New Methods For Electrochemical Bioanalysis Based On Micro/Nanochannels

The electrochemical bioanalysis based on micro/nanochannels has been rapidly developed in the recent years.The micro/nanochannels mainly include protein nanochanels,solid nanochannels,solid nanochannel arrays and microfluidic channels.While the detectable analytes range from small molecules,nucleic acids,peptides,proteins,enzymes to biomolecular complexes.Such electrochemical bioanalysis based on micro/nanochannels provides new approaches for clinical diagnosis,life analysis,environmental and industrial studies.In this dissertation,three solid nanochannel array-based and two microfluidic channel-based novel devices integrated with electrochemical techniques and other analysis methods have been developed for investigation of enzymatic reaction and immunological reaction kinetics in nanospaces,detection of small biomolecules using aptamers,characterization of composition and structure of hydrated protein films and electrochemical intermediate species at electrode-electrolyte interfaces.The details are given below:(1)The construction of nanodevices coupled with a real-time detection system for evaluation of the enzymatic reaction kinetics occurring at the confined nanospaces or interfaces has received growth interest.Thus,a nanochannel array-enzyme system in which the enzymatic reaction could be investigated with an electrochemical method was constructed.Glucose oxidase(GOD)was covalently linked onto the inner wall of the nanochannels of the porous anodic alumina(PAA)membrane.An Au disc was attached at the end of the nanochannels of the PAA membrane as the working electrode for detection of enzymatic reaction product hydrogen peroxide(H2O2).The effects of ionic strength,amount of immobilized enzyme and pore diameter of the nanochannels on the enzymatic reaction kinetics were illustrated.As a result,the GOD confined in nanochannels showed high stability and reactivity.Upon addition of glucose to the nanochannel-enzyme system,the current response had a calibration range span from 0.005 mM to 2 mM of glucose concentration.The apparent Michaelis-Menten constant(Kmapp)of GOD confined in nanochannel was 0.4 mM.This work provided a platform for real-time monitoring of the enzyme reaction kinetics confined in nanospaces.Such a nanochannel-enzyme system could also help design advanced biosensors and enzyme reactors with high sensitivity and efficiency.(2)Evaluating the kinetics of biological reaction occurring in confined nanospaces is of great significance in studying the molecular biological processes in vivo.Thus,a nanochannel array-based electrochemical bioreactor and a kinetic model to investigate the immunological reaction in confined nanochannels simply by the electrochemical method were developed.The PAA nanochannels were modified with single-stranded DNA which each contained two covalently linked digoxin molecules.When antidigoxin flowed into the nanochannels and captured by digoxin,the flux of indicator ions in nanochannels would be modulated,which was also influenced by many general experimental conditions,such as the ionic strength and pH of the solution,the pore size of the nanochannels,and the charge property of the indicator species.On the basis of these,a mathematical model to quantitatively describe the immunological reaction in nanochannels were proposed.As a result,except for the reaction kinetic constant that was previously studied,more insightful kinetic information such as the moving speed of the antibody and the immunological reaction progress in nanochannels were successfully revealed in a quantitative way for the first time.This study would provide a deeper insight for the biological reaction that occurred in confined nanospaces.(3)The development of nanomaterial-based system for facile detection of multiple small biomolecules still remains a great challenge.Thus,a nanochannel array-based electrochemical platform was developed for the quantitative detection of biorelated small molecules such as potassium ions(K+)and adenosine triphosphate(ATP)in a facile way.K+ or ATP G-quadruplex aptamers were covalently assembled onto the inner wall of PAA nanochannels through a Schiff reaction between aldehyde groups(-CHO)in the aptamer and amino groups(-NH2)modified on the inner wall of the PAA nanochannels under mild reaction conditions.Conformational switching of the aptamers confined in the nanochannels occurred in the presence of the target molecules,resulting in increased steric hindrance in the nanochannels.Changes in steric hindrance in the nanochannels were monitored by the anodic current of indicator molecules transported through the nanochannels.As a result,quantitative detection of K+ and ATP was realized with a concentration ranging from 0.005 mM to 1.0 mM for K+and 0.05 mM to 10.0 mM for ATP.The proposed platform displayed significant selectivity,good reproducibility,and universality.Moreover,this platform showed its potential for use in the detection of other aptamer-based analytes,which could promote its development for use in biological detection and clinical diagnosis.(4)Hydration is crucial to keep the conformation and biological activity of proteins.It is particularly important to understand the real composition and structure of the hydrated protein film,especially for the distribution of water molecules in it.Thus,a microfluidic device,System for Analysis at the Liquid Vacuum Interface(SALVI),was developed to enable direct surface analysis of liquid surfaces and solid-liquid interfaces using vacuum-based surface tools such as Time-of-Flight Secondary Ion Mass Spectrometry(ToF-SIMS).In situ liquid SIMS enabled by SALVI was employed to probe the adsorbed hydrated protein film on a solid surface.Five representative hydrated protein films and pure water were investigated.As a result,the semi-quantitative observation of water clusters and amino acid fragments revealed detailed composition of water molecules and amino acids in these hydrated protein films.The 3D images demonstrated their spatial structures,which could provide experimental information to the molecular dynamic simulations of proteins.This study would not only pave the investigation of biomolecular hydration but also be promising to extend to other fields such as life analysis and biological studies.(5)The electrochemical interface between solid electrode and liquid electrolyte has long been studied,however,real-time spatial mapping of ionic and molecular intermediate species at the dynamic interface still remains a key challenge.Thus,an in situ time-resolved dynamic characterization of the electrode-liquid electrolyte interface by imaging mass spectrometry was demonstrated.This unique approach was enabled by a vacuum compatible electrochemical microfluidic reactor.The chemical mechanism of iodide oxidation at the gold electrode surface was revealed using simultaneous cyclic voltammetry(CV)and dynamic SIMS.The results suggested that more complex surface reactions existed concerning the gold-iodine compounds adlayer formation on the electrode surface,providing the discovery of more short-lived transient species and new insights of elementary electrode reactions.Such findings further illustrated the importance of truly observing products and intermediate species resulting from electrochemical reactions in real time with high spatial chemical mapping.This innovated approach was suitable for fundamental kinetic studies in electrochemistry at the solid-liquid interface with many potential applications such as energy storage,material synthesis,electrocatalysis and energy conversion.