Anwei Zhu

Functional Surface Engineering of C-Dots for Fluorescent Biosensing and in Vivo Bioimaging

Nanoparticles are promising scaffolds for applications such as imaging, chemical sensors and biosensors, diagnostics, drug delivery, catalysis, energy, photonics, medicine, and more. Surface functionalization of nanoparticles introduces an additional dimension in controlling nanoparticle interfacial properties and provides an effective bridge to connect nanoparticles to biological systems. With fascinating photoluminescence properties, carbon dots (C-dots), carbon-containing nanoparticles that are attracting considerable attention as a new type of quantum dot, are becoming both an important class of imaging probes and a versatile platform for engineering multifunctional nanosensors. In order to transfer C-dots from proof-of-concept studies toward real world applications such as in vivo bioimaging and biosensing, careful design and engineering of C-dot probes is becoming increasingly important. A comprehensive knowledge of how C-dot surfaces with various properties behave is essential for engineering C-dots with useful imaging properties such as high quantum yield, stability, and low toxicity, and with desirable biosensing properties such as high selectivity, sensitivity, and accuracy. Several reviews in recent years have reported preparation methods and properties of C-dots and described their application in biosensors, catalysis, photovoltatic cells, and more. However, no one has yet systematically summarized the surface engineering of C-dots, nor the use of C-dots as fluorescent nanosensors or probes for in vivo imaging in cells, tissues, and living organisms. In this Account, we discuss the major design principles and criteria for engineering the surface functionality of C-dots for biological applications. These criteria include brightness, long-term stability, and good biocompatibility. We review recent developments in designing C-dot surfaces with various functionalities for use as nanosensors or as fluorescent probes with fascinating analytical performance, and we emphasize applications in bioimaging and biosensing in live cells, tissues, and animals. In addition, we highlight our work on the design and synthesis of a C-dot ratiometric biosensor for intracellular Cu(2+) detection, and a twophoton fluorescent probe for pH measurement in live cells and tissues. We conclude this Account by outlining future directions in engineering the functional surface of C-dots for a variety of in vivo imaging applications, including dots with combined targeting, imaging and therapeutic-delivery capabilities, or high-resolution multiplexed vascular imaging. With each application C-dots should open new horizons of multiplexed quantitative detection, high-resolution fluorescence imaging, and long-term, real-time monitoring of their target.

Carbon Dot‐Based Inorganic–Organic Nanosystem for Two‐Photon Imaging and Biosensing of pH Variation in Living Cells and Tissues

A carbon dot (C-Dot)-based two-photon fluorescent probe has been developed for the monitoring of pH changes across a broad range with high sensitivity and selectivity. The inorganic-organic probe also shows good biocompatibility and cell permeability, and thus can be successfully applied in bioimaging and biosensing of physiological pH in living cells, as well as living tissues at a depth of 65-185 μm.

Carbon-Dot-Based Ratiometric Fluorescent Probe for Imaging and Biosensing of Superoxide Anion in Live Cells

In this article, a ratiometric fluorescent biosensor for O2(•-) was developed, by employing carbon dots (C-Dots) as the reference fluorophore and hydroethidine (HE), a specific organic molecule toward O2(•-), playing the role as both specific recognition element and response signal. The hybrid fluorescent probe CD-HE only emitted at 525 nm is ascribed to C-Dots, while HE was almost nonfluorescent, upon excitation at 488 nm. However, after reaction with O2(•-), a new emission peak ascribed to the reaction products of HE and O2(•-) was clearly observed at 610 nm. Meanwhile, this peak gradually increased with the increasing concentration of O2(•-) but the emission peak at 525 nm stayed constant, leading to a ratiometric detection of O2(•-). The inorganic-organic fluorescent sensor exhibited high sensitivity, a broad dynamic linear range of ~5 × 10(-7)-1.4 × 10(-4) M, and low detection limit down to 100 nM. The present probe also showed high accuracy and excellent selectivity for O2(•-) over other reactive oxygen species (ROS), metal ions, and so on. Moreover, the C-Dot-based inorganic-organic probe demonstrated long-term stability against pH changes and continuous light illumination, good cell-permeability, and low cytotoxicity. Accordingly, the developed fluorescent biosensor was eventually applied for intracellular bioimaging and biosensing of O2(•-) changes upon oxidative stress.

Nanoporous gold film encapsulating cytochrome c for the fabrication of a H2O2 biosensor

A layer-by-layer route to prepare nanoporous Au film materials on transparent ITO substrates is reported by alternatively assembling Au and Ag nanoparticles through 1,5-pentanedithiol as a cross-linker, followed by that Ag nanoparticles are dissolved at room temperature in HAuCl4 solution. Electron transfer of cytochrome c (cyt. c) - an excellent model for investigation of biomolecules, is greatly facilitated at the nanoporous Au film with electron transfer rate constant (ks) of 3.9s(-1). Meanwhile, cyt. c adsorped onto the nanoporous Au film still maintain its enzymatic activity toward H2O2. On the basis of these experimental results, the cyt. c-nanoporous Au film is exploited to an amperometric biosensor for H2O2 with high selectivity, broad linear range, low detection limit, and long stability.

Plasmon‐Driven Selective Oxidation of Aromatic Alcohols to Aldehydes in Water with Recyclable Pt/TiO2 Nanocomposites

Selective oxidation of alcohols to aldehydes is a key reaction for the synthesis of fine chemicals, since aldehyde derivatives are widely used in the flavoring, confectionary, and beverage industries. Efficient aerobic oxidation of alcohols has usually been facilitated by catalysts such as palladium, platinum, and copper. With the development of photocatalysis, selective photocatalytic oxidation of aromatic alcohols to aldehydes in water has been reported on nanostructured rutile, anatase, and brookite TiO2 under UV irradiation. [3] One of the great challenges in catalysis is to develop new photocatalysts with a high activity response to visible light, which will allow the utilization of sunlight, an abundant and clean low-cost energy source. Recently, the selective oxidation of alcohols in organic solvent was carried out under visible-light irradiation in a catalyst system containing dye-sensitized TiO2 and 2,2,6,6tetramethylpiperidinyloxyl (TEMPO). Herein, we report an alternative strategy for selective oxidation of aromatic alcohols to aldehydes in water with high selectivity and relatively long-term stability based on surface plasmon resonance of platinum nanoparticles deposited onto TiO2 (Pt/TiO2) film under visible-light irradiation at ambient temperature. Surface plasmon resonance of noble metal nanoparticles has been afforded much attention of late, due to their unique properties and their wide applications in multicolor imaging, photovoltaic cells, chemical sensors, and biosensors. Charge separation has also been realized on a metal nanoparticles/TiO2 nanofilm and successfully applied to photovoltaic cells, photocatalysis, and photolithography. However, plasmon-driven selective conversion of alcohols to aldehydes has, to our knowledge, never been reported. Furthermore, compared to the power catalysts, such as the TEMPO-based system, it is much easier for the film catalyst to separate from the system and undergo recycling. In addition, the oxidation has been carried out in water, which is considerably safer, cheaper, and more environmentally friendly than organic solvents. More importantly, the products can be separated by simple decantation, and the catalyst solution can be recycled. TiO2 films with different particle sizes and crystalline forms (anatase or rutile) were prepared from various TiO2 sols by spin coating followed by sintering in air atmosphere. Rutile TiO2 film was fabricated by calcination of anatase TiO2 at high temperature. The size and crystalline form of the TiO2 films were characterized by scanning electron microscopy (SEM) and X-ray diffraction (XRD), respectively. The SEM image and XRD pattern of anatase TiO2 STS-21 film sintered at 723 K for 1 h are shown

Plasmon-induced enhancement in analytical performance based on gold nanoparticles deposited on TiO2 film.

This paper demonstrates a novel approach for developing the analytical performance of electrochemical biosensors in which hydrogen peroxide (H(2)O(2)) is selected as a model target, based on surface plasmon resonance of gold nanoparticles (Au NPs) deposited onto a TiO(2) nanoneedle film. Direct electron transfer of cytochrome c (cyt. c) is realized at Au NPs deposited onto a TiO(2) nanoneedle film (Au/TiO(2) film), and both anodic and cathodic currents of the redox reaction at the Au/TiO(2) film upon visible-light irradiation are amplified. Meanwhile, in the presence of oxidized or reduced states of cyt. c, cathodic or anodic photocurrents are generated respectively by the Au/TiO(2) film, suggesting that the amplified anodic and cathodic currents are ascribed to the visible-light excitation. The photocurrent action spectrum obtained at the Au/TiO(2) film in the presence of cyt. c is in a good agreement with the surface plasmon absorption spectrum of Au NPs deposited onto the TiO(2) film, and maximum photocurrent is also consistent with the plasmon absorption peak of Au NPs themselves. It indicates that the enhanced photocurrents generated by visible-light irradiation are attributed to the surface plasmon resonance of Au NPs. On the other hand, experimental results reveal that cyt. c is stably immobilized onto the Au/TiO(2) film and maintains inherent enzymatic activity toward H(2)O(2) even under continuous visible-light illumination. The amplified redox currents of cyt. c produced by surface plasmon resonance of Au NPs, combined with the stability and enzymatic activity of cyt. c confined on the Au/TiO(2) film even after continuous visible-light illumination, subsequently provide the enhanced analytical performance in determination of H(2)O(2). The sensitivity of the present biosensor for H(2)O(2) is 4-fold larger than that obtained without visible-light irradiation, the detection limit is achieved to be 4.5 x 10(-8) M and the dynamic detection linear range extends from 1 x 10(-7) M to 1.2 x 10(-2) M.

Real-Time Electrochemical Monitoring of Cellular H2O2 Integrated with In Situ Selective Cultivation of Living Cells Based on Dual Functional Protein Microarrays at Au−TiO2 Surfaces

This paper demonstrates a novel strategy for site-selective cell adhesion and in situ cultivation of living cells, integrated with real-time monitoring of cellular small biomolecules based on dual functional protein microarrays. The protein microarrays have been produced on the superhydrophobic|philic Au-TiO2 micropatterns, through further modification of L-cysteine (Cys) and followed by successive immobilization of a model protein, cytochrome c (cyt c). Experimental results have revealed that the created cyt c microarrays play dual functions: one is employed as a robust substrate for site-selective cell adhesion and in situ cultivation of living cells, because the protein microarrays exhibit high selectivity and bioaffinity toward cells, as well as long biostability under cell culture condition up to 7 days. Meanwhile, the cyt c microarrays can also serve as sensing elements for hydrogen peroxide (H2O2) due to the inherent enzymatic activity of the heme center in cyt c. Direct electron transfer of cyt c has been enhanced at the Cys-modified Au-TiO2 (Au-TiO2/Cys) microarrays, and the electrochemical behavior can be tuned by varying the width and spacing of the microband arrays. Furthermore, cyt c is stably immobilized on the Au-TiO2/Cys microarrays and maintains its enzymatic activity after confined on the microarrays. Thus, the optimized cyt c microarrays show striking analytical performance for H2O2 determination, e.g., high sensitivity and selectivity, broad linear range from 10(-9) M to 10(-2) M, low detection limit down to 2 nM, and short response time within 5 s. As a result, the excellent analytical properties of the cyt c microarrays, as well as the characteristic of the protein microarrays themselves, including high selectivity, long biostability, and good bioaffinity, opens up a method for selective in situ cultivation of cells integrated with real-time detection of signaling biomolecules such as H2O2 released from living cells, which shows potential for physiological and pathological investigations.

Two-Photon Ratiometric Fluorescent Sensor Based on Specific Biomolecular Recognition for Selective and Sensitive Detection of Copper Ions in Live Cells

In this work, we develop a ratiometric two-photon fluorescent probe, ATD@QD-E2Zn2SOD (ATD = amino triphenylamine dendron, QD = CdSe/ZnSe quantum dot, E2Zn2SOD = Cu-free derivative of bovine liver copper-zinc superoxide dismutase), for imaging and sensing the changes of intracellular Cu(2+) level with clear red-to-yellow color change based on specific biomolecular recognition of E2Zn2SOD for Cu(2+) ion. The inorganic-organic nanohybrided fluorescent probe features two independent emission peaks located at 515 nm for ATD and 650 nm for QDs, respectively, under two-photon excitation at 800 nm. Upon addition of Cu(2+) ions, the red fluorescence of QDs drastically quenches, while the green emission from ATD stays constant and serves as a reference signal, thus resulting in the ratiometric detection of Cu(2+) with high accuracy by two-photon microscopy (TPM). The present probe shows high sensivity, broad linear range (10(-7)-10(-3) M), low detection limit down to ∼10 nM, and excellent selectivity over other metal ions, amino acids, and other biological species. Meanwhile, a QD-based inorganic-organic probe demonstrates long-term photostability, good cell-permeability, and low cytotoxicity. As a result, the present probe can visualize Cu(2+) changes in live cells by TPM. To the best of our knowledge, this is the first report for the development of a QD-based two-photon ratiometric fluorescence probe suitable for detection of Cu(2+) in live cells.

A two-photon ratiometric fluorescence probe for Cupric Ions in Live Cells and Tissues

Development of sensitive and selective probes for cupric ions (Cu2+) at cell and tissue level is a challenging work for progress in understanding the biological effects of Cu2+. Here, we report a ratiometric two-photon probe for Cu2+ based on the organic-inorganic hybrids of graphene quantum dots (GQDs) and Nile Blue dye. Meanwhile, Cu-free derivative of copper-zinc superoxide dismutase (SOD) – E2Zn2SOD is designed as the unique receptor for Cu2+ and conjugated on the surface of GQDs. This probe shows a blue-to-yellow color change in repose to Cu2+, good selectivity, low cytotoxicity, long-term photostability, and insensitivity to pH over the biologically relevant pH range. The developed probe allows the direct visualization of Cu2+ levels in live cells as well as in deep-tissues at 90–180 μm depth through the use of two-photon microscopy. Furthermore, the effect of ascorbic acid is also evaluated on intracellular Cu2+ binding to E2Zn2SOD by this probe.

Development of Au Disk Nanoelectrode Down to 3 nm in Radius for Detection of Dopamine Release from a Single Cell

A Au disk nanoelectrode down to 3 nm in radius was developed by a facile and reliable method and successfully applied for monitoring dopamine release from single living vesicles. A fine etched Au wire was coated with cathodic electrophoretic paint followed by polyimide, which retracted from the tip end during curing to expose the Au nanotip. By cyclic voltammetric scanning the above tip in 0.5 M KCl, the transformation of a core-shaped apex into a geometrically well-defined Au disk nanoelectrode with different dimensions can be controllably and reproducibly achieved. Scanning electron microscopy, transmission electron microscopy, and steady-state voltammetry were used to determine the size of nanoelectrodes. The results showed that the specific etching and insulation method not only avoids the use of toxic etching solution and the uncontrollable treatment to expose the tip but also makes possible the controllable and reproducible fabrication of Au disk nanoelectrode down to 3 nm in radius. The nanoelectrodes with well-demonstrated analytical performance were further applied for amperometrically monitoring dopamine release from single rat pheochromacytoma cells with high spatial resolution.