Showing papers by "Shouzhuo Yao published in 2018"

Fluorescent Ti3C2 MXene quantum dots for an alkaline phosphatase assay and embryonic stem cell identification based on the inner filter effect

Abstract: As an emerging two-dimensional material, MXenes have attracted much attention due to their unique physicochemical properties, but their application in biosensing has been lagging far behind because of their poor salt tolerance. Herein, a titanium carbide MXene quantum dot (Ti3C2 QD)-based fluorescent probe for the alkaline phosphatase (ALP) activity assay and embryonic stem cell (ESC) identification was developed by taking advantage of the inner filter effect (IFE). Ti3C2 QDs with ∼4.2 nm in diameter were prepared from Ti3C2 MXenes by hydrothermal treatment, exhibiting excellent salt tolerance, anti-photobleaching and dispersion stability in aqueous solution. Owing to the remarkable overlap between the absorption spectrum of p-nitrophenol (p-NP) and the excitation and emission spectrum of Ti3C2 QDs, p-NP generated from ALP-catalyzed dephosphorylation of the substrate, p-nitrophenyl phosphate (p-NPP), can effectively quench the fluorescence of Ti3C2 QDs through the IFE. As a result, sensitive fluorometric analysis of ALP activity was achieved without complicating the conventional colorimetric ALP detection system. The proposed assay was successfully applied to determine ALP activity with a low limit of detection (0.02 U L-1) as well as monitoring the enzyme activity in real time. Finally, accurate analysis of ALP, the biomarker of ESC, in ESC lysates was also achieved using this IFE-based method, affording an alternative method for ESC identification.

Cell-Surface-Anchored Ratiometric DNA Tweezer for Real-Time Monitoring of Extracellular and Apoplastic pH.

Abstract: Precise and dynamic imaging of extracellular pH is one crucial yet challenging task for studying cell physiological and pathological processes. Here, we construct a DNA tweezer to dynamically monitor pH changes of cellular microenvironments. The DNA tweezer contains three key elements: a three-strand ssDNA-frame labeled with cholesterol to anchor it on the cell membrane, a pH-sensitive i-motif sequence in the middle to dynamically control the switch between the “open” and “closed” states of the DNA tweezer, and a pair of FRET fluorophores (rhodamine green and rhodamine red) on the two arms of the tweezer to reflect its state. With cholesterol, a natural component of cell membranes, as an anchoring element, the sensor exhibited high cell-membrane-insertion efficiency and low cytotoxicity. Using the i-motif as a sensing element, it can quickly and reversibly respond to extracellular pH in the pH range of 5.0–7.5 and further perform real-time imaging of cell-surface-pH changes with excellent spatial and temp...

Phospholipid-Tailored Titanium Carbide Nanosheets as a Novel Fluorescent Nanoprobe for Activity Assay and Imaging of Phospholipase D.

Abstract: As one of the emerging inorganic graphene analogues, two-dimensional titanium carbide (Ti3C2) nanosheets have attracted extensive attention in recent years because of their remarkable structural and electronic properties. Herein, a sensitive and selective nanoprobe to fluorescently probe phospholipase D activity was developed on the basis of an ultrathin Ti3C2 nanosheets-mediated fluorescence quenching effect. Ultrathin Ti3C2 nanosheets with ∼1.3 nm in thickness were synthesized from bulk Ti3AlC2 powder by a two-step exfoliation procedure and further modified by a natural phospholipid that is doped with rhodamine B-labeled phospholipid (RhB-PL-Ti3C2). The close proximity between RhB and Ti3C2 leads to efficient fluorescence quenching (>95%) of RhB by energy transfer. Phospholipase D-catalyzed lipolysis of the phosphodiester bond in RhB-PL results in RhB moving away from the surface of Ti3C2 nanosheets and subsequent fluorescence recovery of RhB, providing a fluorescent “switch-on” assay for the phospholip...

Transpeptidation-Mediated Assembly of Tripartite Split Green Fluorescent Protein for Label-Free Assay of Sortase Activity.

Abstract: Transpeptidation of surface proteins catalyzed by the transpeptidase sortase plays a critical role in the infection process of Gram-positive pathogen, and probing sortase activity and screening its inhibitors are of great significance to fundamental biological research and pharmaceutical development, especially novel antivirulence drug design. Herein, we developed a novel fluorescent biosensor to detect sortase activity based on a transpeptidation-triggered assembly of tripartite split green fluorescent protein (split GFP). Peptide P1, composed the 10th β-sheet of GFP (GFP10) and the sortase A (SrtA) recognition sequence (LPETX), and peptide P2, the 11th β-sheet of GFP (GFP11) with oligoglycine at N-terminal, were designed and synthesized, respectively. Existence of SrtA enables P1 and P2 to ligate into one peptide, which could spontaneously bind to GFP1-9 (the 1st to 9th β-sheets of GFP) and assemble into functional GFP. Thus, the sortase-catalyzed transpeptidation can switch on the fluorescence signal o...

Simultaneous Monitoring of Cell-surface Receptor and Tumor-targeted Photodynamic Therapy via TdT-initiated Poly-G-Quadruplexes.

Abstract: Cancer cells contain a unique set of cell surface receptors that provide potential targets for tumor theranostics. Here, we propose an efficient approach to construct G-quadruplex-based aptamers that specifically recognize cell-surface receptors and monitor them in an amplified manner. This designed aptamer combined particular sequence for the c-Met on the cell surface and poly-G-quadruplexes structures that allow a rapid and amplified fluorescent readout upon the binding of thioflavin T (ThT). The poly-G-quadruplexes also function as a carrier for photosensitizers such as TMPyP4 in that, the aptamer further trigger the production of reactive oxygen species (ROS) to commit cells to death. This unique c-Met targeting aptamer enabled simultaneous monitoring of c-Met on the cell surface with ThT and photodynamic killing of these lung cancer cells with TMPyP4. This strategy is expected to enhance the development of tumor-targeted diagnosis and drug delivery.

An enzymatic polymerization-activated silver nanocluster probe for in situ apoptosis assay.

Abstract: As an emerging category of fluorophores, nucleic acid-stabilized silver nanoclusters (DNA/AgNCs) have attracted a great deal of interest and have been widely applied for interdisciplinary research. In this work, we have constructed a novel DNA/AgNC probe for cell apoptosis detection and imaging based on an enzyme-polymerized polyadenylic acid (poly-dA) DNA chain and a toehold strand displacement reaction. This method can effectively “tag” intracellular genomic DNA fragments, a biochemical hallmark of apoptosis, with poly-dA DNA chains up to 400-bases produced by terminal deoxynucleotidyl transferase (TdT)-activated polymerization. The strand displacement initiated by the target poly-dA DNA chain releases the quencher labeled-DNA from the DNA/AgNC probe, leading to a significant fluorescence lighting-up of DNA/AgNCs for the sensitive detection of cell apoptosis, with a high signal-to-background ratio (S/B = 58). Using the DNA/AgNC-based assay, as few as 20 apoptotic cells can be detected in vitro. Furthermore, the feasibility of our approach was demonstrated by the in situ quantitative analysis of apoptosis in HepG2 cells without the need for tedious washing and separation steps.