Combining culture and microbead-based immunoassay for the early and generic detection of bacteria in platelet concentrates.

BACKGROUND: Despite current preventive strategies, bacterial contamination of platelets is the highest residual infectious risk in transfusion. Bacteria can grow from an initial concentration of 0.03-0.3 colony-forming units (CFUs)/mL up to 108 to 109 CFUs/mL over the product shelf life. The aim of this study was to develop a cost-effective approach for an early, rapid, sensitive, and generic detection of bacteria in platelet concentrates. STUDY DESIGN AND METHODS: A large panel of bacteria involved in transfusion reactions, including clinical isolates and reference strains, was established. Sampling was performed 24 hours after platelet spiking. After an optimized culture step for increasing bacterial growth, a microbead-based immunoassay allowed the generic detection of bacteria. Antibody production and immunoassay development took place exclusively with bacteria spiked in fresh platelet concentrates to improve the specificity of the test. RESULTS: Antibodies for the generic detection of either gram-negative or gram-positive bacteria were selected for the microbead-based immunoassay. Our approach, combining the improved culture step with the immunoassay, allowed sensitive detection of 1 to 10 CFUs/mL for gram-negative and 1 to 102 CFUs/mL for gram-positive species. CONCLUSION: In this study, a new approach combining bacterial culture with immunoassay was developed for the generic and sensitive detection of bacteria in platelet concentrates. This efficient and easily automatable approach allows tested platelets to be used on Day 2 after collection and could represent an alternative strategy for reducing the risk of transfusion-transmitted bacterial infections. This strategy could be adapted for the detection of bacteria in other cellular products.

Highly labeled methylene blue-ds DNA silica nanoparticles for signal enhancement of immunoassays: application to the sensitive detection of bacteria in human platelet concentrates.

A nanoparticle-based electrochemical sandwich immunoassay was developed for bacteria detection in platelet concentrates. For the assay, magnetic beads were functionalized with antibodies to allow the specific capture of bacteria from the complex matrix, and innovative methylene blue-DNA/nanoparticle assemblies provided the electrochemical response for amplified detection. This nanoparticular system was designed as a temperature-sensitive nano-tool for electrochemical detection. First, oligonucleotide-functionalized nanoparticles were obtained by direct synthesis of the DNA strands on the nanoparticle surface using an automated oligonucleotide synthesizer. Densely packed DNA coverage was thus obtained. Then, DNA duplexes were constructed on the NP surface with a complementary strand bearing a 3 methylene blue tag. This strategy ultimately produced highly functionalized nanoparticles with electrochemical markers. These assemblies enabled amplification of the electrochemical signal, resulting in a very good sensitivity. A proof-of-concept was carried out for E. coli detection in human platelet concentrates. Bacterial contamination of this complex biological matrix is the highest residual infectious risk in blood transfusion. The development of a rapid assay that could reach 10-102 CFU mL-1 sensitivity is a great challenge. The nanoparticle-based electrochemical sandwich immunoassay carried out on a boron doped diamond electrode proved to be sensitive for E. coli detection in human platelets. Two antibody pairs were used to develop either a generic assay against certain Gram negative strains or a specific assay for E. coli. The methylene blue-DNA/nanoparticles amplify sensitivity ×1000 compared with the assay run without NPs for electrochemical detection. A limit of detection of 10 CFU mL-1 in a biological matrix was achieved for E. coli using the highly specific antibody pair.

Interaction of polycationic Ni(II)-salophen complexes with G-quadruplex DNA.

A series of nine Ni(II) salophen complexes involving one, two, or three alkyl-imidazolium side-chains was prepared. The lengths of the side-chains were varied from one to three carbons. The crystal structure of one complex revealed a square planar geometry of the nickel ion. Fluorescence resonance energy transfer melting of G-quadruplex structures in the presence of salophen complex were performed. The G-quadruplex DNA structures were stabilized in the presence of the complexes, but a duplex DNA was not. The binding constants of the complexes for parallel and antiparallel G-quadruplex DNA, as well as hairpin DNA, were measured by surface plasmon resonance. The compounds were selective for G-quadruplex DNA, as reflected by equilibrium dissociation constant KD values in the region 0.1-1 µM for G-quadruplexes and greater than 2 µM for duplex DNA. Complexes with more and shorter side-chains had the highest binding constants. The structural basis for the interaction of the complexes with the human telomeric G-quadruplex DNA was investigated by computational studies: the aromatic core of the complex stacked over the last tetrad of the G-quadruplex with peripherical cationic side chains inserted into opposite grooves. Biochemical studies (telomeric repeat amplification protocol assays) indicated that the complexes significantly inhibited telomerase activity with IC50 values as low as 700 nM; the complexes did not significantly inhibit polymerase activity.

Template-assembled synthetic G-quadruplex (TASQ): a useful system for investigating the interactions of ligands with constrained quadruplex topologies.

A new biomolecular device for investigating the interactions of ligands with constrained DNA quadruplex topologies, using surface plasmon resonance (SPR), is reported. Biomolecular systems containing an intermolecular-like G-quadruplex motif 1 (parallel G-quadruplex conformation), an intramolecular G-quadruplex 2, and a duplex DNA 3 have been designed and developed. The method is based on the concept of template-assembled synthetic G-quadruplex (TASQ), whereby quadruplex DNA structures are assembled on a template that allows precise control of the parallel G-quadruplex conformation. Various known G-quadruplex ligands have been used to investigate the affinities of ligands for intermolecular 1 and intramolecular 2 DNA quadruplexes. As anticipated, ligands displaying a pi-stacking binding mode showed a higher binding affinity for intermolecular-like G-quadruplexes 1, whereas ligands with other binding modes (groove and/or loop binding) showed no significant difference in their binding affinities for the two quadruplexes 1 or 2. In addition, the present method has also provided information about the selectivity of ligands for G-quadruplex DNA over the duplex DNA. A numerical parameter, termed the G-quadruplex binding mode index (G4-BMI), has been introduced to express the difference in the affinities of ligands for intermolecular G-quadruplex 1 against intramolecular G-quadruplex 2. The G-quadruplex binding mode index (G4-BMI) of a ligand is defined as follows: G4-BMI=K(D)(intra)/K(D)(inter), where K(D)(intra) is the dissociation constant for intramolecular G-quadruplex 2 and K(D)(inter) is the dissociation constant for intermolecular G-quadruplex 1. In summary, the present work has demonstrated that the use of parallel-constrained quadruplex topology provides more precise information about the binding modes of ligands.

Construction of anti-parallel G-quadruplexes through sequential templated click.

Biologically relevant DNA sequences were assembled onto addressable cyclopeptide scaffolds through sequential oxime and CuAAc reactions. The resulting conjugates are able to fold into well-defined anti-parallel DNA G-quadruplex structures, which exhibit high stability and reduced polymorphism.

Click-click chemistry on a peptidic scaffold for easy access to tetrameric DNA structures.

The use of a peptidic template and orthogonal oxime ligation and CuAAC reaction allows a stepwise approach for the design of antiparallel tetrameric DNA structure. As a proof of concept template assisted synthesis of i-motif DNA was achieved. As anticipated, a dramatic improvement in the stability of such a tetrameric structure was observed. More interestingly the formation of the i-motif was still observed at neutral pH, which could be attributed to the spatial proximity of the basic centers.

Improvement of porphyrins for G-quadruplex DNA targeting.

G-quadruplex nucleic acids are emerging as therapeutic targets for small molecules referred to as small-molecule G-quadruplex ligands. The porphyrin H(2)-TMPyP4 was early reported to be a suitable motif for G-quadruplex DNA recognition. It probably binds to G-quadruplex nucleic acid through π-π stacking with the external G-quartets. We explored chemical modifications of this porphyrin such as insertion of various metal ions in the centre of the aromatic core and addition of bulky substituents that may improve the specificity of the compound toward G-quadruplex DNA. Porphyrin metallation, affording a G4-ligand with two symmetric faces, allowed the conclusion that the presence of an axial water molecule perpendicular to the aromatic plane lowered but did not hamper π-π stacking interactions between the aromatic parts of the ligand on the one hand and the external G-quartet on the other. The charge introduced in the centre of the porphyrin had little influence on binding. Thus, the ionic channel in the centre of G-quadruplex nucleic acids was not found to provide clear additional molecular clues for G-quadruplex nucleic acids targeting by porphyrins tested in the present study. Furthermore, we confirmed the unique G-quadruplex selectivity of a porphyrin modified with four bulky substituents at the meso positions and showed that although the compound is not "drug-like" it was capable of entering cells in culture and mediated some of the typical cellular effects of small-molecule G-quadruplex ligands.