Facile synthesis and photophysical characterization of luminescent CdTe quantum dots for Forster resonance energy transfer based immunosensing of staphylococcal enterotoxin B.

Aqueous phase synthesis of CdTe quantum dots (QDs) with surface functionalization for bioconjugation remains the best approach for biosensing and bioimaging applications. We present a facile aqueous phase method to prepare CdTe QDs by adjusting precursor and ligand concentrations. CdTe QDs had photoluminescence quantum yield up to ≈33% with a narrow spectral distribution. The powder X-ray diffraction profile elucidated characteristic broad peaks of zinc blende cubic CdTe nanoparticles with 2.5-3 nm average crystalline size having regular spherical morphology as revealed by transmission electron microscopy. Infra-red spectroscopy confirmed disappearance of characteristic absorptions for -SH thiols inferring thiol coordinated CdTe nanoparticles. The effective molar concentration of 1 : 2.5 : 0.5 respectively for Cd(2+)/3-mercaptopropionic acid/HTe(-) at pH 9 ± 0.2 resulted in CdTe quantum dots of 2.2-3.06 nm having band gap in the range 2.74-2.26 eV respectively. Later, QD523 and QD601 were used for monitoring staphylococcal enterotoxin B (SEB; a bacterial superantigen responsible for food poisoning) using Forster resonance energy transfer based two QD fluorescence. QD523 and QD601 were bioconjugated to anti-SEB IgY antibody and SEB respectively according to carbodiimide protocol. The mutual affinity between SEB and anti-SEB antibody was relied upon to obtain efficient energy transfer between respective QDs resulting in fluorescence quenching of QD523 and fluorescence enhancement of QD601. Presence of SEB in the range 1-0.05 µg varied the rate of fluorescence quenching of QD523 , thereby demonstrating efficient use of QDs in the Forster resonance energy transfer based immunosensing method by engineering the QD size.

An immunoreactor-based competitive fluoroimmunoassay for monitoring staphylococcal enterotoxin B using bioconjugated quantum dots.

Extensive research on avian systems has proved hens as an alternate source for polyclonal antibody generation necessary for immunosensing applications. Herein, we present the immobilization of avian antibody raised against staphylococcal enterotoxin B (SEB) and its applicability for a competitive fluoroimmunoassay technique. White leghorn hens immunized with SEB generated high affinity antibodies with a highest yield of 3.2 mg ml(-1) having affinity constant of 0.976 × 10(10) M l(-1). A competitive fluoroimmunoassay format was developed comprising CdTe(557) as a fluorescence detector for monitoring SEB, a bacterial super-antigen. CdTe(557) was bioconjugated to SEB according to the carbodiimide protocol and confirmed by absorption spectral analysis. An immunoreactor column was designed by immobilizing anti-SEB antibodies and was successfully employed as an efficient bio-recognition tool. An immuno-affinity reaction involving competitive binding between free SEB and CdTe(557)-bioconjugated SEB for immobilized antibody was relied upon to attain assay specificity and sensitivity. It was possible to quantify SEB from 1000 to 10 ng based on the integrated fluorescence of the SEB-CdTe(557) bioconjugate eluted from the immunoreactor column with a limit of detection of 8.15 ng and a regression coefficient R(2) = 0.9925. Thus, integration of QDs with immuno-affinity reactions revealed the versatility of nanoparticles as a potential fluorescence label for bioanalytical applications.

Development of dipstick-based immuno-chemiluminescence techniques for the rapid detection of dichlorodiphenyltrichloroethane.

The occurrence of organochlorine pesticides in the environment has been a major concern, due to their high persistence and the possible impacts of their exposure to humans. Dichlorodiphenyltrichloroethane (DDT) is most hazardous and one of the most widely used organochlorine insecticides. DDT and its main metabolites are highly stable to physical, chemical and biological degradation and are therefore still being detected in many parts of the world. The present study describes dipstick-based immuno-chemiluminescence method for the detection of DDT with high sensitivity. Anti-DDT antibodies raised in chicken (IgY) were used as the biological sensing elements by immobilizing onto nitrocellulose membrane strips in a chemiluminescence (CL)-based dipstick technique. The photons generated during the biochemical interaction were directly proportional to the DDT concentration. A mean recovery of 81.2-95.6% was obtained for DDT-spiked fruit juice samples with 2.8-4.6% relative standard deviation (RSD). Using the proposed dipstick-based immuno-CL method, DDT was detected with linearity in the range 0.05-1 ng/mL, having a limit of detection (LOD) of 0.05 ng/mL. This method can be used for the rapid, reliable detection of DDT pesticide.

Photoabsorption and resonance energy transfer phenomenon in CdTe-protein bioconjugates: an insight into QD-biomolecular interactions.

Luminescent quantum dots (QDs) possess unique photophysical properties, which are advantageous in the development of new generation robust fluorescent probes based on Forster resonance energy transfer (FRET) phenomena. Bioconjugation of these QDs with biomolecules create hybrid materials having unique photophysical properties along with biological activity. The present study is aimed at characterizing QD bioconjugates in terms of optical behavior. Colloidal CdTe QDs capped with 3-mercaptopropionic acid (MPA) were conjugated to different proteins by the carbodiimide protocol using N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC) and a coupling reagent like N-hydroxysuccinimide (NHS). The photoabsorption of these QD-protein bioconjugates demonstrated an effective coupling of electronic orbitals of constituents. A linear variation in absorbance of bioconjugates at 330 nm proportionate to conjugation suggests a covalent attachment as confirmed by gel electrophoresis. A red shift in the fluorescence of bovine serum albumin (BSA) due to conjugation inferred a decrease in Stokes shift and solvent polarization effects on protein. A proportionate quenching in BSA fluorescence followed by an enhancement of QD fluorescence point toward nonradiative dipolar interactions. Further, reduction in photobleaching of BSA suggests QD-biomolecular interactions. Bioconjugation has significantly influenced the photoabsorption spectrum of QD bioconjugates suggesting the formation of a possible protein shell on the surface of QD. The experimental result suggests that these bioconjugates can be considered nanoparticle (NP) superstructures for the development of a new generation of robust nanoprobes.

Trace-level detection of atrazine using immuno-chemiluminescence: dipstick and automated flow injection analyses formats.

A sensitive chemiluminescence (CL)-based immunoassay technique based on both dipstick and flow injection analytical formats is reported for the detection of atrazine. In the dipstick-based immunoassay technique, antibody (anti-atrazine) was first immobilized on the nitrocellulose membranes. The dipstick was then treated with atrazine and atrazine-horseradish peroxidase conjugate (atra-HRP) to facilitate the competitive binding. The dipstick was further treated with urea-hydrogen peroxide (U-H202) and luminol to generate photons. The number of photons generated was inversely proportional to the atrazine concentration. In the flow injection analysis (FIA) format, the antibody was immobilized on protein-A sepharose matrix and packed in a glass capillary column, which functioned as an immunoreactor. Competitive binding of antigen and antibody occurred. The CL signals generated during the biochemical reactions were correlated with atrazine concentrations in the analytical samples. By using dipstick technique, it was possible to detect atrazine concentration down to 0.1 ng/mL; with the FIA format, the detection of atrazine was down to 0.01 ng/mL.