Fluorescence Imaging Detection Of Fungi Based On （1,3）-β-D-glucan Aptamer

As the number of fungal pathogens threatening human health increases and the infection rates climb,fungal infections have attracted wide attention.According to the depth of the tissue fungi invade,fungal infections are divided into superficial fungal infections and invasive fungal infections.The detection for superficial fungal infections mainly include direct microscopy,culture-based method and newly developed molecular diagnostic techniques.Among these methods,the culture-based method takes a long time,and the molecular diagnostic technology has not been applied on a large scale.The optical brightener staining method has been widely used in the clinical detection of fungi because of its simple and rapid fluorescence imaging of fungal samples.However,this method has the disadvantage of poor specificity.Therefore,it is necessary to develop a specific method for the fluorescence imaging detection of fungi.Aptamers,as single-stranded nucleic acid sequences synthesized by artificial in vitro screening,could bind to target small molecules,proteins,and even cells with extremely high specificity,which have widespread application prospects in biological analysis.With aptamers targeting（1,3）-β-D-glucan,a characteristic structural substance of fungal cell wall,the specific detection of fungi could be achieved.However,if the fluorescent dye-modified aptamer targets fungi for fluorescence imaging,it may cause misjudgment for the detection results due to low sensitivity.Therefore,it is also necessary to improve the sensitivity for the detection of fungi.Based on this,this work utilized（1,3）-β-D-glucan aptamer to specifically target fungi,and by modifying the aptamer,the enzymatic tyramide signal amplification technology and enzyme-free hybridization chain reaction were introduced respectively to construct specific and highly sensitive methods for fluorescence imaging detection of fungi.The main research contents are as follows:（1）The（1,3）-β-D-glucan aptamer was combined with tyramide signal amplification technology to carry out fluorescence imaging detection of fungi.First,the coupling of（1,3）-β-D-glucan aptamer and horseradish peroxidase was achieved by click chemistry.The fungal biomarker（1,3）-β-D-glucan was specifically targeted by aptamer,so that horseradish peroxidase was labeled on the surface of the fungal cell wall.With the addition of biotinyl tyramide and hydrogen peroxide,biotinyl tyramide was converted into biotinyl radical under the catalysis of horseradish peroxidase,which tagged cell wall protein adjacent to horseradish peroxidase and horseradish peroxide.Cy3-conjugated streptavidin was subsequently introduced with the specific interaction between streptavidin and biotin,and multiple Cy3 was labeled on the fungal cell wall.The fluorescence signal was greatly enhanced with up to 6 times.The presence and morphological characteristics of fungi could be clearly observed under a fluorescence microscope,providing morphological information and diagnostic information for the detection of fungi.（2）The work in the previous chapter utilized horseradish peroxidase to catalyze tyramide substrate to achieve fluorescence signal amplification.However,protease is easily affected by external environmental factors,which reduce the activity of the enzyme and affect the accuracy and reproducibility of detection.In view of this,this work combined（1,3）-β-D-glucan aptamer with hybridization chain reaction to establish an enzyme-free,convenient and highly sensitive method for the fluorescence imaging detection of fungi.With（1,3）-β-D-glucan aptamer specifically targeting the fungal cell wall,the primer modified at one end of the aptamer was labeled on the surface of fungal cell,and the hybridization chain reaction was initiated.The hairpin probes alternately hybridized to form double-stranded DNA,which carried a large amount of ROX fluorescent dye,achieving a fluorescence signal amplification of about 5 times.The morphology of fungi could be observed more conveniently and clearly under mild conditions without enzyme catalysis,achieving highly sensitive fluorescence imaging detection of fungi.