Lysozyme-encapsulated gold nanocluster-based affinity mass spectrometry for pathogenic bacteria.

RATIONALE: Bacterial infections can be difficult to treat and can lead to irreversible damage to patients if proper treatment is not provided in time. Additionally, the emerging threat from antibiotic-resistant bacterial strains makes medical treatment even more difficult. Thus, rapid identification of infected bacterial strains is essential to assist diagnostics and medical treatment. METHODS: Lysozymes are glycoside hydrolases that can bind with peptidoglycans on bacterial cell walls. In this work, we demonstrated that lysozyme-encapsulated gold nanoclusters (lysozyme-AuNCs) with red photoluminescence can be used as affinity probes to concentrate pathogenic bacteria. After bacteria had been probed by the lysozyme-AuNCs in a sample solution, the lysozyme-AuNC-bacteria conjugates were readily spun down at a low centrifugation speed. The red emission from the AuNCs on the conjugates could be visualized with the naked eye under illumination of ultraviolet light. The bacteria in the conjugates can be identified by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) combined with principal component analysis (PCA). RESULTS: We demonstrated that pathogenic bacteria including Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, pandrug-resistant Acinetobacter baumannii, Staphylococcus aureus, Enterococcus faecalis, and vancomycin-resistant Enterococcus faecalis (VRE) can be readily concentrated by the lysozyme-AuNCs and distinguished by the results combining MALDI-MS and PCA. Additionally, the possibility of using the current approach to differentiate E. faecalis from VRE was also demonstrated. The lowest detection concentration for E. coli using the current approach is ~10(6) cells/mL. CONCLUSIONS: The results indicated that the lysozyme-AuNCs are effective affinity probes for Gram-positive and Gram-negative bacteria. By combining the results from MALDI-MS and PCA, different bacteria can be easily distinguished. The current approach can be potentially used to assist the identification of bacteria from biological fluids.

Human serum albumin stabilized gold nanoclusters as selective luminescent probes for Staphylococcus aureus and methicillin-resistant Staphylococcus aureus.

In this work, human serum albumin (HSA) stabilized gold nanoclusters (HSA-AuNCs) with reddish photoluminescence were used as sensing probes for pathogenic bacteria including Enterobacter cloacae, Escherichia coli J96, Pseudomonas aeruginosa, pandrug-resistant Acinetobacter baumannii (PDRAB), Staphylococcus aureus, methicillin-resistant S. aureus (MRSA), Streptococcus pyogenes, and vancomycin-resistant Enterococcus faecalis (VRE). We discovered that HSA-AuNCs have unique affinity with S. aureus and MRSA. In addition to demonstrating the selective sensing ability of HSA-AuNCs toward S. aureus and MRSA, the binding peptide motifs identified from HSA-AuNCs were characterized by mass spectrometry. The identified binding peptides were further used as the reducing and stabilizing agents for generation of peptide-bound AuNCs (Pep-AuNCs). The generated Pep-AuNCs were demonstrated to have the binding affinities with S. aureus and MRSA.

Using gold nanoclusters as selective luminescent probes for phosphate-containing metabolites.

Glutathione-bound gold nanoclusters (AuNCs@GSH) can emit reddish photoluminescence under illumination of ultraviolet light. The luminescence of the AuNCs@GSH is quenched when chelating with iron ions (AuNCs@GSH-Fe(3+)), presumably resulting from the effective electron transfer between the nanoclusters and iron ions. Nevertheless, we found that the luminescence of the gold nanoclusters can be restored in the presence of phosphate-containing molecules, which suggested the possibility of using AuNCs@GSH-Fe(3+) complexes as the selective luminescent switches for phosphate-containing metabolites. Phosphate-containing metabolites such as adenosine-5'-triphosphate (ATP) and pyrophosphate play an important role in biological systems. In this study, we demonstrated that the luminescence of the AuNCs@GSH-Fe(3+) is switched-on when mixing with ATP and pyrophosphate, which can readily be observed by the naked eye. It results from the high formation constants between phosphates and iron ions. When employing fluorescence spectroscopy as the detection tool, quantitative analysis for phosphate-containing metabolites such as ATP and pyrophosphate can be conducted. The linear range for ATP and pyrophosphate is 50 µM to sub-millimolar, while the limit of detection for ATP and pyrophosphate are ∼43 and ∼28 µM, respectively. Additionally, we demonstrated that the luminescence of the AuNCs@GSH-Fe(3+) can also be turned on in the presence of phosphate-containing metabolites from cell lysates and blood plasma.

Optimization of the separation and on-line sample concentration of phenethylamine designer drugs with capillary electrophoresis-fluorescence detection.

Five 2C-series of phenethylamine designer drugs, including 2,5-dimethoxy-4-ethylthio-phenethylamine (2C-T-2), 2,5-dimethoxy-4-(n)-propylthiophenethylamine (2C-T-7), 4-chloro-2,5-dimethoxyphenethylamine (2C-C), 4-bromo-2,5-dimethoxy-phenethylamine (2C-B), 2,5-dimethoxy-4-iodo-phenethylamine (2C-I), were synthesized and standard GC/MS and fluorescence spectra are reported for them. A mixture of the five drugs was separated and detected by means of capillary electrophoresis (CE) with native fluorescence and light emitting diode (LED)-induced fluorescence (LIF) detection, respectively, for comparison. In the former case, exciting at a wavelength of 300 nm from a Xe lamp was used. The detection limits were found to be only in the range of approximately 10(-4) M by the micellar electrokinetic chromatography (MEKC) mode but were improved to approximately 10(-7) M when the sweeping-MEKC mode was used. For a highly sensitive analysis, LED-induced fluorescence detection was examined by derivatizing the compounds with a fluorescent dye, fluorescein isothiocyanate isomer I (FITC). A blue-LED (approximately 2 mW) was used as the fluorescence excitation source. The detection limits were improved to approximately 10(-7) and approximately 10(-8) M, respectively, when the MEKC and stacking-MEKC modes were applied. A mimic urine sample was obtained by spiking urine from a volunteer with the five standards, and after liquid-liquid extraction, the sample was examined by means of the MEKC-LIF mode. The extraction procedures used for the urine sample and the CE conditions for the separation were optimized.

Synthesis of oligomeric mannosides and their structure-binding relationship with concanavalin†A.

Small glycodendrimers with α-mannosyl ligands were synthesized by using copper-catalyzed azide-alkyne coupling chemistry and some of these molecules were used as multivalent ligands to study the induction of concanavalin A (Con A) precipitation. The results showed that the monovalent mannose ligand could induce the precipitation of Con A. This unexpected finding initiated a series of studies to characterize the molecular basis of the ligand-lectin interaction. The atypical precipitation is found to be specific to the mannose, fluorescein moiety (FITC), and Con A. Apparently the mannose ligand binds to Con A through hydrogen-bonding interactions, whereas the binding of FITC is mediated by hydrophobic forces.

Photoluminescent gold nanoclusters as sensing probes for uropathogenic Escherichia coli.

Glycan-bound nanoprobes have been demonstrated as suitable sensing probes for bacteria containing glycan binding sites. In this study, we demonstrated a facile approach for generating glycan-bound gold nanoclusters (AuNCs). The generated AuNCs were used as sensing probes for corresponding target bacteria. Mannose-capped AuNCs (AuNCs@Mann) were generated and used as the model sensors for target bacteria. A one-step synthesis approach was employed to generate AuNCs@Mann. In this approach, an aqueous solution of tetrachloroauric acid and mannoside that functionized with a thiol group (Mann-SH) was stirred at room temperature for 48 h. The mannoside functions as reducing and capping agent. The size of the generated AuNCs@Mann is 1.95±0.27 nm, whereas the AuNCs with red photoluminescence have a maximum emission wavelength of ~630 nm (λexcitation = 375 nm). The synthesis of the AuNCs@Mann was accelerated by microwave heating, which enabled the synthesis of the AuNCs@Mann to complete within 1 h. The generated AuNCs@Mann are capable of selectively binding to the urinary tract infection isolate Escherichia coli J96 containing the mannose binding protein FimH expressed on the type 1 pili. On the basis of the naked eye observation, the limit of detection of the sensing approach is as low as ~2×10(6) cells/mL.

A new approach for the detection of a nonfluorescent compound by CE-resonance Raman spectroscopy based on the sweeping-MEKC mode.

A CE-resonance Raman spectroscopy (CE-RRS) method based on MEKC and sweeping-MEKC modes is described. A nonfluorescent compound, malachite green (MG), and a doubled Nd:YAG laser (532 nm, 300 mW) were selected as model compound and light source, respectively. In order to carry out a quantitative analysis of MG, a monochromator (effective bandwidth, 0.4 nm) was used to collect the specific Raman line at 1616 cm(-1) (N-phi and C-C stretch, corresponding to 582 nm when the wavelength of the exciting source was 532 nm). As a result, the LOD for MG was 10 ppm, based on the MEKC/RRS mode. This could be improved to 5 ppb when the sweeping-MEKC/RRS mode was applied. Furthermore, with the addition of nano-size silver colloids to the CE buffer the detection limits can be further improved, but the data obtained with surface-enhanced resonance Raman spectroscopy (SERRS) are less useful for quantitative purposes.