Direct mass spectrometry analysis of biofluid samples using slug-flow microextraction nano-electrospray ionization.

Direct mass spectrometry (MS) analysis of biofluids with simple procedures represents a key step in the translation of MS techniques to clinical and point-of-care applications. The current study reports the development of a single-step method using slug-flow microextraction and nano-electrospray ionization for MS analysis of organic compounds in blood and urine. High sensitivity and quantitation precision have been achieved in the analysis of therapeutic and illicit drugs in 5 µL samples. Real-time chemical derivatization has been incorporated for analyzing anabolic steroids. The monitoring of enzymatic functions has also been demonstrated with cholinesterase in wet blood. The reported study encourages the future development of disposable cartridges, which function with simple operation to replace the traditional complex laboratory procedures for MS analysis of biological samples.

The construction of a maize-teosinte introgression population and quantitative trait loci analysis of their 21 agronomic traits.

Teosinte is a progenitor species of maize (Zea mays ssp. mays) that retains a significant reservoir of genetic resources unaltered via the domestication process. To harness and explore the genetic reservoirs inherent in teosinte, we used the cultivated publicly inbred line H95 and wild species PI566673 (Zea mays ssp. mexicana) to develop a set of introgression lines (ILs), including 366 BC2F5 lines. Using these lines, 12481 high-quality polymorphic homozygous single nucleotide polymorphisms were converted into 2358 bin markers based on Genotyping by Target Sequencing technology. The homozygous introgression ratio in the ILs was approximately 12.1 % and the heterozygous introgression ratio was approximately 5.7 %. Based on the population phenotypic data across 21 important agronomic traits collected in Sanya and Beijing, 185 and 156 quantitative trait loci (QTLs) were detected in Sanya and Beijing, respectively, with 64 stable QTLs detected in both locations. We detected 12 QTL clusters spanning 10 chromosomes consisting of diverse QTLs related to yield traits such as grain size and weight. In addition, we identified useful materials in the ILs for further gene cloning of related variations. For example, some heterogeneous inbred families with superior genetic purity, shorter target heterozygotes, and some ILs exhibit clear morphological variation associated with plant growth, development, and domestication, manifesting traits such as white stalks, sharp seeds, and cob shattering. In conclusion, our results provide a robust foundation for delving into the genetic reservoirs of teosinte, presenting a wealth of genetic resources and offering insight into the genetic architecture underlying maize agronomic traits.

Assembly of hemoglobin from denatured monomeric subunits: heme ligation effects and off-pathway intermediates studied by electrospray mass spectrometry.

The oxygen binding properties of vertebrate hemoglobins (Hb) have been explored in great detail. In contrast, folding and assembly of these heterotetrameric protein complexes remain poorly understood. Similar to investigations of other multisubunit systems, in vitro Hb refolding experiments are often plagued by aggregation. Here we monitor the refolding of bovine Hb by electrospray mass spectrometry (ESI-MS). This technique allows the observation of coexisting subunit combinations, heme binding states, and protein conformers. Exposure to 40% acetonitrile at pH 10 causes Hb disassembly and extensive subunit unfolding. Hb reassembly is triggered by solvent exchange. Experiments conducted at room temperature provide a low metHb refolding yield. A significantly improved yield is achieved by lowering the temperature to 4 °C and by supplementing the protein solution with KCN prior to denaturation. Comparative studies under "low-yield" and "high-yield" conditions report on the interplay between folding and misfolding. The tendency of ß-globin to undergo aggregation is found to be the key impediment to the formation of native Hb. The α/ß imbalance generated in this way favors the formation of non-native α-globin assemblies. Our data imply that hemin dicyanide formed in the presence of KCN remains weakly bound to denatured ß-globin, thereby counteracting aggregation, such that the refolding yield is enhanced. In the absence of aggregation-related interference, Hb assembly follows a symmetric pathway. Monomeric α- and ß-globin adopt a compact conformation upon heme binding. Heme-bound monomers then form heterodimers, and ultimately heterodimer association results in native Hb. This work highlights the utility of time-resolved ESI-MS investigations for interrogating the kinetic competition between on-pathway events and aberrant side reactions during the self-assembly of biomolecular complexes.

Enabling quantitative analysis in ambient ionization mass spectrometry: internal standard coated capillary samplers.

We describe a sampling method using glass capillaries for quantitative analysis of trace analytes in small volumes of complex mixtures (~1 µL) using ambient ionization mass spectrometry. The internal surface of a sampling glass capillary was coated with internal standard then used to draw liquid sample and so transfer both the analyte and internal standard in a single fixed volume onto a substrate for analysis. The internal standard was automatically mixed into the sample during this process and the volumes of the internal standard solution and sample are both fixed by the capillary volume. Precision in quantitation is insensitive to variations in length of the capillary, making the preparation of the sampling capillary simple and providing a robust sampling protocol. Significant improvements in quantitation accuracy were obtained for analysis of 1 µL samples using various ambient ionization methods.

Nanocluster isotope distributions measured by electrospray time-of-flight mass spectrometry.

Electrospray ionization (ESI) mass spectrometry (MS) is a widely used tool for the characterization of organometallic nanoclusters. By matching experimental mass spectra with calculated isotope distributions it is possible to determine the elemental composition of these analytes. In this work we conduct ESI-MS investigations on M(14)E(13)Cl(2)(tmeda)(6) nanoclusters, where M is a transition metal, E represents a chalcogen, and tmeda is N,N,N',N'-tetramethyl-ethylenediamine. ESI mass spectra of these systems agree poorly with theoretical isotope distributions when data are acquired under standard conditions. This behavior is attributed to dead-time artifacts of the time-of-flight (TOF) analyzer used. It is well-known that excessively high TOF ion count rates lead to dead-time issues. Surprisingly, our data reveal that nanocluster spectra are affected by this problem even at moderate signal intensities that do not cause any problems for other types of analytes. This unexpected vulnerability is attributed to the extremely wide isotope distributions of the nanoclusters studied here. A good match between experimental and calculated nanocluster spectra is obtained only at ion count rates that are more than 1 order of magnitude below commonly used levels. Discrepancies between measured and theoretical isotope distributions have been observed in a number of previous ESI-MS nanocluster investigations. The dead-time issue identified here likely represents a contributing factor to the spectral distortions that were observed in those earlier studies. Using low-intensity ESI-MS conditions we demonstrate the feasibility of analyzing highly heterogeneous nanocluster samples that comprise subpopulations with a wide range of metal compositions.

Quantitative paper spray mass spectrometry analysis of drugs of abuse.

An ambient method for rapid monitoring and quantitation of drugs of abuse in dried blood spots was developed using paper spray tandem mass spectrometry (PS-MS).

Mass spectrometry imaging for biomedical applications.

The development of technologies for mass spectrometry imaging is of substantial research interest. Mass spectrometry is potentially capable of providing highly specific information about the distribution of compounds in tissues, with high sensitivity. The in-situ analysis needed for tissue imaging requires MS to be performed under conditions different from the traditional ones, typically with intensive sample preparation and optimized for pharmaceutical applications. In this paper we critically review the current status of MS imaging with different methods of sample ionization and discuss the 3D and quantitative imaging capabilities which need further development, the importance of the multi-modal imaging, and the balance between the pursuit of high-resolution imaging and the practical application of MS imaging in biomedicine.

Analysis of Biological Samples Using Paper Spray Mass Spectrometry: An Investigation of Impacts by the Substrates, Solvents and Elution Methods.

Paper spray has been developed as a fast sampling ionization method for direct analysis of raw biological and chemical samples using mass spectrometry (MS). Quantitation of therapeutic drugs in blood samples at high accuracy has also been achieved using paper spray MS without traditional sample preparation or chromatographic separation. The paper spray ionization is a process integrated with a fast extraction of the analyte from the raw sample by a solvent, the transport of the extracted analytes on the paper, and a spray ionization at the tip of the paper substrate with a high voltage applied. In this study, the influence on the analytical performance by the solvent-substrate systems and the selection of the elution methods was investigated. The protein hemoglobin could be observed from fresh blood samples on silanized paper or from dried blood spots on silica-coated paper. The on-paper separation of the chemicals during the paper spray was characterized through the analysis of a mixture of the methyl violet 2B and methylene blue. The mode of applying the spray solvent was found to have a significant impact on the separation. The results in this study led to a better understanding of the analyte elution, on-paper separation, as well as the ionization processes of the paper spray. This study also help to establish a guideline for optimizing the analytical performance of paper spray for direct analysis of target analytes using mass spectrometry.

PoolNet+: Exploring the Potential of Pooling for Salient Object Detection.

We explore the potential of pooling techniques on the task of salient object detection by expanding its role in convolutional neural networks. In general, two pooling-based modules are proposed. A global guidance module (GGM) is first built based on the bottom-up pathway of the U-shape architecture, which aims to guide the location information of the potential salient objects into layers at different feature levels. A feature aggregation module (FAM) is further designed to seamlessly fuse the coarse-level semantic information with the fine-level features in the top-down pathway. We can progressively refine the high-level semantic features with these two modules and obtain detail enriched saliency maps. Experimental results show that our proposed approach can locate the salient objects more accurately with sharpened details and substantially improve the performance compared with the existing state-of-the-art methods. Besides, our approach is fast and can run at a speed of 53 FPS when processing a 300 ×400 image. To make our approach better applied to mobile applications, we take MobileNetV2 as our backbone and re-tailor the structure of our pooling-based modules. Our mobile version model achieves a running speed of 66 FPS yet still performs better than most existing state-of-the-art methods. To verify the generalization ability of the proposed method, we apply it to the edge detection, RGB-D salient object detection, and camouflaged object detection tasks, and our method achieves better results than the corresponding state-of-the-art methods of these three tasks. Code can be found at http://mmcheng.net/poolnet/.

Highly sensitive flexible heat flux sensor based on a microhole array for ultralow to high temperatures.

With the growing demand for thermal management of electronic devices, cooling of high-precision instruments, and biological cryopreservation, heat flux measurement of complex surfaces and at ultralow temperatures has become highly imperative. However, current heat flux sensors (HFSs) are commonly used in high-temperature scenarios and have problems when applied in low-temperature conditions, such as low sensitivity and embrittlement. In this study, we developed a flexible and highly sensitive HFS that can operate at ultralow to high temperatures, ranging from -196 °C to 273 °C. The sensitivities of HFSs with thicknesses of 0.2 mm and 0.3 mm, which are efficiently manufactured by the screen-printing method, reach 11.21 µV/(W/m2) and 13.43 µV/(W/m2), respectively. The experimental results show that there is a less than 3% resistance change from bending to stretching. Additionally, the HFS can measure heat flux in both exothermic and absorptive cases and can measure heat flux up to 25 kW/m2. Additionally, we demonstrate the application of the HFS to the measurement of minuscule heat flux, such as heat dissipation of human skin and cold water. This technology is expected to be used in heat flux measurements at ultralow temperatures or on complex surfaces, which has great importance in the superconductor and cryobiology field.

On the formation of highly charged gaseous ions from unfolded proteins by electrospray ionization.

Electrospray ionization (ESI) of native proteins results in a narrow distribution of low protonation states. ESI for these folded species proceeds via the charged residue mechanism. In contrast, ESI of unfolded proteins yields a wide distribution of much higher charge states. The current work develops a model that can account for this effect. Recent molecular dynamics simulations revealed that ESI for unfolded polypeptide chains involves protein ejection from nanodroplets, representing a type of ion evaporation mechanism (IEM). We point out the analogies between this IEM, and the dissociation of gaseous protein complexes after collisional activation. The latter process commences with unraveling of a single subunit, in concert with Coulombically driven proton transfer. The subunit then separates from the residual complex as a highly charged ion. We propose that similar charge equilibration events accompany the IEM of unfolded proteins, thereby causing the formation of high ESI charge states. A bead chain model is used for examining how charge is partitioned as protein and droplet separate. It is shown that protein ejection from differently sized ESI droplets generates a range of protonation states. The predicted behavior agrees well with experimental data.

Influences of RF Magnetron Sputtering Power and Gas Flow Rate on a High Conductivity and Low Drift Rate of Tungsten-Rhenium Thin-Film Thermocouples.

Thin-Film Thermocouples (TFTCs) are characterized by their high spatial resolutions, low cost, high efficiency and low interference on the air flow. However, the thermal stability of TFTCs should be further improved for application since their accuracy is influenced by joule heat and temperature time drift. In this paper, 3D molecular dynamics and finite element analysis are used for structural design. The effects of RF magnetron sputtering power and gas flow rate on conductivity and temperature time drift rate (DT) of high thermal stability tungsten-rhenium (95% W/5% Re vs. 74% W/26% Re) TFTCs were analyzed. According to the experimental results, the average Seebeck coefficient reached 31.1 µV/°C at 900 °C temperature difference (hot junction 1040 °C) with a repeatability error at ±1.37% in 33 h. The conductivity is 17.1 S/m, which is approximately 15.2 times larger than the compared tungsten-rhenium sample we presented, and the DT is 0.92 °C/h (1040 °C for 5 h), which is 9.5% of the old type we presented and 4.5% of compared ITO sample. The lumped capacity method test shows that the response time is 11.5 ms at 300 °C. This indicated an important significance in real-time temperature measurement for narrow spaces, such as the aero-engine combustion chamber.

Biological tissue diagnostics using needle biopsy and spray ionization mass spectrometry.

Needle biopsy is a routine medical procedure for examining tissue or biofluids for the presence of disease using standard methods of pathology. In this work, spray ionization directly from tissue in the biopsy needle is shown to provide highly specific molecular information through mass spectrometry analysis. The data are available within a minute after the tissue biopsy, a time scale that allows immediate medical decisions to be made. This method has been performed for tissues in a variety of organs including brain, liver, kidney, adrenal gland, stomach, and spinal cord. Amino acids, hormones, fatty acids, anesthetics, and phospholipids are detected from the tissues and identified using exact mass measurement and tandem mass spectrometry. Lipid profiles are rich in information and, as in imaging MS methods, they have the potential to serve to distinguish diseased from healthy tissue. Needle biopsies allow a crude form of depth profiling that is demonstrated with the analysis of tissue samples taken by a needle inserted into a porcine kidney at various depths.

Leaf spray: direct chemical analysis of plant material and living plants by mass spectrometry.

The chemical constituents of intact plant material, including living plants, are examined by a simple spray method that provides real-time information on sugars, amino acids, fatty acids, lipids, and alkaloids. The experiment is applicable to various plant parts and is demonstrated for a wide variety of species. An electrical potential is applied to the plant and its natural sap, or an applied solvent generates an electrospray that carries endogenous chemicals into an adjacent benchtop or miniature mass spectrometer. The sharp tip needed to create a high electric field can be either natural (e.g., bean sprout) or a small nick can be cut in a leaf, fruit, bark, etc. Stress-induced changes in glucosinolates can be followed on the minute time scale in several plants, including potted vegetables. Differences in spatial distributions and the possibility of studying plant metabolism are demonstrated.

Rethinking the U-Shape Structure for Salient Object Detection.

The U-shape structure has shown its advantage in salient object detection for efficiently combining multi-scale features. However, most existing U-shape-based methods focused on improving the bottom-up and top-down pathways while ignoring the connections between them. This paper shows that we can achieve the cross-scale information interaction by centralizing these connections, hence obtaining semantically stronger and positionally more precise features. To inspire the newly proposed strategy's potential, we further design a relative global calibration module that can simultaneously process multi-scale inputs without spatial interpolation. Our approach can aggregate features more effectively while introducing only a few additional parameters. Our approach can cooperate with various existing U-shape-based salient object detection methods by substituting the connections between the bottom-up and top-down pathways. Experimental results demonstrate that our proposed approach performs favorably against the previous state-of-the-arts on five widely used benchmarks with less computational complexity. The source code will be publicly available.

High-Performance Temperature Sensor by Employing Screen Printing Technology.

In the present study, a high-performance n-type temperature sensor was developed by a new and facile synthesis approach, which could apply to ambient temperature applications. As impacted by the low sintering temperature of flexible polyimide substrates, a screen printing technology-based method to prepare thermoelectric materials and a low-temperature heat treatment process applying to polymer substrates were proposed and achieved. By regulating the preparation parameters of the high-performance n-type indium oxide material, the optimal proportioning method and the post-treatment process method were developed. The sensors based on thermoelectric effects exhibited a sensitivity of 162.5 µV/°C, as well as a wide range of temperature measurement from ambient temperature to 223.6 °C. Furthermore, it is expected to conduct temperature monitoring in different scenarios through a sensor prepared in masks and mechanical hands, laying a foundation for the large-scale manufacturing and widespread application of flexible electronic skin and devices.

A thin-film temperature sensor based on a flexible electrode and substrate.

Accurate temperature measurements can efficiently solve numerous critical problems and provide key information. Herein, a flexible micro-three-dimensional sensor, with a combination of platinum and indium oxide to form thermocouples, is designed and fabricated by a microfabrication process to achieve in situ real-time temperature measurements. The stability and reliability of the sensor are greatly improved by optimizing the process parameters, structural design, and preparation methods. A novel micro-three-dimensional structure with better malleability is designed, which also takes advantage of the fast response of a two-dimensional thin film. The as-obtained flexible temperature sensor with excellent stability and reliability is expected to greatly contribute to the development of essential components in various emerging research fields, including bio-robot and healthcare systems. The model of the application sensor in a mask is further proposed and designed to realize the collection of health information, reducing the number of deaths caused by the lack of timely detection and treatment of patients.

Optimization on thermoelectric characteristics of indium tin oxide/indium oxide thin film thermocouples based on screen printing technology.

In this work, indium tin oxide (ITO)/indium oxide (In2O3) thin film thermocouples (TFTCs) were prepared based on screen printing technology for high temperature measurement. With terpilenol as solvent, epoxy resin and polyether amine as binders and glass powders as additives, the ITO and In2O3 slurries were printed onto the Al2O3 substrate to form thermocouples. The effect on thermoelectric properties of the TFTCs with heat treatment and different contents of additives was investigated through microstructure observation and thermal cycle test. The static calibration experiment shows that the annealed TFTCs with 7.5 wt. % glass powders additives have the maximum Seebeck coefficient. The thermoelectric voltage output of the TFTCs can reach 126.5 mV at 1275 °C while the temperature difference is 1160 °C and the sensitivity of the TFTCs was 109.1 µV/°C. The drift rate of the TFTCs was 8.34 °C/h at a measuring time of 20 min at 1275 °C. The TFTCs prepared via screen printing technology with excellent thermoelectric properties and thermal stability are aimed to be a viable replacement for practical applications.

Fabrication of microwell arrays based on two-dimensional ordered polystyrene microspheres for high-throughput single-cell analysis.

This paper describes a method of fabricating rounded bottom microwell arrays (MA) in poly(dimethylsiloxane) (PDMS) by molding a monolayer of ordered polystyrene (PS) microspheres. PS microspheres were self-assembled on a glass slide and partially melted mainly from the bottom at 240 °C to increase adhesive force with the substrate. The partially melted PS arrays were used as master to generate MA. Microwell sizes are tunable in the 10-20 µm range with rounded bottoms; such a 3D structure is not readily obtainable through conventional soft lithography. Both adherent and nonadherent cell types can be retained in the microwells with high efficiency. As a demonstration of the advantage of real-time cell screening with this MA, single cell enzyme kinetic analysis was also carried out on trapped single cells. The PDMS MA may find applications in high-throughput drug screening, guided formation of cell clusters, and multicellular communication.

Development, characterization, and application of paper spray ionization.

Paper spray is developed as a direct sampling ionization method for mass spectrometric analysis of complex mixtures. Ions of analyte are generated by applying a high voltage to a paper triangle wetted with a small volume (<10 microL) of solution. Samples can be preloaded onto the paper, added with the wetting solution, or transferred from surfaces using the paper as a wipe. It is demonstrated that paper spray is applicable to the analysis of a wide variety of compounds, including small organic compounds, peptides, and proteins. Procedures are developed for analysis of dried biofluid spots and applied to therapeutic drug monitoring with whole blood samples and to illicit drug detection in raw urine samples. Limits of detection of 50 ng/mL (or 20 pg absolute) are achieved for atenolol in bovine blood. The combination of sample collection from surfaces and paper spray ionization also enables fast chemical screening at high sensitivity, for example 100 pg of heroin distributed on a surface and agrochemicals on fruit peels are detectable. Online derivatization with a preloaded reagent is demonstrated for analysis of cholesterol in human serum. The combination of paper spray with miniature mass spectrometers offers a powerful impetus to wide application of mass spectrometry in nonlaboratory environments.