Ming-Yuan Wei

Development of redox-labeled electrochemical immunoassay for polycyclic aromatic hydrocarbons with controlled surface modification and catalytic voltammetric detection

A redox-labeled direct competitive electrochemical immunoassay for polycyclic aromatic hydrocarbons (PAHs) was developed. A ruthenium tris(bipyridine)-pyrenebutyric acid conjugate was synthesized as the redox-labeled tracer. Its recognition by an anti-PAH monoclonal antibody was confirmed by surface plasmon resonance. In the immunoassay, the antibody was immobilized on (3-glycidoxypropyl)-trimethoxysilane (GPTMS)-modified indium tin oxide (ITO) electrodes. The assay was quantified by measuring the electro-catalytic current of the redox label in an oxalate-containing electrolyte which served as a sacrificial electron donor to amplify the current signal. Formation of GPTMS film on ITO and subsequent antibody immobilization were characterized by X-ray photoelectron spectroscopy (XPS) and electrochemistry. Using a ruthenium tris(bipyridine)-conjugated IgG (IgG-Ru) as the surface-bound redox probe, the highest electrochemical signal was obtained on GPTMS electrodes with 1 h modification. Under the optimized conditions for ITO modification, antibody immobilization and tracer concentration, competition curves for benzo[a]pyrene and pyrenebutyric acid were obtained with a detection limit of 2.4 and 10 ng mL(-1), respectively. The redox-labeled electrochemical immunoassay with signal amplification mechanism offers a potential analytical method for the simultaneous detection of multiple environmental organic pollutants on antibody biochips.

High resolution imaging beyond the acoustic diffraction limit in deep tissue via ultrasound-switchable NIR fluorescence.

Fluorescence imaging in deep tissue with high spatial resolution is highly desirable because it can provide details about tissues structural, functional, and molecular information. Unfortunately, current fluorescence imaging techniques are limited either in penetration depth (microscopy) or spatial resolution (diffuse light based imaging) as a result of strong light scattering in deep tissue. To overcome this limitation, we developed an ultrasound-switchable fluorescence (USF) imaging technique whereby ultrasound was used to switch on/off the emission of near infrared (NIR) fluorophores. We synthesized and characterized unique NIR USF contrast agents. The excellent switching properties of these agents, combined with the sensitive USF imaging system developed in this study, enabled us to image fluorescent targets in deep tissue with spatial resolution beyond the acoustic diffraction limit.

Development of Ultrasound-Switchable Fluorescence Imaging Contrast Agents Based on Thermosensitive Polymers and Nanoparticles

In this paper, we first introduced a recently developed high-resolution, deep-tissue imaging technique, ultrasound-switchable fluorescence (USF). The imaging principles based on two types of USF contrast agents were reviewed. To improve USF imaging techniques further, excellent USF contrast agents were developed based on high-performance thermoresponsive polymers and environment-sensitive fluorophores. Herein, such contrast agents were synthesized and characterized with five key parameters: 1) peak excitation and emission wavelengths (λ_ex and λ_em); 2) the fluorescence intensity ratio between on- and off-states (I_On/I_Off); 3) the fluorescence lifetime ratio between on- and off-states (τ_On/τ_Off); 4) the temperature threshold to switch on fluorophores (T_th); and 5) the temperature transition bandwidth (T_BW). We mainly investigated fluorescence intensity and lifetime changes of four environment-sensitive dyes [7-(2-Aminoethylamino)-N,N-dimethyl-4-benzofurazansulfonamide (DBD-ED), St633, Sq660, and St700] as a function of temperature, while the dye was attached to poly(N-isopropylacrylamide) linear polymers or encapsulated in nanoparticles. Six fluorescence resonance energy transfer systems were invented in which both the donor (DBD-ED or ST425) and the acceptor (Sq660) were adopted. Our results indicate that three Förster resonance energy transfer systems, where both I_On/I_Off and τ_On/τ_Off are larger than 2.5, are promising for application in future surface tissue bioimaging by the USF technique.

Structure‐Dependent Mitochondrial Dysfunction and Hypoxia Induced with Single‐Walled Carbon Nanotubes

Cytotoxicity of nanomaterials on living systems is known to be affected by their size, shape, surface chemistry, and other physicochemical properties. Exposure to a well-characterized subpopulation of specific nanomaterials is therefore desired to reveal more detailed mechanisms. This study develops scalable density gradient ultracentrifugation sorting of highly dispersed single-walled carbon nanotubes (SWNTs) into four distinct bands based on diameter, aggregation, and structural integrity, with greatly improved efficiency, yield, and reproducibility. With guarantee of high yield and stability of four SWNT fractions, it is possible for the first time, to investigate the structure-dependent bioeffects of four SWNT fractions. it is possible Among these, singly-dispersed integral SWNTs show no significant effects on the mitochondrial functions and hypoxia. The aggregated integral SWNTs show more significant effects on the mitochondrial dysfunction and hypoxia compared to the aggregated SWNTs with poor structure integrity. Then, it is found that the aggregated integral SWNTs induced the irregular mitochondria respiratory and pro-apoptotic proteins activation, while aggregated SWNTs with poor structure integrity greatly enhanced reactive oxygen species (ROS) levels. This work supports the view that control of the distinct structure characteristics of SWNTs helps establish clearer structure-bioeffect correlation and health risk assessment. It is also hoped that these results can help in the design of nanomaterials with higher efficiency and accuracy in subcellular translocation.

Ultrasound-modulated fluorescence based on fluorescent microbubbles

Abstract. Ultrasound-modulated fluorescence (UMF) imaging has been proposed to provide fluorescent contrast while maintaining ultrasound resolution in an optical-scattering medium (such as biological tissue). The major challenge is to extract the weakly modulated fluorescent signal from a bright and unmodulated background. UMF was experimentally demonstrated based on fluorophore-labeled microbubble contrast agents. These contrast agents were produced by conjugating N-hydroxysuccinimide (NHS)-ester-attached fluorophores on the surface of amine-functionalized microbubbles. The fluorophore surface concentration was controlled so that a significant self-quenching effect occurred when no ultrasound was applied. The intensity of the fluorescent emission was modulated when microbubbles were oscillated by ultrasound pulses, presented as UMF signal. Our results demonstrated that the UMF signals were highly dependent on the microbubbles’ oscillation amplitude and the initial surface fluorophore-quenching status. A maximum of ∼42% UMF modulation depth was achieved with a single microbubble under an ultrasound peak-to-peak pressure of 675 kPa. Further, UMF was detected from a 500-μm tube filled with contrast agents in water and scattering media with ultrasound resolution. These results indicate that ultrasound-modulated fluorescent microbubble contrast agents can potentially be used for fluorescence-based molecular imaging with ultrasound resolution in the future.

Rapid and efficient sonochemical formation of gold nanoparticles under ambient conditions using functional alkoxysilane

Gold nanoparticles (NPs) are rapidly and efficiently formed under ambient conditions with a novel and highly-efficient sonochemical promoter. Despite of the presence of free oxygen, 3-glycidoxypropyltrimethoxysilane (GPTMS) showed remarkable efficiency in promoting the reduction rate of Au (III) than that of conventional promoters (primary alcohols). This is likely attributed to the formation of a variety of radical scavengers, which are alcoholic products from sonochemical hydrolysis of the epoxide group and methoxysilane moieties of GPTMS under weakly acidic conditions. Interestingly, the promotion is quenched by amine- or thiol-functionalized alkoxysilane, thereby producing marginal amounts of gold NPs. Furthermore, products of hydrolyzed GPTMS were confirmed to attach on the surface of gold NPs by attenuated total reflectance-Fourier transform infrared spectroscopy. However, according to transmission electron microscopy images, gold NPs that were produced in the presence of GPTMS tend to fuse with each other as condensation of silanols occurs, forming worm- or nugget-like gold nanostructures. The use of long chain surfactants (i.e. polyethylene glycol terminated with hydroxyl or carboxyl) inhibited the fusion, leading to mono-dispersed gold NPs. Additionally, the fact that this approach requires neither an ultrasound source with high frequency nor anaerobic conditions provides a huge advantage. These findings could potentially open an avenue for rapid and large-scale green-synthesis of gold NPs in future work.

High-Resolution Ultrasound-Switchable Fluorescence Imaging in Centimeter-Deep Tissue Phantoms with High Signal-To-Noise Ratio and High Sensitivity via Novel Contrast Agents

For many years, investigators have sought after high-resolution fluorescence imaging in centimeter-deep tissue because many interesting in vivo phenomena—such as the presence of immune system cells, tumor angiogenesis, and metastasis—may be located deep in tissue. Previously, we developed a new imaging technique to achieve high spatial resolution in sub-centimeter deep tissue phantoms named continuous-wave ultrasound-switchable fluorescence (CW-USF). The principle is to use a focused ultrasound wave to externally and locally switch on and off the fluorophore emission from a small volume (close to ultrasound focal volume). By making improvements in three aspects of this technique: excellent near-infrared USF contrast agents, a sensitive frequency-domain USF imaging system, and an effective signal processing algorithm, for the first time this study has achieved high spatial resolution (~ 900 μm) in 3-centimeter-deep tissue phantoms with high signal-to-noise ratio (SNR) and high sensitivity (3.4 picomoles of fluorophore in a volume of 68 nanoliters can be detected). We have achieved these results in both tissue-mimic phantoms and porcine muscle tissues. We have also demonstrated multi-color USF to image and distinguish two fluorophores with different wavelengths, which might be very useful for simultaneously imaging of multiple targets and observing their interactions in the future. This work has opened the door for future studies of high-resolution centimeter-deep tissue fluorescence imaging.

A visualized observation of calcium-dependent gelsolin activity upon the surface coverage of fluorescent-tagged actin filaments

Gelsolin regulates the dynamics of F-actin by binding to F-actin to sever and cap. In the present study, a novel approach is introduced to observe gelsolin activity through the coverage of surface-bound F-actin. Gelsolin was immobilized on streptavidin coated surface using biotinylation and, as a result, the interaction between gelsolin and F-actin was visualized. Consequently, the coverage of F-actin reflects the activity of gelsolin as a function of free Ca(2+) concentrations. In order to prevent non-specific binding of F-actin, the combinations of BSA and Tween-20 as blocking agents were investigated. Moreover, the measurement of the length of F-actin with actin-gelsolin mixtures at various ratios provided the verification of gelsolin activity after biotinylation. The data shows the increase in Ca(2+) concentration leads to a proportional increase in F-actin coverage, giving to half-maximal coverage at ~2.9 μM. Furthermore, the length of bound F-actin was found to decrease along with increasing Ca(2+) concentration, and full-length F-actin was rarely observed. This may suggest that severing and capping activities of gelsolin occur without more additional Ca(2+) for subsequent activation after full-length gelsolin binds to a side of F-actin. This finding may provide a key to understand gelsolin activity.

Ultrasound-modulated fluorescence based on donor-acceptor-labeled microbubbles

Abstract. A fluorescence resonance energy transfer (FRET)-based microbubble contrast agent system was designed to experimentally demonstrate the concept of ultrasound-modulated fluorescence (UMF). Microbubbles were simultaneously labeled with donor and acceptor fluorophores on the surface to minimize self-quenching and maximize FRET. In response to ultrasound, the quenching efficiency was greatly modulated by changing the distance between the donor and acceptor molecules through microbubble size oscillations. Both donors and acceptors exhibited UMF on individual microbubbles. The UMF strength of the donor was more significant compared to that of the acceptor. Furthermore, the UMF of the donor was observed from a microbubble solution in a turbid media. This study exploits the feasibility of donor–acceptor labeled microbubbles as UMF contrast agents.

Re-evaluation of biotin-streptavidin conjugation in Förster resonance energy transfer applications.

Bioaffinity conjugation between streptavidin (SA) and biotin has been widely used to link donors and acceptors for investigating the distance-dependent Förster resonance energy transfer (FRET). When studying a commonly used FRET system of (QD-SA)-(biotin-DNA-dye) [donor: quantum dot (QD); acceptor: small organic fluorescent dye; and linker: deoxyribose nucleic acid (DNA) molecule via SA-biotin conjugation], however, a contradictory finding was recently reported in the literature. It was found that the FRET lost its dependence on the number of DNA base pairs when using a phosphate-buffered saline (PBS) solution. We found that the conflicted results were caused by the ionic strength of the adopted buffer solutions. Our results suggest that the dependent FRET on the number of DNA bases is favorable in a low-ionic-strength buffer, whereas in relatively high-ionic-strength buffers, the FRET loses the DNA length dependence. We propose that the independence is mainly caused by the conformational change of DNA molecules from a stretched to a coiled mode when the cations in the high-ionic-strength buffer neutralize the negatively charged backbone of DNA molecules, thereby bringing the acceptors close to the donors.