Shih-Chun Wei

Highly flexible and stable aptamer-caged nanoparticles for control of thrombin activity

In this paper, we have demonstrated that the thymine linker length number (Tn, n = 0–60) and stem pair number (Pm, m = 0–16) in the terminal of thrombin binding aptamers (TBAs) have a strong impact on the flexibility and stability of TBA-modified gold nanoparticles (TBA–Au NPs) and thus the binding strength and inhibitory potency toward thrombin. The anticoagulation of TBA–Au NPs increased with an increase in the linker length from T0 to T30 due to an increase in the flexibility of G-quadruplexes of TBAs on the Au NP surfaces (TBA-Tn–Au NPs). The inhibition of TBA-PmT15–Au NPs increased with an increase in the Pm from P0 to P8 as a result of the increase in the rigidity and the stability of G-quadruplexes of the TBAs on the Au NP surfaces. The best results were observed for multivalent TBA–Au NPs conjugates—TBA15/TBA29-P8T15–Au NPs—which exhibited ultra-high binding affinity toward thrombin (Kd = 8.86 × 10−12 M) and thus extremely high anticoagulant (inhibitory) potency because of their particularly flexible and stable conformation and multivalency. Compared to the case without inhibitors, their measured thrombin clotting time (TCT) was 296 times longer, whereas for TBA15 alone it was only 3.9 times longer. From the dosage dependence of the TCT delay, we further demonstrated the anticoagulation ability of our TBA15/TBA29-P8T15–Au NPs was much better than the commercial drugs (argatroban and hirudin). Moreover, the Au NPs modified with TBA with photocleavable (PC) units allow a reversal in the activity of TBAPC–Au NPs via near-UV light-inducement of TBA release from Au NPs. We believe that our described techniques can be used widely to modify NPs with other anticoagulant DNA or RNA aptamers towards different proteins such as factor IX, activated protein C, and factor VIIa.

Gold nanoparticles modified with self-assembled hybrid monolayer of triblock aptamers as a photoreversible anticoagulant

We demonstrated that thrombin-binding aptamer-conjugated gold nanoparticles (TBA-Au NPs), prepared from a self-assembled hybrid monolayer (SAHM) of triblock aptamers on Au NPs (13 nm), can effectively inhibit thrombin activity toward fibrinogen. The first block poly(adenine) at the end of the triblock TBA was used for the self-assembly on Au NP surface. The second block, in the middle of TBA, was composed of oligonucleotides that could hybridize with each other. The third block, containing TBA15 (15-base, binding to the exosite I of thrombin) and TBA29 (29-base, binding to the exosite II of thrombin) provided bivalent interaction with thrombin. The SAHM triblock aptamers have optimal distances between TBA15 and TBA29, aptamer density, and orientation on the Au NP surfaces. These properties strengthen the interactions with thrombin (Kd=1.5 × 10(-11)M), resulting in an extremely high anticoagulant potency. The thrombin clotting time mediated by SAHM TBA15/TBA29-Au NPs was >10 times longer than that of four commercially available drugs (heparin, argatroban, hirudin, or warfarin). In addition, the rat-tail bleeding assay time further demonstrated that the SAHM TBA15/TBA29-Au NPs were superior to heparin. The SAHM TBA15/TBA29-Au NPs exhibited excellent stability in the human plasma (half-life >14 days) and good biocompatibility (low cytotoxicity and hemolysis). Most interestingly, the inhibition by SAHM TBA15/TBA29-Au NPs was controllable by the irradiation of green laser, via heat transfer-induced TBA release from Au NPs. Therefore, these easily prepared (self-assembled), low cost (non-thiolated aptamer), photo-controllable, multivalent TBA15/TBA29-Au NPs (high density of TBA15/TBA29 on Au NPs) show good potential for the treatment of various diseases related to blood-clotting disorders. Our study opens up the possibility of regulation of molecule binding, protein recognition, and enzyme activity using SAHM aptamer-functionalized nanomaterials.

Graphene oxide membrane as an efficient extraction and ionization substrate for spray-mass spectrometric analysis of malachite green and its metabolite in fish samples

A graphene oxide (GO) nanosheet-modified N+-nylon membrane (GOM) has been prepared and used as an extraction and spray-ionization substrate for robust mass spectrometric detection of malachite green (MG), a highly toxic disinfectant in liquid samples and fish meat. The GOM is prepared by self-deposition of GO thin film onto an N+-nylon membrane, which has been used for efficient extraction of MG in aquaculture water samples or homogenized fish meat samples. Having a dissociation constant of 2.17 × 10-9 M-1, the GOM allows extraction of approximately 98% of 100 nM MG. Coupling of the GOM-spray with an ion-trap mass spectrometer allows quantitation of MG in aquaculture freshwater and seawater samples down to nanomolar levels. Furthermore, the system possesses high selectivity and sensitivity for the quantitation of MG and its metabolite (leucomalachite green) in fish meat samples. With easy extraction and efficient spray ionization properties of GOM, this membrane spray-mass spectrometry technique is relatively simple and fast in comparison to the traditional LC-MS/MS methods for the quantitation of MG and its metabolite in aquaculture products.

DNA Modulates the Interaction of Genetically Engineered DNA-Binding Proteins and Gold Nanoparticles: Diagnosis of High-Risk HPV Infection

Gene detection has an important role in diagnosing several serious diseases and genetic defects in modern clinical medicine. Herein, we report a fast and convenient gene detection method based on the modulation of the interaction between a heat-resistant double-stranded DNA (dsDNA)-binding protein (Sso7d) and gold nanoparticles (Au NPs). We prepared a recombinant Cys-Sso7d, which is Sso7d with an extra cysteine (Cys) residue in the N-terminus, through protein engineering to control the interaction between Sso7d and Au NPs. Cys-Sso7d exhibited a stronger affinity for Au NPs and more easily induced the aggregation of Au NPs than Sso7d. In addition, Cys-Sso7d retained its ability to bind with dsDNA. The aggregation of Au NPs induced by Cys-Sso7d was diminished in the presence of dsDNA, which could be utilized as a transduction mechanism for the detection of the polymerase chain reaction (PCR) products of human papillomavirus (HPV) gene fragments (HPV types 16 and 18). The Cys-Sso7d/Au NP probe could detect as few as 1 copy of the HPV gene. The sensitivity and specificity of the Cys-Sso7d/Au NP probe for Pap smear clinical specimens (n = 52) for HPV 16 and HPV 18 detection were 85.7%/100.0% and 85.7%/91.7%, respectively. Our results demonstrate that the Cys-Sso7d/Au NP probe can be used to diagnose high-risk HPV types in Pap smear samples with high sensitivity, specificity, and accuracy.

Self-Assembled Chiral Gold Supramolecules with Efficient Laser Absorption for Enantiospecific Recognition of Carnitine

Stereospecific recognition of chiral molecules is ubiquitous in chemical and biological systems, thus leading to strong demand for the development of enantiomeric drugs, enantioselective sensors, and asymmetric catalysts. In this study, we demonstrate the ratio of d-Cys and l-Cys playing an important role in determining the optical properties and the structures of self-assembled Cys-Au(I) supramolecules prepared through a simple reaction of tetrachloroaurate(III) with chiral cysteine (Cys). The irregularly shaped -[d-Cys-Au(I)] n- or - [l-Cys-Au(I)] n- supramolecules with a size larger than 500 nm possessing strong absorption in the near-UV region and chiroptical characteristics were only obtained from the reaction of Au(III) with d-Cys or l-Cys. On the other hand, spindle-shaped -[d/l-Cys-Au(I)] n- supramolecules were formed when using Au(III) with mixtures of d/l-Cys. Our results have suggested that Au(I)···Au(I) aurophilic interactions, and stacked hydrogen bonding and zwitterionic interactions between d/l-Cys ligands are important in determining their structures. The NaBH4-mediated reduction induces the formation of photoluminescent gold nanoclusters (Au NCs) embedded in the chiral -[d-Cys-Au(I)] n- or -[l-Cys-Au(I)] n- supramolecules with a quantum yield of ca. 10%. The as-formed Au NCs/-[d-Cys-Au(I)] n- and Au NCs/-[l-Cys-Au(I)] n- are an enantiospecific substrate that can trap l-carnitine and d-carnitine, respectively, and function as a nanomatrix for surface-assisted laser desorption/ionization mass spectrometry (LDI-MS). The high absorption efficiency of laser energy, analyte-binding capacity, and homogeneity of the Au NCs/-[Cys-Au(I)] n- allow for quantitation of enantiomeric carnitine down to the micromolar regime with high reproducibility. The superior efficiency of the Au NCs/-[d-Cys-Au(I)] n- substrate has been further validated by quantification of l-carnitine in dietary supplements with accuracy and precision. Our study has opened a new avenue for chiral quantitation of various analytes through LDI-MS using metal nanocomposites consisting of NCs and metal-ligand complexes.