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A hydrophilic metal-organic network based on Ti4+ and dual natural ligand, tannic acid (TA) and phytic acid (PA), has been developed to enrich phosphopeptides from complex bio-samples prior to liquid chromatography-mass spectrometric analysis. Due to the strong chelation ability of TA and PA, abundant Ti4+ can be immobilized in the material, forming hydrophilic network by one-step coordination-driven self-assembly approach. The sorbent, TA-Ti-PA@Fe3O4, exhibited satisfactory selectivity for the phosphopeptides in the tryptic digest of β-casein, and can eliminate the interference components in 1000-fold excess of bovine serum albumin. The adsorption capacity of the sorbents for phosphopeptides was up to 35.2 mg g−1 and the adsorbing equilibrium can be reached in 5 min. The adsorbing mechanism has been investigated and the results indicated that the Ti4+ in forms of [Ti(f-TA)(H2O)4]2+, [Ti(f-PA)(H2O)4]2+ and Ti(f-PA)2(H2O)2 may play an important role in the adsorption process. The sorbent of the TA-Ti-PA@Fe3O4 has been applied to enrichment of the phosphopeptides in tryptic digest of rat liver lysate, and 3,408 phosphopeptides have been identified, while the numbers of the identified phosphopeptides were 2730 and 1217 when the sample was enriched by the commercial TiO2 and Fe3+-IMAC kit, respectively. This work provides a strategy to enrich phosphopeptides from complex samples and shows great potential application in phosphoproteome research.

s The tyrosine phosphorylation of proteins and peptides plays a vital role in cell signal transduction pathways, and it is very important to assay them for understanding their action mechanism. Due to the low levels of the tyrosine phosphopeptides (pTyr) in cells, it is a challenge to enrich them with traditional sorbents, therefore, development of specific and selective sorbents is urgent and necessary. In this work, the phosphate-imprinted magnetic nanoparticles (PMNPs) to enrich the pTyr with high efficiency and selectivity have been fabricated using the phenylphosphonic acid as a template for the “epitope” of pTyr. The magnetic nanoparticles have been functionalized with TiO2 and then the imprinting silica shells have been coated on the surface of the functional core to obtain the PMNPs sorbents. The PMNPs can obviously shorten the enrichment time and improve the adsorption efficiency for pTyr, and the epitope imprinting films provide an excellent selective recognition ability to target. The recognition capability of PMNPs for pTyr is 90.3 μg/mg and the imprinting factor of the sorbents can reach 24.4. The results indicate that the PMNPs can enrich the pTyr from the tryptic digest of β-casein samples with high specificity, and the spiking recoveries of the pTyr range from 85.1% to 93.8% with the RSD from 0.04 to 3.73. With the high adsorption capacity, rapid separation, excellent specificity and recyclability, the PMNPs sorbents show great potential for analysis of the phosphorylation of peptides in biological and medical fields.

Fluorescent hydroxyapatite nanoparticles (HAP-NPs) were synthesized by introducing trisodium citrate during the HAP-NP synthesis process. The HAP-NPs display strong emission at around 440 nm when excited at 340 nm. In the presence of low levels of biothiols, such as cysteine (Cys) and homocysteine (hCys), the fluorescence intensity of HAP-NPs was found to be enhanced, while for the same concentration of glutathione (GSH), a smaller fluorescence intensity increase was observed. So a selective and sensitive fluorescence turn-on assay for detection of low levels of biothiols is reported. The linear range of the assay for Cys is from 2 to 100 nM, and for hCys is from 2 to 200 nM. The assay was successfully applied to the detection of Cys in human serum samples. The interesting fluorescence properties of HAP-NPs, plus their good biocompatibility, mean that HAP-NPs may find wide application as fluorescence probes in different assays and bio-imaging areas.

The authors describe a carbon dot (CD) based dual-emission ratiometric optical probe for the on-site visual and fluorometric determination of mercury(II) ions. The nanoparticle (NP) probe was obtained by covalently linking the blue emissive carbon dots to the surface of silica nanoparticles containing red-emissive quantum dots (QDs). The red emitting QDs in the silica matrix are inert to Hg(II) and provide a reliable and constant reference signal. They also reduce their toxicity and improve the optical and chemical stabilities, while the blue emission CDs are very sensitive to Hg(II). With increasing concentration of Hg(II), a solution containing the NP probe undergoes a continuous color change from light purple to red. This can be seen with bare eyes or detected instrumentally by measurement of fluorescence intensity under excitation/emission wavelengths of 350/453 and 658 nm. The probe exhibits high sensitivity to Hg(II), with a detection limit of 0.47 nM (at an S/N ratio of 3). This is much lower than the allowable level of mercury (10 nM, ~10 ppb) in drinking water set by the U.S. Environmental Protection Agency. For practical use, the probe was used to quantify Hg(II) in (spiked) tap water where it gave recoveries between 95 and 106% and relative standard deviations between 1.9 and 3.2%. The probe can also be applied in filter paper-based assays, and this paves the way to point-of-care pollution control. This ratiometric probe is nontoxic and easily operated, and therefore shows potential applications for rapid and low-cost visual identification of Hg(II).

The traditional enzyme-linked immunosorbent assay (ELISA) has some disadvantages, such as insufficient sensitivity and low stability of the labeled enzyme, which limit its further applications. In this study, a more stable enzyme, Amp cephalosporinase (AmpC), was selected as the labeled enzyme, and its substrate was designed and synthesized. This substrate contained the cephalosporin ring core as the enzymatic recognition section and the structural motif of the 3-hydroxyflavone (3-HF) as the reporter molecule. AmpC can specifically catalyze the substrate and release 3-HF, which can enter the cavity of β-cyclodextrin (β-CD) on the surface of ZnS quantum dots and form a fluorescence resonance energy transfer (FRET) signal amplification system. An AmpC-catalyzed, FRET-mediated ultrasensitive immunosensor (ACF immunosensor) for procalcitonin (PCT) was developed by combining the signal amplification system of the polystyrene microspheres and effective immune-based magnetic separation. The ACF immunosensor has high sensitivity and specificity for the detection of PCT: its linear range is from 0.1 ng mL

A novel magnetic nanomaterial for use in metal ion based affinity chromatography is described. It is based on the chelation between the phosphate groups of phytic acid (PA) and Ti(IV) ions. Due to the large number (6) of phosphate groups of PA, it has a large capacity for Ti(IV) ions. PA was first immobilized on magnetite nanoparticles (PA-MNPs) and then loaded with Ti(IV) ions to obtain the sorbent (Ti-PA-MNPs). The fraction of Ti(IV) ions on the surface of PA-MNPs that is exposed to the solution binds the phosphate groups of phosphopeptides. The bound phosphopeptides can then be magnetically separated. The method was applied to the enrichment of the phosphopeptides in a β-casein tryptic digest. A tryptic digest of bovine serum albumin (BSA) was added at a molar ratio (β-casein to BSA) of 1:2000 to study selectivity. The phosphopeptides were quantified by mass spectrometry. The limit of detection can be as low as 8 × 10−10 mol L−1. This sorbent has a high absorption capacity (53.5 μg mg-1) and shows good recoveries (90%). As many as 2145 phosphopeptides were isolated from 500 μg tryptic digest of a rat liver lysate after enrichment by Ti-PA-MNPs. This is superior to that (1568 phosphopeptides) of commercial TiO2 kit.

The authors describe a method for highly sensitive and selective determination of the activity of protein kinase (PKA). It is based on the finding that silver nanoclusters (AgNCs) can act as a nucleus to catalyze further deposition of silver nanoparticles. This causes the color of a solution to change from pale yellow to black. In the detection scheme presented here, the substrate peptide is phosphorylated by PKA in the presence of ATP. The resulting phosphopeptides bind to oligonucleotide-stabilized AgNCs in the presence of Zr(IV) ions due to electrostatic interactions between Zr(IV) and the phosphate groups, thereby capping the AgNCs. The silver enhancement process (leading to a color change to black) does not work if the AgNCs are capped. The degree of inhibition is proportional to the activity of the kinase. The color change can be detected visually or photographically in a microplate format by exploiting the changes in the grey values of the digital photos. In addition, the DNA-AgNCs display fluorescence emission at 635 nm when excited at 565 nm. Electrochemical assays were performed (at a working voltage as low as 38 mV vs. Ag/AgCl) by using a glassy carbon electrode modified with a solution containing AgNCs, Zr(IV) ions and the peptide, and immersing it into the silver enhancement solution. The assay is highly sensitive and selective. It was applied to the determination of PKA in lysates of HeLa cells. The detection limits typically are between 32 and 37 U⋅ L-1 based on a signal-to-noise ratio of 3.

The authors describe a fluorometric method for the determination of the activity and inhibition of protein kinase A (PKA). In the presence of ATP, PKA catalyzes the transfer of phosphate groups from ATP to a peptide, and the generated phosphorylated peptide quenches the fluorescence (measured at excitation/emission peaks of 340/440 nm) of the hydroxyapatite nanoparticles (HAP-NPs). A linear logarithmic relationship of PKA concentrations with fluorescence intensity in the range from 1 to 50 U·L−1 was obtained, and the lower limit of detection (LOD) is 0.5 U·L−1. This is much lower than LODs reported in the literature. The PKA inhibitor H-89 was studied, and the inhibition plot has a sigmoidal shape with a half-maximal inhibitory concentration of around 750 nM of H-89. At a 4.5 nM level of H-89, fluorescence of HAP-NPs fell to levels of no PKA controls, demonstrating that the assay is a viable tool to screen for kinase inhibitors. An assay with Hela cell lysates in combination with forskolin (an activator of adenylyl cyclase) and IBMX (a phosphodiesterase inhibitor used to activate the cellular activity of PKA) resulted in decreased fluorescence of HAP-NPs. This suggests that the assay can be applied for testing in vitro cell kinase activity. In our perception, this method will enable high-throughput screening for kinase-related drugs and fluorometric enzymatic detection in various areas.