Effect of dietary supplementation of Bacillus subtilis TLRI 211-1 on laying performance, egg quality and blood characteristics of Leghorn layers.

OBJECTIVE: TLRI 211-1 is a novel Bacillus subtilis strain. This experiment was to investigate dietary supplementation of TLRI 211-1 on laying performance, egg quality and blood characteristics of layers. METHODS: One hundred and twenty 65-wk-old Leghorn layers were divided into four treatment groups for 8 weeks experiment. Each treatment had three replicates. The basal diet was formulated as control group with crude protein 17% and metabolizable energy 2,850 kcal/kg and supplemented with TLRI 211-1 0.1%, 0.3%, and commercial Bacillus amyloliquefaciens 0.1% as treatment 2, 3 and 4 groups, respectively. Both TLRI 211-1 and commercial Bacillus amyloliquefaciens were adjusted to contain 1×109 colony-forming unit (CFU)/mL (g), hence the 0.1% supplemental level was 1×109 CFU/kg. RESULTS: The results showed that TLRI 211-1 0.3% and commercial B. amyloliquefaciens groups had higher weight gain than the other groups; TLRI 211-1 0.1% group had better feed to eggs conversion ratio than the control and commercial B. amyloliquefaciens groups (p<0.05). Bacillus subtilis supplementation increased yolk weight (p<0.05). In egg quality during storage, TLRI 211-1 0.1% had higher breaking strength than the control group at the second week of storage (p<0.05). At the third week of storage, TLRI 211-1 0.3% had higher Haugh unit (p<0.05). Hens fed diets supplemented with TLRI 211-1 0.3% significantly decreased blood triglyceride levels and increased blood calcium levels (p< 0.05). TLRI 211-1 0.3% group had lower H2S (p<0.05) and hence had less unpleasant odor in excreta of hens. CONCLUSION: In conclusion, supplementation with 0.1% TLRI 211-1 can significantly improve feed to eggs conversion ratio. TLRI 211-1 supplementation also can maintain eggs at their optimum quality level during storage. The study showed that B. subtilis TLRI 211-1 can be used as feed additives for improving egg production performance and egg quality.

Quantification of saccharides in honey samples through surface-assisted laser desorption/ionization mass spectrometry using HgTe nanostructures.

Quantification of monosaccharides and disaccharides in five honey samples through surface-assisted laser desorption/ionization mass spectrometry (SALDI-MS) using HgTe nanostructures as the matrix and sucralose as an internal standard has been demonstrated. Under optimal conditions (1× HgTe nanostructure, 0.2 mM ammonium citrate at pH 9.0), the SALDI-MS approach allows detection of fructose and maltose at the concentrations down to 15 and 10 µM, respectively. Without conducting tedious sample pretreatment and separation, the SALDI-MS approach allows determination of the contents of monosaccharides and disaccharides in honey samples within 30 min, with reproducibility (relative standard deviation <15%). Unlike only sodium adducts of standard saccharides detected, sodium adducts and potassium adducts with differential amounts have been found among various samples, showing different amounts of sodium and potassium ions in the honey samples. The SALDI-MS data reveal that the contents of monosaccharides and disaccharides in various honey samples are dependent on their nectar sources. In addition to the abundant amounts of monosaccharides and disaccharides, oligosaccharides in m/z range of 650 - 2700 are only detected in pomelo honey. Having advantages of simplicity, rapidity, and reproducibility, this SALDI-MS holds great potential for the analysis of honey samples.

Gold nanosponges: green synthesis, characterization, and cytotoxicity.

A simply approach for the synthesis of Au nanostructures in tea infusions at room temperature is developed. By controlling the concentrations of tea infusions, Au nanostructures in various shapes and sizes have been prepared. From 1 x (original concentration) and 0.01 x (100 times diluted) tea infusions, 52.2 +/- 8.1 nm Au nanosponges (T-Au NSs) and 23 +/- 2 nm spherical Au nanoparticles (T-Au NPs) were prepared. The phytochemicals present on the surface of T-Au NSs were proved by surface-assisted laser desorption/ionization mass spectrometry, Fourier transform infrared spectrometry and capillary electrophoresis coupled with UV detection. The energy-dispersive X-ray spectroscopy and powder X-ray diffraction data reveal pure crystalline structures of the T-Au NSs. The dark field scattering images observe that the T-Au NSs have significant affinity toward HeLa cells. The cytotoxicity of T-Au NSs on HeLa cells is through caspase-3 activation.

Using surface-assisted laser desorption/ionization mass spectrometry to detect ss- and ds-oligodeoxynucleotides.

We applied surface-assisted laser desorption/ionization mass spectrometry (SALDI-MS) with HgTe nanostructures as the matrix for the detection of single- and double-stranded oligodeoxynucleotides (ss-ODNs and ds-ODNs). The concentrations of surfactant and additives (metal ions, an amine) and the pH and ionic strength of the sample matrix played significantly different roles in the detection of ss- and ds-ODNs with various sequences. In the presence of Brij 76 (1.5 %), Hg(2+) (7.5 µM), and cadaverine (10 µM) at pH 5.0, this SALDI-MS approach allowed the simultaneous detection of T15, T20, T33, and T40, with limits of detection at the femtomole-to-picomole level and sample-to-sample intensity variation <23 %. In the presence of Ag(+) (1 µM) and cadaverine (10 µM) at pH 7.0, this technique allowed the detection of randomly sequenced ss- and ds-ODNs at concentrations down to the femtomole level. To the best of our knowledge, this paper is the first to report the detection of ss-ODNs (up to 50-mer) and ds-ODNs (up to 30 base pairs) through the combination of SALDI-MS with HgTe nanostructures as matrices. We demonstrated the practicality of this approach through analysis of a single nucleotide polymorphism that determines the fate of the valine residue in the ß-globin of sickle cell megaloblasts.

Analysis of the formation process of gold nanoparticles by surface-assisted laser desorption/ionization mass spectrometry.

Chemical reactions of reducing agents in the gold nanoparticle (AuNP) formation process were characterized using surface-assisted laser desorption/ionization mass spectrometry (SALDI-MS). As the reaction of the AuNPs progresses, the produced AuNPs can serve as an efficient SALDI substrate. SALDI-MS revealed that the reducing agents and their oxidation products can be determined in the mass spectra. With respect to the transmission electron microscopic and UV-Vis spectroscopic examination of AuNPs, SALDI-MS results confirm not only the tendency toward AuNPs formation, but also reflect the information of the redox reaction process. Our results provide useful information for developing SALDI-MS methods to explore the chemical information regarding the surface behavior between adsorbates and nanomaterials.

Enzyme mimics of Au/Ag nanoparticles for fluorescent detection of acetylcholine.

We have developed a highly sensitive and selective fluorescent assay for the detection of acetylcholine (ACh) based on enzyme mimics of Au/Ag nanoparticles (NPs). These NPs were prepared via a one-step solution phase reaction between 13 nm Au NPs and Ag(+) ions in the presence of stabilizing agents such as adenosine triphosphate (ATP) and polyethylene glycol (PEG). Our sensing strategy involves reacting ACh with acetylcholinesterase (AChE) to form choline that is in turn oxidized by choline oxidase (ChOx) to produce betaine and H(2)O(2), which reacts with Amplex UltraRed (AUR) in the presence of bimetallic NPs catalyst to form a fluorescent product. The fluorescence intensity (excitation/emission wavelengths of 540/592 nm) is proportional to the concentration of ACh over a range of 1-100 nM (R(2) = 0.998), with a limit of detection of 0.21 nM (signal/noise = 3). When compared with Au NPs and horseradish peroxidase, the Au/Ag NPs provide 150- and 115-fold higher catalytic activity toward the H(2)O(2)-mediated AUR reaction. The practicality of the assay has been validated by determining the concentrations of ACh in plasma and blood samples, with results of 2.69 ± 0.84 nM (n = 5) and 6.75 ± 1.42 nM (n = 5), respectively. Thus, the present assay holds great potential for the analysis of ACh in biological samples.

Catalytic gold nanoparticles for fluorescent detection of mercury(II) and lead(II) ions.

In this study, we developed a fluorescence assay for the highly sensitive and selective detection of Hg(2+) and Pb(2+) ions using a gold nanoparticle (Au NP)-based probe. The Hg-Au and Pb-Au alloys that formed on the Au NP surfaces allowed the Au NPs to exhibit peroxidase-mimicking catalytic activity in the H(2)O(2)-mediated oxidation of Amplex UltraRed (AUR). The fluorescence of the AUR oxidation product increased upon increasing the concentration of either Hg(2+) or Pb(2+) ions. By controlling the pH values of 5mM tris-acetate buffers at 7.0 and 9.0, this H(2)O(2)-AUR-Au NP probe detected Hg(2+) and Pb(2+) ions, respectively, both with limits of detection (signal-to-noise ratio: 3) of 4.0 nM. The fluorescence intensity of the AUR oxidation product was proportional to the concentrations of Hg(2+) and Pb(2+) ions over ranges 0.05-1 µM (R(2)=0.993) and 0.05-5 µM (R(2)=0.996), respectively. The H(2)O(2)-AUR-Au NP probe was highly selective for Hg(2+) (>100-fold) and Pb(2+) (>300-fold) ions in the presence of other tested metal ions. We validated the practicality of this simple, selective, and sensitive H(2)O(2)-AUR-Au NP probe through determination of the concentrations of Hg(2+) and Pb(2+) ions in a lake water sample and of Pb(2+) ions in a blood sample. To the best of our knowledge, this system is the first example of Au NPs being used as enzyme-mimics for the fluorescence detection of Hg(2+) and Pb(2+) ions.

Detection of melamine in infant formula and grain powder by surface-assisted laser desorption/ionization mass spectrometry.

We have developed a method for the determination of melamine (MEL), ammeline (AMN), and ammelide (AMD) by surface-assisted laser desorption/ionization mass spectrometry (SALDI-MS) using gold nanoparticles (Au NPs). The major peaks for MEL, AMN, and AMD at m/z 127.07, 128.05, and 129.04 are assigned to the [MEL + H](+), [AMN + H](+), and [AMD + H](+) ions. Because the three tested compounds adsorb weakly onto the surfaces of the Au NPs through Au-N bonding, they can be easily concentrated from complex samples by applying a simple trapping/centrifugation process. The SALDI-MS method provides limits of detection of 5, 10, and 300 nM for MEL, AMN, and AMD, respectively, at a signal-to-noise ratio of 3. The signal variation for 150-shot average spectra of the three analytes within the same spot was 15%, and the batch-to-batch variation was 20%. We have validated the practicality of this approach by the analysis of these three analytes in infant formula and grain powder. This simple and rapid SALDI-MS approach holds great potential for screening of MEL in foods.

Using surface-assisted laser desorption/ionization mass spectrometry to detect proteins and protein-protein complexes.

In this study, we combined surface-assisted laser desorption/ionization mass spectrometry (SALDI-MS) with HgTe nanostructures as matrix for the detection of several proteins (α1-antitrypsin, trypsin, IgG, protein G) and their complexes. We investigated the effects of several parameters (the concentration and nature of surfactants and metal ions, the pH, and concentration of the analytes in the sample matrixes) on the sensitivity of the detection of these proteins and their complexes. The presence of stabilizing Brij 76 surfactant and Zn(II) ions allowed the detection of weak protein complexes, such as α1-antitrypsin-trypsin and IgG-protein G complexes, at the picomole level. We observed multiply charged states at m/z 72,160 ([α1-antitrypsin + trypsin + H](+)) and 86,585 ([IgG + protein G + 2H](2+)) for the α1-antitrypsin-trypsin and IgG-protein G complexes, respectively. To the best of our knowledge, detection of weak protein complexes and determination of their stoichiometry have not been demonstrated previously when a combination of SALDI-MS and nanostructures were used. This simple and reproducible SALDI-MS approach using HgTe nanostructures holds great potential for the detection of other proteins and their complexes.

Nanoparticle-based mass spectrometry for the analysis of biomolecules.

Nanoparticles (NPs) are useful as matrixes for the analyses of several types of biomolecules (including aminothiols, peptides, and proteins) and for mass spectrometric imaging through surface-assisted laser desorption/ionization mass spectrometry (SALDI-MS), mainly because of their large surface area, strong absorption in the ultraviolet-near-infrared region, and ready functionalization. Metallic NPs, metal oxide NPs, and semiconductor quantum dots, unmodified or functionalized with recognition ligands, have a strong affinity toward analytes; therefore, they allow the enrichment of biomolecules, leading to improved sensitivity with minimal matrix interference in their mass spectra. SALDI-MS using NPs overcomes the two major problems commonly encountered in matrix-assisted laser desorption/ionization mass spectrometry: the presence of "sweet spots" and the high background signals in the low-mass region. In this tutorial review, we discuss the roles played by the nature, size, and concentration of the NPs, the buffer composition, and the laser energy in determining the sensitivity and mass ranges for the analytes. We describe internal standard SALDI-MS methods that allow the concentrations of analytes to be determined with low variation (relative standard deviations: <10%) and we highlight how the simplicity, sensitivity, and reproducibility of SALDI-MS approaches using various NPs allow the analyses of proteins and small analytes and the imaging of cells.

Detection of proteins and protein-ligand complexes using HgTe nanostructure matrixes in surface-assisted laser desorption/ionization mass spectrometry.

We have analyzed peptides, proteins, and protein-drug complexes through surface-assisted laser desorption/ionization mass spectrometry (SALDI-MS) using HgTe nanostructures as matrixes. We investigated the effects of several parameters, including the concentration of the HgTe nanostructures, the pH of the buffer, and the concentration of salt, on the performance of this system. When HgTe nanostructures are used as matrixes, [M + H](+) ions were the dominant signals. Relative to other commonly used nanomaterials, HgTe nanostructures provided lower background signals from metal clusters, fewer fragment ions, less interference from alkali-adducted analyte ions, and a higher mass range (up to 150,000 Da). The present approach provides limits of detection for angiotensin I and bovine serum albumin of 200 pM and 14 nM, respectively, with great reproducibility (RSD: <25%). We validated the applicability of this method through the detections of (i) the recombinant proteins that were transformed in E. coli, (ii) the specific complex between bovine serum albumin and l-tryptophan, and (iii) a carbonic anhydrase-acetazolamide complex. Our results suggest that this novel and simple SALDI-MS approach using HgTe nanostructures as matrixes might open several new ways for proteomics and the analysis of drug-protein complexes.

Exploring the interactions between gold nanoparticles and analytes through surface-assisted laser desorption/ionization mass spectrometry.

Surface-assisted laser desorption/ionization mass spectrometry (SALDI-MS) is applied to provide strong evidence for the chemical reactions of functionalized gold nanoparticles (Au NPs) with analytes--Hg(2+) ions induced MPA-Au NPs aggregation in the presence of 2,6-pyridinedicarboxylic acid (PDCA) and H(2)O(2) induced fluorescence quenching of 11-MUA-Au NDs. PDCA-Hg(2+)-MPA coordination is responsible for Au NPs aggregation, while the formation of 11-MUA disulfide compounds that release into the bulk solution is responsible for H(2)O(2)-induced fluorescence quenching. In addition to providing information about the chemical structures, SALDI-MS is also selective and sensitive for the detection of Hg(2+) ions and H(2)O(2). The limits of detection (LODs) for Hg(2+) ions and H(2)O(2) by SALDI-MS were 300 nM and 250 microM, respectively. The spot-to-spot variations in the two studies were both less than 18% (50 sample spots). Our results reveal that SALDI-MS can be used to study analyte-induced changes in the surface properties of nanoparticles.

Accurate quantitation of glutathione in cell lysates through surface-assisted laser desorption/ionization mass spectrometry using gold nanoparticles.

We developed a method for the determination of three aminothiols--cysteine, glutathione (GSH), and homocysteine--using surface-assisted laser desorption/ionization mass spectrometry (SALDI-MS). The analytes were first captured using the unmodified 14-nm gold nanoparticles; N-2-mercaptopropionylglycine-modified gold nanoparticles serving as internal standard were sequentially added, and then the sample was analyzed using SALDI-MS. This approach provided good quantitative linearity of the three analytes (R(2) = approximately 0.99), with good reproducibility (relative standard deviations: <10%), in the analyses of GSH in the lysates of human red blood cells and MCF-7 cancer breast cells in the presence and absence of the anti-inflammatory drug sulfasalazine. The internal-standard SALDI-MS approach provides simplicity, accuracy, and precision to the determination of GSH in cells under drug invasion, to open an avenue for SALDI-MS to be used for the precise quantitative determination of a variety of analytes. From the clinical editor: This paper reports the development of a surface assisted laser desorption/ionization mass spectrometry method to precisely determine aminothiols-cysteine (Cys), glutathione (GSH), and homocysteine (HCys).

Quantification of captopril in urine through surface-assisted laser desorption/ionization mass spectrometry using 4-mercaptobenzoic acid-capped gold nanoparticles as an internal standard.

We have developed a new internal standard method for the determination of the concentration of captopril (CAP) through surface-assisted laser desorption/ionization mass spectrometry (SALDI-MS) using gold nanoparticles (Au NPs). This approach provided linearity for CAP over the concentration range 2.5-25 microM (R(2) = 0.987), with a limit of detection (signal-to-noise ratio = 3) of 1.0 microM. The spot-to-spot variations in the concentration of CAP through SALDI-MS analyses performed in the absence and presence of the internal standard were 26% and 9%, respectively (15 measurements). This approach provides simplicity, accuracy, precision, and great reproducibility to the determination of the levels of CAP in human urine samples.

Determination of perchlorate in river by ion-pair hollow-fiber liquid-phase microextraction coupled with electrospray ionization tandem mass spectrometry.

This paper describes the analysis of perchlorate (ClO(4)(-)) in surface water samples by a rapid and reliable ion-pair hollow-fiber liquid-phase microextraction (HF-LPME) method coupled with flow-injection electrospray ionization tandem mass spectrometry (ESI-MS-MS) technique. The effects of the type and concentration of ion-pairing reagents, extraction time, temperature and pH value on the quantitative extraction of perchlorate by ion-pair HF-LPME were investigated and optimized. Di-n-hexyl ammonium acetate (DHAA) was employed to form an extractable ion-pair complex with aqueous perchlorate. The characteristic ions [ClO(4)-ClO(4)-DHA](-) at m/z 384.6 and 386.7 were observed in the ESI negative-ionization mode. The predominant product ions [ClO(4)](-) at m/z 99 and 101 were used for quantitation and to maximize the detection selectivity and sensitivity. The limit of detection (LOD) was 0.5 microg/L. The reliability and precision of the standard addition method of ion-pair HF-LPME for the determination of trace levels of perchlorate in surface water were demonstrated.