In Situ Sensing of Reactive Oxygen Species on Dye-Stained Single DNA Molecules under Illumination.

Reactive oxygen species (ROS) are a necessary evil in many biological systems and have been measured with fluorescent probes at the ensemble levels both in vitro and in vivo. Measuring ROS generated from a single molecule is important for mechanistic studies, yet measuring ROS near a dye-labeled single-molecule under illumination has been challenging. Here, we use CellROX, a group of ROS probes, to sense ROS near dye-stained DNA that has been flow-stretched and immobilized on a surface. ROS is responsible for the photodamage of DNA molecules under this circumstance. In this report, we confirmed the ROS sensing reaction in bulk solutions and optimized the conditions for single-molecule experiments including the selection of substrates, dye concentrations, probes in the CellROX series, excitation lasers, and emission filter-sets. We observed a correlation between ROS and the dye-labeled DNA and localized the ROS-activated CellROX probe molecules at both the ensemble level and the single-molecule level.

Ultrafast Transient Absorption Spectra of Photoexcited YOYO-1 molecules call for additional investigations of their fluorescence quenching mechanism.

In this report, we observed that YOYO-1 immobilized on a glass surface is much brighter when dried (quantum yield 16±4% in the ambient air) or in hexane than in water (quantum yield ~%).YOYO-1 is a typical cyanine dye that has a photo-isomerization reaction upon light illumination. In order to understand this quenching mechanism, we use femtosecond transient absorption spectroscopy to measure YOYO-1's electron dynamics after excitation directly. By deconvoluting the hot-ground-state absorption and the stimulated emission, the dynamics of electronic relaxation and balance are revealed. The results support the intermolecular charge transfer mechanism better than the intramolecular relaxation mechanism that has been widely believed before. We believe that the first step of the relaxation involves a Dexter charge transfer between the photo-excited YOYO-1 molecule and another guest molecule that is directly bound to the YOYO-1 giving two radicals with opposite signs of charges. The charges are recombined either directly between these two molecules, or both molecules start to rotate and separate from each other. Eventually, the two charges recombined non-radiatively via various pathways. These pathways are reflected on the complicated multi-exponential decay curves of YOYO-1 fluorescence lifetime measurements. This charge transfer mechanism suggests that (1) electrical insulation may help improve the quantum yield of YOYO-1 in polar solutions significantly and (2) a steric hindrance for the intramolecular rotation may have a less significant effect.

Reusable Chemically-Micropatterned Substrates via Sequential Photoinitiated Thiol-Ene Reactions as Template for Perovskite Thin-Film Microarrays.

Patterning semiconducting materials are important for many applications such as microelectronics, displays, and photodetectors. Lead halide perovskites are an emerging class of semiconducting materials that can be patterned via solution-based methods. Here we report an all-benchtop patterning strategy by first generating a patterned surface with contrasting wettabilities to organic solvents that have been used in the perovskite precursor solution then spin-coating the solution onto the patterned surface. The precursor solution only stays in the area with higher affinity (wettability). We applied sequential sunlight-initiated thiol-ene reactions to functionalize (and pattern) both glass and conductive fluorine-doped tin oxide (FTO) transparent glass surfaces. The functionalized surfaces were measured with the solvent contact angles of water and different organic solvents and were further characterized by XPS, selective fluorescence staining, and selective DNA adsorption. By simply spin-coating and baking the perovskite precursor solution on the patterned substrates, we obtained perovskite thin-film microarrays. The spin-coated perovskite arrays were characterized by XRD, AFM, and SEM. We concluded that patterned substrate prepared via sequential sunlight-initiated thiol-ene click reactions is suitable to fabricate perovskite arrays via the benchtop process. In addition, the same patterned substrates can be reused several times until a favorable perovskite microarray is acquired. Among a few conditions we have tested, DMSO solvent and modified FTO surfaces with alternatively carboxylic acid and alkane is the best combination to obtain high-quality perovskite microarrays. The solvent contact angle of DMSO on carboxylic acid-modified FTO surface is nearly zero and 65±3° on octadecane modified FTO surface.

Single-Particle Organolead Halide Perovskite Photoluminescence as a Probe for Surface Reaction Kinetics.

Photoluminescence (PL) of organolead halide perovskites (OHPs) is sensitive to OHPs' surface conditions and is an effective way to report surface states. Literature has reported that at the ensemble level, the PL of photoexcited OHP nanorods declines under an inert nitrogen (N2) atmosphere and recovers under subsequent exposure to oxygen (O2). At the single-particle level, we observed that OHP nanorods photoblink at rates dependent on both the excitation intensity and the O2 concentration. Combining the two sets of information with the charge-trapping/detrapping mechanism, we are able to quantitatively evaluate the interaction between a single surface defect and a single O2 molecule using a new kinetic model. The model predicts that the photodarkening of OHP nanorods in the N2 atmosphere has a different mechanism than conventional PL quenching, which we call photo-knockout. This model provides fundamental insights into the interactions of molecular O2 with OHP materials and helps design a suitable OHP interface for a variety of applications in photovoltaics and optoelectronics.

Molecular design of near-infrared fluorescent Pdots for tumor targeting: aggregation-induced emission versus anti-aggregation-caused quenching.

Semiconducting polymer dots (Pdots) have recently emerged as a new type of ultrabright fluorescent probe that has been proved to be very useful for biomedical imaging. However, Pdots often suffer from serious fluorescence aggregation-caused quenching (ACQ) especially for near-infrared (NIR) fluorescent Pdots. This article compared two strategies to overcome the ACQ effect in near-infrared emissive Pdot systems: aggregation-induced emission (AIE) and anti-aggregation-caused quenching (anti-ACQ). The results show that the anti-ACQ platform outperforms the AIE system. The fluorescence quantum yield of anti-ACQ-based Pdots can be over 50% and the average per-particle brightness of the Pdots is about 5 times higher than that of the commercially available quantum dots. To help understand why the monomer conformations could greatly affect the optical properties of Pdots, molecular dynamics simulations were performed for the first time in such complicated Pdot systems. To demonstrate applications for in vivo fluorescence imaging, both microangiography imaging on living zebrafish embryos and specific tumor targeting on mice were performed. We anticipate that these studies will pave the way for the design of new highly fluorescent Pdot systems.

Correction: Photobleaching of YOYO-1 in super-resolution single DNA fluorescence imaging.

[This corrects the article DOI: 10.3762/bjnano.8.229.].

Kinetic Model under Light-Limited Condition for Photoinitiated Thiol-Ene Coupling Reactions.

Thiol-ene click chemistry has become a powerful paradigm in synthesis, materials science, and surface modification in the past decade. In the photoinitiated thiol-ene reaction, an induction period is often observed before the major change in its kinetic curve, for which a possible mechanism is proposed in this report. Briefly, light soaking generates radicals following the zeroth-order reaction kinetics. The radical is the reactant that initializes the chain reaction of thiol-ene coupling, which is a first-order reaction. Combining both and under the light-limited conditions, a surprising kinetics represented by a Gaussian-like model evolves that is different from the exponential model used to describe the first-order reaction of the final product. The experimental data are fitted well with the new model, and the reaction kinetic constants can be pulled out from the fitting.

Photobleaching of YOYO-1 in super-resolution single DNA fluorescence imaging.

Super-resolution imaging of single DNA molecules via point accumulation for imaging in nanoscale topography (PAINT) has great potential to visualize fine DNA structures with nanometer resolution. In a typical PAINT video acquisition, dye molecules (YOYO-1) in solution sparsely bind to the target surfaces (DNA) whose locations can be mathematically determined by fitting their fluorescent point spread function. Many YOYO-1 molecules intercalate into DNA and remain there during imaging, and most of them have to be temporarily or permanently fluorescently bleached, often stochastically, to allow for the visualization of a few fluorescent events per DNA per frame of the video. Thus, controlling the fluorescence on-off rate is important in PAINT. In this paper, we study the photobleaching of YOYO-1 and its correlation with the quality of the PAINT images. At a low excitation laser power density, the photobleaching of YOYO-1 is too slow and a minimum required power density was identified, which can be theoretically predicted with the proposed method in this report.

Molecular Engineering and Design of Semiconducting Polymer Dots with Narrow-Band, Near-Infrared Emission for in Vivo Biological Imaging.

This article describes the design and synthesis of donor-bridge-acceptor-based semiconducting polymer dots (Pdots) that exhibit narrow-band emissions, ultrahigh brightness, and large Stokes shifts in the near-infrared (NIR) region. We systematically investigated the effect of π-bridges on the fluorescence quantum yields of the donor-bridge-acceptor-based Pdots. The Pdots could be excited by a 488 or 532 nm laser and have a high fluorescence quantum yield of 33% with a Stokes shift of more than 200 nm. The emission full width at half-maximum of the Pdots can be as narrow as 29 nm, about 2.5 times narrower than that of inorganic quantum dots at the same emission wavelength region. The average per-particle brightness of the Pdots is at least 3 times larger than that of the commercially available quantum dots. The excellent biocompatibility of these Pdots was demonstrated in vivo, and their specific cellular labeling capability was also approved by different cell lines. By taking advantage of the durable brightness and remarkable stability of these NIR fluorescent Pdots, we performed in vivo microangiography imaging on living zebrafish embryos and long-term tumor monitoring on mice. We anticipate these donor-bridge-acceptor-based NIR-fluorescent Pdots with narrow-band emissions to find broad use in a variety of multiplexed biological applications.

A Two-Step Method for smFRET Data Analysis.

We demonstrate a two-step data analysis method to increase the accuracy of single-molecule Förster Resonance Energy Transfer (smFRET) experiments. Most current smFRET studies are at a time resolution on the millisecond level. When the system also contains molecular dynamics on the millisecond level, simulations show that large errors are present (e.g., > 40%) because false state assignment becomes significant during data analysis. We introduce and confirm an additional step after normal smFRET data analysis that is able to reduce the error (e.g., < 10%). The idea is to use Monte Carlo simulation to search ideal smFRET trajectories and compare them to the experimental data. Using a mathematical model, we are able to find the matches between these two sets, and back guess the hidden rate constants in the experimental results.