Pear metal transport protein PbMTP8.1 confers manganese tolerance when expressed in yeast and Arabidopsis thaliana.

Manganese (Mn) is demonstrated to be essential for plants. Ion homeostasis is maintained in plant cells by specialized transporters. PbMTP8.1, which encodes a putative Mn-CDF transporter in Pyrus bretschneideri Rehd, was expressed mainly in leaves and complemented the Mn hypersensitivity of the Mn-sensitive yeast mutant △pmr1 in previous research conducted by our laboratory. In the present study, we report that the expression of PbMTP8.1 can enhance Mn tolerance and accumulation in Saccharomyces cerevisiae. Subcellular localization analysis of the PbMTP8.1-GFP fusion protein indicated that PbMTP8.1 was targeted to the pre-vacuolar compartment (PVC). In addition, the overexpression of PbMTP8.1 in Arabidopsis thaliana conferred increased resistance to plants under toxic Mn levels, as indicated by increased fresh and dry weights of shoots and roots. Mn accumulation in vacuoles of PbMTP8.1-overexpressing plants was significantly increased when compared with that in wild-type plants under Mn stress. This suggests that a considerable proportion of Mn enters into the vacuoles through a PbMTP8.1-dependent mechanism. Taken together, these results indicate PbMTP8.1 is a Mn-specific transporter that is localized to the PVC, and confers Mn tolerance by sequestering Mn into the vacuole.

Colocalized Particle Counting Platform for Zeptomole Level Multiplexed Quantification.

For multiplexed detection, it is important yet challenging to simultaneously meet the requirement of sensitivity, throughput, and implementation convenience for practical applications. Using the detection of DNAs and miRNAs for illustration, we present a colocalized particle counting platform that can realize the separation-free multiplexed detection of 6 nucleic acid targets with a zeptomole sensitivity and a dynamic range of up to 5 orders of magnitude. The presence of target induces the formation of a sandwich nanostructure via hybridization; thus, there is an occurrence of colocalization of two microbeads with two different colors. The sequence specific coding is realized by an arbitrary combination of two fluorescence channels with different emitting colors. The platform presents robustness in detecting multiple nucleic acid targets with a minimal cross talk and matrix effect as well as the ability to distinguish the specific miRNA from members of the same family. The results of simultaneous detection of 3 miRNAs in 3 different cell lines present straight consistency with that of the standard qRT-PCR. This platform can be adapted to other multiplexing designs such as the "turn-off" mode, in which the proportion of colocalized microbeads is decreased due to the strand-displacement reaction initiated by the specific target. This separation-free platform offers the possibility to achieve the on-site multiplexed detection with compatibility to different experimental designs and extensibility to other signal sources for enumeration.

Nanomaterial-based multiplex optical sensors.

The drive for a simultaneous analysis of multiple targets with excellent accuracy and efficiency, which is often required in both basic biomedical research and clinical applications, demands the development of multiplexed bioassays with desired throughput. With the development of nanotechnologies, innovative multiplex optical bioassays have been achieved. Nanomaterials exhibit unique physical and chemical properties such as easily tunable size, large surface-to-volume ratio, excellent catalysis and the desired signal transduction mechanism, which makes them excellent candidates for the fabrication of novel optical nanoprobes. This mini review summarizes nanomaterial-based optical multiplex sensors from the last 5 years. Specific optical techniques covered in this review are fluorescence, surface-enhanced Raman scattering (SERS), localized surface plasmon resonance (LSPR), chemiluminescence (CL), and the multimodality with fundamentals and examples.

Competitive aptasensor for the ultrasensitive multiplexed detection of cancer biomarkers by fluorescent nanoparticle counting.

Cancer biomarker quantification in human serum is of great importance for accurate patient diagnosis and informed clinical management. To date, ultrasensitive multiplexed detection of proteins without amplification is still a major challenge. Herein, we proposed a competitive aptasensor strategy for ultrasensitive multiplexed cancer biomarker detection by fluorescent nanoparticle (FNP) counting. The sequences are designed such that the binding abilities of linker DNA (L-DNA) with DNA-functionalized FNPs (DNA-FNPs) and aptamer are comparable. As long as one target binds with one molecule of aptamer, a signalling FNP forms a sandwich-structured nanocomposite, which was subsequently observed and enumerated with a fluorescence microscope. This 1 : 1 target-to-signal FNP production assured an improved sensitivity, benefiting from the reasonably good brightness and photostability of FNPs. For both singleplexed and multiplexed detection, this proposed strategy achieved an approximately 1000-fold improved limit of detection than the conventional method with the detection volume of 3.2 µL. Notably, the results for carcinoembryonic antigen (CEA) detection obtained directly from 9 human serum samples (colorectal/lung/healthy individuals) were consistent with that obtained by ELISA, showing potential application in clinical diagnosis.

Direct Observation of Nanoparticles within Cells at Subcellular Levels by Super-Resolution Fluorescence Imaging.

Direct observation of nanoparticles with high spatial resolution at subcellular levels is of great importance to understand the nanotoxicology and promote the biomedical applications of nanoparticles. Super-resolution fluorescence microscopy can break the diffraction resolution limit to achieve spatial resolution of tens of nanometers, making it ideal for highly accurate observation of nanoparticles in the cellular world. In this study, we introduced the employment of super-resolution fluorescence imaging for monitoring nanoparticles within cells. Carbocyanine dyes Alexa Flour 647 labeled mesoporous silica nanoparticles (designated as MSNs-AF647) were constructed as the super-resolution imaging nanoplatform in this work as proof of concept. The MSNs-AF647 were incubated with Hela cells, and the nanoparticles within cells were further monitored by super-resolution fluorescence microscopy. The fluorescence images of MSNs-AF647 within cells captured with the super-resolution fluorescence microscopy showed a much higher spatial resolution than that obtained using conventional fluorescence microscopy, showing that super-resolution fluorescence images can provide more accurate information to locate the nanoparticles at the subcellular levels. Moreover, other functional molecules can be easily loaded into the MSNs-AF647 super-resolution imaging nanoplatform, which suggested that super-resolution fluorescence imaging can further be applied to various bioimaging-related areas, such as imaging-guided therapy, with the aid of the MSNs-AF647 nanoplatform. This study demonstrates that super-resolution fluorescence microscopy offers a highly accurate method to study nanoparticles in the cellular world. We anticipate this strategy may further be applied to research areas such as studying the nanotoxicology and optimization of nanoparticle-based bioprobes or drugs by designing new nanostructured materials with multifunctional properties based on MSNs-AF647.

An on-site bacterial detection strategy based on broad-spectrum antibacterial ε-polylysine functionalized magnetic nanoparticles combined with a portable fluorometer.

A sensitive on-site bacterial detection strategy is presented that integrates the broad-spectrum capturing feature of ε-polylysine-functionalized magnetic nanoparticles with an in-house built portable fluorometer. Based on the electrostatic interaction, the functionalized magnetic nanoparticles (ε-PL-MNPs) were prepared for Gram-positive and Gram-negative bacterial separation and subsequent viable release. ε-PL-MNPs show a broad reactivity towards bacteria with the high capture efficiency from real-world sample media. They also enable controlled viable bacterial release with pH adjustment. Detection of bacteria is based on a combination of broad-spectrum capture with colorimetric and fluorimetric immunoassays. A portable fluorometer is built to enhance the applicability for sensitive on-site detection. A limit of detection of 98 CFU·mL-1 is achieved that is comparable to that of a known spectrofluorometric method for E. coli DH5α. Graphical abstract Schematic presentation of bacterial capture using cationic polymer functionalized magnetic nanoparticles and general fluorometric immunoassay with portable fluorometer. The limit of detection is 98 CFU·mL-1 for E. coli DH5α.

Expression of a Brassica napus metal transport protein (BnMTP3) in Arabidopsis thaliana confers tolerance to Zn and Mn.

The essential micronutrient elements zinc (Zn) and manganese (Mn) are crucial for plant growth and development. As an important oil crop, the yield and quality of rapeseed are affected by Zn and Mn toxicity. The cation diffusion facilitator (CDF) family of proteins play significant roles in maintaining intracellular ionic homeostasis and tolerance in plants. However, research on CDF proteins in rapeseed is lacking. In this study, the function of a Brassica napus cation diffusion facilitator/ metal tolerance protein (CDF/MTP) was investigated. The protein, abbreviated BnMTP3 is homologous to the Arabidopsis thaliana MTP3 (AtMTP3). Heterologous expression of BnMTP3 in yeast enhanced tolerance and intracellular sequestration of Zn and Mn. Expression of BnMTP3 in A. thaliana increased Zn and Mn tolerance and markedly increased Zn accumulation in roots. Quantitative RT-PCR analysis showed that BnMTP3 is primarily expressed in roots, and subcellular localization suggested that BnMTP3 is localized in the trans-Golgi network (TGN) and the prevacuolar compartment (PVC) in Arabidopsis and rape protoplast. After treatment with Zn and Mn, BnMTP3 was observed on the vacuolar membrane in transgenic Arabidopsis lines. These findings suggest that BnMTP3 confers Zn and Mn tolerance by sequestering Zn and/or Mn into the vacuole.