Detection of Pathogens Using Luminescent CdSe/ZnS Dendron Nanocrystals and a Porous Membrane Immunofilter

Abstract

A biosensor system for detection of pathogens was developed by using CdSe/ZnS core/shell dendronnanocrystals with high efficiency and stability as fluorescence labels and a flowing chamber with a micro-porous immunofilter. The antibody-immobilized immunofilter captured the targeted pathogens, E. coli O157:H7 as an example for bacteria and Hepatitis B being a model system for viruses. The CdSe/ZnS core/shell dendron-nanocrystals were conjugated with the corresponding antibodies and then passed through the micro-porous membrane where they attached to the membrane-antigen-antibody. The efficient and stable photoluminescence (PL) of the CdSe/ZnS nanocrystals on the formed "sandwich" structure complexes (membrane-antigen-antibody conjugated with the nanocrystals) was used as the detection means. The effects of the pore size of the membranes, buffer pH, and assay time on the detection of E. coli O157:H7 were investigated and optimized. The detectable level of this new system was as low as 2.3 CFU/ml for E. coli O157:H7 and 5ng/ml for Hepatitis B surface Ag (HBsAg). The assay time was shortened to 30 min without any enrichment and incubation. KeywordsCdSe/ZnS nanocrystals; photoluminescence; HBsAg detection; E. coli O157:H7 detection; immunoassay; biosensor; rapid detection Fluorescence measurements are the most widely used readout modalities in the life sciences today, from research at academic institutions to high-throughput screening by pharmaceutical companies. Colloidal semiconductor nanocrystals, or quantum dots (QDs), have recently been explored as a new generation of fluorescent labels in the field of bio-medical science owing to their unique optical properties, i.e., a broad absorption spectrum and a narrow, symmetric emission peak [1]. However, most QDs are synthesized in organic solvents and coated with a monolayer of hydrophobic ligands. Consequently, they have to be converted to water-soluble/ biocompatible nanoparticles for biological applications, which should have functional groups for bioconjugation such that the optical properties of the resulting nanocrystals are not diminished. *Corresponding authors: Yongcheng Liu, email: xpeng@uark.edu In the recent years, quite a few of strategies have been, and are still being, designed to make QDs water-soluble and biocompatible. A recent review provided a summary of them [2]. These strategies can be separated into two different approaches: surface-ligand exchange by hydrophilic ligands and coating of the QDs with additional layers of water-soluble molecules. In the former, the initial ligand molecules are replaced with bifunctional molecules, such as cysteines [3], thiol-containing molecules with a hydrophilic terminal group (such as mercapto alkanoic acids) [5][6], and more sophisticated ones such as oligomeric phosphines [7], peptides [8], and dendrons [9][10][11]. The common nature in all of these new ligands is that the outer surface of the nanocrystal-ligand complex is terminated with some hydrophilic groups, such as carboxyl, amine, PEG units, or...

Semiconductor Quantum Dots for Bioanalysis

“…[42,43] Recently, QDs were stabilized by exchanging ODA with water-soluble dendrons to yield water-soluble QDs with quantum yields of 36 %. [44] Interestingly, internally facing carboxylate groups on the dendrons retained a higher Figure 2. Modification of semiconductor QDs with functional encapsulating layers for water solubilization and preservation of luminescence properties and/or secondary covalent modification of the surface with biomolecules.…”

Preparation of gold nanoparticles by microwave heating and application of spectroscopy to study conjugate of gold nanoparticles with antibody E. coli O157:H7

“…For example, some groups have combined ligandmagnetic nanoparticles (LMNPs) enrichment with a fluorescent silica nanoparticles (FSiNPs) based two-color flow cytometry assay for the detection of E. coli O157:H7 [16]. Besides, luminescent CdSe/ZnS nanocrystals was applied to detect low levels of E. coli O157:H7 [17].…”

Application of Nanoparticles for the Detection and Sorting of Pathogenic Bacteria by Flow-Cytometry

“…During the last decade fl uorescent q-dots have been chemically modifi ed on their surface for the introduction in biological matrices (Gerion et al 2001 ;Goldman et al 2002 ;Michalet et al 2005 ) . The potential of using q-dots as fl uorescent labels for biological systems was studied by several research groups which are aiming for developing new tool for cell studying (Bruchez et al 1998 ) , cell staining (Yitzhaki et al 2005 ) , fl uoro-immunoassays (Goldman et al 2002 ) , pathogen immunodetection (Hahn et al 2008 ;Liu et al 2007 ;Su and Li 2004 ) , in-vivo imaging (Michalet et al 2005 ) , immunophenotyping by fl ow cytometry analysis (Chattopadhyay et al 2006 ;Summers et al 2010 ;Tarnok 2010 ;Zahavy et al 2010 ;Godfrey et al 2009 ;Jaron and Godfrey 2009 ) , FRET biological sensor (Geisler et al 2010 ;Medintz and Mattoussi 2009 ) , and multiplexed analysis for DNA analysis (Hahn et al 2001 ) . In here we will show that the use of the fl uorescent q-dots can contribute to current immunofl uorescence analysis of biological hazards in two ways: (1) Different conjugates can be used for multi-parameter analysis by FCM in order to achieve fast, sensitive and accurate analysis of a single biological threat, such as Bacillus anthracis ( B. anthracis ) spores.…”