Shape-controlled synthesis of NIR absorbing branched gold nanoparticles and morphology stabilization with alkanethiols.

Gold nanoparticles are ideal candidates for clinical applications if their plasmon absorption band is situated in the near infrared region (NIR) of the electromagnetic spectrum. Various parameters, including the nanoparticle shape, strongly influence the position of this absorption band. The aim of this study is to produce stabilized NIR absorbing branched gold nanoparticles with potential for biomedical applications. Hereto, the synthesis procedure for branched gold nanoparticles is optimized varying the different synthesis parameters. By subsequent electroless gold plating the plasmon absorption band is shifted to 747.2 nm. The intrinsic unstable nature of the nanoparticles' morphology can be clearly observed by a spectral shift and limits their use in real applications. However, in this article we show how the stabilization of the branched structure can be successfully achieved by exchanging the initial capping agent for different alkanethiols and disulfides. Furthermore, when using alkanethiols/disulfides with poly(ethylene oxide) units incorporated, an increased stability of the gold nanoparticles is achieved in high salt concentrations up to 1 M and in a cell culture medium. These achievements open a plethora of opportunities for these stabilized branched gold nanoparticles in nanomedicine.

Impact of pre-concentration to covalently biofunctionalize suspended nanoparticles.

The effective biofunctionalization of nanoparticles is crucial for biomedical applications. In this study we investigated the covalent biofunctionalization of magnetic nanoparticles based on carbodiimide activation. An important aspect in the covalent biofunctionalization of nanoparticles has been neglected, namely pre-concentration. Exploiting the electrostatic attraction forces between a protein and the nanoparticle surface will favor the covalent immobilization. We showed that low ionic strength buffers with a pH slightly lower than the pI of the selected biomolecules is needed to increase the yield of covalent immobilization. Additionally, it is demonstrated that the covalently immobilized proteins are bioactive, relying on a sandwich assay using gold nanoparticles as reporter labels.

Increased stability of mercapto alkane functionalized Au nanoparticles towards DNA sensing.

The use of gold nanoparticles (GNPs) in bioassays is often hampered by their colloidal stability. In this study, gold nanoparticles coated with different mercapto alkanes were investigated towards their stability. Hereto, the effects of the alkane chain length (5-11 methylene groups), the type of functional end-group (-OH or -COOH) and the amount of incorporated poly-ethylene oxide units (none, 3 or 6) on the GNP stabilization was evaluated. Based on these results, an optimal mercapto alkane (HS(CH(2))(11)PEO(6)COOH) was selected to increase the colloidal stability up to 2 M NaCl. Furthermore, it was proved that this mercapto alkane is ideally suited to enhance the stability of DNA functionalized GNPs in high electrolytic hybridization buffers. The effectiveness of these DNA functionalized GNPs was demonstrated in a sandwich assay using a surface plasmon resonance biosensor. The superior stability of these nanoparticles during hybridization may lead to enhanced biosensor technologies.

Multiplex PCR detection of slowly-evolving trypanosomatids and neogregarines in bumblebees using broad-range primers.

AIMS: The aims of this study were to design universal markers for different protozoan parasites of Bombus spp. based on the phylogenetic position of two important bumblebee parasites Crithidia bombi and Apicystis bombi. METHODS AND RESULTS: Standard PCR and extraction techniques were used to amplify and sequence 18S rDNA. Phylogenetic analysis of the rDNA was performed in order to predict the parasite range of the primers. CONCLUSIONS: Crithidia bombi phylogenetically clusters with the trypanosomatids with slowly-evolving SSU-rRNA sequences (SE), while A. bombi is the closest sister group of Mattesia. A multiplex was designed containing an internal control and two broad-range primer pairs, detecting C. bombi and other SE trypanosomatids and also A. bombi and other neogregarines. SIGNIFICANCE AND IMPACT OF THE STUDY: Sequence data generated will further improve the current systematics of insect trypanosomatids and gregarines that remain troublesome. Broad-range markers for bumblebee parasites are necessary tools enabling the screening of commercially imported colonies and thus controlling their worldwide distribution and to discover related emerging parasites.

Chemical and biological characterization of thiol SAMs for neuronal cell attachment.

Cellular adhesion and growth on solid-state surfaces is the central theme in the development of cell-based biosensors and implantable medical devices. Suitable interface techniques must be applied to construct stable and well-organized thin films of biologically active molecules that would control the development of neuronal cells on chips. Peptides such as RGD fragments, poly-L-lysine (PLL), or basal lamina proteins, such as laminin or fibronectin, are often used in order to promote cellular adhesion on surfaces. In this paper we describe the characterization of several self-assembled monolayers (SAMs) for their ability to anchor a laminin-derived synthetic peptide, PA22-2, a peptide known to promote neuronal attachment and stimulate neurite outgrowth. We have evaluated the immobilization of PA22-2 onto 16-mercaptohexadecanoic acid, 4-maleimide-N-(11-undecyldithio)butanamide, and 2-(maleimide)ethyl-N-(11-hexaethylene oxide-undecyldithio)acetamide SAM functionalized Au substrates. The neuronal attachment and outgrowth have been evaluated in embryonic mouse hippocampal neuron cultures up to 14 days in vitro. Our results show that differences in the cell morphologies were observed on the surfaces modified with various SAMs, despite the minor differences in chemical composition identified using standard characterization tools. These different cell morphologies can most probably be explained when investigating the effect of a given SAM layer on the adsorption of proteins present in the culture medium. More likely, it is the ratio between the specific PA22-2 adsorption and nonspecific medium protein adsorption that controls the cellular morphology. Large amounts of adsorbed medium proteins could screen the PA22-2 sites required for cellular attachment.

Peptide-functionalized microfabricated structures for improved on-chip neuronal adhesion.

Extracellular, high signal-to-noise ratio recordings from electrogenic cells require a tight coupling between the cellular membrane and the recording electrode. Self assembled monolayers (SAMs) of alkanethiols functionalized with peptides were used in combination with micro- and nano-structured features on the sensor surface. This combination of surface chemistry and topography triggers a phagocytosis-like engulfment and ensures tight coupling. In this paper we report the results concerning usage of different SAMs and the influence of the peptide concentration towards cell adhesion and outgrowth. Later on, the optimized peptide functionalized SAMs were applied on micro- and nano-structured sensor surfaces. As a result, phagocytosis-like events could be shown using focused ion beam SEM and confocal fluorescence imaging.

High-throughput microplate enzymatic assays for fast sugar and acid quantification in apple and tomato.

In this article, we report on the use of miniaturized and automated enzymatic assays as an alternative technology for fast sugar and acid quantification in apples and tomatoes. Enzymatic assays for d-glucose, d-fructose, sucrose, D-sorbitol/xylitol, L-malic acid, citric acid, succinic acid, and L-glutamic acid were miniaturized from the standard 3 mL assays in cuvettes into assays of 200 microL or lower in 96 or 384 well microplates. The miniaturization and the automation were achieved with a four channel automatic liquid handling system in order to reduce the dispensing errors and to obtain an increased sample throughput. Performance factors (limit of detection, linearity of calibration curve, and repeatability) of the assays with standard solutions were proven to be satisfactory. The automated and miniaturized assays were validated with high-pressure liquid chromatography (HPLC) analyses for the quantification of sugars and acids in tomato and apple extracts. The high correlation between the two techniques for the different components indicates that the high-throughput microplate enzymatic assays can serve as a fast, reliable, and inexpensive alternative for HPLC as the standard analysis technique in the taste characterization of fruit and vegetables. In addition to the analysis of extracts, the high-throughput microplate enzymatic assays were used for the direct analysis of centrifuged and filtered tomato juice with an additional advantage that the sample preparation time and analysis costs are reduced significantly.

A new diabetic with complications: primary nursing care.

Martha, an elderly patient, presented a real challenge to our nursing staff. As a new diabetic, she needed a great deal of education in addition to extensive nursing care for her multiple bleeding leg ulcers, dehydration and malnutrition due to uncontrolled diabetes, and reactive depression. Despite these problems, in approximately one month's time Martha was able to return to her home in control of her diabetes and her emotions and ambulating without pain. Her successful return to normal life was enhanced by holistic nursing management. The nursing staff found that the use of a problem list and a diabetes educational plan assisted them in individualizing their patient care.