Ordered porous alumina geometries and surface metals for surface-assisted laser desorption/ionization of biomolecules: possible mechanistic implications of metal surface melting

Platinum- or gold-coated porous alumina with submicrometer structures is a potential substrate for surface-assisted laser desorption/ionization (SALDI) mass spectrometry of biomolecules, not requiring sample matrixes. In this study, a highly ordered porous alumina substrate was fabricated to study the geometric factors allowing good SALDI performance. Evaluation was based on the signal-to-noise ratio of protonated angiotensin I ions in the mass spectrum obtained by 337-nm ultraviolet laser irradiation. Varying the geometries, including pore densities and diameters, revealed the laser intensity required to generate ions to be related to surface porosity. Surface platinum was melted upon laser irradiation at the fluence sufficient to generate peptide ions as confirmed by scanning electron microscopy. Moreover, a thin (5-20 nm) platinum coat requires a low intensity of laser light for desorption/ionization. Considering the size effect on the melting of metals, our findings suggest the surface platinum melting to be involved in ion generation from this SALDI substrate type. Indeed, tantalum, which has a higher melting point, required more laser fluence to generate ions. The porous alumina layer beneath surface metals probably worked as a thermal insulator. This double-layer-type substrate allowed ionization of angiotensin I and verapamil at low femtomole levels. Moreover, small proteins and glycoproteins such as 24-kDa trypsinogen and 15-kDa ribonuclease B could be ionized with sufficient sensitivity on this target. Taking advantage of matrix-free methods, concentrating the sample solution in the target concavity or widening the laser beam focus enhanced the signal-to-noise ratio for analyte ions in the mass spectrum. Activity is maintained for months in air.     

Experimental factors controlling analyte ion generation in laser desorption/ionization mass spectrometry on porous silicon

Desorption/ionization on porous silicon (DIOS) is a relatively new laser desorption/ionization technique for the direct mass spectrometric analysis of a wide variety of samples without the requirement of a matrix. Porous silicon substrates were fabricated using the recently developed nonelectrochemical H2O2-metal-HF etching as a versatile platform for investigating the effects of morphology and physical properties of porous silicon on DIOS-MS performance. In addition, laser wavelength, mode of ion detection, pH, and solvent contributions to the desorption/ionization process were studied. Other porous substrates such as GaAs and GaN, with similar surface characteristics but differing in thermal and optical properties from porous silicon, allowed the roles of surface area, optical absorption, and thermal conductivities in the desorption/ionization process to be investigated. Among the porous semiconductors studied, only porous silicon has the combination of large surface area, optical absorption, and thermal conductivity required for efficient analyte ion generation under the conditions studied. In addition to these substrate-related factors, surface wetting, determined by the interaction of deposition solvent with the surface, and charge state of the peptide were found to be important in determining ion generation efficiency.     

Ordered silicon nanocavity arrays in surface-assisted desorption/ionization mass spectrometry

We report here a simple method to generate ordered nanocavity arrays on a Si wafer and use it in surface-assisted laser desorption/ionization mass spectrometry (SALDI-MS). A close-packed SiO2 nanosphere array was first deposited on a low-resistivity Si wafer using a convective self-assembly method. The nanoparticle array was then used as a mask in a reactive ion etching (RIE) process to selectively remove portions of the Si surface. Subsequent sonication removed those physically adsorbed SiO2 nanoparticles and exposed an ordered nanocavity array underneath. The importance of this approach is its capability of systematically varying surface geometries to achieve desired features, which makes detailed studies of the impacts of surface features on the desorption/ionization mechanism feasible. We demonstrated that the in-plane width and out-of-plane depth of the cavities were adjustable by varying etching times, and the intercavity spacing was controllable by varying the number of particle layers deposited. MS detection of small peptides on these substrates showed comparable sensitivity to conventional porous Si substrates (DIOS, desorption/ ionization on porous silicon). The desorption and ionization efficiency of these roughened surfaces exhibited a nonmonotonic relationship to the increased total surface area. Several possible factors contributing to the observed phenomenon are speculated upon. The application of this arrayed surface in metabolite detection of Arabidopsis thaliana root extracts is also demonstrated.     

Infrared laser desorption/ionization on silicon

Laser desorption/ionization from a single-crystal silicon surface was performed using a laser operating in the 3-microm region of the mid-infrared. Analyte molecules up to 6 kDa were ionized with no added matrix. As with ultraviolet desorption/ionization from porous silicon (DIOS), IR laser desorption from silicon does not produce matrix ions that can interfere with analysis of low-mass analytes. However, in contrast to UV DIOS, silicon porosity or roughness is not required for ionization using an IR laser. Mass spectra were obtained in the wavelength range between 2.8 and 3.5 microm, which is consistent with energy absorption by a hydrogen-bonded OH group. A mechanism based on desorption of adsorbed solvent molecules is postulated.     

Adjustable fragmentation in laser desorption/ionization from laser-induced silicon microcolumn arrays

Laser-induced silicon microcolumn arrays (LISMA) were developed as matrix-free substrates for soft laser desorption/ionization mass spectrometry (SLDI-MS). When low-resistivity silicon wafers were irradiated in air, sulfur hexafluoride, or water environment with multiple pulses from a 3 x omega mode-locked Nd:YAG laser, columnar structures were formed on the surface. The radii of curvature of the column tips varied with the processing environment, ranging from approximately 120 nm in water, to <1 mum in SF6, and to approximately 2 mum in air. In turn, these microcolumn arrays were used as matrix-free soft laser desorption substrates. In SLDI-MS experiments with a nitrogen laser, the microcolumn arrays obtained in water environment readily produced molecular ions for peptides and synthetic polymers at low laser fluence. These surfaces demonstrated the best ion yield among the three arrays. The threshold laser fluence and ion yield were comparable to those observed in matrix-assisted laser desorption/ionization. Low-femtomole sensitivity and approximately 6000 Da mass range were achieved. At elevated laser fluence, efficient in-source decay was observed and extensive peptide sequence information was extracted from the resulting mass spectra. The versatility of LISMA was attributed to confinement effects due to the submicrometer morphology and to the surface, thermal, and optical properties of processed silicon.     

A facile preparation route for netlike microstructures on a stainless steel using an ethanol-mediated femtosecond laser irradiation

Netlike or porous microstructures are highly desirable in metal implants and biomedical monitoring applications. However, realization of such microstructures remains technically challenging. Here, we report a facile and environmentally friendly method to prepare netlike microstructures on a stainless steel by taking the full advantage of the liquid-mediated femtosecond laser ablation. An unordered netlike structure and a quasi-ordered array of holes can be fabricated on the surface of stainless steel via an ethanol-mediated femtosecond laser line-scan method. SEM analysis of the surface morphology indicates that the porous netlike structure is in the micrometer scale and the diameter of the quasi-ordered holes ranges from 280 nm to 320 nm. Besides, we find that the obtained structures are tunable by altering the laser processing parameters especially scanning speed.     

High sensitivity and analyte capture with desorption/ionization mass spectrometry on silylated porous silicon

Silylation chemistry on porous silicon provides for ultrahigh sensitivity and analyte specificity with desorption/ionization on silicon mass spectrometry (DIOS-MS) analysis. Here, we report that the silylation of oxidized porous silicon offers a DIOS platform that is resistant to air oxidation and acid/base hydrolysis. Furthermore, surface modification with appropriate hydrophobic silanes allows analytes to absorb to the surface via hydrophobic interactions for direct analyte extraction from complex matrixes containing salts and other nonvolatile interferences present in the sample matrix. This enables rapid cleanup by simply spotting the sample onto the modified DIOS target and removing the liquid phase containing the interferences. This approach is demonstrated in the analysis of protein digests and metabolites in biofluids, as well as for the characterizing of inhibitors from their enzyme complex. An unprecedented detection limit of 480 molecules (800 ymol) for des-Arg(9)-bradykinin is reported on a pentafluorophenyl-functionalized DIOS chip.     

Layer-by-layer self-assembled mutilayer films of gold nanoparticles for surface-assisted laser desorption/ionization mass spectrometry

Layer-by-layer (LBL) self-assembled multilayer films of gold nanoparticles (AuNPs) on a silicon wafer were demonstrated to be promising substrates for surface-assisted laser desorption/ionization (SALDI) mass spectrometry (MS) of peptides and environmental pollutants for the first time. LBL multilayer films, (AuNPs/PAHC)n, consisting of alternating layers of ammonium citrate capped AuNPs and poly(allylamine hydrochloride) (PAHC) were prepared on a silicon surface. Silicon plates with aggregated AuNPs were more suitable than those with dispersed AuNPs for the SALDI-MS of peptides. The number of particle layers had a significant effect on the laser desorption/ionization of angiotensin I; the peak intensity of the peptide (molecular ion amount) increased with an increase in the number of layers of AuNPs. As a result, the (AuNPs/PAHC)5 multilayer films increased the sensitivity of the angiotensin I to subfemtomoles and raised the useful analyte mass range, thus making it possible to detect small proteins (a 12 kDa cytochrome c). The signal enhancement when using (AuNPs/PAHC)5 may be due to (i) the high absorption of the UV laser light at 337 nm by the AuNP layers, (ii) the low thermal conductivity due to the AuNPs being covered with a thin monolayer of PAHC, and (iii) the increase in the surface roughness (approximately 100 nm) with the number of AuNP layers. Thus, laser-induced rapid high heating of AuNPs for effective desorption/ionization of peptides is possible. In addition, it was found that (AuNPs/PAHC)5 could be used to extract environmental pollutants (pyrene and dimethyldistearylammonium chloride) from very dilute aqueous solutions with concentrations less than 10(-10) mg/mL, and the analytes trapped in the LBL film could be identified by introducing the film directly into the SALDI mass spectrometer without needing to elute the analytes out of the film.     

Matrix-free laser desorption/ionization-mass spectrometry using self-assembled germanium nanodots

A novel ionization platform for matrix-free laser desorption/ionization-mass spectrometry (LDI-MS) was developed using self-assembled germanium nanodots (GeNDs) of uniform size (approximately 150 - 200-nm width and approximately 50-nm height) grown on a silicon wafer produced by molecular beam epitaxy. The performance of LDI-MS using GeNDs (GeND-MS) was investigated through measurements of a broad range of analytes, including peptides, proteins, synthetic oligomers, and polymer additives. Mass spectra of tryptic digests were clearly observed even for the mass range lower than m/z 800 without obstructive peaks. A detection limit of subfemtomole level was achieved for angiotensin-I. The upper limit of detectable mass range was approximately 17 kDa (myoglobin). GeND-MS also has potential for application to the characterization of industrial compounds. Almost accurate molecular weight distribution was obtained for a nonionic surfactant (Triton X-100) and for poly(ethylene glycol) oligomer. Furthermore, a brominated flame retardant, tetrabromobisphenol-A bis(2,3-dibromopropyl ether), was successfully ionized with less fragmentation, a result not obtainable by matrix-assisted laser desorption/ionization-mass spectrometry or desorption/ionization on porous silicon-mass spectrometry.     

Dual desorption electrospray ionization-laser desorption ionization mass spectrometry on a common nanoporous alumina platform for enhanced shotgun proteomic analysis

A gold coated nanoporous alumina surface was used for dual ionization mode mass spectrometric analysis using desorption electrospray ionization (DESI) and laser desorption ionization (LDI). DESI and LDI mass spectrometry (MS) from the nanoporous alumina surface were compared with conventional electrospray ionization (ESI) mass spectrometry and matrix assisted laser desorption ionization (MALDI) for analysis of tryptic digests of proteins. Combined use of DESI and LDI offer greater peptide coverage than either method alone and comparable peptide coverage as with dual MALDI and ESI. This dual ionization technique using a common platform with same sample spot demonstrates a potential time and cost-effective tool for improved shotgun proteomic analysis.     

Enzyme kinetics by directly imaging a porous silicon microfluidic reactor using desorption/ionization on silicon mass spectrometry

Enzyme kinetics were obtained in a porous silicon microfluidic channel by combining an enzyme and substrate droplet, allowing them to react and deposit a small amount of residue on the channel walls, and then analyzing this residue by directly ionizing the channel walls using a matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS) laser source. The porous silicon of the channel walls functions in a manner analogous to the matrix in MALDI-MS, and is referred to as a desorption/ionization on silicon mass spectrometry (DIOS-MS) target when used in this configuration. Mass spectrometry signal intensity of substrate residue correlates with relative concentration, and position in the microchannel correlates with time, thus allowing determination of kinetic parameters. The system is especially suitable for initial reaction velocity determination. This microreactor is broadly applicable to time-resolved kinetic assays as long as at least one substrate or product of the reaction is ionizable by DIOS-MS.     

Desorption ionization of biomolecules on metals

Direct desorption ionization of various types of biomolecules on metal substrates without the need of matrices was observed by a time-of-flight mass spectrometer. It provides a new convenient method for detection of small biomolecules without the confusion of ion peaks from matrix compounds. Simple commercial Al foil can be used as the substrate to obtain mass spectra of biomolecules without the need of an etching process to produce a porous surface such as with direct ionization on silicon (DIOS). The desorption and ionization mechanism is also discussed.     

Temperature-programmed GC using silicon microfabricated columns with integrated heaters and temperature sensors

Columns were fabricated in silicon substrates by deep reactive-ion etching. The channels were sealed with a glass wafer anodically bonded to the silicon surface. Heaters and temperature sensors were fabricated on the back side of each column chip. A microcontroller-based temperature controller was used with a PC for temperature programming. Temperature programming, with channel lengths of 3.0 and 0.25 m, is described. The 3.0-m-long channel was fabricated on a 3.2 cmx3.2 cm chip. Four columns were fabricated on a standard 4-in. silicon wafer. The 0.25-m-long channel was fabricated on a 1.1 cmx1.1 cm chip, and approximately 40 columns could be fabricated on a 4-in. wafer. All columns were coated with a nonpolar poly(dimethylsiloxanes) stationary phase. A static coating procedure was employed. The 3.0-m-long column generated about 12000 theoretical plates, and the 0.25-m-long channel generated about 1000 plates at optimal carrier gas velocity. Linear temperature ramps as high as 1000 degrees C/min when temperature programmed from 30 to 200 degrees C were obtained with the shorter column. With the 0.25-m-long column, normal alkanes from n-C5 through n-C15 were eluted in less than 12 s using a temperature ramp rate of 1000 degrees C/min. Temperature uniformity over the column chip surface was measured with infrared imaging. A variation of about 2 degrees C was obtained for the 3.0-m-long channel. Retention time reproducibility with temperature programming typically ranged from +/-0.15% to +/-1.5%. Design of the columns and the temperature controller are discussed. Performance data are presented for the different columns lengths.     

硅基含能材料爆炸性能研究

采用电化学双槽腐蚀法在P型单晶硅片表面生长多孔硅膜。通过扫描电镜（SEM）、能量色谱（EDS）对多孔硅结构参数以及多孔硅含能材料性能进行了分析,同时进行了爆炸性能测试。结果表明：采用电化学腐蚀法可以制备出20nm左右孔径的多孔硅膜;通过原位装药技术形成的多孔硅含能材料在开放空间以及热能、机械撞击、电能、激光能量刺激下发生猛烈爆炸作用。     

Development and characterization of an ionization technique for analysis of biological macromolecules: liquid matrix-assisted laser desorption electrospray ionization

We have developed an atmospheric pressure ionization technique called liquid matrix-assisted laser desorption electrospray ionization (liq-MALDESI) for the generation of multiply charged ions by laser desorption from liquid samples deposited onto a stainless steel sample target biased at a high potential. This variant of our previously reported MALDESI source does not utilize an ESI emitter to postionize neutrals. Conversely, we report desorption and ionization from a macroscopic charged droplet. We demonstrate high mass resolving power single-acquisition FT-ICR-MS analysis of peptides and proteins ranging from 1 to 8.6 kDa at atmospheric pressure. The liquid sample acts as a macroscopic charged droplet similar to those generated by electrospray ionization, whereby laser irradiation desorbs analyte from organic matrix containing charged droplets generating multiply charged ions. We have observed a singly charged radical cation of an electrochemically active species indicating oxidation occurs for analytes and therefore water; the latter would play a key role in the mechanism of ionization. Moreover, we demonstrate an increase in ion abundance and a concurrent decrease in surface tension with an increase in the applied potential.     

Desorption/ionization on silicon nanowires

Dense arrays of single-crystal silicon nanowires (SiNWs) have been used as a platform for laser desorption/ionization mass spectrometry of small molecules, peptides, protein digests, and endogenous and xenobiotic metabolites in biofluids. Sensitivity down to the attomole level has been achieved on the nanowire surfaces by optimizing laser energy, surface chemistry, nanowire diameter, length, and growth orientation. An interesting feature of the nanowire surface is that it requires lower laser energy as compared to porous silicon and MALDI to desorb/ionize small molecules, therefore reducing background ion interference. Taking advantage of their high surface area and fluid wicking capabilities, SiNWs were used to perform chromatographic separation followed by mass analysis of the separated molecules providing a unique platform that can integrate separation and mass spectrometric detection on a single surface.     

Diffraction-Based Strategy for Monitoring Topographical Features Fabricated by Direct Laser Interference Patterning

Process monitoring in laser-based manufacturing has become a forward-looking strategy for industrial-scale laser machines to increase process reliability, efficiency, and economic profit. Moreover, monitoring techniques are successfully used in laser surface texturing workstations to improve and guarantee the quality of the produced workpieces by analyzing the resulting surface topography. Herein, dot-like periodic surface structures are fabricated on stainless steel samples by direct laser interference patterning (DLIP) using a 70 ps-pulsed laser system at an operating wavelength of 532 nm. A scatterometry-based measurement device is utilized to indirectly determine the mean depth and spatial period of the produced topography by analyzing the recorded diffraction patterns. As a result, the average depth and the spatial period of the dot-like structures can be estimated with a relative error below 15% and 2%, respectively. This new process monitoring approach enables a significant improvement in quality assurance in DLIP processing.     

Macro-/nanoporous silicon as a support for high-performance protein microarrays

The present work demonstrates the possibilities of using macroporous silicon as a substrate for highly sensitive protein chip applications. The formation of 3D porous silicon structures was performed by electrochemical dissolution of monocrystalline silicon. The fabricated macroporous silicon network has a rigid spongelike structure showing high uniformity and mechanical stability. The microfluidic properties of the substrates were found to be essential for a good bioassay performance. Small spot area, good spot reproducibility, and homogeneous spot profiles were demonstrated on the substrates for immobilized aRIgG. Water contact angles were measured on the porous surface and compared to that of planar silicon, silanized glass, and ordinary microscope glass slides. The effect of the porous surface on the performance of a model IgG-binding immunoassay is presented. aRIgG was microdispensed onto the chip surface forming a microarray of spots with high affinity for the target analyte. The dispensing was performed using an in-house-developed piezoelectric flow-through dispenser. Each spot was formed by a single droplet (100 pL) at each position. The macroporous silicon allowed a high-density microarraying with spot densities up to 4400 spots/cm2 in human plasma samples without cross-talk and consumption of only 0.6 pmol of antibodies/1-cm2 array. Antigen levels down to 70 pM were detected.     

Emitting far-field multicolor patterns and characters through plastic diffractive micro-optics elements illuminated by common Gaussian lasers in the visible range

Far-field multicolor patterns and characters are emitted effectively in a relatively wide and deep spatial region by plastic diffractive micro-optics elements (DMOEs), which are illuminated directly by common Gaussian lasers in the visible range. Phase-only DMOEs are composed of a large number of fine step-shaped phase microstructures distributed sequentially over the plastic wafer selected. The initial DMOEs in silicon wafer are fabricated by an innovative technique with a combination of a single-mask ultraviolet photolithography and low-cost and rapid wet KOH etching. The fabricated silicon DMOEs are further converted into a nickel mask by the conventional electrochemical method, and they are finally transferred onto the surface of the plastic wafer through mature hot embossing. Morphological measurements show that the surface roughness of the plastic DMOEs is in the nanometer range, and the feature height of the phase steps in diffractive elements is in the submicrometer scale, which can be designed and adjusted flexibly according to requirements. The dimensions of the DMOEs can be changed from the order of millimeters to centimeters. A large number of pixel phase microstructures with a square microappearance employed to construct the phase-only DMOEs are created by the Gerchberg-Saxton algorithm, according to the target patterns and characters and common Gaussian lasers manipulated by the DMOEs fabricated.     

Small-molecule analysis with silicon-nanoparticle-assisted laser desorption/ionization mass spectrometry

Silicon nanopowder (5-50 nm) was applied as a matrix for the analysis of small molecules in laser desorption/ionization mass spectrometry. In contrast with conventional matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry, the matrix background interference in the low mass range was significantly reduced. Effects of the particle size and sample preparation procedures on the background mass spectra and the analyte signal intensity have been investigated, and an optimized powder and sample preparation protocol was established. Several surface characterization tools have been applied as well. Both positive mode and negative mode laser desorption/ionization have been applied to different analytes including drugs, peptides, pesticides, acids, and others. Detection limits down to the low femtomole per microliter levels were achieved for propafenone and verapamil drugs. The method developed was found relatively tolerant to salt contamination, which allowed the direct analysis of morphine and propaphenone in untreated urine and triazine herbicides in a soil extract. The new silicon-nanoparticle-assisted laser desorption ionization method was found to be highly selective, which may be due to analyte-dependent precharging in solution, prior to vacuum laser desorption. Some aspects of the charge-transfer mechanism have been studied and discussed. In comparison with standard MALDI matrixes, the silicon nanopowder requires much lower laser fluence (contributing to a reduced background) has much better surface homogeneity, and is more tolerant to salt interference, which makes it an easily applicable practical tool at a potentially low cost.