Quantification of quantum dot concentration using inductively coupled plasma-mass spectrometry (ICP-MS).

As quantum dot (QD) bioconjugates are increasingly being used for biomedical in vitro and in vivo studies, validated methods for the quantitative determination of QD concentration are of considerable potential value. In this work, we have assessed inductively coupled plasma mass spectrometry (ICP-MS) as a method for the quantitative detection of QDs and QD bioconjugates. We have established a linear relationship between the concentration of unconjugated QD and the mass of cadmium, selenium and zinc detected by ICP-MS. Furthermore, ICP-MS was used to quantitatively estimate the unknown concentration of a QD-antibody bioconjugate. Quantitative measurement of QD bioconjugate concentration was also attempted by optical methods, including fluorescence and absorbance, and compared to ICP-MS. Consistent with previous literature, the fluorescence of the nanoparticle construct was reduced after functionalization with a biomolecule (biotin or streptavidin). Optical absorbance of the QD is unaffected by chemical modifications in this study and is a reliable method to determine the concentration. Optical absorption in this application achieves nanomolar concentrations but is not suitable for most biomedical studies that require a nanoparticle detection limit in the sub-nanomolar region. Unlike optical absorbance and fluorescence, ICP-MS can reliably detect the concentration of QD bioconjugates in the nanomolar range, making ICP-MS a quantitative, sensitive method for QD concentration measurements even after surface conjugation and consequent changes in fluorescence characteristics.

Proximity-activated nanoparticles: in vitro performance of specific structural modification by enzymatic cleavage.

The development and in vitro performance of a modular nanoscale system capable of specific structural modification by enzymatic activity is described in this work. Due to its small physical size and adaptable characteristics, this system has the potential for utilization in targeted delivery systems and biosensing. Nanoparticle probes were synthesized containing two distinct fluorescent species including a quantum dot base particle and fluorescently labeled cleavable peptide substrate. Activity of these probes was monitored by gel electrophoresis with quantitative cleavage measurements made by fluorometric analysis. The model proximity-activated nanoparticles studied here exhibit significant susceptibility to cleavage by matrix metalloprotease-7 (MMP-7) at physiologically relevant concentrations, with nearly complete cleavage of available substrate molecules after 24 hours. This response is specific to MMP-7 enzyme activity, as cleavage is completely inhibited with the addition of EDTA. Utilization of enzyme-specific modification is a sensitive approach with broad applications for targeted therapeutics and biosensing. The versatility of this nanoparticle system is highlighted in its modular design, as it has the capability to integrate characteristics for detection, biosensing, targeting, and payload delivery into a single, multifunctional nanoparticle structure.

Versatile biomimetic dendrimer templates used in the formation of TiO2 and GeO2.

Biomimetic synthesis is emerging as an advantageous alternative to the harsh synthetic conditions traditionally used in metal oxide syntheses techniques. Silaffins, proteins from the C. fusiformis diatom, form silica in an aqueous environment under benign conditions. Amine terminated PAMAM and PPI dendrimers are effective mimics of silaffins and other silica precipitating polyamines. We have expanded the scope of dendrimer mediated metal oxide formation to include titanium dioxide, a photocatalyst, and germanium dioxide, a blue photoluminescent material. The nanoparticles were characterized using scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (IR), and X-ray diffraction patterns (XRD). A variable temperature XRD analysis of TiO(2) nanoparticles was conducted to study the transition from anatase to rutile. TiO(2) nanoparticles synthesized in phosphate buffer showed a 200 degrees C decrease in the anatase to rutile transition temperature relative to TiO(2) templated in water. XRD analysis of GeO(2) nanoparticles in either water or phosphate buffer reveal crystalline alpha-phase germanium oxide. To our knowledge, this is the first report of the synthesis of crystalline GeO(2) under ambient conditions.

Piezoelectric ink jet processing of materials for medical and biological applications.

Many advanced medical and biological devices require microscale patterning of cells, proteins, and other biological materials. This article describes the use of piezoelectric ink jet processing in the fabrication of biosensors, cell-based assays, and other microscale medical devices. A microelectromechanical system-based piezoelectric transducer was used to develop uniform fluid flow through nozzles and to prepare well-defined microscale patterns of proteins, monofunctional acrylate ester, sinapinic acid, deoxyribonucleic acid (DNA), and DNA scaffolds on relevant substrates. Our results demonstrate that piezoelectric ink jet deposition is a powerful non-contact, non-destructive additive process for developing biosensors, cell culture systems, and other devices for medical and biological applications.

The basis of the immunomodulatory activity of malaria pigment (hemozoin).

The most common and deadly form of the malaria parasite, Plasmodium falciparum, is responsible for 1.5-2.7 million deaths and 300-500 million acute illnesses annually [Bremen in J. Trop. Med. Hyg. 64:1-11 (2001); World Health Organization (2002)]. Hemozoin, the biomineral formed to detoxify the free heme produced during parasitic hemoglobin catabolism, has long been suspected of contributing to the pathological immunodeficiencies that occur during malarial infection. While there is a growing consensus in the literature that native hemozoin maintains immunosuppressive activity, there is considerable controversy over the reactivity of the synthetic form, beta-hematin (BH). Given the emerging importance of hemozoin in modulating a host immune response to malarial infection, a careful examination of the effects of the constitutive components of the malaria pigment on macrophage response has been made in order to clarify the understanding of this process. Herein, we present evidence that BH alone is unable to inhibit stimulation of NADPH oxidase and inducible nitric oxide synthase, the key enzymes involved in oxidative burst, and is sensitive to the microbicidal agents of these enzymes both in vitro and in vivo. Further, by systematically examining each of the malaria pigment's components, we were able to dissect their impact on the immune reactivity of a macrophage model cell line. Reactions between BH and red blood cell (RBC) ghosts effectively reconstituted the observed immunomodulatory reactivity of native hemozoin. Together, these results suggest that the interaction between hemozoin and the RBC lipids results in the generation of toxic products and that these products are responsible for disrupting macrophage function in vivo.

Size control of dendrimer-templated silica.

One of the most significant challenges facing the biomimetic synthesis of materials is achieving the requisite level of dimensional and spatial control. Typical reaction conditions for biomimetic silica synthesis allow for continued growth and ripening leading to the formation of larger nanospheres on the order of 200-600 nm in diameter. Herein, we have used polyamidoamine and polypropylenimine dendrimers as templates to expand the reaction conditions of biogenic silica production to produce a more robust synthesis leading to size-selective precipitation of silica nanospheres. Through the use of defined concentrations of phosphate buffer and main group metal chloride salts, we have shown that the biomimetic silica growth process is controlled by cationic neutralization of the anionic silica nanosphere surface. Neutralization minimizes electrostatic repulsions, allowing for agglomerization and continued growth of nanospheres. By controlling these concentrations, we can selectively produce silica nanospheres of desired dimensions between 30 and 300 nm without adversely affecting the template's activity.