Autobiography of an Analytical Chemist.

Most of my research directions were opportunistic. Having worked with lasers in the early stages of laser applications in analytical chemistry, attending conferences, workshops, and administrative meetings that were not exactly aligned with our own research, locating to a building or in a department that housed scientists with different backgrounds, having certain specialized equipment at the right time, and having funding agencies that were broad-minded clearly contributed to my ventures into diverse fields. Most of all, it had to be the many eager minds that I have had the fortune to work with. I have always tried to suggest research topics that might be interesting to the individual coworker rather than something straight out of my own research proposals. Only then did each person actually own the project rather than consider it a chore. After all, we work in the field of analytical chemistry, in which almost anything we do can fit in.

Sequence-Modulated Interactions between Single Multivalent DNA-Conjugated Gold Nanoparticles.

DNA-conjugated gold nanoparticle (AuNP) is an attractive building block to construct elegant plasmonic nanomaterials by self-assembly but the complicated interaction between multivalent nanoconjugates governing the assembly process and the properties of assembled structures remains poorly understood. Herein, with an in situ kinetic single-particle imaging method, we report the dynamic interaction between single multivalent DNA-conjugated AuNPs quantitatively depends on the nucleic acid sequence in nanoconjugates. From the binding dynamics analysis, it was found that the binding of nanoconjugates with DNA length longer than nine bases is kinetically irreversible and the binding rate is dependent on both the sequence length and GC content, enabling us to predict the rational modulation of binding rates of individual building blocks for stepwise assembly. Moreover, the reversibility for the multivalent interaction between single nanoconjugates at constant temperature can be reinstated by adopting the DNA sequence with single-nucleotide mismatch and the lifetime for nanoconjugates at bound state can be tailored by changing the mismatch positions in DNA strands, providing new opportunity to create active nanostructures with controlled dynamic properties. All these findings provide new insights for understanding the multivalent interaction during the assembly process at the single-nanoconjugate level and predicting the programmable self-assembly of engineered nanoconjugates for the fabrication of dynamic nanomaterials.

Direct Imaging of Single Plasmonic Metal Nanoparticles in Capillary with Laser Light-Sheet Scattering Imaging.

Understanding the heterogeneous distribution of the physical and chemical properties of plasmonic metal nanoparticles is fundamentally important to their basic and applied research. Traditionally, they are obtained either indirectly via bulk spectroscopic measurements plus electron microscopic characterizations or through single molecule/particle imaging of nanoparticles immobilized on planar substrates. In this study, by using light-sheet scattering microscopy with a supercontinuum white laser, highly sensitive imaging of individual metal nanoparticles (MNPs) flowing inside a capillary, driven by either pressure or electric field, was achieved for the first time. We demonstrate that single plasmonic nanoparticles with different size or chemical modification could be differentiated through their electrophoretic mobility in a few minutes. This technique could potentially be applied to high throughput characterization and evaluation of single metal nanoparticles as well as their dynamic interactions with various local environments.

Single Plasmonic Nanosprings for Visualizing Reactive-Oxygen-Species-Activated Localized Mechanical Force Transduction in Live Cells.

Mechanical force signaling in cells has been regarded as the biological foundation of various important physiological functions. To understand the nature of these biological and physiological processes, imaging and determining the mechanical signal transduction dynamics in live cells are required. Herein, we proposed a strategy to determine mechanical force as well as its changes with single-particle dark-field spectral microscopy by using a single plasmonic nanospring as a mechanical sensor, which can transfer force-induced molecular extension/compression into spectral responses. With this robust plasmonic nanospring, we achieved the visualization of activation of localized mechanical force transduction in single live cells triggered by reactive-oxygen-species (ROS) stimulation. The successful demonstration of a biochemical ROS signal to mechanical signal conversion suggested this strategy is promising for studying mechanical force signaling and regulation in live biological systems.

Direct quantitative screening of influenza A virus without DNA amplification by single-particle dual-mode total internal reflection scattering.

Quantitative screening of influenza A (H7N9) virus without DNA amplification was performed based on single-particle dual-mode total internal reflection scattering (SD-TIRS) with a transmission grating (TG). A gold nanopad was utilized as a substrate for the hybridization of probe DNA molecules with the TIRS nanotag (silver-nanoparticle). The TG effectively isolated the scattering signals in first-order spectral images (n=+1) of the nanotag from that of the substrate, providing excellent enhancement of signal-to-noise and selectivity. By using single-DNA molecule/TIRS nanotag hybridization, target DNA molecules of H7N9 were detected down to 74 zM, which is at least 100,000 times lower than the current detection limit of 9.4fM. By simply modifying the design of the probe DNA molecules, this technique can be used to directly screen other viral DNAs in various human biological samples at the single-molecule level without target amplification.

A novel scattering switch-on detection technique for target-induced plasmon-coupling based sensing by single-particle optical anisotropy imaging.

We reported a novel scattering switch-on detection technique using flash-lamp polarization darkfield microscopy (FLPDM) for target-induced plasmon-coupling based sensing in homogeneous solution. With this method, we demonstrated sub-nM sensitivity for hydrogen sulfide (H2S) detection over a dynamic range of five orders of magnitude. This robust technique holds great promise for applications in toxic environmental pollutants and biological molecules.

Single Particle Tracking of Peptides-Modified Nanocargo on Lipid Membrane Revealing Bulk-Mediated Diffusion.

Understanding the detailed diffusion behavior of the nanocargo on lipid membrane can afford deep insight into the surface chemistry controlled translocation mechanism for the rational design of an efficient delivery system. By tracking the diffusion trajectory of transacting activator of transcription (TAT, a cell penetrating peptide) peptides-modified nanocargo on lipid membrane, bulk-mediated (intermittent hopping) diffusion was observed for the first time after a blended modification of TAT peptides and polyethylene glycol (PEG) molecules onto the nanoparticle surface. In contrast to random walk or confined diffusion, the nanoparticles could be temporarily confined for random waiting times between surface displacements produced by excursions through the bulk fluid, which was not noted before. Non-Gaussian distributed step length (with a stretched power law like tail) was observed, making large displacements much more probable than one would predict for regular Gaussian decay. This kind of larger displacement would therefore significantly facilitate a kinetically controlled surface searching process like heterogeneous penetration site recognition on a fluidic membrane with suitable spatial orientation.

Selective Colorimetric Detection of Hydrogen Sulfide Based on Primary Amine-Active Ester Cross-Linking of Gold Nanoparticles.

Hydrogen sulfide (H2S) is a highly toxic environmental pollutant and also an important gaseous transmitter. Therefore, selective detection of H2S is very important, and visual detection of it with the naked eye is preferred in practical applications. In this study, thiolated azido derivates and active esters functionalized gold nanoparticles (AE-AuNPs)-based nanosensors have been successfully prepared for H2S perception. The sensing principle consists of two steps: first, H2S reduces the azide group to a primary amine; second, a cross-linking reaction between the primary amine and active ester induces the aggregation of AuNPs. The AE-AuNPs-based nanosensors show high selectivity toward H2S over other anions and thiols due to the specific azide-H2S chemistry. Under optimal conditions, 0.2 µM H2S is detectable using a UV-vis spectrophotometer, and 4 µM H2S can be easily detected by the naked eye. In addition, the practical application of the designed nanosensors was evaluated with lake water samples.

Fluorescent gold nanodots based sensor array for proteins discrimination.

A series of dual-ligand cofunctionalized fluorescent gold nanodots with similar fluorescence and diverse surface properties has been designed and synthesized to build a protein sensing array. Using this "chemical nose/tongue" strategy, fluorescence response patterns can be obtained on the array and identified via linear discriminant analysis (LDA). Eight proteins have been well distinguished at low concentration (A280 = 0.005) based on this sensor array. The practicability of this sensor array was further validated by high accuracy (100%) examination of 48 unknown protein samples.

Optical analysis of biological hydrogen sulphide: an overview of recent advancements.

Determining the levels of endogenous hydrogen sulphide in real time has become increasingly crucial because of its important biological roles in various physiological and pathological processes. Optical methods allowing sensitive, multiplex and dynamic analysis in a non-invasive manner have attracted much attention in biological and biomedical analysis. This review provides an overview of recent advancements in optical analysis of biological hydrogen sulphide, with a focus on fluorescence and non-fluorescence optical strategies for sensing and imaging subcellular hydrogen sulphide in living biosystems.

Color difference amplification between gold nanoparticles in colorimetric analysis with actively controlled multiband illumination.

Spectral chemical sensing with digital color analysis by using consumer imaging devices could potentially revolutionize personalized healthcare. However, samples with small spectral variations often cannot be differentiated in color due to the nonlinearity of color appearance. In this study, we address this problem by exploiting the color image formation mechanism in digital photography. A close examination of the color image processing pipeline emphasizes that although the color can be represented digitally, it is still a reproducible subjective perception rather than a measurable physical property. That makes it possible to physically manage the color appearance of a nonradiative specimen through engineered illumination. By using scattering light imaging of gold nanoparticles (GNPs) as a model system, we demonstrated via simulation that enlarged color difference between spectrally close samples could be achieved with actively controlled illumination of multiple narrow-band light sources. Experimentally, darkfield imaging results indicate that color separation of single GNPs with various sizes can be significantly improved and the detection limit of GNP aggregation-based colorimetric assays can be much reduced when the conventional spectrally continuous white light was replaced with three independently intensity-controlled laser beams, even though the laser lines were uncorrelated with the LSPR maxima of the GNPs. With low-cost narrow-band light sources widely available today, this actively controlled illumination strategy could be utilized to replace the spectrometer in many spectral sensing applications.

Determination of sterols using liquid chromatography with off-line surface-assisted laser desorption/ionization mass spectrometry.

A new method, reversed phase liquid chromatography with off-line surface-assisted laser desorption/ionization mass spectrometry (RPLC-SALDI MS) for the determination of brassicasterol (BR), cholesterol (CH), stigmasterol (ST), campesterol (CA) and ß-sitosterol (SI) in oil samples has been developed. The sample preparation consisted of alkaline saponification followed by extraction of the unsaponificable fraction with diethyl ether. The recovery of the sterols ranged from 91 to 95% with RSD less than 4%. Separation of the five major sterols on a C18 column using methanol-water gradient was achieved in about 10min. An on-line UV detector was employed for the initial sterol detection prior to effluent deposition using a laboratory-built spotter with 1:73 splitter. Off-line SALDI MS was then applied for mass determination/identification and quantification of the separated sterols. Ionization of the nonpolar analytes was achieved by silver ion cationization with silver nanoparticles used as the SALDI matrix providing limits of detection 12, 6 and 11fmol for CH, ST and SI, respectively. Because of the incorporated splitter, the effective limits of detection of the RPLC-SALDI MS analysis were 4, 3 and 4pmol (or 0.08, 0.06 and 0.08µg/mL) for CH, ST and SI, respectively. For quantification, 6-ketocholestanol (KE) was used as the internal standard. The method has been applied for the identification and quantification of sterols in olive, linseed and sunflower oil samples. The described off-line coupling of RPLC to SALDI MS represents an alternative to GC-MS for analysis of nonpolar compounds.

Pericellular matrix plays an active role in retention and cellular uptake of large-sized nanoparticles.

As the outmost coating of cells, the pericellular matrix (PCM) involved in various cellular functions has been exploited previously to be able to accumulate 120 nm Au nanoparticles (NPs), adjust their diffusion coefficient similar to that of membrane receptors, and enhance their uptake efficiency. In this study, the interactions between PCM and NPs with different sizes and materials were systematically investigated. We found that PCM can selectively enhance the retention and cellular uptake of NPs with diameters from 50 to 180 nm, but has no enhancement effect for 20 nm NPs. Identical behaviors of PCM was observed for both Au NPs and polystyrene NPs, indicating that this unique phenomenon is more related to the dimensions of the NPs. The study of single-particle tracking of 50-180 nm NPs on the surface of thick PCM cells revealed that PCM actively adjusts the diffusion coefficient of NPs to ∼0.1 µm(2)/s regardless of their sizes. By blocking the receptor-mediated endocytosis (RME) pathway with four different inhibitors, this active role of PCM can be effectively suppressed, further confirming that the trapping and retention of NPs by PCM is an inherent biological function. These findings provided new insights for better understanding of the RME pathway and may have promising NP-based applications for controlled drug delivery and therapy in biomedicine.

High-throughput sulfide sensing with colorimetric analysis of single Au-Ag core-shell nanoparticles.

We present a high-throughput strategy for sensitive detection of H2S by using individual spherical Au-Ag core-shell plasmonic nanoparticles (PNPs) as molecular probes. This method is based on quantification of color variation of the single PNPs resulting from formation of Ag2S on the particle surface. The spectral response range of the 51 nm PNP was specifically designed to match the most sensitive region of color cameras. A high density of immobilized PNPs and rapid color RGB (red/green/blue) analysis allow a large number of individual PNPs to be monitored simultaneously, leading to reliable quantification of color change of the PNPs. A linear logarithmic dependence on sulfide concentrations from 50 nM to 100 µM was demonstrated by using this colorimetric assay. By designing PNPs with various surface chemistries, similar strategies could be developed to detect other chemically or biologically important molecules.

Optical imaging of individual plasmonic nanoparticles in biological samples.

Imaging of plasmonic nanoparticles (PNP) with optical microscopy has aroused considerable attention in recent years. The unique localized surface plasmon resonance (LSPR) from metal nanoparticles facilitates the transduction of a chemical or physical stimulus into optical signals in a highly efficient way. It is therefore possible to perform chemical or biological assays at the single object level with the help of standard optical microscopes. Because the source of background noise from different samples is different, distinct imaging modalities have been developed to discern the signals of interest in complex surroundings. With these convenient yet powerful techniques, great improvements in chemical and biological assays have been demonstrated, and many interesting phenomena and dynamic processes have also been elucidated. Further development and application of optical imaging methods for plasmonic probes should lead to many exciting results in chemistry and biology in the future.

Direct observation of the orientation dynamics of single protein-coated nanoparticles at liquid/solid interfaces.

We have observed the rotational dynamics of single protein-coated gold nanorods (AuNRs) on C18-modified silica surfaces in real time by dual-channel polarization dark-field microscopy. Four different rotational states were identified, depending on the apparent strength of interactions between the AuNRs and the surface. The distributions of the states could be regulated by adjusting the salt concentration, and the state transitions were verified by monitoring the entire desorption process of a single AuNR. Our study provides insight into the interfacial orientation and dynamics of nanoparticles and could be useful for in vitro biophysics and the separation of proteins.

Direct imaging of transmembrane dynamics of single nanoparticles with darkfield microscopy: improved orientation tracking at cell sidewall.

Investigation of the cellular internalization processes of individual nanoparticles (NPs) is of great scientific interest with implications to drug delivery and NP biosafety. Herein, by using dual-channel polarization darkfield microcopy (DFM) and single gold nanorods (AuNRs) as orientation probes, we developed a method that is capable of monitoring AuNR orientation dynamics during its transmembrane process. With annular oblique illumination and a birefringent prism to split AuNR plasmonic scattering into two channels of orthogonal polarizations, the AuNR azimuth and polar angles are obtained from their intensity difference and intensity sum. By placing the focal plane of the microscope objective at the elevated cell sidewall rather than at the flat cell top, interference from cellular background is reduced and the signal-to-noise ratio of AuNR orientation sensing is improved significantly, especially for AuNRs inserting into the membrane at a large out-of-plane angle. As a result, we were able to capture the complete membrane-crossing dynamics of single AuNRs. This powerful method could be utilized to obtain valuable insights on NP endocytosis mechanisms of various cells.

Subdiffraction-limited plasmonic imaging with anisotropic metal nanoparticles.

We have developed a high-resolution nonfluorescent imaging method based on superlocalization of gold nanorods (AuNRs). By taking advantage of their anisotropic optical property of the plasmonic scattering of AuNRs, selective imaging of only a fraction of AuNRs can be achieved by rotating the sample relative to the linear polarized illumination under cross-polarization microscopy with a high NA objective. The AuNR positions obtained from a series of images could then be used to reconstruct the overall image. Two AuNRs with center-to-center distances of 80 nm were successfully resolved. This simple but deterministic super-resolution imaging technique can potentially be used to fingerprint optically anisotropic metal nanoparticles and their assemblies for labeling, sensing, and encryption applications.

Preventing UV induced cell damage by scavenging reactive oxygen species with enzyme-mimic Au-Pt nanocomposites.

We have prepared enzyme-mimic Au-Pt nanocomposites (NCs) for catalyzing the decomposition of reactive oxygen species. After surface modification, the Au-Pt NCs can be readily internalized and retained by human skin cells and also can effectively reduce cellular oxidative stress. We have demonstrated that the active and biocompatible Au-Pt nanocomposites can be applied for preventing cell damages by scavenging cellular reactive oxygen species induced by ultraviolet irradiation, indicating potential uses for the prevention and therapy of ROS-mediated diseases.