| Biological enzymes are essential active substances in human metabolic activities,and their specific and efficient catalytic effects maintain the normal functioning of the body.They also play an important role in the digestive process and participate in various pathological processes.Chymotrypsin(CHT)is one of the serine proteases present in the human digestive system,which is involved in various metabolic processes in organisms.CHT plays a crucial role in the metabolism of living organisms and affects cell proliferation and apoptosis.Abnormal levels of CHT in organisms can lead to various diseases such as rheumatoid arthritis,nephritis,diabetes,pharyngitis,indigestion,and pancreatic cancer.Therefore,designing an effective method for detecting CHT in the living system is of great significance.In this paper,we propose a highly sensitive detection method for CHT by combining fluorescence and photoacoustic imaging.Activatable near-infrared(NIR)imaging probes are widely used in the fields of detection and biological imaging due to their strong tissue penetration,high signal-to-background ratio,high sensitivity and specificity.In this paper,we constructed three kinds of chymotrypsin activated imaging fluorescent-photoacoustic probes near-infrared absorption and emission wavelengths based on structurally highly adjustable phthalocyanine derivatives and isophorone platform,applies them to the detection of CHT levels and fluorescence(FL)and photoacoustic(PA)imaging in cell and mouse disease models.Specifically:(1)We synthesized a specific deep-red non-peptide probe DT by effectively combining isophorone and p-hydroxybenzaldehyde for the detection of CHT using 3-phenylpropionyl chloride as recognition group based on protection-deprotection strategy.The probe DT exhibited an emission range of525-700 nm and showed excellent photostability,high sensitivity,and selectivity for CHT detection.The cellular experiments demonstrated that DT could sensitively recognize CHT activity in three cell lines and the content of CHT was much higher in P815 cells than that in MCF-7 and 3T3 cells.Also,DT was successfully used to visualize the endogenous CHT in zebrafish.Notably,the probe DT provided an intuitive way to visualize endogenous CHT in mouse pancreas for the first time,demonstrating the potential application in the future clinical diagnosis of pancreatic diseases.Therefore,the small-molecule probe DT is expected to be a useful molecular tool for CHT-related disease diagnosis and drug discovery.(2)We first proposed a 3D-PA/NIRF dual-modality strategy triggered by CHT to image deep tumor with high spatial resolution and deep penetration.The non-peptide probe HDC was established with hemicyanine as dual signal platform and 4-bromobutyryl as reaction site of CHT.The probe HDC showed good sensitivity,selectivity,and affinity in response to CHT in vitro.Significantly,the probe HDC enabled 3D-PA imaging of CHT activated deep tumor with high spatial resolution and deep penetration.Meanwhile,owing to the NIR fluorescence in tumor site is strong with higher contrast than that of surrounding healthy tissue,it is convenient to depict tumor margin for anatomical analysis.Thus,CHT activated 3D-PA/NIRF dual-modality imaging strategy in vivo is expected to be used to further guide the clinical treatment and clinical therapy effect.(3)Taking advantage of cyanine structure,we have designed and synthesized a novel near-infrared fluorescent probe(Cy G)for the detection of CHT in organisms by recognizing the group 4-bromo-butyryl chloride through a simple reactive link.Cy G showed high selectivity and sensitivity to CHT.Most importantly,the probe was successfully used to detect CHT in cell imaging and in vivo mouse imaging,indicating that it provides a new way to detect CHT in clinic. |
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