Preparation of Microkernel-Based Mesoporous (SiO2-CdTe-SiO2)@SiO2 Fluorescent Nanoparticles for Imaging Screening and Enrichment of Heat Shock Protein 90 Inhibitors from Tripterygium Wilfordii.

The currently utilized ligand fishing for bioactive molecular screening from complex matrixes cannot perform imaging screening. Here, we developed a new solid-phase ligand fishing coupled with an in situ imaging protocol for the specific enrichment and identification of heat shock protein 90 (Hsp 90) inhibitors from Tripterygium wilfordii, utilizing a multiple-layer and microkernel-based mesoporous nanostructure composed of a protective silica coating CdTe quantum dot (QD) core and a mesoporous silica shell, i.e., microkernel-based mesoporous (SiO2-CdTe-SiO2)@SiO2 fluorescent nanoparticles (MMFNPs) as extracting carries and fluorescent probes. The prepared MMFNPs showed a highly uniform spherical morphology, retention of fluorescence emission, and great chemical stability. The fished ligands by Hsp 90α-MMFNPs were evaluated via the preliminary bioactivity based on real-time cellular morphology imaging by confocal laser scanning microscopy (CLSM) and then identified by mass spectrometry (MS). Celastrol was successfully isolated as an Hsp 90 inhibitor, and two other specific components screened by Hsp 90α-MMFNPs, i.e., demecolcine and wilforine, were preliminarily identified as potential Hsp 90 inhibitors through the verification of strong affinity to Hsp 90 and antitumor bioactivity. The approach based on the MMFNPs provides a strong platform for imaging screening and discovery of plant-derived biologically active molecules with high efficiency and selectivity.

Single-step, homogeneous and sensitive detection for microRNAs with dual-recognition steps based on luminescence resonance energy transfer (LRET) using upconversion nanoparticles.

A single-step, homogeneous and sensitive LRET assay is presented for the detection of miRNAs. The amplification-free assay provides a unique combination of high specificity with dual-recognition approach of different hybridization and ligation steps and preventing background auto-fluorescence in biological samples using upconversion nanoparticles (UCNPs) as signal-producing nanoprobes. The assay probe is composed of signal-producing unit (a pair of homogeneous upconversion luminescence resonance energy transfer (UC-LRET)-based oligonucleotides) and recognition unit (two adaptor oligonucleotides). In the presence of target miRNAs, the probe and target miRNAs leads to the formation of stable double-strands and semi-stable adaptor-miRNAs complexes with an adaptor nick. Ligation of the nick using ligase cause the formation of stable double-strands, resulting in UCNPs-to-dye UC-LRET for detection of the miRNAs with near-infrared radiation (980nm). Sensitive detection of miRNA-21 at concentrations of 200pM to 1.4nM and detection limits of 0.095nM with good precision of 3.9% (RSD) for seven repeated measurements of 500pM miRNAs demonstrate the feasibility of both high throughput and point-of-care clinical diagnostics. The homogeneous UC-LRET assay without any washing can be extended to the application in other important types of nucleic acid analysis.

Layer-by-layer functionalized porous Zinc sulfide nanospheres-based solid-phase extraction combined with liquid chromatography time-of-flight/mass and gas chromatography-mass spectrometry for the specific enrichment and identification of alkaloids from Crinum asiaticum var. sinicum.

The current widely utilized polymer or C8, C18 end-capped material-based sorbents for solid-phase extraction could not capture alkaloids well only based on "like dissolves like" principle. In this paper, a layer-by-layer functionalized porous Zinc sulfide nanospheres-based solid-phase extraction (SPE) combined with liquid chromatography time-of-flight/mass spectrometry (LC-TOF/MS) and gas chromatography-mass spectrometry (GC-MS) was developed for the specific enrichment and identification of alkaloids from complex matrixes, Crinum asiaticum var. sinicum crude extracts. The functionalized porous Zinc sulfide nanospheres were prepared by the amidation reaction of poly-(acrylic acid) (PAA) homopolymer with amino groups onto the porous ZnS nanospheres. Tandem LC-TOF/MS spectrometry presented that the almost all of the twenty-three main peaks in elution fraction from the SPE could be inferred as alkaloids with ion of mass according to the nitrogen rule and hit formula with Peak View1.2@software from AB SCIEX, and seven alkaloids including two new found chemical entities were directly identified from their GC-MS spectra and retention indices. We believe that this SPE protocol can also be utilized in the future to selectively enrich alkaloids from extracts of other plant species.

Robust nanoplasmonic substrates for aptamer macroarrays with single-step detection of PDGF-BB.

An aptamer macroarray on a robust nanoplasmonic substrate with fluorescence enhancement is developed for a single-step sensitive detection of human platelet-derived growth factor-BB (PDGF-BB), a predominant cancer biomarker in cancer angiogenesis. A hybrid Au-nanoparticles-poly (dimethylsiloxane) (PDMS) as nanoplasmonic substrate is prepared via the in-situ reduction of AuCl4(-) ions in PDMS matrixes onto 96 or 384 well plates. In the absence of target molecules, unfolded PDGF-BB aptamer conjugated with dye TAMRA is electrostatically bound to a positively charged poly-L-lysine (PLL)-coated Au nanocomposites film surface, and the fluorescence enhancement effects can be optimized by varying the distance between TAMRA and the Au nanocomposites film, which is easily adjusted by varying the thickness of the biocompatible poly-(acrylic acid) (PAA/PLL) multilayers, and thus metal-enhanced fluorescence of dye TAMRA conjugated with the aptamer is generated up to 15.2-fold. The interaction of the aptamer to its target induces the reversible conformation change of the aptamer, and consequently, the electrostatic potential is overcome by binding force. As a result, the target-binding interaction of the aptamer causes the irreversible detachment of the aptamer from the nanostructured Au film surface to decrease fluorescence of TAMRA. The aptamer macroarray provides not only the appropriate sensitivity for clinical diagnostics with a wide range of linear detection from 10pg/mL to 10µg/mL, high specificity for PDGF-BB against VEGF-165, VEGF-121, NaCl and IgG, and temporal biological stability, but also a single-step detection. We envision that the efficient and robust aptamer macroarray can be extended to the detection of other biomarkers.

Silver nanoparticles-enhanced time-resolved fluorescence sensor for VEGF(165) based on Mn-doped ZnS quantum dots.

A silver nanoparticles (AgNPs)-enhanced time-resolved fluorescence (TR-FL) sensor based on long-lived fluorescent Mn-doped ZnS quantum dots (QDs) is developed for the sensitive detection of vascular endothelial growth factor-165 (VEGF165), a predominant cancer biomarker in cancer angiogenesis. The aptamers bond with the Mn-doped ZnS QDs and the BHQ-2 quencher-labelling strands hybridized in duplex are coupled with streptavidin (SA)-functionalized AgNPs to form the AgNPs-enhanced TR-FL sensor, showing lower fluorescence intensity in the duplex state due to the fluorescence resonance energy transfer (FRET) between the Mn-doped ZnS QDs and quenchers. Upon the addition of VEGF165, the BHQ-2 quencher-labelling strands of the duplex are displaced, leading to the disruption of the FRET. As a result, the fluorescence of the Mn-doped QDs within the proximity of the AgNPs is recovered. The FL signal can be measured free of the interference of short-lived background by setting appropriate delay time and gate time, which offers a signal with high signal-to-noise ratio in photoluminescent biodetection. Compared with the bare TR-FL sensor, the AgNPs-based TR-FL sensor showed a huge improvement in fluorescence based on metal-enhanced fluorescence (MEF) effect, and the sensitivity increased 11-fold with the detection limit of 0.08 nM. In addition, the sensor provided a wide range of linear detection from 0.1 nM to 16 nM.