A Near-infrared Two-photon Fluorescent Probe For Detecting Carboxylesterase 2 And Its Application In Evaluating Anti-tumor Drugs

Two-photon excited fluorescence imaging is by confining the excitation and emission of the fluorophore to the focal plane of the microscope,by providing a thinner optical section from the depth of a thicker scattering sample.Compared to singlephoton confocal microscopy,the spatial limitation of two-photon excitation yields many advantages.First,it will increase the penetration depth of the excitation beam;second,it will never produce defocused fluorescence,so pinholes are not needed in the detection path of the microscope,so the fluorescence collection efficiency will be improved.Third,two-photon excitation significantly reduces the overall photobleaching and photodamage of the sample,thereby extending the viability of the biological specimen during long-term imaging.Here we have constructed a two-photon fluorescent probe for detecting carboxylesterase 2 in living cells,tissues,and living organisms.As one of the essential enzymes that can catalyze diverse substrates,carboxylesterase 2(CES2)has attracted increasing attention over the past decades.In this work,a long-wavelength two-photon off-on fluorescent probe has been rationally devised for sensing of CES2 with high selectivity and sensitivity.When reacting with CES2,a NIR-excited fluorophore(DCM)in probe DCM-CES2 will be released and simultaneously emerged a characteristic fluorescence peak in 685 nm,accompanied by obvious enhancement in intensity according to the gradually increasing substrate.It shows a fast(within 10 min)and specific fluorescence response toward CES2 with a large stokes shift of 125 nm and a limit of detection(LOD)as low as 0.087 ?g/mL.Furthermore,the probe DCM-CES2 with low cytotoxicity and improved penetrating ability can be utilized to evaluate real-time activity of CES2 in living cells,deep tissues and mice with little background interference.Under physiological conditions,DCMCES2 has been successfully applied to continuously monitor the tumor region during the process of cancer treatment with anticancer drugs including Irinotecan(CPT-11)and Capecitabine(CAPE),thus providing influential instructions for drug evaluation.Therefore,DCM-CES2 can serve as an effective tool for the specific investigation of CES2 and at the same time guide the pathological and physiological processes related to CES2.