Construction Of New Polymer Chemiluminescence Nanoprobes For Imaging Of Bioactive Molecules In Vivo

The bioactive molecules can regulate cellular functions and be closely related in occurrence and development of diseases in biological systems. However, the intrinsic concentrations or native fluctuations of some bioactive molecules are extremely subtle. In the meantime, the high background fluorescence in complicated biological materials greatly disturbs the accurate detection of biological molecules. Therefore, developing a nolve method for ultrasensitive monitoring of their actual levels or native fluxes in biological systems becomes an attractive avenue for elucidating their biological functions. Compared to traditional photoluminescence detection, chemiluminescence?CL? sensors, without external excitation source, provide prominent advantages, including lower background signal interference and photodamage, avoidance of the noise caused by light scattering, rapid response time and relatively broad linearity range, and so on. Therefore, CL-based sensing system is widely applied to monitor biological molecules. However, short CL lifetime, low emission intensity, and short emission wavelength seriously obstruct the application of CL-based sensors. In view of the above problems, due to providing high sensitivity, dramatically prolonged luminescence time as well as red shift of wavelength, chemiluminescence resonance energy transfer?CRET? has received growing attention, which is considered to construct a new probe. Meanwhile, owing to distinguished signal amplification along the backbone and adjustable optical performance, conjugated polymers?CPs? are often chosen as the ideal energy receptors and are successfully used in imaging detection.Based on the above issues, here, we utilized CRET to construct novel conjugated polymer CL nanoprobes for monitoring intrinsic levels of superoxide anion(O2^·-) and Alkaline phosphatase?ALP? in vivo, respectively. Two specific aspects carried out are as follows:1. O2^·- as the preliminary reactive oxygen species acts as a sink for many other radicals generated intracellularly during oxidative metabolism. Also, O2^·- plays critical roles in various significant biological events and diseases, such as cell signal transduction, inflammation, cancer and neurodegeneration. However, actual O2^·- concentration is very low in biological systems. Up to now, without exogenous stimulation, a new probe for visualizing O2^·- native level in vivo has not been emerged. Herein, based on CRET between imidazopyrazinone and conjugated polymers, we constructed innovatively a polymer nanoprobe PCLA-O2^·- for detection of O2^·-. Structurally, the designed probe PCLA-O2^·- consists of three segments: imidazopyrazinone moiety termed CLA acting as the recognition unit of O2^·- that is capable of CL as the energy donor; CPs?PFBT? with signal amplification matrix as the energy acceptor; the covalent bond between CLA and PFBT that can powerfully control the close proximity of donor/acceptor. Using “nanoprecipitation” technique, the compact spherical morphology of polymer nanoparticles were formed in aqueous solution. Meanwhile, CLA parts were embedded in a hydrophobic interior of nanoparticles, which can significantly enhance CL intensities and prolong luminescence time. After adding O2^·-, the energy produced?490 nm? by specific reaction between CLA and O2^·- is transferred to PFBT through CRET, then PFBT acceptor emits 560 nm magnified light. Experiments results in vitro clearly substantiated that the attractive probe is endowed with ultrahigh sensitivity at the picomolar level, substantially extended luminescence time, excellent selectivity and biocompatibility. CL intensities of PCLA-O2^·- increased linearly with the concentrations of O2^·- in the range from 0 to 950 pM, and the correlation coefficient was 0.9924. The detection limit of PCLA-O2^·- is estimated to be as low as 19.3 pM. Without an external excitation source, the attractive probe is applied in mice to selectively visualize O2^·- in normal/inflammation tissues. More importantly, PCLA-O2^·- is utilized for the first time in situ to visualize the native variance of O2^·- in normal/tumor cells and tissues without exogenous stimulation. These exceptional features ensure that PCLA-O2^·- as a self-luminescing probe is an alternative in vivo imaging approach for ultralow level O2^·-.2. As reported, an abnormal level of ALP is often implicated in the development of numerous diseases, such as bone diseases, liver dysfunction, breast cancer, prostatic cancer, and so on. And, ALP has long been recognized as a significant biomarker in clinical analysis. To our knowledge, without stimulation, monitoring of endogenous ALP in vivo using molecular probes or nanoprobes has been absent so far. Herein, we present the rational design and construction of a new polymer nanoprobe for ultrasensitive monitoring of ALP in vivo based on CRET. Structurally, this probe contains three moieties, AMPPD as both a substrate of ALP and the energy donor; CPs with light-amplifying matrix as the energy acceptor; ?-cyclodextrin moiety controlling close distance of CPs to AMPPD and affording a hydrophobic interior environment. Once ALP is added, AMPPD and ALP occur fast reaction. Afterwards, the CPs can be excited and emits magnified 660 nm light through the CRET process. Based on the above principle, the background fluorescence interference of biological materials is significantly reduced and the CRET efficiency is elevated. Therefore, a supersensitive imaging nanoprobe for ALP can be realized.