Integration of fluorescence and MALDI imaging for microfluidic chip-based screening of potential thrombin inhibitors from natural products.

The microfluidic technology provides an ideal platform for in situ screening of enzyme inhibitors and activators from natural products. This work described a surface-modified ITO glass-PDMS hybrid microfluidic chip for evaluating thrombin interaction with its potential inhibitors by fluorescence imaging and matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI). The fluorescence-labeled substrate was immobilized on a conductive ITO glass slide coated with gold nanoparticles/thiol-ß-cyclodextrin modified TiO2 nanowires (Au-ß-CD@TiO2 NWs) via Au-S bonds. A PDMS microchannel plate was placed on top of the modified ITO slide. The premixed solutions of thrombin and candidate thrombin inhibitors were infused into the microchannels to form a microreactor environment. The enzymatic reaction was rapidly monitored by fluorescence microscopy, and MALDI MS was used to validate and quantify the enzymatic hydrolysate of thrombin to determine the enzyme kinetic process and inhibitory activities of selected flavonoids. The fluorescence and MALDI MS results showed that luteolin, cynaroside, and baicalin have good thrombin inhibitory activity and their half-maximal inhibitory concentrations (IC50) were below 30 µM. The integration of fluorescence imaging and MALDI MSI for in situ monitoring and quantifying the enzymatic reaction in a microfluidic chip is capable of rapid and accurate screening of thrombin inhibitors from natural products.

Monolithic Gold Nanoparticles/Thiol-ß-cyclodextrin-Functionalized TiO2 Nanowires for Enhanced SALDI MS Detection and Imaging of Natural Products.

Surface-assisted laser desorption/ionization mass spectrometry (SALDI MS) has been successfully applied in the analysis of various small molecules. In this work, gold nanoparticles/thiol-ß-cyclodextrin-functionalized TiO2 nanowires (AuNPs/SH-ß-CD-TiO2 NWs) were prepared to enhance the performance of SALDI MS and mass spectrometry imaging (MSI). A monolithic TiO2 film was first grown on an indium tin oxide (ITO) glass slide via a modified sol-gel method and treated in an alkaline environment to form nanowires. TiO2 NWs were chemically modified by SH-ß-CD for immobilizing AuNPs densely and strongly. Compared with the conventional organic matrix 2,5-dihydroxybenzoic acid (DHB), the prepared AuNPs/SH-ß-CD-TiO2 NWs showed superior performances on detection sensitivity, repeatability, and analyte coverage. Analytes typically detectable with negative-ion matrix-assisted laser desorption/ionization (MALDI) MS could also be observed using AuNPs/SH-ß-CD-TiO2 NWs in the positive ion mode. Its successful usage efficiently enhanced the SALDI MS detection of various small molecules such as carbohydrates, fatty acids, and bile acids in the positive ion mode. The developed SALDI substrate was further used to characterize and discriminate the natural and in vitro cultured Calculus Bovis, as well as natural and artificial Moschus. Furthermore, the spatial distribution of several natural products in spearmint leaves and potato tubers was explored by tissue imprinting and deposition on the AuNPs/SH-ß-CD-TiO2 NW surface for SALDI MSI in dual-polarity mode, respectively. The wide application and satisfied detection sensitivity make AuNPs/SH-ß-CD-TiO2 NWs ideal for SALDI MS and MSI of various natural products.

Porous TiO2 Film Immobilized with Gold Nanoparticles for Dual-Polarity SALDI MS Detection and Imaging.

Surface-assisted laser desorption/ionization (SALDI) mass spectrometry (MS) has become an attractive complementary approach to matrix-assisted laser desorption/ionization (MALDI) MS. SALDI MS has great potential for the detection of small molecules because of the absence of applied matrix. In this work, a functionalized porous TiO2 film immobilized with gold nanoparticles (AuNPs-FPTDF) was prepared to enhance SALDI MS performance. The porous TiO2 films were prepared by the facile sol-gel method and chemically functionalized for dense loading of AuNPs. The prepared AuNPs-FPTDF showed superior performance in the detection and imaging of small molecules in dual-polarity modes, with high detection sensitivity in the low pmol range, good repeatability, and low background noise compared to common organic MALDI matrixes. Its usage efficiently enhanced SALDI MS detection of various small molecules, such as amino acids and neurotransmitters, fatty acids, saccharides, alkaloids, and flavonoids, as compared with α-cyano-4-hydroxycinnamic acid, 9-aminoacridine, and the three precursor substrates of AuNPs-FPTDF. In addition, the blood glucose level in rats was successfully determined from a linearity concentration range of 0.5-9 mM, as well as other biomarkers in rat serum with SALDI MS. More importantly, the spatial distribution of metabolites from the intact flowers of the medicinal plant Catharanthus roseus was explored by using the AuNPs-FPTDF as an imprint SALDI MS substrate in dual-polarity modes. These results demonstrate wide applications and superior performances of the AuNPs-FPTDF as a multifunctional SALDI surface with enhanced detection sensitivity and imaging capabilities.