New Methods For Determination Of Enzyme Activity By Fluorescence Correlation Spectroscopy

Enzymes are biomacromolecules with biological catalysis?proteins or RNA?.The abnormal enzyme activity is often associated with some major diseases.So the determination of enzyme activity has become an important method for basic research,early diagnosis,drug screening and targeted therapy.The traditional enzyme activity determination method requires a large amount of reagent,high cost and long analysis time.Therefore,it is necessary to develop sensitive,fast and micro analytical methods.Fluorescence correlation spectroscopy?FCS?is a single molecule method with the advantages of high sensitivity,short analysis time,and small detection volume.FCS is very suitable for the determination of enzyme activity and screening of enzyme inhibitors.In this dissertation,the FCS detection method of telomerase activity was established.The new microwell chip-fluorescence correlation spectroscopy?MC-FCS?method was developed and was applied to Caspase-3 enzyme activity analysis.The main research work includes the following aspects:?1?We proposed an ultrasensitive and robust method for quantitative determination of telomerase activity by combining single molecule fluorescence correlation spectroscopy?FCS?with telomerase repeat amplification protocol?TRAP?.Telomerase is a key enzyme for maintaining the telomere length.It is regarded as a versatile cancer biomarker and a potential drug target due to its important role in cancer and aging.It is necessary to develop a sensitive and reliable method for detecting telomerase activity due to its very low level in cells.The principle of this new method?FCS-TRAP?is based on measurement of the change in characteristic diffusion time and molecule number of TRAP products by FCS.The characteristic diffusion time is related to the length of TRAP products,and the molecule number represents the concentration of TRAP products.We optimized the conditions of TRAP procedure and FCS measurements.We observed that the telomerase activities are positively correlated to characteristic diffusion time and molecule number of TRAP products at optimal conditions.This method was successfully applied for detecting telomerase activity of different cell lines.Meanwhile,the detection of a single cell was realized.This method was also used to evaluate the inhibition efficiency of inhibitors.In addition,the IC₅₀ values obtained were in good agreement with the references.Compared to current TRAP methods,this method without separation shows reliable quantification,ultrahigh sensitivity,and short detection time.We believe that the FCS-TRAP method will have a well prospective application in clinical diagnosis and screening of telomerase inhibitors.?2?The PDMS/glass microwell chip?MC?was successfully fabricated and coupled with FCS detection method.The evaporation of sample in microwell chip was solved by thin film encapsulation.Then the non-specific adsorption of sample was aslo solved by dynamic modification of surfactant.After optimization,the results of the samples on the cover slide and in the microwell chip were basically consistent.This result proved that the microwell chip-fluorescence correlation spectrometry system was successful.We figured out that the best well diameter of microwell chip was 0.9 mm.Then we also optimized the required sample volume.It was significantly reduced to 1?L level.Compared with the current FCS method,MC-FCS system dramatically reduced the volume of sample and reagent.It could lead to the cost saving of enzyme activity detection and drug screening.?3?We proposed a new method for determination of Caspase-3activity and its inhibition constant by combining single molecule fluorescence correlation spectroscopy with a microwell chip.Caspase-3 is a key enzyme executing apoptosis during ontogenesis and homeostasis of multicellular organisms.It is a very important and potential drug target in the treatment of apoptosis disturbance.So far,there are no available commercial drugs for Caspase-3 so that it is urgently necessitated to develop an effective method for Caspase-3 activity assay and its inhibitor screening.Its principle is based on measurement of the enzyme reaction kinetics and homogeneous detection of the reaction product by FCS.This MC-FCS system can reduce the required sample volume to 1?L level.The Caspase-3 substrates are doubly labeled with fluorophore and Biotin.The enzyme reaction can be quickly terminated in the presence of Streptavidin.The reaction products can also be selectively detected by FCS due to the enlargement of molecular weight by Streptavidin.We established the model of Caspase-3 inhibitor screening by combining the dynamics of enzyme reaction with FCS theory.This new method was successfully used for determination of inhibition constants of certain inhibitors and assay of drug-induced apoptosis.Compared to current methods,this method shows high sensitivity,small reagent dosage and short analysis time.We believe that this method will become an efficient platform for screening of Caspase-3 inhibitors and detection of apoptosis.