanopores in detail.Next,three detection approaches based on nanopores are summarized and the strategies of fluorescent nanomaterials to maintain their fluorescence properties at the solid-liquid interface are discussed.Finally,the main content and research significance of this paper are clarified.Chapter 2: Fluorescence and Electrochemical Analysis Based on Carbon Dot Functionalized Glass NanoporesIn this chapter,we developed a strategy based on terbium ion-functionalized carbon dots(CDs-Tb)modified glass nanopores to detect pp Gpp.This method not only maintains the fluorescence properties of carbon dots but also retains the coordination ability of terbium ions.The fluorescence efficiency of terbium ion is very low,but they can emit strong green fluorescence due to the antenna effect when chelating with the ligand pp Gpp.Within a certain range,the green fluorescence gradually increased with the increase of pp Gpp concentration.At the same time,the surface charge change of CDs-Tb film after combination with pp Gpp were studied by the Zeta potential,and the charge change triggered the ion current response.Based on this,we established a dual signal output model(fluorescence and ion current)for the detection of pp Gpp.This mode provides additional correction for interference caused by a single signal,which improves the accuracy and selectivity of the method.In addition,we achieved the detection of pp Gpp in E.coli,which would enable a deeper understanding of the mechanisms related to pp Gpp in bacterial.Chapter 3: Fluorescence and electrochemical analysis of functionalized glass nanopores based on gold nanoclusters.In this article,using cysteine(Cys)and glutathione(GSH)determination as a model,we designed a fluorescence/ion current dual-signal sensing platform based on BSA-Au NCs modified glass nanopore.First,fourier transform infrared spectroscopy(FT-IR)and X-ray photoelectron spectroscopy(XPS)were used to explain fluorescence enhancement of BSA-Au NCs.Meanwhile,surface charge changes of BSA-Au NCs films after binding with Cys were studied by Zeta potential.Through the specific interaction between biothiols and BSA-Au NCs,the system not only performed well in aqueous fluorescent detection,but also can be developed into a more selective and sensitive nanopore sensor.The recognition process of biothiols not only enhances the gold cluster film fluorescence,but also changes the gold cluster surface charge,triggering the ionic current response.The dual-signal-output platform can offer an additional correction for false signals in the single detection modal,further improving the reliability and practicability of our method.Importantly,this strategy can be used to detect biothiols in human serum and holds great significance for practical application.