Enhancing Hydrogen Production from the Photoreforming of Lignin.

Photoreforming of lignocellulose biomass is widely recognised as a challenging but key technology for producing value-added chemicals and renewable hydrogen (H2 ). In this study, H2 production from photoreforming of organosolv lignin in a neutral aqueous solution was studied over a 0.1 wt % Pt/TiO2 (P25) catalyst with ultraviolet A (UVA) light. The H2 production from the system employing the lignin (~4.8 µmol gcat -1 h-1 ) was comparable to that using hydroxylated/methoxylated aromatic model compounds (i. e., guaiacol and phenol, 4.8-6.6 µmol gcat -1 h-1 ), being significantly lower than that from photoreforming of cellulose (~62.8 µmol gcat -1 h-1 ). Photoreforming of phenol and reaction intermediates catechol, hydroquinone and benzoquinone were studied to probe the mechanism of phenol oxidation under anaerobic photoreforming conditions with strong adsorption and electron transfer reactions lowering H2 production from the intermediates relative to that from phenol. The issues associated with catalyst poisoning and low photoreforming activity of lignins demonstrated in this paper have been mitigated by implementing a process by which the catalyst was cycled through anaerobic and aerobic conditions. This strategy enabled the periodic regeneration of the photocatalyst resulting in a threefold enhancement in H2 production from the photoreforming of lignin.

Label-Free Evaluation of Maturation and Hepatotoxicity of Human iPSC-Derived Hepatocytes Using Hyperspectral Raman Imaging.

To promote the clinical application of human induced pluripotent stem cell (hiPSC)-derived hepatocytes, a method capable of monitoring regenerative processes and assessing differentiation efficiency without harming or modifying these cells is important. Raman microscopy provides a powerful tool for this as it enables label-free identification of intracellular biomolecules in live samples. Here, we used label-free Raman microscopy to assess hiPSC differentiation into hepatocyte lineage based on the intracellular chemical content. We contrasted these data with similar phenotypes from the HepaRG and from commercially available hiPSC-derived hepatocytes (iCell hepatocytes). We detected hepatic cytochromes, lipids, and glycogen in hiPSC-derived hepatocyte-like cells (HLCs) but not biliary-like cells (BLCs), indicating intrinsic differences in biomolecular content between these phenotypes. The data show significant glycogen and lipid accumulation as early as the definitive endoderm transition. Additionally, we explored the use of Raman imaging as a hepatotoxicity assay for the HepaRG and iCell hepatocytes, with data displaying a dose-dependent reduction of glycogen accumulation in response to acetaminophen. These findings show that the nondestructive and high-content nature of Raman imaging provides a promising tool for both quality control of hiPSC-derived hepatocytes and hepatotoxicity screening.

Surfactant-free Synthesis of Spiky Hollow Ag-Au Nanostars with Chemically Exposed Surfaces for Enhanced Catalysis and Single-Particle SERS.

Spiky/hollow metal nanoparticles have applications across a broad range of fields. However, the current bottom-up methods for producing spiky/hollow metal nanoparticles rely heavily on the use of strongly adsorbing surfactant molecules, which is undesirable because these passivate the product particles' surfaces. Here we report a high-yield surfactant-free synthesis of spiky hollow Au-Ag nanostars (SHAANs). Each SHAAN is composed of >50 spikes attached to a hollow ca. 150 nm diameter cubic core, which makes SHAANs highly plasmonically and catalytically active. Moreover, the surfaces of SHAANs are chemically exposed, which gives them significantly enhanced functionality compared with their surfactant-capped counterparts, as demonstrated in surface-enhanced Raman spectroscopy (SERS) and catalysis. The chemical accessibility of the pristine SHAANs also allows the use of hydroxyethyl cellulose as a weakly bound stabilizing agent. This produces colloidal SHAANs that remain stable for >1 month while retaining the functionalities of the pristine particles and allows even single-particle SERS to be realized.

Highly sensitive Fourier-transform coherent anti-Stokes Raman scattering spectroscopy via genetic algorithm pulse shaping.

We report highly sensitive Fourier-transform coherent anti-Stokes Raman scattering spectroscopy enabled by genetic algorithm (GA) pulse shaping for adaptive dispersion compensation. We show that the non-resonant four-wave mixing signal from water can be used as a fitness indicator for successful GA training. This method allows GA adaptation to sample measurement conditions and offers significantly improved performance compared to training using second-harmonic generation from a nonlinear crystal in place of the sample. Results include a 3× improvement to peak signal-to-noise ratio for 2-propanol measurement, as well as a 10× improvement to peak intensities from the high-throughput measurement of polystyrene microbeads under flow.

High-Throughput Raman Flow Cytometry and Beyond.

Flow cytometry is a powerful tool with applications in diverse fields such as microbiology, immunology, virology, cancer biology, stem cell biology, and metabolic engineering. It rapidly counts and characterizes large heterogeneous populations of cells in suspension (e.g., blood cells, stem cells, cancer cells, and microorganisms) and dissociated solid tissues (e.g., lymph nodes, spleen, and solid tumors) with typical throughputs of 1,000-100,000 events per second (eps). By measuring cell size, cell granularity, and the expression of cell surface and intracellular molecules, it provides systematic insights into biological processes. Flow cytometers may also include cell sorting capabilities to enable subsequent additional analysis of the sorted sample (e.g., electron microscopy and DNA/RNA sequencing), cloning, and directed evolution. Unfortunately, traditional flow cytometry has several critical limitations as it mainly relies on fluorescent labeling for cellular phenotyping, which is an indirect measure of intracellular molecules and surface antigens. Furthermore, it often requires time-consuming preparation protocols and is incompatible with cell therapy. To overcome these difficulties, a different type of flow cytometry based on direct measurements of intracellular molecules by Raman spectroscopy, or "Raman flow cytometry" for short, has emerged. Raman flow cytometry obtains a chemical fingerprint of the cell in a nondestructive manner, allowing for single-cell metabolic phenotyping. However, its slow signal acquisition due to the weak light-molecule interaction of spontaneous Raman scattering prevents the throughput necessary to interrogate large cell populations in reasonable time frames, resulting in throughputs of about 1 eps. The remedy to this throughput limit lies in coherent Raman scattering methods such as stimulated Raman scattering (SRS) and coherent anti-Stokes Raman scattering (CARS), which offer a significantly enhanced light-sample interaction and hence enable high-throughput Raman flow cytometry, Raman imaging flow cytometry, and even Raman image-activated cell sorting (RIACS). In this Account, we outline recent advances, technical challenges, and emerging opportunities of coherent Raman flow cytometry. First, we review the principles of various types of SRS and CARS and introduce several techniques of coherent Raman flow cytometry such as CARS, multiplex CARS, Fourier-transform CARS, SRS, SRS imaging flow cytometry, and RIACS. Next, we discuss a unique set of applications enabled by coherent Raman flow cytometry, from microbiology and lipid biology to cancer detection and cell therapy. Finally, we describe future opportunities and challenges of coherent Raman flow cytometry including increasing sensitivity and throughput, integration with droplet microfluidics, utilizing machine learning techniques, or achieving in vivo flow cytometry. This Account summarizes the growing field of high-throughput Raman flow cytometry and the bright future it can bring.

Large-scale label-free single-cell analysis of paramylon in Euglena gracilis by high-throughput broadband Raman flow cytometry.

Microalga-based biomaterial production has attracted attention as a new source of drugs, foods, and biofuels. For enhancing the production efficiency, it is essential to understand its differences between heterogeneous microalgal subpopulations. However, existing techniques are not adequate to address the need due to the lack of single-cell resolution or the inability to perform large-scale analysis and detect small molecules. Here we demonstrated large-scale single-cell analysis of Euglena gracilis (a unicellular microalgal species that produces paramylon as a potential drug for HIV and colon cancer) with our recently developed high-throughput broadband Raman flow cytometer at a throughput of >1,000 cells/s. Specifically, we characterized the intracellular content of paramylon from single-cell Raman spectra of 10,000 E. gracilis cells cultured under five different conditions and found that paramylon contents in E. gracilis cells cultured in an identical condition is given by a log-normal distribution, which is a good model for describing the number of chemicals in a reaction network. The capability of characterizing distribution functions in a label-free manner is an important basis for isolating specific cell populations for synthetic biology via directed evolution based on the intracellular content of metabolites.

Ultrafast Simultaneous Raman-Fluorescence Spectroscopy.

Raman and fluorescence spectroscopies offer complementary approaches in bioanalytical chemistry, particularly in microbiological assays. The former method is used to detect lipids, metabolites, and nonspecific proteins and nucleic acids in a label-free manner, while the latter is used to investigate targeted proteins, nucleic acids, and their interactions via labeling or transfection. Despite their complementarity, these regimes are seldom used in conjunction due to fluorescent signals overwhelming inherently weak Raman signals by more than several orders of magnitude. Here we report a multimodal spectrometer that simultaneously performs Raman and fluorescence spectroscopies at high speed. It is made possible by Fourier-transform-coherent anti-Stokes Raman scattering (FT-CARS) and Fourier-transform-two-photon excitation (FT-TPE) measurements powered by a femtosecond pulse laser coupled to a homemade rapid-scan Michelson interferometer, operating at 24 000 spectra per second. As a proof-of-principle demonstration, we validate the ultrafast fluoRaman spectrometer by measuring coumarin dyes in organic solvents. To show its potential for applications that require rapid fluoRaman spectroscopy, we also demonstrate fluoRaman flow cytometry of Haematococcus pluvialis cells under varying culture conditions with a high throughput of ∼10 events per second to perform large-scale single-cell analysis of their metabolic stress response.