David A. Russell

Functional, biochemical and genetic diversity of prokaryotic nitrate reductases

Abstract. Prokaryotic nitrate reduction can serve a number of physiological roles and can be catalysed by a number of biochemically distinct nitrate reductases. Three distinct nitrate reductase classes can be indentified in prokaryotes, NAS, NAR and NAP. NAS is located in the cytoplasmic compartment and participates in nitrogen assimilation. NAR is usually a three-subunit complex anchored to the cytoplasmic face of the membrane with its active site located in the cytoplasmic compartment and is involved in anaerobic nitrate respiration. NAP is a two-subunit complex, located in the periplasmic compartment, that is coupled to quinol oxidation via a membrane anchored tetraheme cytochrome. It shows considerable functional flexibility by participating in anaerobic respiration or redox energy dissipation depending on the organism in which it is found. The members of all three classes of enzymes bind the bis-molybdopterin guanine dinucleotide cofactor at the active site, but they differ markedly in the number and nature of cofactors used to transfer electrons to this site. Analysis of prokaryotic genome sequences available at the time of writing reveals that the different nitrate reductases are phylogenetically widespread.

Self-assembled monolayers: a versatile tool for the formulation of bio-surfaces

The recent developments in self-assembly technology applied to protein surface modification are described. The simplicity and adaptability of self-assembled monolayers (SAMs) and the control over biomolecule surface orientation suggest that SAMs will play an important role in the construction of artificial biomolecular recognition surfaces and particularly in the future development of biosensors.

Advances in Fingerprint Analysis

Fingerprints have been used in forensic investigations for the identification of individuals since the late 19th century. However, it is now clear that fingerprints can provide significantly more information about an individual. Here, we highlight the considerable advances in fingerprinting technology that can simultaneously provide chemical information regarding the drugs ingested and the explosives and drugs handled by a person as well as the identity of that individual.

The in vivo efficacy of phthalocyanine–nanoparticle conjugates for the photodynamic therapy of amelanotic melanoma

The efficiency of a Zn(II)-phthalocyanine disulphide (C11Pc), a compound with both phthalocyanine units bearing seven hexyl chains and a sulphur terminated C11 chain, as a photodynamic therapy (PDT) agent was investigated in C57 mice bearing a sub-cutaneously transplanted amelanotic melanoma. The phthalocyanine was intravenously injected at a dose of 1.5 micromol/kg body weight either free or bound to gold nanoparticles, using a Cremophor emulsion as a delivery vehicle. Biodistribution studies at selected post-injection times showed that the nanoparticle-associated C11Pc was recovered in significantly larger amounts from all the examined tissues and the serum and yielded a greater selectivity of tumour targeting: thus, the ratio between the amount of phthalocyanine recovered from the amelanotic melanoma and the skin (peritumoural tissue) increased from 2.3 to 5.5 from the free to the gold nanoparticle-bound C11Pc at 24 h after injection. PDT studies with the C11Pc-loaded amelanotic melanoma showed a markedly more significant response of the tumour in the mice that had received the nanoparticle-bound photosensitiser; the PDT effect was especially extensive if the irradiation was performed at 3h after C11Pc injection when large phthalocyanine amounts were still present in the serum. This suggests that the PDT promoted by C11Pc predominantly acts via vascular damage at least in this specific animal model. This hypothesis was fully confirmed by electron microscopy observations of tumour specimens obtained at different times after the end of PDT, showing an extensive damage of the blood capillaries and the endothelial cells.

Imaging of Latent Fingerprints through the Detection of Drugs and Metabolites

Magnetic particles functionalised with anti-cotinine antibody have been used to image latent fingermarks through the detection of the cotinine antigen in the sweat deposited within the fingerprints of smokers. The antibody–magnetic particle conjugates are readily applied to latent fingerprints while excess reagents are removed through the use of a magnetic wand. The results have shown that drug metabolites, such as cotinine, can be detected and used to image the fingermark to establish the identity of an individual within 15 minutes.

Colorimetric detection of Ricinus communis Agglutinin 120 using optimally presented carbohydrate-stabilised gold nanoparticles.

Ricin is a toxic lectin which presents a potential security threat. Its rapid detection is highly desirable. Here we present a colorimetric bioassay based on the aggregation of carbohydrate-stabilised gold nanoparticles which has been used to detect Ricinus communis Agglutinin 120 (RCA(120)) - a ricin surrogate. To achieve a stable and robust sensing system the anchor chain length and the density of the assembled carbohydrates on the gold particle surface has been examined to determine the optimal coverage for maximal aggregation with both RCA(120) and Concanavalin A (Con A) lectins. Gold nanoparticles were stabilised with either a thiolated galactose derivative (9-mercapto-3,6-diaoxaoctyl-beta-d-galactoside) or a thiolated mannose derivative (9-merapto-3,6-dioxaoctyl-alpha-d-mannoside), for RCA(120) and Con A respectively, diluted in each instance with varying ratios of a thiolated triethylene glycol derivative. Aggregation was induced with the respective cognate lectin with the reaction monitored by UV-visible spectrophotometry. The results obtained show that a particle surface with at least 7.5% galactose is required for aggregation with RCA(120) and 6% mannose coverage is required for aggregation with Con A. For each lectin the sensitivity of the assay could be controlled by adjustment of the carbohydrate density on the gold nanoparticles, but with differing results. Maximal aggregation with Con A was achieved with a monolayer consisting of 100% mannose, whereas for RCA(120) maximal aggregation occurred with 70% coverage of galactose. The limit of detection for RCA(120) using the optimally presented galactose-stabilised nanoparticles was 9 nM.

A versatile gold surface approach for fabrication and interrogation of glycoarrays

Glycoarrays on gold: A designer gold surface incorporating a self-assembled monolayer with weak protein absorption properties has been optimised for rapid display and interrogation of both native and derivatised glycans in array formats. This rapid, facile approach has diverse applications in glycomics, through exploitation of fluorescence, SPR and MALDI-ToF MS detection methods.

Multiplexed Detection of Metabolites of Narcotic Drugs from a Single Latent Fingermark

An immunoassay based technique is used for the detection of psychoactive substances in the sweat deposited within fingermarks of a narcotic drug user. Magnetic particles functionalized with antimorphine and antibenzoylecgonine antibodies were used for the detection of a metabolite of heroin (morphine) and a metabolite of cocaine (benzoylecgonine), respectively. The drug metabolites were detected individually as well as simultaneously from a single fingermark. The images of the fingermarks obtained using brightfield and fluorescence microscopy were of high evidential quality with resolution to enable identification of an individual in addition to providing information on drug usage.

Targeting the Oncofetal Thomsen-Friedenreich Disaccharide Using Jacalin-PEG Phthalocyanine Gold Nanoparticles for Photodynamic Cancer Therapy

Photodynamic therapy (PDT) is an emerging therapeutic modality for cancer treatment during which a photoactive drug, termed a photosensitizer, is used to destroy tumors upon activation with red or near-infrared light through the production of cytotoxic singlet oxygen. PDT has shown significant efficacy with photosensitizers, such as Photofrin and Foscan, which are in current clinical use for the treatment of a number of cancers. The “ideal” photosensitizer has been shown to possess hydrophobic characteristics and consequently suffers from limited aqueous solubility, thus impairing its systemic biodistribution. An additional drawback is that some photosensitizers exhibit limited selectivity towards cancerous tissue, thus resulting in nonspecific photodamage of peripheral tissue. To overcome these limitations there have been considerable efforts to develop nanoparticle-based systems for use as photosensitizer drug delivery vehicles. Such systems maintain the stability and activity of hydrophobic photosensitizers in aqueous environments, while the nanoparticle itself provides a platform for further functionality with targeting moieties for cancer-specific PDT. The targeting of tumors using nanoparticle systems can be achieved either passively or actively. Passive targeting with nanoparticles involves the enhanced permeability and retention (EPR) effect that is invariably observed in tumors. Active targeting of nanoparticles can be achieved through the conjugation of biological ligands, such as antibodies, peptides, carbohydrates, and folic acid, each with an affinity for a specific surface receptor expressed by cancer cells. During the transformation of healthy to malignant cells aberrations in the glycosylation of cell-surface glycoproteins occur. It has been suggested that these glycosylation patterns play roles in cancer cell metastasis, survival, and evasion of immune surveillance. One of the most therapeutically attractive cancer-associated carbohydrates is the Thomsen– Friedenreich disaccharide antigen (T antigen). The T antigen is a truncated O-glycan (Galb1-3GalNAc-O-serine/ threonine) that is expressed in more than 90 % of primary human carcinomas, but is usually cryptic in normal cells. This antigen is often referred to as being oncofetal, as its exposure at the cell surface is only observed in developing embryonic cells or cancer cells. 12] Since the T antigen is expressed in such a large number of cancers and its exposure on non-developmental cells is restricted to malignant tissue, it is clearly an appealing molecular target for cancer therapies, such as PDT. Herein we present, for the first time, the synthesis and application of gold nanoparticles that are stabilized by a mixed monolayer of a hydrophobic zinc phthalocyanine photosensitizer and a water-soluble thiol-functionalized poly(ethylene glycol) (PEG) to which jacalin (a lectin) is covalently conjugated to specifically target the T antigen disaccharide. Previously, lectin-functionalized nanoparticles have been used for the targeted delivery of drugs to the brain, lungs, and colon, while other lectin–nanoparticle conjugates, based on gold nanoparticles and quantum dots, have been used for bioimaging. Lectins (specifically, wheat germ agglutinin loaded on polymeric nanoparticles and fluorescently labeled nanospheres functionalized with peanut agglutinin) have been used to enhance drug delivery to colon cancer cells and to detect implanted colorectal tumors expressing the T antigen, respectively. Within PDT, wheat germ agglutinin has been used with liposomes carrying the photosensitizer temoporphin for antimicrobial studies, and Poiroux et al. directly coupled the Morniga G lectin to a water-soluble porphyrin photosensitizer to target the T antigen and the Tn antigen (another cancer-associated carbohydrate) expressed on Jurkat leukaemia cells. We have now demonstrated that the jacalin-PEG phthalocyanine gold nanoparticles described herein target the T antigen expressed on HT-29 colon adenocarcinoma cells. The jacalin is shown to promote cellular adhesion and uptake of the nanoparticle conjugates. As a result of jacalin targeting, the specificity of which was confirmed through inhibition studies, enhanced cellular uptake of the nanoparticles was achieved. Exceptional PDT efficacy with the light-activated jacalin–nanoparticle conjugates for the destruction of the colon cancer cells has been demonstrated. Finally, it has been established that necrosis was the mechanism of jacalintargeted photodynamic toxicity. The results highlight that jacalin-PEG phthalocyanine nanoparticles can selectively target the important T antigen on the surface of colon [*] G. Obaid, Dr. I. Chambrier, Prof. M. J. Cook, Prof. D. A. Russell School of Chemistry, University of East Anglia Norwich, Norfolk, NR4 7TJ (UK) E-mail: d.russell@uea.ac.uk

Localized Intracellular pH Measurement Using a Ratiometric Photoinduced Electron‐Transfer‐Based Nanosensor

Hydrogen ions are an important intracellular species. In eukaryotic cells the function of several organelles is dependent on the pH in each of those individual organelles. The pH in the cytosol and the nucleus is typically 7.2–7.4, the secretory and the endocytic pathways have a lower pH, while the acidic organelles, such as endosomes and lysosomes, typically have a pH as low as 4.0–5.5. Disruption of the pH within the different organelles may lead to dysfunction of the affected organelle and ultimately to a diseased state. For example, tumor cells have been shown to have an abnormal cellular pH and disturbances in the pH of the acidic organelles have been associated with renal failure, oncological processes, and with the so called lysosomal storage disorders. With consideration of the large number of diseases related to abnormal values of pH in the acidic organelles it is important to develop new tools, to supplement those molecular probes that already exist, to quantify the hydrogen ion concentration. Ratiometric fluorescence-based nanosensors are an example of a robust tool for the sensing and quantification of ions of biological interest. Kopelman and co-workers designed PEBBLEs (probes encapsulated by biologically localized embedding), ratiometric fluorescence nanosensors, for the intracellular measurement of pH. Since then, a number of ratiometric fluorescence nanosensors for pH have been reported based on polymeric nanoparticles, silica nanoparticles, quantum dots, cellulose nanocrystals, latex nanobeads, and zeolite-based nanoparticles. There have been some reports on the internalization of ratiometric fluorescence pH nanosensors by cells, with localization of the nanoprobes in the cytoplasm or within acidic organelles. Other studies have reported measurements of a change in intracellular pH values using dual ratiometric fluorescence nanoprobes. Recently, a ratiometric polymeric nanoprobe composed of three fluorophores has been reported for intracellular pH measurements. Fluorescent photoinduced electron-transfer (PET) based pH molecular probes have been widely studied. Previously, we have reported an anthracene-based PET pH probe with a readily tunable pKa that accumulated in acidic organelles within Raw 264.7 macrophage cells. The self-assembly of such PET-based pH probes onto gold nanoparticles has not been previously described. Herein, we report a fluorescence pH nanosensor that provides, for the first time, precise localized determination of pH from within acidic organelles. The ratiometric nanosensor consists of a thiolated anthracene molecule as a fluorescent PET-based pH ligand (1) and a thiolated rhodamine ligand (2), both self-assembled onto the surface of a gold nanoparticle (3, Figure 1). The synthesized fluorescence-based pH