Attomole sensitivity of staphylococcal enterotoxin B detection using an aptamer-modified surface-enhanced Raman scattering probe.

In this report, we present a new homogeneous detection method for staphylococcal enterotoxin B (SEB) utilizing core-shell-structured iron-gold magnetic nanoparticles and a gold nanorod surface-enhanced Raman scattering (SERS) probe in solution. Peptide ligand (aptamer) functionalized magnetic gold nanorod particles were used as scavengers for target SEB. After the SEB molecules were separated from the matrix, the sandwich assay procedure was tested by gold nanorod particles that act as SERS probes. The binding constant between SEB and peptide-nanoparticle complex was determined as 8.0 × 10(7) M(-1). The correlation between the SEB concentration and SERS signal was found to be linear within the range of 2.5 fM to 3.2 nM. The limit of detection for the homogeneous assay was determined as 224 aM (ca. 2697 SEB molecules/20 µL sample volume). Also, gold-coated surfaces were used as capture substrates and performances of the two methods were compared. Furthermore, the developed method was evaluated for investigating the SEB specificity on bovine serum albumin (BSA) and avidin and detecting SEB in artificially contaminated milk, blood, and urine.

Comparison of sensing strategies in SPR biosensor for rapid and sensitive enumeration of bacteria.

Rapid and sensitive detections of microorganisms are very important for biodefence, food safety, medical diagnosis and pharmaceutics. The present study aims to find out the most proper bioactive surface preparation method to develop rapid, sensitive and selective bacteria biosensor, based on surface plasmon resonance (SPR) spectroscopy. Escherichia coli (E. coli) was used as a model bacterium and four sensing strategies in SPR were tested. Three of these strategies are antibody immobilization methods that are non-specific adsorption, specific adsorption via the avidin-biotin interaction, and immobilization of antibodies via self-assembled monolayer formation. The fourth strategy is a novel method for bacteria enumeration based on the combination of the SPR spectroscopy and immunomagnetic separation with using gold-coated magnetic nanoparticles. According to results, the most efficient SPR method is the one based on gold-coated magnetic nanoparticles. This method allows to specifically separate E. coli from the environment and to quantify rapidly without any labeling procedure. The developed method has a linear range between 30 and 3.0 × 10(4)cfu/ml, and a detection limit of 3 cfu/ml. The selectivity of the method was examined with Enterobacter aerogenes and Enterobacter dissolvens, which did not produce any significant response. The usefulness of the method to detect E. coli in real water samples was also investigated, and the results were compared with the results from plate-counting method. There was no significant difference between the methods (p>0.05).

SERS-based sandwich immunoassay using antibody coated magnetic nanoparticles for Escherichia coli enumeration.

A method combining immunomagnetic separation (IMS) and surface-enhanced Raman scattering (SERS) was developed to enumerate Escherichia coli (E. coli). Gold-coated magnetic spherical nanoparticles were prepared by immobilizing biotin-labeled anti-E. coli antibodies onto avidin-coated magnetic nanoparticles and used in the separation and concentration of the E. coli cells. Raman labels have been constructed using rod shaped gold nanoparticles coated with 5,5-dithiobis-(2-nitrobenzoic acid) (DTNB) and subsequently with a molecular recognizer. Then DTNB-labeled gold nanorods were interacted with gold-coated magnetic spherical nanoparticle-antibody-E. coli complex. The capture efficiency and calibration graphs were obtained and examined in different E. coli concentrations (10(1)-10(7) cfu mL(-1)). The correlation between the concentration of bacteria and SERS signal was found to be linear within the range of 10(1)-10(4) cfu mL(-1) (R(2) = 0.992). The limit of detection (LOD) and limit of quantification (LOQ) values of the developed method were found to be 8 and 24 cfu mL(-1), respectively. The selectivity of the developed immunoassay was examined with Enterobacter aerogenes, Enterobacter dissolvens, and Salmonella enteriditis which did not produce any significant response. The ability of the immunoassay to detect E. coli in real water samples was also investigated and the results were compared with the experimental results from plate-counting methods. There was no significant difference between the methods that were compared (p > 0.05). This method is rapid and sensitive to target organisms with a total analysis time of less than 70 min.

A high sensitive assay platform based on surface-enhanced Raman scattering for quantification of protease activity.

In this study, a new, sensitive, and rapid assay was developed to quantitatively measure the proteolytic enzyme activity using the surface-enhanced Raman scattering (SERS) probe. Two different shapes of gold nanoparticles, gold nanosphere and nanorod particles were produced. SERS label, comprising self-assembled monolayers (SAMs) of Raman reporter molecule (5,5-Dithiobis (2-Nitrobenzoic acid), DTNB), was coated on the surface of the nanoparticles. Two different SERS-based analysis platforms were designed using gold-coated glass slide and polystyrene microtiter plate. The calibration curves were obtained by plotting the intensity of the SERS signal of symmetric NO(2) stretching of DTNB at 1326 cm(-1) vs. the protease concentration. The effects of nanoparticle geometry and assay platform on the protease assay were investigated and the best working combination of the parameters was selected as rod shaped SERS probe and gold-coated glass slide. The correlation between the protease activity and SERS signal was found to be linear within the range of 0.1-2 mU/mL (R(2)=0.979). The limit of detection (LOD) and limit of quantification (LOQ) values of the validated method were found as 0.43 and 1.30 mU/mL, respectively. The intra-day and inter-day precisions of the method, as relative standard deviation (RSD), were determined as 2.5% and 3.6%, respectively. The developed method was successfully applied for quantitative analysis of the commercial enzyme preparate that is used in cheese making process. It was also used for investigation of substrate specificity of protease enzyme towards the casein and bovine serum albumin. The proposed method has a flexibility to try different substrates for the detection of various enzyme activities.

A highly sensitive detection platform based on surface-enhanced Raman scattering for Escherichia coli enumeration.

A very sensitive and highly specific heterogeneous immunoassay system, based on surface-enhanced Raman scattering (SERS) and gold nanoparticles, was developed for the detection of bacteria and other pathogens. Two different types of gold nanoparticles (citrate-stabilized gold nanosphere and hexadecyltrimethylammonium bromide (CTAB)-stabilized gold nanorod particles) were examined and this immunoassay was applied for the detection of Escherichia coli. Raman labels were constructed by using these spherical and rod-shaped gold nanoparticles which were first coated with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) and subsequently with a molecular recognizer. The working curve was obtained by plotting the intensity of the SERS signal of the symmetric NO(2) stretching of DTNB at 1,333 cm(-1) versus the concentration of the E. coli. The analytical performance of gold particles was evaluated via a sandwich immunoassay, and linear calibration graphs were obtained in the E. coli concentration range of 10(1)-10(5) cfu/mL with a 60-s accumulation time. The sensitivity of the Raman label fabricated with gold nanorods was more than three times higher than spherical gold nanoparticles. The selectivity of the developed sensor was examined with Enterobacter aerogenes and Enterobacter dissolvens, which did not produce any significant response. The usefulness of the developed immunoassay to detect E. coli in real water samples was also demonstrated.