Syntheses and characterization of lisinopril-coated gold nanoparticles as highly stable targeted CT contrast agents in cardiovascular diseases

Lisinopril was used as the targeting moiety to prepare gold nanoparticle-based functional CT contrast agents. Pure lisinopril, thioctic acid-lisinopril conjugate, and reduced thioctic acid-lisinopril conjugate were used to obtain GNP-Lis, GNP-TA-Lis, and GNP-RTA-Lis, respectively, via ligand exchange reaction on citrate-coated gold nanoparticles (GNPs). These lisinopril-decorated GNPs were fully characterized, and their chemical stabilities in biological relevant media and in high salt concentration were compared. Their relative stabilities toward lyophilization and against cyanide-induced decomposition were also investigated. Because of their higher stability, GNP-TA-Lis were used to assess the targeting of angiotensin converting enzyme (ACE) using X-ray computed tomography (CT). The images obtained displayed high contrast in the region of the lungs and heart, clearly indicating the targeting of ACE, whose overexpression is associated with development of cardiac and pulmonary fibrosis. Thus, the new nanoprobes prepared here will serve as very useful tools for the monitoring of cardiovascular pathophysiologies using CT imaging.     

Macromolecular contrast agents for optical imaging of tumors: comparison of indotricarbocyanine-labeled human serum albumin and transferrin

Macromolecules accumulate in solid tumors and can thus be used as carriers for the delivery of attached contrast agents to tumors. We report the synthesis and use of serum protein-dye conjugates consisting of transferrin (Tf) or human serum albumin (HSA) and an indotricarbocyanine (ITCC) derivative as contrast agents for the optical imaging of tumors. The compounds were characterized with respect to their photophysical properties and tested in vitro for their ability to bind to tumor cells and in vivo for their potential to delineate experimental tumors. In contrast to HAS-ITTC, Tf-ITCC showed receptor-mediated uptake by HT29 human colon cancer cells in vitro. After intravenous injection into HT29 tumor-bearing nude mice both compounds induced increased fluorescence contrast of tumors in vivo. After 24 h the contrast between tumor and normal tissue was significantly higher for Tf-ITCC than for HAS-ITCC. Dye-induced fluorescence was found to be predominantly located in perinecrotic areas of the tumor. Furthermore, Tf-ITCC produced fluorescence of viable tumor cells, whereas HAS-ITCC fluorescence was recorded along connective tissue. We conclude that ITCC-labeled Tf and HSA can serve as macromolecular contrast agents for the optical imaging of tumors, with Tf-ITCC showing higher efficiency.     

Multimodal radio- (PET/SPECT) and fluorescence imaging agents based on metallo-radioisotopes: current applications and prospects for development of new agents

This perspective focuses on complexes of radioactive metal ions applied in multimodal radio- and optical imaging. The application of metal ions in radioimaging techniques such as Positron Emission Tomography (PET) and Single Photon Emission Computed Tomography (SPECT) has advantages over lighter nuclei in terms of half-life, but there are particular issues related to their speciation (particularly leaching from complexes) and with the combination of certain ions with fluorescent systems. The basic coordination chemistry of the ions involved and issues relating to biological conditions and their compatibility with optical imaging techniques are reviewed, the current literature presented in context, and the prospect of exploiting the intrinsic luminescence of certain metal-ligand complexes is discussed.     

Statistical mixture design development of digestion methods for Oyster tissue using inductively coupled plasma optical emission spectrometry for the determination of metallic ions

The quantitative determination of chemical elements in organic or biological samples is an important analytical problem. Normally the elements to be determined in the organic matrix must be transformed into a simple inorganic form. A digestion method by heating on a block digestor has been developed for the determination of Al, As, Ba, Ca, Cd, Co, Cu, Fe, Mg, Mn, V and Zn in Oyster tissue by ICP OES. A simplex centroid statistical mixture design has been used to study the effects of changing HNO₃, HCl and H₂O₂ reagent proportions on the digestion of these samples. Response surface and principal component analyses show that the species Ca, Cd, Cu, Fe, Mg, Mn and Zn have very similar analytical tendencies under this experiment. By means of mixture modeling maximum recoveries for these ions were predicted using 19%, 18% and 63% of the HCl, HNO₃ and H₂O₂ pseudocomponent mixtures, respectively. This corresponds to 21.4%, 30.8% and 47.8% of the HCl, HNO₃ and H₂O₂ commercial solutions. Furthermore the As, Co and V ions present large recoveries for these mixtures as well. The Al and Ba ion recoveries are seen to be independent of the mixture proportions. The analysis of Oyster tissue reference material (SRM 1566b - NIST) under optimized conditions at the selected wavelengths resulted in ion recoveries between 90% and 100%.     

Centrifugal enrichment of nickel isotopes and their application to the development of new technologies

This work contains the results of the research on centrifugal enrichment of all stable nickel isotopes. Non-steady-state separation methods were used for the effective extraction of isotopes ⁶²Ni and ⁶⁴Ni. The results of the experimental batches of the enriched isotopes of nickel are presented in the article. The technique of synthesis of tetrafluophosphine—Ni(PF₃)₄ is considered. The results of the research of the process of thermo decomposition of Ni(PF₃)₄ in metal form are presented. As a result of this work, for the first time in the world, we obtained (by centrifuge enrichment process) tens of grams quantities of all isotopes of nickel at high enrichment.     

Method development and measurements of endogenous serine/threonine Akt phosphorylation using capillary electrophoresis for systems biology

Signal transduction studies have indicated that Akt is essential for transducing the signals originating from extracellular stimuli. An exploration of the Akt signal transduction mechanism depends on the ability to assay its activation states by determining the ability of Akt to phosphorylate various substrates. This paper describes a CE-based kinase assay for Akt using a UV detection method. The RPRAATF peptide was used as the specific substrate to determine the Akt activity. Under the CE separation conditions used, the phosphorylated and nonphosphorylated forms of the RPRAATF peptide were rapidly resolved in the Akt reaction mixture within 20 min. Using this method for measuring the Akt activity, the incubation time for the Akt reactions as well as the kinetic parameters (KM) were examined. Furthermore, the developed method was applied to a PC12 cell system to assess the dynamics of the Akt activity by examining the effectiveness of the RPRAATF peptide substrate under various cytokine-stimulated environments. These results highlight the feasibility of the CE method, which is a simple and reliable technique for determining and characterizing various enzyme reactions particularly kinase enzymes.     

Development of impedimetric and optical calcium biosensor by using modified gold electrode with porcine S100A12 protein

We describe the development of a label free method to analyze the interactions between Ca(2+) and the porcine S100A12 protein immobilized on polyvinyl butyral (PVB). The modified gold electrodes were characterized using cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), scanning electron microscopy (SEM) and surface plasmon resonance (SPR) techniques. SEM analyses of PVB and PVB-S100A12 showed a heterogeneous distribution of PVB spherules on gold surface. EIS and CV measurements have shown that redox probe reactions on the modified gold electrodes were partially blocked due the adsorption of PVB-S100A12, and confirm the existence of a positive response of the immobilized S100A12 to the presence of calcium ions. The biosensor exhibited a wide linear response to Ca(2+) concentrations ranging from 12.5 to 200mM. The PVB-S100A12 seems to be bound to the gold electrode surface by physical adsorption; we observed an increase of 1184.32m° in the SPR angle after the adsorption of the protein on the PVB surface (in an indication that 9.84ng of S100A12 are adsorbed per mm(2) of the Au-PVB electrode), followed by a further increase of 581.66m° after attachment of the Ca(2+) ions. In addition, no SPR response is obtained for non-specific ions. These studies might be useful as a platform for the design of new reusable and sensitive biosensing devices that could find use in the clinical applications.     

From nano to micro: synthesis and optical properties of homogeneous spheroidal gold particles and their superlattices

Iodide ions have been used as an additive to fabricate homogeneous gold spheres with a la carte dimensions, ranging from the nano- (50 nm) to the microscale (ca. 1 μm). Due to the high uniformity and surface functionalization of the produced materials, they undergo spontaneous assembly into organized superlattices upon solvent drying. Thus, optical properties of the particles including localized surface plasmon resonances and surface enhanced Raman scattering (SERS) response, both in solution and organized into superlattices, are also reported.     

Theoretical study of the electronic and optical properties of photochromic dithienylethene derivatives connected to small gold clusters

In the course of developing electronic devices on a molecular scale, dithienylethenes photochromic molecules constitute promising candidates for optoelectronic applications such as memories and switches. There is thus a great interest to understand and control the switching behavior of photochromic compounds deposited on metallic surfaces or nanoparticles. Within the framework of the density functional theory, we studied the effect of small gold clusters (Au3 and Au9) on the electronic structure and absorption spectrum of a model dithienylethene molecule. The molecular orbital interactions between the photochromic molecule and the gold cluster made it possible to rationalize some experimental findings (Dulic, D.; van der Molen, S. J.; Kudernac, T.; Jonkman, H. T.; de Jong, J. J. D.; Bowden, T. N.; van Esch, J.; Feringa, B. L.; van Wees, B. J. Phys. Rev. Lett. 2003, 91, 207402). For the closed-ring isomer, grafting a photochromic molecule on a small gold cluster does not change the characteristics of the electronic transition involved in the ring-opening reaction. On the opposite, the absorption spectrum of the photochromic open-ring isomer is strongly modified by the inclusion of the metallic cluster. In agreement with experimental results, our study thus showed that the cycloreversion reaction which involves the closed-ring isomer should be still possible, whereas the ring-closure reaction which involves the open-ring isomer should be inhibited. Connecting a dithienylethene molecule to a small gold cluster hence provides a qualitative comprehension of the photochromic activities of dithienylethenes connected to a gold surface.     

Determination of ATP by resonance light scattering using a binuclear uranyl complex and aptamer modified gold nanoparticles as optical probes

Microchimica Acta - We describe a resonance light scattering (RLS) method for the direct detection of adenosine triphosphate (ATP) in aqueous solution without the need for separation. The binuclear...     

Enhanced mass resolution method development, validation and assay application to support preclinical studies of a new drug candidate

A very highly sensitive and highly selective liquid chromatographic/tandem mass spectrometric (LC/MS/MS) method was developed to evaluate and quantify a new drug candidate in different biological matrices. Following a simple plasma protein precipitation using acetonitrile, the post-treatment samples were analyzed on a C18 column interfaced with a new generation of triple-quadrupole mass spectrometer. The recently introduced triple-quadrupole mass spectrometer, the TSQ Quantum Ultra, with enhanced mass-resolution capability, demonstrated improved sensitivity (0.05 ng/mL), coupled with suitable accuracy and precision, over a broad linear dynamic range (0.05-1000 ng/mL). A comparison of the assay performance data (dynamic range, calibration curve equation, precision and accuracy) of the enhanced resolution method against a unit resolution method under optimized conditions showed the performance improvement of the enhanced mass resolution method for bioanalytical high-throughput applications. The enhanced mass resolution method herein described was successfully applied to the evaluation of the pharmacokinetic profile of a new drug candidate in rat, rabbit and dog plasma samples.     

Real-time size discrimination and elemental analysis of gold nanoparticles using ES-DMA coupled to ICP-MS

We report the development of a hyphenated instrument with the capacity to quantitatively characterize aqueous suspended gold nanoparticles (AuNPs) based on a combination of gas-phase size separation, particle counting, and elemental analysis. A customized electrospray-differential mobility analyzer (ES-DMA) was used to achieve real-time upstream size discrimination. A condensation particle counter and inductively coupled plasma mass spectrometer (ICP-MS) were employed as downstream detectors, providing information on number density and elemental composition, respectively, of aerosolized AuNPs versus the upstream size selected by ES-DMA. A gas-exchange device was designed and optimized to improve the conversion of air flow (from the electrospray) to argon flow required to sustain the ICP-MS plasma, the key compatibility issue for instrumental hyphenation. Our work provides the proof of concept and a working prototype for utilizing this construct to successfully measure (1) number- and mass-based distributions; (2) elemental compositions of nanoparticles classified by size, where the size classification and elemental analysis are performed within a single experiment; (3) particle concentrations in both solution (before size discrimination) and aerosol (after size discrimination) phases; and (4) the number of atoms per nanoparticle or the nanoparticle density.     

Detection of analyte binding to microarrays using gold nanoparticle labels and a desktop scanner

Microarray hybridization or antibody binding can be detected by many techniques, however, only a few are suitable for widespread use since many of these detection techniques rely on bulky and expensive instruments. Here, we describe the usefulness of a simple and inexpensive detection method based on gold nanoparticle labeled antibodies visualized by a commercial, office desktop flatbed scanner. Scanning electron microscopy studies showed that the signal from the flatbed scanner was proportional to the surface density of the bound antibody-gold conjugates, and that the flatbed scanner could detect six attomoles of antibody-gold conjugates. This detection system was used in a competitive immunoassay to measure the concentration of the pesticide metabolite 2,6-dichlorobenzamide (BAM) in water samples. The results showed that the gold labeled antibodies functioned comparably with a fluorescent based immunoassay for detecting BAM in water. A qualitative immunoassay based on gold-labeled antibodies could determine if a water sample contained BAM above and below 60-70 ng L(-1), which is below the maximum allowed BAM concentration for drinking water (100 ng L(-1)) according to European Union legislation.     

Scanning thermal microscopy techniques for polymeric thin films using temperature contrast mode to measure thermal diffusivity and a novel approach in conductivity contrast mode to the mapping of thermally conductive particles

Advances in production are leading to increasing use of polymeric thin films in applications such as automotive bearings. Two approaches have been developed to study the thermophysical properties of these thin films: The first technique based on Flash theory uses a scanning thermal microscopy (SThM) tip in temperature contrast mode to measure thermal diffusivity over a nano-scale area. The SThM tip is in contact with the upper surface of the film to detect a heat pulse delivered by a microelectromechanical heater platform from the lower surface. The second technique is a conductivity contrast mode SThM based approach for measuring the size and distribution of thermally conducting particles in thin film polymeric coatings. Topographical and thermal conductivity data are combined to produce a “correlation analysis value” 3D particle map of the coating. Good practice and a case study are highlighted.     

Top-down identification of endogenous peptides up to 9 kDa in cerebrospinal fluid and brain tissue by nanoelectrospray quadrupole time-of-flight tandem mass spectrometry

Recent work on protein and peptide biomarker patterns revealed the difficulties in identifying their molecular components, which is indispensable for validation of the biological context. Cerebrospinal fluid and brain tissue are used as sources to discover new biomarkers, e.g. for neurodegenerative diseases. Many of these biomarker candidates are peptides with a molecular mass of <10 kDa. Their identification is favourably achieved with a 'top-down' approach, because this requires less purification and an enzymatic cleavage will often not yield enough specific fragments for successful database searches. Here, we describe an approach using quadrupole time-of-flight mass spectrometry (TOFMS) as a highly efficient mass spectrometric purification and identification tool after off-line decomplexation of biological samples by liquid chromatography. After initial peptidomic screening with matrix-assisted laser desorption/ionization (MALDI) TOFMS, the elution behaviour in chromatography and the exact molecular mass were used to locate the same signals in nanoelectrospray measurements. Most of the peaks detected in MALDI-TOFMS could be retrieved in nanoelectrospray quadrupole TOFMS. Suitable collision energies for informative fragment spectra were investigated for different parent ions, charge states and molecular masses. After collision-induced dissociation, the resulting fragmentation data of multiply charged ions can become much more complicated than those derived from tryptic peptide digests. However, the mass accuracy and resolution of quadrupole TOF instruments results in high-quality data suitable for determining peptide sequences. The protein precursor, proteolytic processing and post-translational modifications were identified by automated database searches. This is demonstrated by the exemplary identifications of thymosin beta-4 (5.0 kDa) and NPY (4.3 kDa) from rat hypothalamic tissue and ubiquitin (8.6 kDa) from human cerebrospinal fluid. The high data quality should also allow for de novo identification. This methodology is generally applicable for peptides up to a molecular mass of about 10 kDa from body fluids, tissues or other biological sources.     

In situ synthesis of gold and silver nanoparticles by using redox-active amphiphiles and their phase transfer to organic solvents

An in situ reduction approach to synthesizing gold and silver nanoparticles by using a series of newly designed, redox-active amphiphiles at basic pH is described. These amphiphiles are the conjugates of a fatty acid (e.g., oleic acid, stearic acid, and lauric acid) and a redox-active amino acid (e.g., tryptophan or tyrosine). The amphiphile-coated nanoparticles are then efficiently transferred from water to different nonpolar organic media (such as benzene, toluene, xylene, cyclohexane, and hexane) simply by acid treatment. The phase-transfer process was monitored by UV/visible spectroscopy and transmission electron microscopy, and the results showed that the average particle size and size distribution remain almost unchanged after transferring to the organic media. The anchoring of the amphiphile to the nanoparticle surface was confirmed by FTIR spectroscopy and thermogravimetric analysis. A mechanism is proposed to describe the stability of colloidal Au and Ag nanoparticles formed in situ and their phase transfer to organic solvents. The presence of the amphiphile increases the thermal stability of the colloidal gold nanoparticle conjugates in organic solvents.