Supramolecular Aptamers on Graphene Oxide for Efficient Inhibition of Thrombin Activity

Graphene oxide (GO), a two-dimensional material with a high aspect ratio and polar functional groups, can physically adsorb single-strand DNA through different types of interactions, such as hydrogen bonding and π-π stacking, making it an attractive nanocarrier for nucleic acids. In this work, we demonstrate a strategy to target exosites I and II of thrombin simultaneously by using programmed hybrid-aptamers for enhanced anticoagulation efficiency and stability. The targeting ligand is denoted as Supra-TBA15/29 (supramolecular TBA15/29), containing TBA15 (a 15-base nucleotide, targeting exosite I of thrombin) and TBA29 (a 29-base nucleotide, targeting exosite II of thrombin), and it is designed to allow consecutive hybridization of TBA15 and TBA29 to form a network of TBAs (i.e., supra-TBA15/29). The programmed hybrid-aptamers (Supra-TBA15/29) were self-assembled on GO to further boost anticoagulation activity by inhibiting thrombin activity, and thus suppress the thrombin-induced fibrin formation from fibrinogen. The Supra-TBA15/29-GO composite was formed mainly through multivalent interaction between poly(adenine) from Supra-TBA15/29 and GO. We controlled the assembly of Supra-TBA15/29 on GO by regulating the preparation temperature and the concentration ratio of Supra-TBA15/29 to GO to optimize the distance between TBA15 and TBA29 units, aptamer density, and aptamer orientation on the GO surfaces. The dose-dependent thrombin clotting time (TCT) delay caused by Supra-TBA15/29-GO was >10 times longer than that of common anticoagulant drugs including heparin, argatroban, hirudin, and warfarin. Supra-TBA15/29-GO exhibits high biocompatibility, which has been proved by in vitro cytotoxicity and hemolysis assays. In addition, the thromboelastography of whole-blood coagulation and rat-tail bleeding assays indicate the anticoagulation ability of Supra-TBA15/29-GO is superior to the most widely used anticoagulant (heparin). Our highly biocompatible Supra-TBA15/29-GO with strong multivalent interaction with thrombin [dissociation constant (Kd) = 1.9 × 10−11 M] shows great potential as an effective direct thrombin inhibitor for the treatment of hemostatic disorders

Nanoparticle-based laser desorption/ionization mass spectrometric analysis of drugs and metabolites

Nanoparticle-assisted laser desorption/ionization mass spectrometry (LDI-MS) is a powerful tool for the analysis of a wide range of molecules. Many of the drawbacks in the matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) can be avoided with the application of nanomaterials as matrices as well as substrates for the LDI-MS to achieve a low background noise in low m/z region and high reproducibility. Surface-assisted LDI (SALDI)-MS, especially the nanoparticle-based LDI-MS, has emerged as a promising technique for the analysis of trace amounts of substances in various biological samples due to their high surface area for analyte enrichment, efficient desorption/ionization, and homogeneous crystallization of sample. Therefore, it is highly useful in clinical, forensic, medical, food and drug analyses, disease diagnosis, and various other fields. In this review, we briefly discuss the application of various nanomaterials, which include metal-based, carbon-based, silicon-based nanomaterials and nanocomposites, as matrices and substrates for LDI-MS based drug and metabolite analyses and possible detection strategies. Also, we discuss the idea of using “mass tag” for signal amplification for drug and metabolite detection using nanoparticle assisted LDI-MS. Keywords: Drugs, Laser desorption and ionization, Mass spectrometry, Matrix, Metabolites, Nanoparticle

Monitoring Thrombin Generation and Screening Anticoagulants through Pulse Laser-Induced Fragmentation of Biofunctional Nanogold on Cellulose Membranes

Thrombin generation (TG) has an important part in the blood coagulation system, and monitoring TG is useful for diagnosing various health issues related to hypo-coagulability and hyper-coagulability. In this study, we constructed probes by using mixed cellulose ester membranes (MCEMs) modified with gold nanoparticles (Au NPs) for monitoring thrombin activity using laser desorption/ionization mass spectrometry (LDI–MS). The LDI process produced Au cationic clusters ([Au_<i>n</i>]⁺; <i>n</i> = 1–3) that we detected through MS. When thrombin reacted with fibrinogen on the Au NPs–MCEMs, insoluble fibrin was formed, hindering the formation of Au cationic clusters and, thereby, decreasing the intensity of their signals in the mass spectrum. Accordingly, we incorporated fibrinogen onto the Au NPs–MCEMs to form Fib–Au NPs–MCEM probes to monitor TG with good selectivity (>1000-fold toward thrombin with respect to other proteins or enzymes) and sensitivity (limit of detection for thrombin of ca. 2.5 pM in human plasma samples). Our probe exhibited remarkable performance in monitoring the inhibition of thrombin activity by direct thrombin inhibitors. Analyses of real samples using our new membrane-based probe suggested that it will be highly useful in practical applications for the effective management of hemostatic complications

Gold-Nanoparticles-Modified Cellulose Membrane Coupled with Laser Desorption/Ionization Mass Spectrometry for Detection of Iodide in Urine

We report an efficient method for the determination of iodide (I^–) ions by using gold–iodide hybrid cluster ions on gold nanoparticles (Au NPs) modified mixed cellulose ester membrane (Au NPs-MCEM) by pulsed laser desorption/ionization mass spectrometry (LDI-MS). When I^– ions were deposited and concentrated on the surfaces of Au NPs (32 nm) via strong Au⁺–I^– interaction on the MECM, the Au NPs-MCEM was observed to function as an efficient surface-assisted LDI substrate with very low background noise. When pulsed laser radiation (355 nm) was applied, I^– binding to Au NPs ions induced the enhancement of the desorption and ionization efficiency of gold–iodide hybrid cluster ions from the Au NPs surfaces. The reproducibility of the probe for both shot-to-shot and sample-to-sample (both less than 10%) ion production was also improved by the homogeneous nature of the substrate surface. Thus, it allows the accurate and precise quantification of I^– ions in high-salinity real samples (i.e., edible salt samples and urine) at the nanomolar range. This novel LDI-MS approach provides a simple route for the high-speed analysis of I^– ions with high sensitivity and selectivity in real biological samples

Membrane-Based Assay for Iodide Ions Based on Anti-Leaching of Gold Nanoparticles

We report a label-free colorimetric strategy for the highly selective and sensitive detection of iodide (I^–) ions in human urine sample, seawater and edible salt. A poly­(<i>N</i>-vinyl-2-pyrrolidone)-stabilized Au nanoparticle (34.2-nm) was prepared to detect I^– ions using silver (Ag⁺) and cyanide (CN^–) ions as leaching agents in a glycine–NaOH (pH 9.0) solution. For the visual detection of the I^– ions by naked eye, and for long time stability of the probe, Au nanoparticles (NPs) decorated mixed cellulose ester membrane (MCEM) was prepared (Au NPs/MCEM). The Au NPs-based probe (CN^–/Ag⁺–Au NPs/MCEM) operates on the principle that Ag⁺ ions form a monolyar silver atoms/ions by aurophilic/argentophilic interactions on the Au NPs and it accelerates the leaching rate of Au atoms in presence of CN^– ions. However, when I^– is introduced into this system, it inhibits the leaching of Au atoms because of the strong interactions between Ag/Au ions and I^– ions. Inductively coupled plasma mass spectrometry, surface-assisted laser desorption/ionization time-of-flight mass spectrometry were used to characterize the surface properties of the Au NPs in the presence of Ag⁺ and I^–. Under optimal solution conditions, the CN^–/Ag⁺–Au NPs/MCEM probe enabled the detection of I^– by the naked eye at nanomolar concentrations with high selectivity (at least 1000-fold over other anions). In addition, this cost-effective probe allowed the determination of I^– ions in complex samples, such as urine, seawater, and edible salt samples

Carbon Dot-Mediated Synthesis of Manganese Oxide Decorated Graphene Nanosheets for Supercapacitor Application

In this work, we demonstrate that carbon dots (CDs) can be used as a dispersing agent for graphene as well as a reducing agent for KMnO₄ for the synthesis of manganese oxide (MnO_<i>x</i>)–graphene hybrid nanocomposites for supercapacitor applications. CDs obtained from the pyrolysis of ammonium citrate under dry heating possess excellent solubility in water due to their oxygen- and nitrogen-containing functional groups. In addition, the sp²-carbon-rich CDs exhibited strong interaction with graphene through π–π stacking for self-immobilizing on graphene in the preparation of water-soluble CD/graphene nanocomposites (CDGs). Interestingly, MnO_<i>x</i> could be grown in situ on CDGs after reaction with KMnO₄ in aqueous solution under a mild reaction temperature (75 °C). Under the mild reaction conditions, CDs undergo sacrificial oxidation for the formation of MnO_<i>x</i> nanoparticles on graphene, whereas the graphene’s graphitic carbons are protected. The as-formed nanostructured MnO_<i>x</i> on CDGs (MnO_<i>x</i>–CDGs) was employed to fabricate flexible solid-state supercapacitor which exhibited good capacitance properties (specific capacitance ∼280 F g^–1) with very high charge–discharge cyclic stability (>10 000 cycles) and good capacitance retention at 90° bending angle. Compared to other graphene-based nanocomposites, our one-pot synthesis route for MnO_<i>x</i>–CDGs is relatively green, simple, rapid, and cost-effective and has a great potential for the synthesis of different metal oxide-decorated graphene nanocomposites for energy conversion and storage application