Discriminative detection of soman or VX exposure using europium chelated microparticle-based immunofluorescence microfluidic chip.

The retrospective detection of organophosphorus nerve agents (OPNAs) exposure has been achieved by the off-site analysis of OPNA-human serum albumin (HSA) adducts using mass spectrometry-based detection approaches. However, few specific methods are accessible for on-site detection. To address this, a novel immunofluorescence microfluidic chip (IFMC) testing system combining europium chelated microparticle (EuCM) with self-driven microfluidic chip assay has been established to unambiguously determine soman (GD) and VX exposure within 20 min, respectively. The detection system was based on the principle of indirect competitive enzyme-linked immunosorbent assay. The specific monoclonal antibodies that respectively recognized the phosphonylated tyrosine 411 of GD-HSA and VX-HSA adducts were labeled by EuCM to capture corresponding adducts in the exposed samples. The phosphonylated peptides in the test line and goat-anti-rabbit antibody in the control line were utilized to bind the EuCM-labeled antibodies for signal exhibition. The developed IFMC chip could discriminatively detect exposed HSA adducts with high specificity, demonstrating a low limit of detection at exposure concentrations of 0.5 × 10-6 mol/L VX and 1.0 × 10-6 mol/L GD. The exposed serum samples can be qualitatively detected following an additional pretreatment procedure. This is a novel rapid detection system capable of discriminating GD and VX exposure, providing an alternative method for rapidly identifying OPNA exposure.

A protein standard absolute quantification strategy for enhanced absolute quantification of ricin in complex matrices using in vitro synthesized mutant holoprotein as internal standard by ultra-high-performance liquid chromatography-tandem mass spectrometry.

Ricin is a highly toxic protein toxin that poses a potential bioterrorism threat due to its potency and widespread availability. However, the accurate quantification of ricin through absolute mass spectrometry (MS) using a protein standard absolute quantification (PSAQ) strategy is not widely practiced. This limitation primarily arises from the presence of interchain disulfide bonds, which hinder the production of full-length isotope-labeled ricin as an internal standard (IS) in vitro. In this study, we have developed a novel approach for the absolute quantification of ricin in complex matrices using recombinant single-chain and full-length mutant ricin as the protein IS, instead of isotope-labeled ricin, in conjunction with ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). The amino acid sequence of the ricin mutant internal standard (RMIS) was designed by introducing site mutations in specific amino acids of trypsin/Glu-C enzymatic digestion marker peptides of ricin. To simplify protein expression, the A-chain and B-chain of RMIS were directly linked to replace the original interchain disulfide bonds. The RMISs were synthesized using an Escherichia coli expression system. An appropriate RMIS was selected as the protein IS based on consistent digestion efficiency, UHPLC-MS/MS behavior, antibody recognition function, lectin activity, and proper depurination activity with intact ricin. The RMIS was utilized to simultaneously quantify A- and B-chain marker peptides of ricin through UHPLC-MS/MS. This method was thoroughly validated using a milk matrix. By employing internal protein standards, this quantitative strategy overcomes the challenges posed by variations in extraction recoveries, matrix effects, and digestion efficiency encountered when working with different matrices. Consequently, calibration curves generated from milk matrix-spiked samples were utilized to accurately and precisely quantify ricin in river water and plasma samples. Moreover, the established method successfully detected intact ricin in samples obtained from the sixth Organization for the Prohibition of Chemical Weapons (OPCW) exercise on biotoxin analysis. This study presents a novel PSAQ strategy that enables the accurate quantification of ricin in complex matrices.

Folic acid modified copper nanoclusters for fluorescent imaging of cancer cells with over-expressed folate receptor.

Water-soluble and functional copper nanoclusters (CuNCs) were prepared by using folic acid (FA) that serves both as a reducing reagent and a stabilizer. FA also acts as a functional ligand on the surface of the CuNCs, and this can be exploited to target the folate receptor which is over-expressed on the surface of HeLa cells. The FA-modified CuNCs nanoclusters have an average size of ca. 0.9 nm and are stable in aqueous medium for 30 days. Under photoexcitation at λex 270 and 350 nm, the FA-CuNCs display strong blue fluorescence with an emission peak at 440 nm. The FA-CuNCs exhibit low cytotoxicity and favorable biocompatibility as demonstrated by an MTT assay. A cell viability of >80% is found when incubating HeLa cells for 20 h with FA-CuNCs at levels of up to 200 µg mL-1. The targeting capability of the FA-CuNCs is demonstrated by live cell imaging. It is shown that HeLa cells with over-expressed folate receptor are much brighter than A549 cells where the receptor is not over-expressed. This is further corroborated by the fact that the copper content in HeLa cells (1.5 pg/cell) is 6.5-fold higher than that of A549 cells (0.23 pg/cell), both measured after the same incubation time of 3 h. If free FA is introduced into the cell culture medium, the folate receptors will be preoccupied with FA, and this results in a significant decrease in the cellular uptake of the FA-CuNCs by HeLa cells. Graphical Abstract Biocompatible copper nanoclusters (CuNCs) coated with folic acid (FA) were prepared and are shown to be viable probes for the differentiation between FR-positive HeLa cells and FR-negative A549 cells.

Protein-Stabilized Gadolinium Oxide-Gold Nanoclusters Hybrid for Multimodal Imaging and Drug Delivery.

A protein-stabilized multifunctional theranostic nanoplatform, gadolinium oxide-gold nanoclusters hybrid (Gd2O3-AuNCs), is constructed for multimodal imaging and drug delivery. The Gd2O3-AuNCs nanohybrid is developed by integrating Gd2O3 nanocrystals and gold nanoclusters into bovine serum albumin scaffold as a stabilizer. The nanohybrid exhibits favorable biocompatibility and is capable of enhancing the contrast in magnetic resonance and X-ray computed tomography imaging. Meanwhile, the integrated AuNCs component not only endows the nanohybrid to produce red fluorescence, but also sensitizes the generation of singlet oxygen (1O2) upon near-infrared laser stimulation at 808 nm. Bovine serum albumin surrounding the nanoparticles makes Gd2O3-AuNCs a brilliant carrier for the delivery of indocyanine green (ICG). ICG loading endows the Gd2O3-AuNCs-ICG nanocomposite with a near-infrared fluorescence imaging capability, and improves its photodynamic property and photothermal capability. Ultimately, further experiments have demonstrated that Gd2O3-AuNCs-ICG nanocomposite is a promising theranostic agent for image guided cancer therapy.

Hydrophobic Carbon Nanodots with Rapid Cell Penetrability and Tunable Photoluminescence Behavior for in Vitro and in Vivo Imaging.

Tunable fluorescent emission and applications in both in vitro and in vivo imaging of hydrophobic carbon nanodots (CNDs) with rapid penetration capability are reported. The hydrophobic CNDs are prepared via hydrothermal treatment of ionic liquid 1-ethyl-3-methylimidazolium bromide and exhibit excitation-dependent photoluminescence behavior along with a red-shift in the excitation/emission maxima with concentration. The quantum yields of the as-prepared CNDs are in the range of 2.5-4.8% at an excitation wavelength of 300-600 nm. The rapid penetration behavior (within 1 min) of CNDs into the cell membrane significantly reduces the sample treatment time and avoids potential fluorescence quenching induced by the interaction between CNDs and samples. A co-location study reveals that the hydrophobic CNDs are distributed mainly in the lysosome. The potentials of the hydrophobic CNDs as fluorescent probe in in vitro and in vivo imaging are well demonstrated by the labeling of HeLa cells, MCF-7 cells, A549 cells, and Kunming mice.

Green preparation of carbon dots for intracellular pH sensing and multicolor live cell imaging.

Carbon dots are prepared using a green hydrothermal approach with dehydrated shiitake mushroom as the sole carbon source without any additives (these carbon dots are shortly termed as MCDs). Carbonization, surface functionalization and nitrogen doping are involved in the hydrothermal treatment and no further modification or surface passivation is necessary. The derived MCDs are nitrogen-doped, oxygen-rich with hydroxyl, carboxyl and amine groups, with a diameter of ca. 4.2 nm. MCDs exhibit cell permeable properties and a distinct pH-sensitive/excitation-dependent photoluminescence emission feature within pH 4.0-8.0, providing an optical probe for intracellular pH sensing and multicolor imaging of live HeLa cells. MCDs also show a strong fluorescence response to hemin, which facilitates sensitive fluorescent sensing of hemin with a detection limit of 120 nmol L-1.