Detection and Quantification of Tightly Bound Zn2+ in Blood Serum Using a Photocaged Chelator and a DNAzyme Fluorescent Sensor.

DNAzymes have emerged as a powerful class of sensors for metal ions due to their high selectivity over a wide range of metal ions, allowing for on-site and real-time detection. Despite much progress made in this area, detecting and quantifying tightly bound metal ions, such as those in the blood serum, remain a challenge because the DNAzyme sensors reported so far can detect only mobile metal ions that are accessible to bind the DNAzymes. To overcome this major limitation, we report the use of a photocaged chelator, XDPAdeCage to extract the Zn2+ from the blood serum and then release the chelated Zn2+ into a buffer using 365 nm light for quantification by an 8-17 DNAzyme sensor. Protocols to chelate, uncage, extract, and detect metal ions in the serum have been developed and optimized. Because DNAzyme sensors for other metal ions have already been reported and more DNAzyme sensors can be obtained using in vitro selection, the method reported in this work will significantly expand the applications of the DNAzyme sensors from sensing metal ions that are not only free but also bound to other biomolecules in biological and environmental samples.

Blended gold/MnO2@BSA nanoparticles for fluorometric and magnetic resonance determination of ascorbic acid.

A fluorometric and magnetic resonance (MR) dual-modal detection scheme is presented for determination of ascorbic acid (AA). It is based on the use of a blended Au/MnO2@BSA mixture that was prepared via a biomimetic strategy, using bovine serum albumin (BSA) as the template at physiological temperature. The MnO2@BSA fraction (one part of the composite) is not susceptible to MR but can be degraded to MR-active compounds upon a redox reaction with even ultralow concentrations of AA. In parallel, the blended Au/MnO2@BSA recovers its fluorescence because MnO2@BSA acts as a quencher of the fluorescence of circumjacent Au@BSA (the other part of the composite). Fluorescence typically is measured at excitation/emission wavelengths of 470/625 nm. Leveraging on this redox reaction between MnO2 and AA, a dual-mode detection scheme for AA was developed. Both the fluorescence and the MR signal increase with the concentration of AA. The lowest limit for the detection of AA is 0.6 µM in the fluorometric mode and 0.4 µM in the MR mode. Analysis of AA-spiked serum samples showed that the recoveries obtained by either the fluorometric and MR mode can reach 94%. This is the first report of the use of blended nanoparticles with their inherent cross-validation regularity. Graphical abstract Schematic presentation of the biomimetic synthesis of blended Au/MnO2@BSA nanoprobes and fluorometric/MR cross-validation dual-modal detection of ascorbic acid.

[Determination of furosine in liquid milk by high performance liquid chromatography-quadrupole time-of-flight mass spectrometry].

Furosine is often used both domestically and internationally as an indicator of the degree of heating to evaluate milk quality. However, in actual detection, the complexity of the milk matrix may lead to the inaccurate quantification of furosine in liquid milk. Therefore, in this study, an efficient and accurate method based on high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (HPLC-Q-TOF/MS) was established to determine furosine in liquid milk. A 2.00 mL milk sample was hydrolyzed with 5 mL 12.00 mol/L hydrochloric acid solution and 1 mL water at 110 ℃ for 12 h. After hydrolysis, vortex-mixing and filtration were performed. The filtrate was diluted six times with 6.00 g/L ammonium acetate solution and then analyzed. Gradient elution was performed with 0.20% formic acid aqueous solution and acetonitrile solution as mobile phases, followed by chromatographic separation on an AQ-C18 column (150 mm×3.5 mm, 5 µm). The data were collected by Q-TOF/MS with an electrospray ionization source operated in positive-ion mode. The accuracy of the quantification of furosine in milk was assessed by investigating the effects of the hydrochloric acid concentration (0.30, 1.25, and 3.00 mol/L) in the furosine solution on the MS response. The results showed that high hydrochloric acid concentrations inhibited the response signals. A good linear relationship was obtained in the mass concentration range of 0.05-2.00 mg/L, with a correlation coefficient (r) of 0.994. The limit of detection of the method was 0.50 mg/100 g, which meets the requirements of actual sample detection. The average recoveries of furosine ranged from 79.9% to 119.7% at three spiked levels of 1.52, 3.03, and 15.17 mg/100 g, with relative standard deviations of 1.4%-2.6%. The method was applied to detect 303 samples from 101 batches of pasteurized milk sold in the market, and the contents of furosine in these samples ranged from 5.1 to 11.9 mg/100 g. The proposed method is characterized with high efficiency, recovery, sensitivity, and accuracy. Thus, it can be used for the determination of large quantities of samples and provides technical support for the continuous promotion of the high-quality development of the whole dairy industry chain.

Tissue distribution study of perfluorooctanoic acid in exposed zebrafish using MALDI mass spectrometry imaging.

Perfluorooctanoic acid (PFOA) as an emerging environmental contaminant, has become ubiquitous in the environment. It is of significance to study bioconcentration and tissue distribution of aquatic organisms for predicting the persistence of PFOA and its adverse effects on the environment and human body. However, the distribution of PFOA in different tissues is a complex physiological process affected by many factors. It is difficult to be accurately described by a simple kinetic model. In present study, a new strategy was introduced to research the PFOA distribution in tissues and estimate the exposure stages. Zebrafish were continuously exposed to 25 mg/L PFOA for 30 days to simulate environmental process. Matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) method was used to monitor the spatio-temporal distribution of PFOA in zebrafish tissues. By analyzing the law of change obtained from the high spatial resolution MSI data, two different enrichment trends in ten tissues were summarized by performing curve fitting. Analyzing the ratio of two types of curves, a new "exposure curve" was defined to evaluate the exposure stages. With this model, three levels (mild, moderate, and deep pollution stage) of PFOA pollution in zebrafish can be simply evaluated.

[Research progress of solid phase extraction materials in the application of metal ion pretreatment].

Monitoring of trace heavy metal pollutants released during industrial and agricultural processes is essential because of their widespread distribution in the environment and health hazards. Several techniques, including inductively coupled plasma-mass spectrometry (ICP-MS), inductively coupled plasma-optical emission spectrometry (ICP-OES), electrothermal atomic absorption (ETAAS), and flame atomic absorption spectrometry (FAAS), have been proposed for the determination of heavy metals in serum, plasma, whole blood, and food. All these techniques have earned robust recognition in the field of trace heavy metals and have many advantages such as multi-elemental analysis capability, large dynamic linear range, low detection limits, and high productivity. Nevertheless, most of the recommended techniques require digestion of the sample and extraction with an organic solvent for isolation of the metal ion from the sample solution prior to analysis. Despite improvements in the performance of modern analytical instruments, the direct determination of heavy metal ions in real samples is difficult because of their low concentration levels and matrix interference. Thus, extraction and clean-up steps are required for pre-concentration of the analyte, so that detection and elimination of the interfering matrix component are possible. Solid-phase extraction (SPE) is one of the popular metal ion pretreatment methods. The advantages of SPE include easy cartridge/column regeneration, high analytical frequency, and high preconcentration factors for sorbents with high adsorption capacities. On the other hand, when the analytes are extracted from a complex matrix such as serum and meat samples, large amounts of proteins from the samples can be retained on the sorbent surface, obstructing the binding sites on the sorbent and leading to poor precision and accuracy. The key to metal ion detection is the development of new SPE materials with high efficiency and enrichment factors as well as an effective pretreatment technology. Nanomaterials such as restricted-access carbon nanotubes, nanoadsorbents, nanoparticle carriers, and magnetic nanoparticles have shown great promise in advancing biomedical and environmental analysis because of the unique properties originating from their ultrafine dimensions. Nanomaterials can provide large specific surface areas and tunable functional groups to facilitate metal ion absorption. They could also possess superior optical properties and allow for high sensitivity in simple fluorescent or colorimetric detection methods. Owing to their excellent mechanical and chemical stability, polymer materials have been of great interest as adsorbents for the SPE of metal ions from solution. Moreover, a designed polymeric material can show triple functionality such as physical adsorption, chelate formation, and ion exchange for the target metal ions. A dual-functional nanomaterial-DNAzyme platform can simultaneously allow for the sensitive detection and effective removal of heavy metal ions in water. Thus, this platform can serve as a simple, cost-effective tool for rapid and accurate metal quantification in the determination of human metal exposure and inspection of environmental contamination. Furthermore, the new photocaged chelator can uncage and release the combined metal ions into an aqueous solution that is free of the other components of the matrix. In this manner, we can develop diagnostic tests for metal ions that are often difficult to detect using other methods. In this paper, the characteristics of new SPE materials, including nanomaterials, polymer materials, and functional materials as well as advances in their applications to the preparation of complex samples are summarized, and the direction for future development is proposed.

Development of an amplified luminescent proximity homogeneous assay for the detection of sulfonamides in animal-derived products.

In this study, we carried out an amplified luminescent proximity homogeneous assay (AlphaLISA) to detect sulfonamides (SAs) antibiotic residues in plasma, milk, pork, chicken, and fish. The SAs AlphaLISA method can detect 13 SAs with half-inhibitory concentration (IC50) 2.11-29.77 ng/ml. The detection level of those SAs was 0.3-41.12 ng/ml in matrices, which satisfied the maximum residue limit (MRL) of the European Union, United States, and China. Our recoveries are in the range of 88% to 116.8% with a coefficient of variation less than 9.3% for different spiked food samples. We observed a good correlation between the AlphaLISA and liquid chromatography-tandem mass spectrometry (LC-MS/MS) with blood samples from injected rabbits. The established AlphaLISA method provided a no-washing, rapid, high-throughput screening tool for SAs in food quality control, which is suitable for small-volume samples.

Development of neurons on micropatterns reveals that growth cone responds to a sharp change of concentration of laminin.

In this report we fabricated laminin (LN) stripes on a background of poly-L-lysine as substrates for the growth of rat hippocampal neurons, and found that a sharp change of the concentration of LN guides the growth of neurites by leading the growth cones in a time- and space-dependent manner. The percentage of neurites that grow along the edge of LN stripes (where there is a sharp change of concentration) decreases as a function of the concentration of LN under a threshold value. The actin cytoskeleton plays an important role in the process of growth cone's response to the sharp change of concentration of LN on micropatterns. We believe that the findings here are useful for not only fundamental studies in neuroscience, but also helpful for the design of devices or chips for nervous prosthesis.

Change of laminin density stimulates axon branching via growth cone myosin II-mediated adhesion.

Axon branching enables neurons to contact with multiple targets and respond to their microenvironment. Owing to its importance in neuronal network formation, axon branching has been studied extensively during the past decades. The chemical properties of extracellular matrices have been proposed to regulate axonal development, but the effects of their density changes on axon branching are not well understood. Here, we demonstrate that both the sharp broadening of substrate geometry and the sharp change of laminin density stimulate axon branching by using microcontact printing (µCP) and microfluidic printing (µFP) techniques. We also found that the change of axon branching stimulated by laminin density depends on myosin II activity. The change of laminin density induces asymmetric extensions of filopodia on the growth cone, which is the precondition for axon branching. These previously unknown mechanisms of change of laminin density-stimulated axon branching may explain how the extracellular matrices regulate axon branching in vivo and facilitate the establishment of neuronal networks in vitro.

Laminin/ß1 integrin signal triggers axon formation by promoting microtubule assembly and stabilization.

Axon specification during neuronal polarization is closely associated with increased microtubule stabilization in one of the neurites of unpolarized neuron, but how this increased microtubule stability is achieved is unclear. Here, we show that extracellular matrix (ECM) component laminin promotes neuronal polarization via regulating directional microtubule assembly through ß1 integrin (Itgb1). Contact with laminin coated on culture substrate or polystyrene beads was sufficient for axon specification of undifferentiated neurites in cultured hippocampal neurons and cortical slices. Active Itgb1 was found to be concentrated in laminin-contacting neurites. Axon formation was promoted and abolished by enhancing and attenuating Itgb1 signaling, respectively. Interestingly, laminin contact promoted plus-end microtubule assembly in a manner that required Itgb1. Moreover, stabilizing microtubules partially prevented polarization defects caused by Itgb1 downregulation. Finally, genetic ablation of Itgb1 in dorsal telencephalic progenitors caused deficits in axon development of cortical pyramidal neurons. Thus, laminin/Itgb1 signaling plays an instructive role in axon initiation and growth, both in vitro and in vivo, through the regulation of microtubule assembly. This study has established a linkage between an extrinsic factor and intrinsic cytoskeletal dynamics during neuronal polarization.

Surface coating as a key parameter in engineering neuronal network structures in vitro.

By quantitatively comparing a variety of macromolecular surface coating agents, we discovered that surface coating strongly modulates the adhesion and morphogenesis of primary hippocampal neurons and serves as a switch of somata clustering and neurite fasciculation in vitro. The kinetics of neuronal adhesion on poly-lysine-coated surfaces is much faster than that on laminin and Matrigel-coated surfaces, and the distribution of adhesion is more homogenous on poly-lysine. Matrigel and laminin, on the other hand, facilitate neuritogenesis more than poly-lysine does. Eventually, on Matrigel-coated surfaces of self-assembled monolayers, neurons tend to undergo somata clustering and neurite fasciculation. By replacing coating proteins with cerebral astrocytes, and patterning neurons on astrocytes through self-assembled monolayers, microfluidics and micro-contact printing, we found that astrocyte promotes soma adhesion and astrocyte processes guide neurites. There, astrocytes could be a versatile substrate in engineering neuronal networks in vitro. Besides, quantitative measurements of cellular responses on various coatings would be valuable information for the neurobiology community in the choice of the most appropriate coating strategy.