Xiaoding Lou

Real-Time, Quantitative Lighting-up Detection of Telomerase in Urines of Bladder Cancer Patients by AIEgens

As a biomarker for early cancer diagnosis, telomerase are one of the promising targets for cancer therapeutics. Inspired by the fluorescent emission principle of aggregation-induced emission fluorogens, we creatively designed an AIE-based turn-on method to detect telomerase activity from cell extracts. A positively charged fluorogen (TPE-Z) is not fluorescent when freely diffused in solution. The fluorescence of TPE-Z is enhanced with the elongation of the DNA strand which could light up telomere elongation process. By exploitation of it, we can detect telomerase activity from different cell lines (E-J, HeLa, MCF-7, and HLF) with high sensitivity and specificity. Moreover, our method is successfully employed to demonstrate the applications in bladder cancer diagnosis (41 urine specimens from bladder cancer patients and 15 urine specimens from normal people are detected). The AIE-based method provides a simple one-pot technique for quantification and monitoring of the telomerase activity and shows great potential for future use in clinical tests.

Biocompatible Green and Red Fluorescent Organic Dots with Remarkably Large Two-Photon Action Cross Sections for Targeted Cellular Imaging and Real-Time Intravital Blood Vascular Visualization.

Fluorescent organic dots are emerging as promising bioimaging reagents because of their high brightness, good photostability, excellent biocompatibility, and facile surface functionalization. Organic dots with large two-photon absorption (TPA) cross sections are highly desired for two-photon fluorescence microscopy. In this work, we report two biocompatible and photostable organic dots fabricated by encapsulating tetraphenylethene derivatives within DSPE-PEG matrix. The two organic dots show absorption maxima at 425 and 483 nm and emit green and red fluorescence at 560 and 645 nm, with high fluorescence quantum yields of 64% and 22%, respectively. Both organic dots exhibit excellent TPA property in the range of 800-960 nm, affording upon excitation at 820 nm remarkably large TPA cross sections of 1.2×10(6) and 2.5×10(6) GM on the basis of dot concentration. The bare fluorophores and their organic dots are biocompatible and have been used to stain living cells for one- and two-photon fluorescence bioimagings. The cRGD-modified organic dots can selectively target integrin αvβ3 overexpressing breast cancer cells for targeted imaging. The organic dots are also applied for real-time two-photon fluorescence in vivo visualization of the blood vasculature of mouse ear, providing the spatiotemporal information about the whole blood vascular network. These results demonstrate that the present fluorescent organic dots are promising candidates for living cell and tissue imaging.

A Selective Glutathione Probe based on AIE Fluorogen and its Application in Enzymatic Activity Assay

In this work, we design and synthesize a malonitrile-functionalized TPE derivative (TPE-DCV), which can react with thiol group through thiol-ene click reaction, leading to the fluorescence change of the system. Combined with the unique AIE property, TPE-DCV can selectively detect glutathione (GSH) but not cysteine or homocysteine. As the cleavage of GSSG with the aid of glutathione reductase produces GSH, which turns on the fluorescence of TPE-DCV, the ensemble of TPE-DCV and GSSG can thus serve as a label-free sensor for enzymatic activity assay of glutathione reductase. We also apply TPE-DCV for the detection of intracellular GSH in living cells.

Sensitive and Bidirectional Detection of Urine Telomerase Based on the Four Detection-Color States of Difunctional Gold Nanoparticle Probe

Telomerase, a valuable biomarker, is highly correlated with the development of most of human cancers. Here, we develop a bidirectional strategy for telomerase activity detection and bladder cancer diagnosis based on four detection-color states of difunctional gold nanoparticle (GNP) probes such as blue, purple, red, and precipitate. Specifically, we define the red GNP probe as origin, which represents urine extracts with inactive telomerase and implies normal individuals. The forward direction is corresponding to the detection of a relatively high concentration of active telomerase, in which system GNP probes assemble obviously and precipitate, predicting bladder cancer samples. The negative direction is corresponding to extracts with a relatively low concentration (purple) and without any telomerase (blue), which can be differentiated by naked eyes or UV-vis spectrum, indicating bladder cancer and normal individuals, respectively. More importantly, this noninvasive strategy shows great sensitivity and selectivity when tested by 18 urine specimens from bladder cancer patients, inflammation, and normal individuals.

Organic Dots Based on AIEgens for Two-Photon Fluorescence Bioimaging

Two-photon fluorescence imaging technique is a powerful bioanalytical approach in terms of high photostability, low photodamage, high spatiotemporal resolution. Recently, fluorescent organic dots comprised of organic emissive cores and a polymeric matrix are emerging as promising contrast reagents for two-photon fluorescence imaging, owing to their numerous merits of high and tunable fluorescence, good biocompatibility, strong photobleaching resistance, and multiple surface functionality. The emissive core is crucial for organic dots to get high brightness but many conventional chromophores often encounter a severe problem of fluorescence quenching when they form aggregates. To solve this problem, fluorogens featuring aggregation-induced emission (AIE) can fluoresce strongly in aggregates, and thus become ideal candidates for fluorescent organic dots. In addition, two-photon absorption property of the dots can be readily improved by just increase loading contents of AIE fluorogen (AIEgen). Hence, organic dots based on AIEgens have exhibited excellent performances in two-photon fluorescence in vitro cellular imaging, and in vivo vascular architecture visualization of mouse skin, muscle, brain and skull bone. In view of the rapid advances in this important research field, here, we highlight representative fluorescent organic dots with an emissive core of AIEgen aggregate, and discuss their great potential in bioimaging applications.

Facile, Fast-Responsive, and Photostable Imaging of Telomerase Activity in Living Cells with a Fluorescence Turn-On Manner

In situ detecting and monitoring intracellular telomerase activity is significant for cancer diagnosis. In this work, we report a facile and fast-responsive bioprobe for in situ detection and imaging of intracellular telomerase activity with superior photostability. After transfected into living cells, quencher group labeled TS primer (QP) can be extended in the presence of intracellular telomerase. Positive charged TPE-Py molecules (AIE dye) will bind to the primer as well as extension repeated units, producing a telomerase activity-related turn-on fluorescence signal. By incorporating positive charged AIE dye and substrate oligonucleotides, in situ light-up imaging and detection of intracellular telomerase activity were achieved. This strategy exhibits good performance for sensitive in situ tracking of telomerase activity in living cells. The practicality of this facile and fast-responsive telomerase detection method was demonstrated by using it to distinguish tumor cells from normal cells and to monitor the change of telomerase activity during treatment with antitumor drugs, which shows its potential in clinical diagnostic and therapeutic monitoring.

High Fluorescence Efficiencies and Large Stokes Shifts of Folded Fluorophores Consisting of a Pair of Alkenyl-Tethered, π-Stacked Oligo-p-phenylenes

A series of pure hydrocarbon fluorophores containing a pair of π-stacked oligo-p-phenylenes have been synthesized and analyzed by NMR and X-ray crystallography. They show good fluorescence in solutions and enhanced fluorescence in the aggregated state. Large Stokes shifts (up to 214 nm) have been achieved in these folded fluorophores in virtue of intramolecular energy transfer, and balanced structural rigidity and flexibility. These folded fluorophores provide perfect models for understanding the energy and charge transfer process in π-stacked systems.

Rational Designed Bipolar, Conjugated Polymer-DNA Composite Beacon for the Sensitive Detection of Proteins and Ions

Nature owns remarkable capabilities in sensing target molecules, while the artificial biosensor lags far behind nature. Inspired by nature, we devise a new sensing platform that can specifically bind the molecules and synchronously initiate a specific signal response. We rationally designed a type of bipolar probe that is comprised of a hydrophilic DNA part and a hydrophobic conjugated polymer (CP) unit. In aqueous solution, they can form micelles with a hydrophobic CP core and a hydrophilic DNA shell. The aggregation-caused quenching suppresses the fluorescence of CP. Adding telomerase, the hydropathical profile of the bipolar probes is drastically regulated that results in the collapse of micelles and liberates fluorescence simultaneously. The probe has been used in both mimic systems and real urine samples (38 samples). We achieve sensitive and specific detection of telomerase and obtain clearly classification for normal people and cancer patients. It can also be used in a signal off sensor that is used to detect mercury ions.

Lab in a Tube: Sensitive Detection of MicroRNAs in Urine Samples from Bladder Cancer Patients Using a Single-Label DNA Probe with AIEgens

We demonstrate an ultrasensitive microRNA detection method based on an extremely simple probe with only fluorogens but without quencher groups. It avoids complex and difficult steps to accurately design the relative distance between the fluorogens and quencher groups in the probes. Furthermore, the assay could accomplish various detection limits by tuning the reaction temperature due to the different activity of exonuclease III corresponding to the diverse temperature. Specifically, 1 pM miR-21 can be detected in 40 min at 37 °C, and 10 aM (about 300 molecules in 50 μL) miR-21 could be discriminated in 7 days at 4 °C. The great specificity of the assay guarantees that the real 21 urine samples from the bladder cancer patients are successfully detected by our method.

Protease-Responsive Prodrug with Aggregation-Induced Emission Probe for Controlled Drug Delivery and Drug Release Tracking in Living Cells

Controlled drug delivery and real-time tracking of drug release in cancer cells are essential for cancer therapy. Herein, we report a protease-responsive prodrug (DOX-FCPPs-PyTPE, DFP) with aggregation-induced emission (AIE) characteristics for controlled drug delivery and precise tracking of drug release in living cells. DFP consists of three components: AIE-active tetraphenylethene (TPE) derivative PyTPE, functionalized cell penetrating peptides (FCPPs) containing a cell penetrating peptide (CPP) and a short protease-responsive peptide (LGLAG) that can be selectively cleaved by a cancer-related enzyme matrix metalloproteinase-2 (MMP-2), and a therapeutic unit (doxorubicin, DOX). Without MMP-2, this prodrug cannot go inside the cells easily. In the presence of MMP-2, DFP can be cleaved into two parts. One is cell penetrating peptides (CPPs) linked DOX, which can easily interact with cell membrane and then go inside the cell with the help of CPPs. Another is the PyTPE modified peptide which will self-aggregate because of the hydrophobic interaction and turn on the yellow fluorescence of PyTPE. The appearance of the yellow fluorescence indicates the release of the therapeutic unit to the cells. The selective delivery of the drug to the MMP-2 positive cells was also confirmed by using the intrinsic red fluorescence of DOX. Our result suggests a new and promising method for controlled drug delivery and real-time tracking of drug release in MMP-2 overexpression cells.