Desorption/ionization on silicon (DIOS) mass spectrometry: background and applications

Abstract

Desorption/ionization on silicon mass spectrometry (DIOS-MS) is a matrix-less laser vaporization method for generating gas-phase ions. The physical properties of the silicon surfaces are crucial to DIOS-MS performance and are controlled by the selection of silicon type and the silicon etching conditions. DIOS-MS has been examined for its applicability to small-molecule analysis, quantitative studies, reaction monitoring, chromatography, protein identification, and protein functional characterization. In organic chemistry, DIOS has been applied to the analysis of reactions directed toward development of new catalysts and transformations. Because DIOS offers a chip-based format, it is capable of being used to raster the silicon surface for biological and chemical screening applications, such as enzymatic activity assays. DIOS-MS extends the mass analysis capabilities of laser desorption to small biomolecules and thus offers a platform on which multiple experiments can be performed on a wide variety of molecules.

Introduction

Since the original desorption/ionization experiments performed in 1910 [1], desorption mass spectrometry (MS) has undergone significant improvements. In the early 1980s, the most dramatic change occurred with the introduction of an organic matrix which served as a medium for energy transfer to the analyte. Matrix-assisted desorption/ionization MS was originally applied with high-energy particle beams, such as those employed in secondary ion mass spectrometry (SIMS) [2] and fast atom/ion bombardment (FAB) [3]. The broad success of matrix-assisted desorption/ionization is related to the ability of the matrix to incorporate and transfer energy to the analyte [3], [4], [5], [6]. As a result, mass measurements could be made to exceed 10,000 Da, but the sensitivity required for biomolecular analysis was not achieved until a matrix medium [5] was used in conjunction with laser desorption; the resulting MALDI technique has revolutionized biomolecular analysis [4].

Direct laser desorption/ionization (without a matrix) has been extensively studied on a variety of surfaces [7], [8], [9], [10], [11], [12], yet has not been widely used due to the rapid molecular degradation usually observed upon direct exposure to laser radiation. However, just as SIMS has had a profound effect on surface science [13], the utility of direct laser desorption/ionization for biomolecular analysis could be highly beneficial due to the dramatically simplified sample preparation, the elimination of matrix background ions, and the potential for rapid analyses.

Since the matrix serves to trap analyte molecules as well as absorb UV radiation, nanoporous materials were investigated, such as porous polystyrene beads because they could potentially mimic the features of the MALDI matrix. While porous polystyrene was not found to be useful, porous silicon was found to be an effective medium for desorbing compounds and generating intact ions in the gas phase [14]. A nano-structured silicon thin film prepared by plasma-enhanced chemical vapor deposition also recently yielded very similar laser-desorption/ionization properties as porous silicon [15]. We speculate that this is, in part, due to the ability of its nanometer-sized pores to trap analyte molecules while the silicon surface, well known to be photoactive and even photoluminescent, effectively absorbs UV light (vide infra) [16]. Thus, desorption/ionization on porous silicon (DIOS) (Fig. 1) has demonstrated characteristics similar to MALDI in that intact molecules are observed at the femtomole and attomole level with little or no fragmentation [14]. This stands in contrast to what is typically observed with other direct desorption/ionization approaches [7], [8], [9], [10], [11], [12]. Most importantly, the absence of matrix material [3], [5], [6], [17] allows the technique to be applied to small molecules. With MALDI it is also possible to perform small-molecule analysis [18] and matrix suppression can be achieved under certain circumstances [19], but matrix interference presents limitations that a matrix-free technique such as DIOS does not encounter.

Optimal performance of DIOS-MS is typically obtained for molecules less than 3000 Da (Fig. 2) and its application to whole proteins is currently being developed. The following factors help make DIOS a technique of practical interest. Since there is no matrix deposition, samples may be deposited in spot sizes of less than 1 mm in diameter, and, thus, detection limits are improved because it is easier to localize a sample into a very small region on the plate. Most existing MALDI mass spectrometers can be used to perform DIOS simply by changing the sample plate; no spectrometer modification is necessary. The rate of data acquisition with DIOS is fast. We normally average 128 spectra obtained over 50 s with a laser firing rate of 3 Hz, yet good spectral data may be obtained with as little as 20 pulses (7 s). Silicon wafers are inexpensive and their conversion to porous silicon is relatively simple. Signals from both porous silicon hydride surfaces and porous silicon surfaces capped with covalently bound organic monolayers, especially 2-phenethyl groups [14], are strong, suggesting that significant improvements can be made to DIOS-MS through further investigation of surface modifications (vide infra). SiH termination can be selectively functionalized to make DIOS chips an integrated affinity capture and detection device. Because of its use in the semiconductor computer chip industry [20], a great deal is known about the properties [21], [22], [23], [24], [25], lithographic micropatterning, and micromachining [21], [26], [27] of silicon wafers; presently the interests of many chemists have been captured by the prospects of miniaturized microfluidic chemical reactors which are lithographically etched into crystalline silicon chips [22], [28]. Therefore, significant potential does exist for the construction of chemical devices that employ direct desorption/ionization MS for rapid and informative readout.

Microns-thick porous silicon is a photoluminescent semiconducting material with a high surface area (up to hundreds of square meters per cubic centimeter), produced from crystalline silicon by a straightforward electrochemical etching process (Fig. 3) [23], [29], [30]. Typically, n-type (phosphorus doped) silicon is etched under white-light illumination; p-type (boron doped) material is etched without special irradiation. For both, etching is accomplished by passing current in an electrochemical cell using an electrolyte with 20% aqueous HF in ethanol, in which the silicon wafer is the anode (anodization). This process produces a network of nanometer-scale Si structures, principally cylindrical pores [31], [32], in which surface silicon groups are terminated by SiH bonds. Although photoluminescence of porous silicon is usually attributed to quantum effects of nanometer-sized silicon domains [23], [30], it has also been proposed that the etching process creates a layer of material at the surface resembling siloxene [(Si₆H₆O₃)_n], which is photoluminescent in bulk [33]. Siloxene prepared by the literature method does not give laser-desorption MS when tested on the standard gold MALDI plate. In our case, photoluminescence is not important; indeed, most of the photoluminescent materials that we prepared in our early investigations were not optimal supports for DIOS, and our now-standard DIOS surfaces (see the following description) are not photoluminescent. Similar conclusions were found independently by Sweedler and coworkers [34]. The structure, thickness, porosity, resistivity, and other characteristics of the material are sensitive to the choice of silicon wafer precursor and etching conditions (such as HF concentration, etching duration, current density, light exposure, and dopant type and concentration) [23], [29], [32], [35], [36], [37], [38], [39], [40], [41]. Porous silicon materials are generally divided into microporous (pore diameters≤2 nm), mesoporous (2–50 nm), and macroporous (>50 nm) categories [25], [29], [35]. We have investigated several of the variables in the etching procedure, with the following results:

• Porous silicon generated from heavily-doped n-type silicon wafers (resistivity 0.008–0.05 Ω cm) at low etching current densities (4 mA/cm²) for short times (1–2 min) under moderate white-light intensity produce the best DIOS-MS results thus far. A material giving very similar DIOS results is also obtained with low light intensity, using less heavily doped silicon wafers (0.5–2 Ω cm) etched at larger current densities (20 mA/cm²) for slightly longer times (5 min). The porous silicon samples appear to be fairly robust towards fracturing or peeling of the porous layer, perhaps because their porosities appear to be well below 50% [42].

• Storage in air for extended periods of time, or brief exposure to ozone or aqueous hydrogen peroxide, results in oxidation of surface groups to oxide (SiOSi) and hydroxide (SiOH) moieties, which we characterize by IR spectroscopy and dramatic changes in wetting properties. In general, DIOS performance degrades with increasing surface oxidation, yet some hydrophilic compounds can be detected with better sensitivity on lightly oxidized surfaces or surfaces derivatized with polar functional groups.

• While pore depth generally increases with increasing HF concentration in the etching solution [43], we observed little effect on DIOS performance when the HF concentration was lowered (to 15%) or raised (to 35%) from the standard mixture (25%). In general, lowering the amount of ethanol below 30% leads to irreproducible results, probably because the formation of H₂ bubbles at the surface under such conditions is too vigorous.

• We have observed similar performance in DIOS-MS analysis of standard peptide samples (vide infra) on porous silicon prepared from both <1 0 0> and <1 1 1> silicon wafers using the same standard etching conditions (4 mA/cm², 1 min, with irradiation), suggesting that Si crystal orientation does not play an important role in DIOS performance.

• DIOS surfaces may be photopatterned by etching n⁺-type silicon with illumination through a mask. Note that the low current densities employed here facilitate the creation of sharp boundaries since hydrogen bubble formation is minimized; such bubbles can stick to the surface and induce heterogeneity in both the lateral and vertical dimensions. A complementary type of photopatterning has been demonstrated by Stewart and Buriak [44] in the covalent derivatization of porous silicon following the etching step.