Highly Bright Silica-Coated InP/ZnS Quantum Dot-Embedded Silica Nanoparticles as Biocompatible Nanoprobes.

Quantum dots (QDs) have outstanding optical properties such as strong fluorescence, excellent photostability, broad absorption spectra, and narrow emission bands, which make them useful for bioimaging. However, cadmium (Cd)-based QDs, which have been widely studied, have potential toxicity problems. Cd-free QDs have also been studied, but their weak photoluminescence (PL) intensity makes their practical use in bioimaging challenging. In this study, Cd-free QD nanoprobes for bioimaging were fabricated by densely embedding multiple indium phosphide/zinc sulfide (InP/ZnS) QDs onto silica templates and coating them with a silica shell. The fabricated silica-coated InP/ZnS QD-embedded silica nanoparticles (SiO2@InP QDs@SiO2 NPs) exhibited hydrophilic properties because of the surface silica shell. The quantum yield (QY), maximum emission peak wavelength, and full-width half-maximum (FWHM) of the final fabricated SiO2@InP QDs@SiO2 NPs were 6.61%, 527.01 nm, and 44.62 nm, respectively. Moreover, the brightness of the particles could be easily controlled by adjusting the amount of InP/ZnS QDs in the SiO2@InP QDs@SiO2 NPs. When SiO2@InP QDs@SiO2 NPs were administered to tumor syngeneic mice, the fluorescence signal was prominently detected in the tumor because of the preferential distribution of the SiO2@InP QDs@SiO2 NPs, demonstrating their applicability in bioimaging with NPs. Thus, SiO2@InP QDs@SiO2 NPs have the potential to successfully replace Cd-based QDs as highly bright and biocompatible fluorescent nanoprobes.

High-quantum yield alloy-typed core/shell CdSeZnS/ZnS quantum dots for bio-applications.

BACKGROUND: Quantum dots (QDs) have been used as fluorophores in various imaging fields owing to their strong fluorescent intensity, high quantum yield (QY), and narrow emission bandwidth. However, the application of QDs to bio-imaging is limited because the QY of QDs decreases substantially during the surface modification step for bio-application. RESULTS: In this study, we fabricated alloy-typed core/shell CdSeZnS/ZnS quantum dots (alloy QDs) that showed higher quantum yield and stability during the surface modification for hydrophilization compared with conventional CdSe/CdS/ZnS multilayer quantum dots (MQDs). The structure of the alloy QDs was confirmed using time-of-flight medium-energy ion scattering spectroscopy. The alloy QDs exhibited strong fluorescence and a high QY of 98.0%. After hydrophilic surface modification, the alloy QDs exhibited a QY of 84.7%, which is 1.5 times higher than that of MQDs. The QY was 77.8% after the alloy QDs were conjugated with folic acid (FA). Alloy QDs and MQDs, after conjugation with FA, were successfully used for targeting human KB cells. The alloy QDs exhibited a stronger fluorescence signal than MQD; these signals were retained in the popliteal lymph node area for 24 h. CONCLUSION: The alloy QDs maintained a higher QY in hydrophilization for biological applications than MQDs. And also, alloy QDs showed the potential as nanoprobes for highly sensitive bioimaging analysis.

Synthesis and Application of Silica-Coated Quantum Dots in Biomedicine.

Quantum dots (QDs) are semiconductor nanoparticles with outstanding optoelectronic properties. More specifically, QDs are highly bright and exhibit wide absorption spectra, narrow light bands, and excellent photovoltaic stability, which make them useful in bioscience and medicine, particularly for sensing, optical imaging, cell separation, and diagnosis. In general, QDs are stabilized using a hydrophobic ligand during synthesis, and thus their hydrophobic surfaces must undergo hydrophilic modification if the QDs are to be used in bioapplications. Silica-coating is one of the most effective methods for overcoming the disadvantages of QDs, owing to silica's physicochemical stability, nontoxicity, and excellent bioavailability. This review highlights recent progress in the design, preparation, and application of silica-coated QDs and presents an overview of the major challenges and prospects of their application.

Silver-Assembled Silica Nanoparticles in Lateral Flow Immunoassay for Visual Inspection of Prostate-Specific Antigen.

Prostate-specific antigen (PSA) is the best-known biomarker for early diagnosis of prostate cancer. For prostate cancer in particular, the threshold level of PSA <4.0 ng/mL in clinical samples is an important indicator. Quick and easy visual detection of the PSA level greatly helps in early detection and treatment of prostate cancer and reducing mortality. In this study, we developed optimized silica-coated silver-assembled silica nanoparticles (SiO2@Ag@SiO2 NPs) that were applied to a visual lateral flow immunoassay (LFIA) platform for PSA detection. During synthesis, the ratio of silica NPs to silver nitrate changed, and as the synthesized NPs exhibited distinct UV spectra and colors, most optimized SiO2@Ag@SiO2 NPs showed the potential for early prostate cancer diagnosis. The PSA detection limit of our LFIA platform was 1.1 ng/mL. By applying each SiO2@Ag@SiO2 NP to the visual LFIA platform, optimized SiO2@Ag@SiO2 NPs were selected in the test strip, and clinical samples from prostate cancer patients were successfully detected as the boundaries of non-specific binding were clearly seen and the level of PSA was <4 ng/mL, thus providing an avenue for quick prostate cancer diagnosis and early treatment.

Recent Advances in Surface-Enhanced Raman Scattering Magnetic Plasmonic Particles for Bioapplications.

The surface-enhanced Raman scattering (SERS) technique, that uses magnetic plasmonic particles (MPPs), is an advanced SERS detection platform owing to the synergetic effects of the particles' magnetic and plasmonic properties. As well as being an ultrasensitive and reliable SERS material, MPPs perform various functions, such as aiding in separation, drug delivery, and acting as a therapeutic material. This literature discusses the structure and multifunctionality of MPPs, which has enabled the novel application of MPPs to various biological fields.

Synthesis of Densely Immobilized Gold-Assembled Silica Nanostructures.

In this study, dense gold-assembled SiO2 nanostructure (SiO2@Au) was successfully developed using the Au seed-mediated growth. First, SiO2 (150 nm) was prepared, modified by amino groups, and incubated by gold nanoparticles (ca. 3 nm Au metal nanoparticles (NPs)) to immobilize Au NPs to SiO2 surface. Then, Au NPs were grown on the prepared SiO2@Au seed by reducing chloroauric acid (HAuCl4) by ascorbic acid (AA) in the presence of polyvinylpyrrolidone (PVP). The presence of bigger (ca. 20 nm) Au NPs on the SiO2 surface was confirmed by transmittance electronic microscopy (TEM) images, color changes to dark blue, and UV-vis spectra broadening in the range of 450 to 750 nm. The SiO2@Au nanostructure showed several advantages compared to the hydrofluoric acid (HF)-treated SiO2@Au, such as easy separation, surface modification stability by 11-mercaptopundecanoic acid (R-COOH), 11-mercapto-1-undecanol (R-OH), and 1-undecanethiol (R-CH3), and a better peroxidase-like catalysis activity for 5,5'-Tetramethylbenzidine (TMB) and hydrogen peroxide (H2O2) reaction. The catalytic activity of SiO2@Au was two times better than that of HF-treated SiO2@Au. When SiO2@Au nanostructure was used as a surface enhanced Raman scattering (SERS) substrate, the signal of 4-aminophenol (4-ATP) on the surface of SiO2@Au was also stronger than that of HF-treated SiO2@Au. This study provides a potential method for nanoparticle preparation which can be replaced for Au NPs in further research and development.

Bioapplications of Nanomaterials.

Nanobiotechnology is known as the application of nanoscaled techniques in biology which bridges natural science to living organism for improving the quality of life of humans. Nanotechnology was first issued in 1959 and has been rapidly developed, supplying numerous benefits to basic scientific academy and to clinical application including human healthcare, specifically in cancer therapy. This chapter discusses recent advances and potentials of nanotechnology in pharmaceutics, therapeutics, biosensing, bioimaging, and gene delivery that demonstrate the multifunctionality of nanotechnology.

Carbon Nanomaterials for Biomedical Application.

The use of carbon-based nanomaterials (CNs) with outstanding properties has been rising in many scientific and industrial application fields. These CNs represent a tunable alternative for applications with biomolecules, which allow interactions in either covalent or noncovalent way. Diverse carbon-derived nanomaterial family exhibits unique features and has been widely exploited in various biomedical applications, including biosensing, diagnosis, cancer therapy, drug delivery, and tissue engineering. In this chapter, we aim to present an overview of CNs with a particular interest in intrinsic structural, electronic, and chemical properties. In particular, the detailed properties and features of CNs and its derivatives, including carbon nanotube (CNT), graphene, graphene oxide (GO), and reduced GO (rGO) are summarized. The interesting biomedical applications are also reviewed in order to offer an overview of the possible fields for scientific and industrial applications of CNs.

Au-Ag assembled on silica nanoprobes for visual semiquantitative detection of prostate-specific antigen.

BACKGROUND: Blood prostate-specific antigen (PSA) levels are widely used as diagnostic biomarkers for prostate cancer. Lateral-flow immunoassay (LFIA)-based PSA detection can overcome the limitations associated with other methods. LFIAbased PSA detection in clinical samples enables prognosis and early diagnosis owing to the use of high-performance signal reporters. RESULTS: Here, a semiquantitative LFIA platform for PSA detection in blood was developed using Au-Ag nanoparticles (NPs) assembled on silica NPs (SiO2@Au-Ag NPs) that served as signal reporters. Synthesized SiO2@Au-Ag NPs exhibited a high absorbance at a wide wavelength range (400-800 nm), with a high scattering on nitrocellulose membrane test strips. In LFIA, the color intensity of the test line on the test strip differed depending on the PSA concentration (0.30-10.00 ng/mL), and bands for the test line on the test strip could be used as a standard. When clinical samples were assessed using this LFIA, a visual test line with particular color intensity observed on the test strip enabled the early diagnosis and prognosis of patients with prostate cancer based on PSA detection. In addition, the relative standard deviation of reproducibility was 1.41%, indicating high reproducibility, and the signal reporter showed good stability for 10 days. CONCLUSION: These characteristics of the signal reporter demonstrated the reliability of the LFIA platform for PSA detection, suggesting potential applications in clinical sample analysis.

Lateral Flow Immunoassay with Quantum-Dot-Embedded Silica Nanoparticles for Prostate-Specific Antigen Detection.

Prostate cancer can be detected early by testing the presence of prostate-specific antigen (PSA) in the blood. Lateral flow immunoassay (LFIA) has been used because it is cost effective and easy to use and also has a rapid sample-to-answer process. Quantum dots (QDs) with very bright fluorescence have been previously used to improve the detection sensitivity of LFIAs. In the current study, a highly sensitive LFIA kit was devised using QD-embedded silica nanoparticles. In the present study, only a smartphone and a computer software program, ImageJ, were used, because the developed system had high sensitivity by using very bright nanoprobes. The limit of PSA detection of the developed LFIA system was 0.138 ng/mL. The area under the curve of this system was calculated as 0.852. The system did not show any false-negative result when 47 human serum samples were analyzed; it only detected PSA and did not detect alpha-fetoprotein and newborn calf serum in the samples. Additionally, fluorescence was maintained on the strip for 10 d after the test. With its high sensitivity and convenience, the devised LFIA kit can be used for the diagnosis of prostate cancer.

Development of a Polo-like Kinase-1 Polo-Box Domain Inhibitor as a Tumor Growth Suppressor in Mice Models.

Polo-like kinase-1 (Plk1) plays a key role in mitosis and has been identified as an attractive anticancer drug target. Plk1 consists of two drug-targeting sites, namely, N-terminal kinase domain (KD) and C-terminal polo-box domain (PBD). As KD-targeting inhibitors are associated with severe side effects, here we report on the pyrazole-based Plk1 PBD inhibitor, KBJK557, which showed a remarkable in vitro anticancer effect by inducing Plk1 delocalization, mitotic arrest, and apoptosis in HeLa cells. Further, in vivo optical imaging analysis and antitumorigenic activities in mouse xenograft models demonstrate that KBJK557 preferentially accumulates in cancer cells and selectively inhibits cancer cell proliferation. Pharmacokinetic profiles and partition coefficients suggest that KBJK557 was exposed in the blood and circulated through the organs with an intermediate level of clearance (t1/2, 7.73 h). The present investigation offers a strategy for specifically targeting cancer using a newly identified small-molecule inhibitor that targets the Plk1 PBD.

Amphiphilic Triazine Polymer Derivatives as Antibacterial And Anti-atopic Agents in Mice Model.

Considering the emergence of bacterial resistance and low proteolytic stability of antimicrobial peptides (AMPs), herein we developed a series of ultra-short triazine based amphipathic polymers (TZP) that are connected with ethylene diamine linkers instead of protease sensitive amide bond. The most potent oligomers, TZP3 and TZP5 not only displayed potent antibacterial action on various drug-resistant pathogens but also exhibited a strong synergic antibacterial activity in combination with chloramphenicol against multidrug-resistant Pseudomonas aeruginosa (MDRPA). Since most of atopic dermatitis (AD) infections are caused by bacterial colonization, we evaluated the potency of TZP3 and TZP5 on AD in vitro and in vivo. In vitro AD analysis of these two polymers showed significant inhibition against the release of ß-hexosaminidase and tumor necrosis factor (TNF-α) from RBL-2H3 cells. In AD-like skin lesions in BALB/c mice model, these two polymers displayed significant potency in suppressing dermal and epidermal thickness, mast cell infiltration and pro-inflammatory cytokines expression. Moreover, these polymers exhibited remarkable efficacy over the allergies caused by the imbalance of Th1/Th2 by regulating total IgE and IgG2a. Finally, the impact of treatment effects of these polymers was examined through analyzing the weights and sizes of spleen and lymph node of AD-induced mice.

Two-dimensional SERS encoding method for on-bead peptide sequencing in high-throughput bioanalysis.

We developed a ready-to-read on-bead peptide encoding method for high-throughput screening bioassays. With two-dimensional surface-enhanced Raman scattering nano-identifiers (2D-SERS IDs) which are concurrently labelled with two SERS codes (coupling steps and kinds of amino acid), we could possibly generate more than 10 trillion codes with only 30 Raman label compounds.

Preparation of tri(ethylene glycol) grafted core-shell type polymer support for solid-phase peptide synthesis.

A core-shell type polymer support for solid-phase peptide synthesis has been developed for high coupling efficiency of peptides and versatile applications such as on-bead bioassays. Although various kinds of polymer supports have been developed, they have their own drawbacks including poor accessibility of reagents and incompatibility in aqueous solution. In this paper, we prepared hydrophilic tri(ethylene glycol) (TEG) grafted core-shell type polymer supports (TEG SURE) for efficient solid-phase peptide synthesis and on-bead bioassays. TEG SURE was prepared by grafting TEG derivative on the surface of AM PS resin via biphasic diffusion control method and subsequent acetylation of amine groups which are located at the core region of AM PS resin. The performance of TEG SURE was evaluated by synthesizing several peptides. Three points can be highlighted: (1) easy control of loading level of TEG, (2) improved efficiency of peptide synthesis compared with the conventional resins, and (3) applicability of on-bead bioassays.

Direct identification of on-bead peptides using surface-enhanced Raman spectroscopic barcoding system for high-throughput bioanalysis.

Recently, preparation and screening of compound libraries remain one of the most challenging tasks in drug discovery, biomarker detection, and biomolecular profiling processes. So far, several distinct encoding/decoding methods such as chemical encoding, graphical encoding, and optical encoding have been reported to identify those libraries. In this paper, a simple and efficient surface-enhanced Raman spectroscopic (SERS) barcoding method using highly sensitive SERS nanoparticles (SERS ID) is presented. The 44 kinds of SERS IDs were able to generate simple codes and could possibly generate more than one million kinds of codes by incorporating combinations of different SERS IDs. The barcoding method exhibited high stability and reliability under bioassay conditions. The SERS ID encoding based screening platform can identify the peptide ligand on the bead and also quantify its binding affinity for specific protein. We believe that our SERS barcoding technology is a promising method in the screening of one-bead-one-compound (OBOC) libraries for drug discovery.

Development of high-throughput phosphorylation profiling method for identification of Ser/Thr kinase specificity.

Identification of substrate specificity of kinases is crucial to understand the roles of the kinases in cellular signal transduction pathways. Here, we present an approach applicable for the discovery of substrate specificity of Ser/Thr kinases. The method, which is named as the 'high-throughput phosphorylation profiling (HTPP)' method was developed on the basis of a fully randomized one-bead one-compound (OBOC) combinatorial ladder type peptide library and MALDI-TOF MS. The OBOC ladder peptide library was constructed by the 'split and pool' method on a HiCore resin. The peptide library sequence was Ac-Ala-X-X-X-Ser-X-X-Ala-BEBE-PLL resin. The substrate specificity of murine PKA (cAMP-dependent protein kinase A) and yeast Yak1 kinase was identified using this method. On the basis of the result, we identified Ifh1, which is a co-activator for the transcription of ribosomal protein genes, as a novel substrate of Yak1 kinase. The putative Yak1-dependent phosphorylation site of Ifh1 was verified by in vitro kinase assay.

Antibody-free peptide substrate screening of serine/threonine kinase (protein kinase A) with a biotinylated detection probe.

Being different from anti-phosphotyrosine antibodies, anti-phosphoserine- or anti-phosphothreonine-specific antibodies with high affinity for the detection of serine/threonine kinase substrates are not readily available. Therefore, chemical modification methods were developed for the detection of phosphoserine or threonine in the screening of protein kinase substrates based on ß-elimination and Michael addition. We have developed a biotin-based detection probe for identification of the phosphorylated serine or threonine residue. A biotin derivative induced a color reaction using alkaline phosphate-conjugated streptavidin that amplified the signal. It was effective for the detection and separation of the target peptide on the resin. The detection probe was successfully used in identifying PKA substrates from peptide libraries on resin beads. The peptide library was prepared as a ladder-type, such that the active peptides on the colored resin beads were readily sequenced with the truncated peptide fragments by MALDI-TOF/MS analysis after releasing the peptides from the resin bead through photolysis.

Automated maskless photolithography system for peptide microarray synthesis on a chip.

Maskless photolithographic peptide synthesis was performed on a glass chip using an automated peptide array synthesizer system. The peptide array synthesizer was built in a closed box, which contained optical and fluidic systems. The conditions for peptide synthesis were fully controlled by a computer program. For the peptide synthesis on a glass chip, 20 NVOC-protected amino acids were synthesized. The coupling efficiencies of two model peptide sequences were examined on ACA/APTS and PEG/CHI/GPTS chips. PEG/CHI/GPTS chip gave higher average stepwise yields of GIYWHHY (94%) and YIYGSFK (98%) than those of ACA/APTS chip. To quantify peptide-protein binding affinity, HPQ- or HPM-containing pentapeptides were synthesized on a PEG/CHI/GPTS chip and the binding event of Cy3 labeled-streptavidin was quantified. The peptide sequence of IQHPQ showed highest binding affinity with Cy3 labeled-streptavidin. The results demonstrated that the photolithographic peptide array synthesis method efficiently quantified the binding activities of protein-peptide interactions and it can be used for additional biological assay applications.

Substrate screening of protein kinases: detection methods and combinatorial peptide libraries.

The study of protein kinases has become a matter of great importance in the development of new drugs for the treatment of diseases, including cancer and inflammation. Substrate screening is the first step in the fundamental investigation of protein kinases and the development of inhibitors for use in drug discovery. Towards this goal, various studies have been reported regarding the development of phospho-peptide detection methods and the screening of phosphorylated peptide sites by protein kinases. This review introduces the detection methods for phosphorylation events using the reagents with (γ(32)P)ATP, ligand-linked ATP, phospho-peptide-specific antibodies and metal chelating compounds. Chemical modification methods using ß-elimination for the detection of phospho-Ser/Thr peptides are introduced as well. In addition, the implementations of combinatorial peptide libraries for screening peptide substrates of protein kinases are discussed. The phage display approach has been suggested as an alternative method of using synthetic peptides for screening the substrate specificities of protein kinase. However, a solid phase assay using a peptide library-bound polymer resin or a peptide-arrayed glass chip is preferred for high throughput screening (HTS).

The association between current Helicobacter pylori infection and coronary artery disease.

BACKGROUND: The role of Helicobacter pylori (H. pylori) in the pathogenesis of coronary artery disease (CAD) is still controversial, and the relation between current H. pylori infection and CAD has not been fully examined. This study evaluated the relation between H. pylori infection as confirmed by gastroduodenoscopic biopsy and CAD. METHODS: We determined the presence of H. pylori infections, via gastroduodenoscopy, in 88 patients of the normal coronary angiographic group and also in 175 patients of the CAD group, and the latter patients had more than 50% coronary stenosis angiographically demonstrated. We excluded those patients with a history of previous H. pylori eradication and/or malignancy. A small piece of tissue from the antrum, which was obtained by gastroduodenoscopic biopsy, was stained by Warthin-starry silver stain. We defined a negative staining result that there was no stained tissue in the sample and the stained tissue was also positive for H. pylori infection. RESULTS: There was no significant difference, except for gender, age, smoking and high density lipoprotein cholesterol (HDL-c), of the demographic and laboratory characteristics between the groups. Twenty seven (30.7%) patients of the normal control group and 71 (40.6%) patients of the CAD group were positive of H. pylori infection, yet there was no statistical difference. We angiographically followed up the 80 patients of the CAD group who were treated by percutaneous coronary intervention (PCI) at 6 to 9 months after their primary intervention. Twenty two (37.9%) of the 58 patients of the H. pylori negative group and 10 (45.5%) of the 22 patients of the H. pylori positive group were treated with reintervention, but reintervention was also not significantly different between the group with H. pylori infection and the group without the infection. CONCLUSIONS: These data indicated that H. pylori infection had a modest influence on CAD and progressive atheroma, but the showed a tendency to increase. Further studies are needed to evaluate the relationship between H. pylori infection and CAD.