Study On Biosensing Based On Gold Nanoclusters And Gold Carbon Dots

Pathogenic microorganisms and cancer have the significant impact on human health.It is significant to develop a series of experimental methods to detect them.Gold nanoclusters,gold nanoparticles,gold nanocomposite have good optical and chemical properties,it can be used as a cancer biomarker and a pathogenic microorganism detection material.Take advantage of the excellent bioadhesive properties of chitosan,the properties of gold nanoclusters peroxidase,and the special fluorescence properties of chitosan gold nanoclusters and gold-containing carbon dots.A series of high-sensitivity colorimetric stratagy and fluorescence detection methods was constructed,and the experimental conditions were further optimized to achieve the detection of staphylococcus aureus,staphylococcal enterotoxin B,prostate specific antigen(PSA),breast cancer specific antigen(CA15-3),glutathione(GSH),Cysteine(Cys),Fe³⁺detection in cells.The main tasks as follows:(1)GNPs/Au NCs/chitosan composite membranes with peroxidase mimic enzyme properties were prepared and used as label-free colorimetric biosensors for detection of disease biomarkers.The biosensor can detect the analytes by combining the catalytic GNPs/Au NCs/chitosan composite membrane with the imuune reaction to adjust the properties of the layered nanostructured membrane to simulate the peroxide enzyme.As a detection signal amplified by colorimetricl readings,the color change of GNPs depends on the catalytic activity of the composite membrane.By using new capture probes to detect new analytes to expand their function,different cancer biomarkers such as prostate specific antigen PSA and breast cancer antigen CA15-3 can be sensitively determined from cancer serum samples.The measurement results of this method are very consistent with the results determined by the standard method adopted by the hospital,which shows that the biosensor has high accuracy in the detection process and is expected to play an important role in practical applications.(2)Gold nanoclusters(Au NCs)in the presence of chitosan were prepared by ultraviolet irradiation,H₂O₂and 3'-3'-5'-5'-tetramethylbenzidine(TMB)were used to test the properties of peroxide mimic enzymes.Au NCs-chitosan composite membrane also has the properties of peroxide mimic enzyme,It was characterized by TEM,AFM and SEM.The results showed that the formed film was uniform,Au NCs was spherical and well dispersed,and there was no agglomeration in the film.Combined with simulating the immune response of Au NCs-chitosan composite membrane,a colorimetric method with gold nanoparticles of different colors as the output signal was established.Three kinds of bacteria such as Staphylococcus aureus,Escherichia coli and Bacillus subtilis were selected as model bacteria to evaluate the selectivity of the sensor to Staphylococcus aureus.This colorimetric sensing is an extensible strategy because the recognition element of the probe can be replaced on the catalytic Au NCs-chitosan composite membrane.The detection limit of Staphylococcus aureus enterotoxin B(SE-B)is as low as 1.0×10^-12g/m L,and can be read by the naked eye.The developed colorimetric method was used for a rapid on-site screening strategy for Staphylococcus aureus and to detect SE-B in food samples.This colorimetric method provides a platform for rapid screening of pathogens and intestinal toxins in food safety and environmental monitoring.(3)Glutathione(GSH)plays many important roles in biological systems.Growth-related changes in GSH concentration in cells may be critical for cell survival,The monitoring of GSH in living cells is of great significance for understanding the dynamic relationship between GSH and certain diseases.In this work,chitosan gold nanoclusters(CM-Au NCs)that emit red fluorescence were prepared by a simple and fast method.The change in the size of the chitosan micelles triggers the reverse process of the aggregation-induced fluorescence(AIE)phenomenon.Based on the AIE reverse process of CM-Au NCs induced by GSH,a unique CM-Au NCs was used to develop a fluorescent probe for detecting GSH in human serum and live cells.GSH can be distinguished from other biological thiols(cysteine and homocysteine),and it can quantitatively detect the concentration of GSH in the serum of healthy people and cancer patients with high sensitivity.The practical application of fluorescent CM-Au NCs in cell imaging and detection of GSH concentration not only monitors the ultra-trace changes of GSH concentration in cells,but also can monitor cells at different growth stages.It was found that the concentration of GSH in cancer cells was always high.Compared with commercial GSH analysis kits for detecting GSH in human serum and live cells,this method has higher accuracy and precision.The experimental results not only reflect the changes in glutathione concentration at different stages during cell growth,but also prove the feasibility of CM-Au NCs AIE reverse process for detecting glutathione.It provides a research platform for understanding the relationship between the dynamic changes of GSH and disease.(4)The changes of biothiols including glutathione(GSH)and cysteine are closely related to various diseases and cell functions.Real-time monitoring of the dynamic changes of intracellular GSH and cysteine in living cells is important for understanding the pathophysiological process.In this study,fluorescent gold-containing carbon dots(GCDs)composed of a carbon skeleton and gold nanoclusters were prepared,which emitted blue fluorescence.The blue fluorescence of the prepared GCDs does not respond to bio-mercaptans including glutathione and cysteine,but red fluorescence will appear as the bio-mercaptan reacts with GCDs.Therefore,it is possible to establish colorimetric fluorescent bio-imaging based on GCDs to detect GSH and cysteine levels in living cells.In addition,real-time monitoring of GSH and cysteine concentrations in living cells at different growth stages is achieved.It was found that the concentration of GSH in cancer cells is higher than that of normal cells,but the concentration of cysteine in normal cells is always higher than that of cancer cells at different growth stages.Because of their ability to quantify GSH and cysteine in real time,fluorescent GCDs probes provide a promising platform for tracking GSH and cysteine.(5)Iron death is a form of iron-dependent regulated cell death.When glutathione-dependent lipid peroxide repair systems are destroyed,fatal accumulation of lipid-based reactive oxygen species(ROS)leads to iron death.Cell death caused by iron death differs from other forms of death(such as apoptosis,necrosis,and autophagy)in morphology,biochemistry,and genetics.Although iron death plays an important role in maintaining the survival of normal cells and tissues,it is increasingly recognized that certain carcinogenic pathways are related to iron death,making cancer cells extremely vulnerable to death caused by iron death.Fluorescent GCDs were successfully prepared using microwave-assisted heating.GCDs have excellent resistance to photobleaching,and their fluorescence is reversible for the detection of thiol compounds,including the quantification of GSH and cysteine and real-time fluorescence imaging of changes in the biothiol content in cells.The fluorescence of GSH-GCDs no longer responds to bio-mercaptans,but Fe³⁺has an obvious quenching effect on its fluorescence.According to this property,we can detect the quantitative changes in intracellular Fe³⁺content during the iron death of cancer cells,and It was subjected to cell imaging.The results show that GCDs can track the intracellular biothiol changes and iron metabolism in the process of cancer cell iron death.During the process of cancer cell iron death,the content of thiol compounds in the cell decreases and the Fe³⁺content increases accordingly.