Sublimation as a method of matrix application for mass spectrometric imaging

Common organic matrix-assisted laser desorption/ionization (MALDI) matrices, 2,5-dihydroxybenzoic acid, 3,5-dimethoxy-4-hydroxycinnamic acid, and alpha-cyano-4-hydroxycinnamic acid, were found to undergo sublimation without decomposition under conditions of reduced pressure and elevated temperature. This solid to vapor-phase transition was exploited to apply MALDI matrix onto tissue samples over a broad surface in a solvent-free application for mass spectrometric imaging. Sublimation of matrix produced an even layer of small crystals across the sample plate. The deposition was readily controlled with time, temperature, and pressure settings and was highly reproducible from one sample to the next. Mass spectrometric images acquired from phospholipid standards robotically spotted onto a MALDI plate yielded a more intense, even signal with fewer sodium adducts when matrix was applied by sublimation relative to samples where matrix was deposited by an electrospray technique. MALDI matrix could be readily applied to tissue sections on glass slides and stainless steel MALDI plate inserts as long as good thermal contact was made with the condenser of the sublimation device. Sections of mouse brain were coated with matrix applied by sublimation and were imaged using a Q-q-TOF mass spectrometer to yield mass spectral images of very high quality. Image quality is likely enhanced by several features of this technique including the microcrystalline morphology of the deposited matrix, increased purity of deposited matrix, and evenness of deposition. This inexpensive method was reproducible and eliminated the potential for spreading of analytes arising from solvent deposition during matrix application.     

Matrix pre‐coated targets for high throughput MALDI imaging of proteins

We have developed matrix pre‐coated targets for imaging proteins in thin tissue sections by matrix‐assisted laser desorption/ionization mass spectrometry. Gold covered microscope slides were coated with sinapinic acid (SA) in batches in advance and were shown to be stable for over 6 months when kept in the dark. The sample preparation protocol using these SA pre‐coated targets involves treatment with diisopropylethylamine (DIEA)‐H₂O vapor, transforming the matrix layer to a viscous ionic liquid. This SA‐DIEA ionic liquid layer extracts proteins and other analytes from tissue sections that are thaw mounted to this target. DIEA is removed by the immersion of the target into diluted acetic acid, allowing SA to co‐crystallize with extracted analytes directly on the target. Ion images (3–70 kDa) of sections of mouse brain and rat kidney at spatial resolution down to 10 µm were obtained. Use of pre‐coated slides greatly reduces sample preparation time for matrix‐assisted laser desorption/ionization imaging while providing high throughput, low cost and high spatial resolution images. Copyright © 2014 John Wiley & Sons, Ltd.     

Automated MALDI Matrix Coating System for Multiple Tissue Samples for Imaging Mass Spectrometry

Uniform matrix deposition on tissue samples for matrix-assisted laser desorption/ionization (MALDI) is key for reproducible analyte ion signals. Current methods often result in nonhomogenous matrix deposition, and take time and effort to produce acceptable ion signals. Here we describe a fully-automated method for matrix deposition using an enclosed spray chamber and spray nozzle for matrix solution delivery. A commercial air-atomizing spray nozzle was modified and combined with solenoid controlled valves and a Programmable Logic Controller (PLC) to control and deliver the matrix solution. A spray chamber was employed to contain the nozzle, sample, and atomized matrix solution stream, and to prevent any interference from outside conditions as well as allow complete control of the sample environment. A gravity cup was filled with MALDI matrix solutions, including DHB in chloroform/methanol (50:50) at concentrations up to 60 mg/mL. Various samples (including rat brain tissue sections) were prepared using two deposition methods (spray chamber, inkjet). A linear ion trap equipped with an intermediate-pressure MALDI source was used for analyses. Optical microscopic examination showed a uniform coating of matrix crystals across the sample. Overall, the mass spectral images gathered from tissues coated using the spray chamber system were of better quality and more reproducible than from tissue specimens prepared by the inkjet deposition method.     

A solid-phase bioreactor with continuous sample deposition for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

We report the development of a solid‐phase proteolytic digestion and continuous deposition microfluidic chip platform for low volume fraction collection and off‐line matrix‐assisted laser desorption/ionization time‐of‐flight (MALDI‐TOF) mass spectrometry. Tryptic peptides were formed in an on‐chip bioreactor and continuously deposited onto a MALDI target plate using a motor‐driven xyz stage. The bioreactor consisted of a 4 cm × 200 µm × 50 µm microfluidic channel with covalently immobilized trypsin on an array of 50 µm diameter micropost structures with a 50 µm edge‐to‐edge inter‐post spacing. A 50 µm i.d. capillary tube was directly attached to the end of the bioreactor for continuous sample deposition. The MALDI target plate was modified by spin‐coating a nitrocellulose solution containing a MALDI matrix on the surface prior to effluent deposition. Protein molecular weight standards were used for evaluating the performance of the digestion and continuous deposition system. Serpentine sample traces 200 µm wide were obtained with a 30 fmol/mm quantity deposition rate and a 3.3 nL/mm volumetric deposition rate. Copyright © 2011 John Wiley & Sons, Ltd.     

Enhancement of protein sensitivity for MALDI imaging mass spectrometry after chemical treatment of tissue sections

MALDI imaging mass spectrometry (IMS) has become a valuable tool for the investigation of the content and distribution of molecular species in tissue specimens. Numerous methodological improvements have been made to optimize tissue section preparation and matrix deposition protocols, as well as MS data acquisition and processing. In particular for proteomic analyses, washing the tissue sections before matrix deposition has proven useful to improve spectral qualities by increasing ion yields and the number of signals observed. We systematically explore here the effects of several solvent combinations for washing tissue sections. To minimize experimental variability, all of the measurements were performed on serial sections cut from a single mouse liver tissue block. Several other key steps of the process such as matrix deposition and MS data acquisition and processing have also been automated or standardized. To assess efficacy, after each washing procedure the total ion current and number of peaks were counted from the resulting protein profiles. These results were correlated to on-tissue measurements obtained for lipids. Using similar approaches, several selected washing procedures were also tested for their ability to extend the lifetime as well as revive previously cut tissue sections. The effects of these washes on automated matrix deposition and crystallization behavior as well as their ability to preserve tissue histology were also studied. Finally, in a full-scale IMS study, these washing procedures were tested on a human renal cell carcinoma biopsy.     

Immersion by rotation‐based application of the matrix for fast and reproducible sample preparations and robust results in mass spectrometry imaging

Automated matrix deposition for matrix‐assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) is crucial for producing reproducible analyte ion signals. Here we report an innovative method employing an automated immersion apparatus, which enables a robust matrix deposition within 5 minutes and with scalable throughput by using MAPS matrix and non‐polar solvents. MSI results received from mouse heart and rat brain tissues were qualitatively similar to those from nozzle sprayed samples with respect to peak number and quality of the ion images. Overall, the immersion‐method enables a fast and careful matrix deposition and has the future potential for implementation in clinical tissue diagnostics.     

Investigation of the surface topography for the characterization of microstructures of amorphous SiNx-coatings

SiN(x)-coatings have been deposited by reactive magnetron sputtering. Gas pressure and film thickness have been varied. Scanning electron microsopic views of the cross sections show a columnar structure as well as polycrystalline films varying with deposition parameters. For quantitative comparisons of the film morphology an average column diameter has been used as a characteristic value obtained from TEM images. Similar results have been obtained by scanning tunneling microscope avoiding a large expenditure of preparation. Scanning tunneling microscopy is suitable for investigations of the fractal nature of top surface of thin films and to determine the height function of thin SiN(x)-coatings on silicon wafers directly. Computer simulations of sputter processes allow to discuss the evolution of microstructures qualitatively.     

Matrix-assisted laser desorption/ionization imaging mass spectrometry for direct measurement of clozapine in rat brain tissue

Matrix-assisted laser desorption/ionization hyphenated with quadrupole time-of-flight (QTOF) mass spectrometry (MS) has been used to directly determine the distribution of pharmaceuticals in rat brain tissue slices which might unravel their disposition for new drug development. Clozapine, an antipsychotic drug, and norclozapine were used as model compounds to investigate fundamental parameters such as matrix and solvent effects and irradiance dependence on MALDI intensity but also to address the issues with direct tissue imaging MS technique such as (1) uniform coating by the matrix, (2) linearity of MALDI signals, and (3) redistribution of surface analytes. The tissue sections were coated with various matrices on MALDI plates by airspray deposition prior to MS detection. MALDI signals of analytes were detected by monitoring the dissociation of the individual protonated molecules to their predominant MS/MS product ions. The matrices were chosen for tissue applications based on their ability to form a homogeneous coating of dense crystals and to yield greater sensitivity. Images revealing the spatial localization in tissue sections using MALDI-QTOF following a direct infusion of (3)H-clozapine into rat brain were found to be in good correlation with those using a radioautographic approach. The density of clozapine and its major metabolites from whole brain homogenates was further confirmed using fast high-performance liquid chromatography/tandem mass spectrometry (HPLC-MS/MS) procedures.     

High-resolution scanning tunneling microscopy images of molecular overlayers prepared by a new molecular beam deposition apparatus with spray-jet technique

We developed a new molecular beam deposition apparatus using a spray-jet technique for high-quality thin film preparation of nonsublimable molecules. The apparatus was used to deposit chloro[tri-tert-butyl-subphthalocyaninato]boron(III) (TBSubPc) molecules on an Au(111) surface for analysis by low-temperature scanning tunneling microscopy (STM). Highly resolved images, in which tert-butyl groups in a TBSubPc molecule were clearly identifiable, were obtained. The image quality and the resolution of these images compared favorably well to STM images taken on reference samples which were sublimed onto Au (111) from a heated crucible.     

Electrospray sample deposition for matrix-assisted laser desorption/ionization (MALDI) and atmospheric pressure MALDI mass spectrometry with attomole detection limits

Electrospray sample deposition was explored for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). In this method, nanoliter volumes of matrix/analyte mixture were electrosprayed from a high voltage biased (1-2 kV) fused-silica capillary onto a grounded MALDI plate mounted 100-500 microm from the capillary outlet. Electrospray deposition with these conditions produced sample spots 200-300 microm in diameter thus matching the laser spot size. Varying spray voltage and distance resulted in different crystal sizes and volatilization rates for alpha-cyano-4-hydroxycinnamic acid matrix. Best results were obtained when the sample was deposited as wet droplets as opposed to deposition as dried solid. Under 'wet-spray' conditions, 2-4 microm diameter crystals were formed and detection limits for several neuropeptides were 0.7-25 amol. Samples could be pre-concentrated on the plate by spraying continuously and allowing sample to evaporate in a small spot. Sample volumes as large as 580 nL were deposited yielding a detection limit of 35 pM for neurotensin 1-11. Electrospray sample deposition yielded similar results when using atmospheric pressure-MALDI coupled with a quadrupole ion trap mass spectrometer, except that the sensitivity was approximately seven-fold worse.     

Two-dimensional self-assemblies of silica nanoparticles formed using the "bubble deposition technique"

Two-dimensional silica nanoparticle assemblies were obtained by deposition of bubble made from a surfactant solution containing nanoparticles onto hydrophobic silicon substrate. The morphologies of the nanoparticle assemblies can be finely controlled by several experimental parameters, including surfactant concentration, nanoparticle concentration, and deposition time. Monolayer of nanoparticles with surface coverage of about 100% can be obtained under appropriate conditions. The method can also be applied to another hydrophobic substrate, HMDS (hexamethyldisilazane)-modified silicon substrate. Furthermore, it can be applied directly to lithography patterned substrates, meaning a high compatibility with the well-developed conventional top-down approaches to nanodevices. This bubble deposition technique is expected to be a promising method in the field of nano-object assembly and organization and has great application potentials.     

Investigation of the surface topography for the characterization of microstructures of amorphous SiNx-coatings

SiN_x-coatings have been deposited by reactive magnetron sputtering. Gas pressure and film thickness have been varied. Scanning electron microsopic views of the cross sections show a columnar structure as well as polycrystalline films varying with deposition parameters. For quantitative comparisons of the film morphology an average column diameter has been used as a characteristic value obtained from TEM images. Similar results have been obtained by scanning tunneling microscope avoiding a large expenditure of preparation. Scanning tunneling microscopy is suitable for investigations of the fractal nature of top surface of thin films and to determine the height function of thin SiN_x-coatings on silicon wafers directly. Computer simulations of sputter processes allow to discuss the evolution of microstructures qualitatively.     

Spray-Coating of Catalytically Active MOF–Polythiourea through Postsynthetic Polymerization

A UiO-66-NCS MOF was formed by postsynthetic modification of UiO-66-NH₂. The UiO-66-NCS MOFs displays a circa 20-fold increase in activity against the chemical warfare agent simulant dimethyl-4-nitrophenyl phosphate (DMNP) compared to UiO-66-NH₂, making it the most active MOF materials using a validated high-throughput screening. The −NCS functional groups provide reactive handles for postsynthetic polymerization of the MOFs into functional materials. These MOFs can be tethered to amine-terminated polypropylene polymers (Jeffamines) through a facile room-temperature synthesis with no byproducts. The MOFs are then crosslinked into a MOF–polythiourea (MOF–PTU) composite material, maintaining the catalytic properties of the MOF and the flexibility of the polymer. This MOF–PTU hybrid material was spray-coated onto Nyco textile fibers, displaying excellent adhesion to the fiber surface. The spray-coated fibers were screened for the degradation of DMNP and showed durable catalytic reactivity.     

ITO上电沉积Pd的成核机理及电催化性质

采用循环伏安技术和计时电流技术, 研究了ITO上电沉积Pd的过程, 发现Pd在ITO表面的电沉积是过电位成核且为不可逆的扩散控制过程; 根据Cottrell方程计算得到[PdCl₄]^2-的扩散系数为2.19×10^-5 cm²/s; 根据Scharifker的理论模型, 归一化处理电流-时间曲线, 与理论成核曲线对照, 判断Pd 的成核机理. 通过场发射扫描电子显微镜(FESEM)对Pd 的形貌进行分析, 讨论了沉积电位和沉积时间对Pd纳米粒子形貌的影响. 用X射线粉末衍射(XRD)对Pd纳米粒子进行结构分析, 并在0.5 mol/L H₂SO₄溶液中研究了其电化学性质及在碱性条件下乙醇分子的电催化性质.     

MALDI imaging mass spectrometry of lipids by adding lithium salts to the matrix solution

Mass spectrometry imaging of lipids using MALDI–TOF/TOF mass spectrometers is of growing interest for chemical mapping of organic compounds at the surface of tissue sections. Many efforts have been devoted to the best matrix choice and deposition technique. Nevertheless, the identification of lipid species desorbed from tissue sections remains problematic. It is now well-known that protonated, sodium- and potassium-cationized lipids are detected from biological samples, thus complicating the data analysis. A new sample preparation method is proposed, involving the use of lithium salts in the matrix solution in order to simplify the mass spectra with only lithium-cationized molecules instead of a mixture of various cationized species. Five different lithium salts were tested. Among them, lithium trifluoroacetate and lithium iodide merged the different lipid adducts into one single lithium-cationized species. An optimized sample preparation protocol demonstrated that the lithium trifluoroacetate salt slightly increased desorption of phosphatidylcholines. Mass spectrometry images acquired on rat brain tissue sections by adding lithium trifluoroacetate showed the best results in terms of image contrast. Moreover, more structurally relevant fragments were generated by tandem mass spectrometry when analyzing lithium-cationized species.     

Synthesis of new microsensor coatings and their response to test vapors 2,4,6-trisubstituted-1,3,5-triazine derivatives

Novel 1,3,5-triazine derivatives were spray-coated onto surface acoustic wave (SAW) devices and exposed to vapors of dimethyl methylphosphonate (DMMP), chloroethyl ethyl sulfide (CEES) and water. Changes in chemiresistor and SAW responses were monitored and recorded by computer-controlled data-acquisition techniques. All the derivatives tested showed little or no chemiresistor or SAW responses to water vapor. The largest reversible chemiresistor response to DMMP vapor was observed with the dicarboxylic acid derivative. The largest SAW response to DMMP was with the dithione, and the dichloro-octylthio derivative showed the largest response to CEES.     

Bioluminescent Flow Sensors: L-Alanine Determination in Serum and Urine

Abstract

A continuous flow bioluminescent method for L-alanine analysis in serum and urine has been developed. Serum can be analyzed directly after simple filtration. Response is linear from 50 to 1500 pmoles in biological matrix. Alanine dehydrogenase is immobilized onto a nylon coil separated from the reactor coil containing bioluminescent enzymes. The stability of nylon immobilized enzymes is high (over three months) and more than 900 samples can be analyzed with few mg of enzymes. The results obtained with the bioluminescent sensor agree well with those obtained by ion exchange chromatography (amino acid analyzer).     

Bioluminescence flow system for determination of branched-chain l-amino acids in serum and urine

A continuous-flow bioluminescence method for determinations of branched-chain l-amino acids in serum and urine is described. Serum can be analyzed directly after simple filtration. Response is linear for 20-2000 pmol in the biological matrix. Leucine dehydrogenase is immobilized onto a nylon coil separated from the reactor coil containing bacterial bioluminescence enzymes. The stability of the immobilized is high (lifetime > two months) and more than 900 samples can be analyzed with the use of a few mg of enzymes. The results obtained agree well with those obtained by ion-exchange chromatography (amino acid analyzer).     

Underpotential deposition of copper on electrodes modified with colloidal gold

This communication is concerned with the electrochemical addressability of gold colloidal particles deposited on a conducting substrate. Cyclic voltammetry of electrodes modified with gold colloid layers indicates that an underpotential deposition of copper onto the gold surface takes place. Analysis of the charge associated with the underpotential deposition permits the electroactive gold area to be calculated. The total gold area may be determined from transmission electron microscopy (TEM) images. Comparison of the geometric and electroactive areas obtained indicates that electrochemically all the gold particles are addressable and the entire colloid surface is accessible.     

The response of some organic microsensor coatings to a variety of vapors

Six organic compounds were spray-coated onto surface acoustic wave devices which were then exposed to vapors of acetone, diethyl ether, dichloromethane, chlorobenzene, benzene, and acetonitrile. Changes in the resonant frequency of the device or in the resistance of the coating were collected by computer-controlled data acquisition. Different patterns of response to the six vapors were observed for each of the coatings.