Patent Publication Number: US-2007114387-A1

Title: Matrix assisted laser desorption ionization (MALDI) support structures and methods of making MALDI support structures

Description:
CROSS-REFERENCE TO RELATED APPLICATION  
      This application claims priority to U.S. provisional application entitled, “Matrix assisted laser desorption Ionization (MALDI) support structures and methods of making MALDI support structures,” having Ser. No. 60/731,711, filed on Oct. 31, 2005, which is entirely incorporated herein by reference. 
    
    
     BACKGROUND  
      A variety of instruments can be used for analyzing analytes such as biomolecules. More recently, mass spectrometry has gained prominence because of its ability to handle a wide variety of analytes with high sensitivity and rapid throughput. A variety of ion sources have been developed for use in mass spectrometry. Many of these ion sources include some type of mechanism that produces ions in accordance with an ionization process. One particular type of ionization process that is used is Matrix Assisted Laser Desorption Ionization (“MALDI”). MALDI is a technique used to produce ions for mass spectrometry. One benefit of MALDI is its ability to produce ions from a wide variety of analytes, including biomolecules such as proteins, peptides, oligosaccharides, oligonucleotides, and the like. Another benefit of MALDI is its ability to produce ions with reduced fragmentation, thus facilitating identification of analytes from which the ions are produced.  
      Typically, MALDI produces ions from a co-precipitate of an analyte and a matrix. The matrix can include organic molecules that exhibit a strong absorption of light at a particular wavelength or a particular range of wavelengths, such as in the ultraviolet range. Examples of the matrix include 2,5-dihydroxybenzoic acid, 3,5-dimethoxy-4-hydroxycinnamic acid, α-cyano-4-hydroxycinnamic acid, and the like. For a conventional MALDI mass spectrometry system, an analyte and a matrix are dissolved in a solvent to form a solution, and the solution is then applied to or positioned on a sample support. As the solvent evaporates, the analyte and the matrix form a co-precipitate on the sample support. The co-precipitate is then irradiated with a short laser pulse that induces an accumulation of energy in the co-precipitate through electronic excitation or molecular vibration of the matrix. As the matrix dissipates the energy by desorption, the matrix carries the analyte into a gaseous phase. During this desorption process, ions are produced from the analyte by charge transfer between the matrix and the analyte.  
      During operation of a conventional MALDI mass spectrometry system, absorption of light by a matrix or by an analyte can affect ionization efficiency for the analyte, which, in turn, can affect sensitivity of mass spectrometric analyses. Accordingly, it is desirable to enhance absorption of light by the matrix or by the analyte, such that mass spectrometric analyses have a desired level of sensitivity.  
     SUMMARY  
      Matrix assisted laser desorption ionization (MALDI) sample substrates, methods of fabricating MALDI sample substrates, methods of ionizing a sample, and mass spectrometry systems including MALDI sample substrates, are disclosed.  
      Briefly described, one embodiment of the MALDI sample substrate, among others, includes: a MALDI substrate, a metal nanostructure catalyst layer disposed on the substrate, wherein the metal nanostructure catalyst layer includes a discrete set of nanostructures, a silicon nanostructure layer disposed on the metal nanostructure catalyst layer, wherein the silicon nanostructure layer includes a discrete set of nanostructures, and a silicon dioxide (SiO 2 ) layer formed on the silicon nanostructure layer.  
      An embodiment of a method of fabricating MALDI sample substrates, among others, includes: providing a sample support as described above, positioning the sample on the sample support, and ionizing the sample.  
      An embodiment of a method of ionizing a sample, among others, includes: providing a sample support comprising a sample support as described above, positioning the sample on the sample support, and ionizing the sample.  
      An embodiment of a method of ionizing a sample, among others, includes: an ion source configured to produce ions and comprising: a light source; and a sample support, as described above, adjacent to the light source and configured to support a sample, and a detector downstream with respect to the ion source and configured to detect the ions.  
      Other systems, methods, features, and advantages of the present disclosure will be or become apparent to one with skill in the art upon examination of the following drawings and detailed description. It is intended that all such additional systems, methods, features, and advantages be included within this description, be within the scope of the disclosure, and be protected by the accompanying claims. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
      Aspects of the disclosure can be better understood with reference to the following drawings. The components in the drawings are not necessarily to scale, emphasis instead being placed upon clearly illustrating the principles of the present disclosure. Moreover, in the drawings, like reference numerals designate corresponding parts throughout the several views.  
       FIG. 1  is a cross sectional view of a matrix assisted laser desorption ionization (MALDI) sample substrate.  
       FIGS. 2A through 2D  illustrate cross sectional views that illustrate an embodiment of a method for forming the MALDI sample substrate shown in  FIG. 1 .  
       FIG. 3  illustrates a mass spectrometry system  20  implemented in accordance with embodiments of the MALDI sample substrate.  
       FIG. 4  is an AFM picture of a nanostructured Au/Si/SiO 2  surface.  
       FIG. 5  illustrates a MALDI measurement using a titanium nitride surface, while  FIG. 6  illustrates a MALDI measurement on nanostructured Au/Si/SiO 2  surface. 
    
    
     DETAILED DESCRIPTION  
      Embodiments of the present disclosure will employ, unless otherwise indicated, techniques of synthetic organic chemistry, biochemistry, molecular biology, semiconductor manufacturing techniques, and the like, that is within the skill of the art. Such techniques are explained fully in the literature.  
      The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to perform the methods and use the compositions disclosed and claimed herein. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.) but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in ° C., and pressure is at or near atmospheric. Standard temperature and pressure are defined as 20° C. and 1 atmosphere.  
      Before the embodiments of the present disclosure are described in detail, it is to be understood that, unless otherwise indicated, the present disclosure is not limited to particular materials, reagents, reaction materials, manufacturing processes, or the like, as such can vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting. It is also possible in the present disclosure that steps can be executed in different sequence where this is logically possible.  
      It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a support” includes a plurality of supports. In this specification and in the claims that follow, reference will be made to a number of terms that shall be defined to have the following meanings unless a contrary intention is apparent.  
      As used herein, the term “set” refers to a collection of one or more elements. Thus, for example, a set of nanostructures can comprise a single nanostructure or multiple nanostructures. Elements of a set can also be referred to as members of the set. Elements of a set can be the same or different. In some instances, elements of a set can share one or more common characteristics.  
      As used herein, the term “adjacent” refers to being near or adjoining. Adjacent structures can be spaced apart from one another or can be in actual contact with one another. In some instances, adjacent structures can be coupled to one another or can be formed integrally with one another.  
      As used herein, the term “ionization efficiency” refers to a ratio of the number of ions produced in an ionization process and the number of electrons or photons used in the ionization process.  
      As used herein, the term “ultraviolet range” refers to a range of wavelengths from about 150 nanometer (nm) to about 400 nm.  
      As used herein, the term “nanometer range” or “nm range” refers to a range of sizes from about 0.1 nm to about 1,000 nm, such as from about 0.1 nm to about 500 nm, from about 0.1 nm to about 100 nm, from about 0.1 nm to about 50 nm, or from about 0.1 nm to about 110 nm.  
      As used herein, the term “aspect ratio” refers to a ratio of a largest dimension of a structure and an average of remaining dimensions of the structure, which remaining dimensions are orthogonal with respect to one another and with respect to the largest dimension. In some instances, remaining dimensions of a structure can be substantially the same, and an average of the remaining dimensions can substantially correspond to either of the remaining dimensions. Thus, for example, an aspect ratio of a cylinder refers to a ratio of a length of the cylinder and a cross-sectional diameter of the cylinder. As another example, an aspect ratio of a spheroid refers to a ratio of a major axis of the spheroid and a minor axis of the spheroid.  
      As used herein, the terms “reflective,” “reflecting,” and “reflection” refer to a bending or a deflection of light. A bending or a deflection of light can be substantially in a single direction, such as in the case of specular reflection, or can be in multiple directions, such as in the case of diffuse reflection or scattering. Reflective materials typically correspond to those materials that produce reflected light when those materials are irradiated with incident light. The reflected light and the incident light can include wavelengths that are the same or different.  
      As used herein, the terms “inert” and “inertness” refer to a lack of interaction. Inert materials typically correspond to those materials that exhibit little or no tendency to interact with a sample under typical operating conditions, such as typical operating conditions of the sample supports described herein. Typically, inert materials also exhibit little or no tendency to interact with ions produced from a sample in accordance with an ionization process. While a material is sometimes referred to herein as being inert, it is contemplated that the material can exhibit some detectable tendency to interact with a sample under certain conditions. One measure of inertness of a material is its chemical reactivity. Typically, the material is considered to be inert if it exhibits little or no chemical reactivity with respect to a sample.  
      As used herein, the term “nanostructure” refers to a structure that includes at least one dimension in the nm range. A nanostructure can include any of a wide variety of shapes and can be formed from any of a wide variety of materials. Examples of nanostructures include, but are not limited to, nanoparticles.  
      As used herein, the term “nanoparticle” refers to a spheroidal nanostructure. Typically, a nanoparticle includes dimensions in the nm range and an aspect ratio that is less than about 2. Thus, for example, a nanoparticle can include a major axis and a minor axis that are both in the nm range. Nanoparticles can be formed using any of a wide variety of techniques, such as aqueous synthetic routes, electron beam evaporation, chemical vapor deposition, and the like.  
      As used herein, the term “metal nanostructure catalyst layer” refers to a material that includes or is formed from a set of nanostructures. One example of a metal nanostructure catalyst layer is one that includes or is formed from a set of nanoparticles, namely a nanoparticle material. In some instances, a metal nanostructure catalyst layer can include a substantially ordered array or arrangement of nanostructures and, thus, can be referred to as being substantially ordered. For example, a metal nanostructure catalyst layer can include an array of nanostructures that are substantially aligned with respect to one another or with respect to a certain axis, direction, plane, surface, or three-dimensional shape. As another example, a metal nanostructure catalyst layer can include an array of nanostructures that are substantially regularly spaced with respect to one another or with respect to a certain lattice, such as any of a wide variety of two-dimensional lattices and three-dimensional lattices.  
      It should be noted that ratios, concentrations, amounts, and other numerical data may be expressed herein in a range format. It is to be understood that such a range format is used for convenience and brevity, and thus, should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. To illustrate, a concentration range of “about 0.1% to about 5%” should be interpreted to include not only the explicitly recited concentration of about 0.1 wt % to about 5 wt %, but also include individual concentrations (e.g., 1%, 2%, 3%, and 4%) and the sub-ranges (e.g., 0.5%, 1.1%, 2.2%, 3.3%, and 4.4%) within the indicated range.  
      All patents, patent applications, and publications mentioned herein are hereby incorporated by reference in their entireties.  
      Discussion  
      Matrix assisted laser desorption ionization (MALDI) sample substrates, mass spectrometry systems including the MALDI sample substrates, methods of ionization, and methods of fabricating MALDI sample substrates are provided. In general, the MALDI sample substrates includes a substrate, a metal nanostructure catalyst layer, a silicon nanostructure layer, and a silicon dioxide (SiO 2 ) layer. The metal nanostructure catalyst layer and the silicon nanostructure layer each include a set of discrete nanostructures. The metal nanostructure catalyst layer is disposed on the substrate, while the silicon nanostructure layer is disposed on the metal nanostructure catalyst layer. The silicon dioxide layer is formed on top of the silicon nanostructure layer. The metal nanostructure catalyst layer can reflect light incident upon the metal nanostructure catalyst layer. Moreover, the metal nanostructure catalyst layer is buried underneath the silicon nanoparticle layer, thus the interaction between the analytes and metal is prohibited. In this way, a sample disposed on the silicon dioxide layer can be desorbed/ionized by the light directed at the sample as well as the light reflected from the metal nanostructure catalyst layer. Use of this type of MALDI sample substrates can result in enhanced desorption/ionization efficiency. Furthermore, the MALDI sample substrates can be formed using standard semiconductor microprocessing techniques, which provides a less expensive method of fabrication.  
       FIG. 1  is a cross sectional view of a MALDI sample substrate  10 . The MALDI substrate  10  includes, but is not limited to, a substrate  12 , a metal nanostructure catalyst layer  14 , a silicon nanostructure layer  16 , and a silicon dioxide (SiO 2 ) layer  18 . It should be noted that the metal nanostructure catalyst layer  14  and the silicon nanostructure layer  16  are composed of discrete nanostructures. The metal nanostructure catalyst layer  14  is disposed on the substrate  12 , while the silicon nanostructure layer  16  is disposed on the metal nanostructure catalyst layer  14 . The silicon dioxide layer  18  is formed on the silicon nanostructure layer  16  via oxidation by oxygen in air, for example.  
      The substrate  12  can be made of a material such as, but not limited to, siliceous materials (e.g., silicon dioxide, glasses, fused silica, ceramics, and the like), metals, polymers, and others that are able to withstand the rigors of metal catalyst deposition and silicon nanoparticle growth condition for MALDI substrate manufacturing. The dimensions can be of those typically used in MALDI processes.  
      The metal nanostructure catalyst layer  14  includes a set of nanostructures, where the nanostructures are adjacent one another. The nanostructures can include, but are not limited to, nanoparticles. In particular the nanostructure can be made of materials such as, but not limited to, gold, silver, titanium, nickel, cobalt, oxides of each, and combinations of each. The metal nanostructure catalyst layer  14  can have a thickness of about 2 to 50 nanometers (nm), about 5 to 20 nm, and about 10 to 20 nm. The metal nanostructure catalyst layer  14  can include about 1 to 2 layers of nanostructures, each layer being about 5 to 9 nm. The nanostructures can have a diameter of about 5 to 9 nm.  
      The metal nanostructure catalyst layer  14  can be formed by techniques such as, but not limited to, electron-beam evaporation and the like. The spacing (e.g., density of the nanostructures) of the nanostructures adjacent one another can be controlled, at least in part, by the fabrication conditions. In this regard, the spacing can be controlled to produce a metal nanostructure catalyst layer appropriate for a particular MALDI application. The metal nanostructure catalyst layer  14  acts as nucleation sites for the subsequent Si nanoparticles deposition, as discussed below.  
      The silicon nanostructure layer  16  includes a set of silicon nanostructures, where the nanostructures are adjacent one another. The silicon nanostructure layer  16  can have a thickness of about 2 to 50 nm, about 5 to 20 nm, about 10 to 20 nm, and about 10 nm. It should be noted that the thickness of the silicon nanostructure layer  16  could be controlled by modifying fabrication conditions. Therefore, the thickness can be optimized for particular uses of the MALDI sample plate  10 . The nanostructures can have a diameter of about 8 to 12 nm.  
      In general, the deposition of silicon nanostructure layer  16  is performed in a chemical vapor deposition reactor. Typically, the substrate  12  with the metal nanostructure catalyst layer  14  is mounted on a susceptor and is heated to a deposition temperature (about 400-700° C.) and a gaseous precursor mixture is passed over the substrate  12  and nanostructure catalyst layer  14 . The gaseous precursor mixture contains a Si precursor such as silane (SiH 4 ) or disilane (Si 2 H 6 ), for example, and carrier gas, such as H 2  or N 2 . As molecules of the gaseous precursor contact the nanoparticles of the metal nanostructure catalyst layer  14 , they are catalytically decomposed and a layer of close-packed Si nanoparticles is deposited on the surface of the substrate and metal nanostructure catalyst layer (silicon nanostructure layer  16 ). Our analysis indicates that the metal nanostructure catalyst layer  14  is substantially buried underneath the silicon nanoparticles layer, where “substantially buried” refers to interaction between the analytes and metal being prohibited or substantially prohibited.  
      The silicon nanostructure layer  16  can be formed by techniques such as, but not limited to, chemical vapor deposition (CVD) and the like. The spacing (e.g., density of the nanostructures) of the nanostructures adjacent one another can be controlled, at least in part, by the fabrication conditions and the metal nanostructure catalyst layer  14 . In this regard, the spacing can be controlled to produce a silicon nanostructure layer appropriate for a particular MALDI application.  
      The silicon dioxide layer  18  is formed on the silicon nanostructure layer  16 . The silicon dioxide layer  18  can have a thickness of about 2 to 10 angstroms (Å), about 2 to 6 Å, and about 2 to 4 Å. The silicon dioxide layer  18  is formed by oxidation of silicon by oxygen in air. In this regard, the silicon dioxide layer appropriate for a particular MALDI application can be controlled while being formed.  
       FIGS. 2A through 2D  illustrate cross sectional views that illustrate an embodiment of a method  100  for forming the MALDI sample substrate  10  shown in  FIG. 1 .  FIG. 2A  illustrates a cross sectional view of the substrate  12 .  FIG. 2B  illustrates the formation of the metal nanostructure catalyst layer  14  on the substrate  12 . The metal nanostructure catalyst layer  14  can be formed by techniques such as, but not limited to, electron-beam evaporation and the like.  
       FIG. 2C  illustrates the formation of the silicon nanostructure layer  16 . The silicon nanostructure layer  16  can be formed by techniques such as, but not limited to, chemical vapor deposition (CVD), and the like. The silicon nanostructure layer  16  is formed in a manner consistent with the description above.  
       FIG. 2D  illustrates the formation of the silicon dioxide (SiO 2 ) layer  18  on the silicon nanostructure layer  52 . The silicon dioxide layer  18  can be formed by the oxidation of silicon by oxygen in air.  
       FIG. 3  illustrates a mass spectrometry system  20  implemented in accordance with embodiments of the MALDI sample substrate. The mass spectrometry system  20  includes an ionization source  22 , which operates to produce ions. In the illustrated embodiment, the ionization source  22  produces ions using MALDI. However, it is contemplated that the ionization source  22  can be implemented to produce ions using any other ionization process, such as vacuum MALDI or Atmospheric Pressure-Matrix Assisted Laser Desorption Ionization (“AP-MALDI”), Atmospheric Pressure Photo Ionization (“APPI”), and the like. It is also contemplated that the ionization source  22  can be implemented as a multi-mode ion source that produces ions using a combination of ionization processes. As illustrated in  FIG. 3 , the mass spectrometry system  20  also includes a detector system  60 , which is positioned downstream with respect to the ionization source  22  to receive ions. The detector system  60  operates to detect ions as a function of mass to charge ratio.  
      As illustrated in  FIG. 3 , the ionization source  22  includes a light source  26 , which operates to produce incident light  16 . In the illustrated embodiment, the light source  26  is implemented as a laser that produces the incident light  52  in the form of a laser beam. Typically, the laser beam is pulsed and comprises a wavelength or a range of wavelengths in the ultraviolet range. However, it is contemplated that the laser beam need not be pulsed and can include any other wavelength or range of wavelengths.  
      In the illustrated embodiment, the ionization source  22  also includes a housing  46  that defines an ionization region  48  within which ions are produced. For certain implementations, the ionization region  48  can be maintained at a low pressure, such as under high vacuum conditions. As illustrated in  FIG. 3 , the ionization source  22  also includes a sample support  10 , which is positioned within the ionization region  48  and is optically coupled to the light source  26  via a reflector  34 . The MALDI sample substrate  10  operates to support or hold a sample  44  that contains an analyte to be analyzed by the mass spectrometry system  20 . For example, the sample  44  can include a co-precipitate of the analyte and a matrix, and the matrix can exhibit a strong absorption of the incident light  52 . During operation, the light source  26  produces the incident light  52 , which is directed into the ionization region  48  and reaches the MALDI sample substrate  10  via the reflector  34 . The incident light  52  interacts with the sample  44  to produce ions from the analyte. The ions are released into the ionization region  48  and eventually reach the detector system  60 .  
      The detector system  60  includes a mass analyzer  54 , which operates to separate or select ions by mass-to-charge ratio. In the illustrated embodiment, the mass analyzer  54  is implemented as a time-of-flight analyzer. However, it is contemplated that other types of mass analyzers can be used, such as ion trap devices, quadrapole mass spectrometers, magnetic sector spectrometers, and the like. As illustrated in  FIG. 3 , the mass analyzer  54  includes a capillary  28 , which defines an internal passageway  38 . During operation, ions are produced by the ionization source  22 , and the ions pass through the capillary  28  via the internal passageway  38 .  
      As illustrated in  FIG. 3 , the mass analyzer  54  also includes a gas source  32  and a gas conduit  36  that encloses the capillary  28 . The gas conduit  36  is fluidly coupled to the gas source  32  and operates to supply an inert gas to the ionization region  48 . Referring to  FIG. 3 , the detector system  60  also includes a detector  58 , which is positioned with respect to the mass analyzer  54  to receive ions. During operation, ions pass through the capillary  28  and eventually reach the detector  58 , which operates to detect the abundance of the ions and to produce a mass spectrum.  
      During operation of the mass spectrometry system  20 , absorption of light by the sample  44  can affect ionization efficiency for the analyte, which, in turn, can affect sensitivity of mass spectrometric analyses. Accordingly, it is desirable to enhance absorption of light by the sample  44 , such that mass spectrometric analyses have a desired level of sensitivity.  
      The MALDI sample substrate  10  includes the substrate  12 , the metal nanostructure catalyst layer  14 , the silicon nanostructure layer  16 , and the silicon dioxide (SiO 2 ) layer  18 . Advantageously, the metal nanostructure catalyst layer  14  can enhance absorption of light by the sample  44  by reflecting the incident light  52  back towards the sample  44 . During operation, a portion of the incident light  52  that is not initially absorbed by the sample  44  passes through the sample  44  and eventually reaches the metal nanostructure catalyst layer  14 . In turn, the metal nanostructure catalyst layer  14  can reflect this portion of the incident light  16  back towards the sample  44 . In such manner, the metal nanostructure catalyst layer  14  can provide multi-path irradiation of the sample  44  to enhance a capture cross-section of the incident light  52 , thus promoting production of ions from the analyte. At the same time, there would be little or no interaction between analytes and metal nanostructure catalyst since the catalyst layer is underneath the silicon nanoparticle layer.  
      In conjunction with enhancing absorption of light by the sample  44 , the MALDI sample substrate  10  can exhibit a number of other characteristics that are desirable for mass spectrometry. For example, another benefit of the MALDI sample substrate  10  is that it can be highly robust. Thus, the MALDI sample substrate  10  can exhibit little or no tendency to degrade under typical operating conditions, thus reducing undesirable chemical background noise in a mass spectrum. Robustness of the MALDI sample substrate  10  can also allow the sample support  10  to be readily cleaned and to be reused for multiple tests. Another benefit of the MALDI sample substrate  10  is that it can be highly inert with respect to typical analytes for mass spectrometry. Accordingly, use of the MALDI sample substrate  10  can reduce undesirable interaction with an analyte for a current test as well as reduce contamination of the MALDI sample substrate  10  with a residual analyte from a previous test.  
      Another advantage is that the disclosed MALDI substrate provides high reproducibility. As can be perceived from the manufacturing process, e-beam evaporation and CVD can generate very homogeneous catalyst deposition and silicon nanoparticle deposition. At the scale of typical spots for MALDI measurement (mm scale), the reproducibility provided by this substrate is crucial for quantitative measurement.  
      It should be emphasized that the above-described embodiments of the present disclosure, are merely possible examples of implementations, merely set forth for a clear understanding of the principles of the disclosure. Many variations and modifications may be made to the above-described embodiment(s) of the disclosure without departing substantially from the principles of the disclosure. All such modifications and variations are intended to be included herein within the scope of this disclosure and the present disclosure and protected by the following claims.  
     EXAMPLES  
      An example of the AFM pictures of a nanostructured Au/Si/SiO 2  surface is shown in  FIG. 4 . The surface contains closely packed Si nanoparticles (with height about 5-9 nm). The amount of Au detected by XPS is negligible (about 0.3%), indicating that the amount of Si deposited on Au is of the order of about 10 nm. It has been verified that the surface characteristics and layer structures remain unchanged after standard process flow for surface chemical modification.  
       FIG. 5  illustrates a MALDI measurement using a titanium nitride surface, while  FIG. 6  illustrates a MALDI measurement on nanostructured Au/Si/SiO 2  surface. AP Maldi experiments were carried out with Agilent ion trap LC/MSD Ion Trap Plus. The nitrogen laser at 337 nm (10 Hz) was used as source. Ion trap accumulation was synchronized with laser firing. Ions from two laser shots were accumulated for each microscan. Each spectrum was an average of 16 microscans. 0.5 min of data ( 8  spectra was collected, representing total of 256 laser shots. Both Agilent TiN (Agilent G1972-60025) substrate and the nanostructured Au/Si/SiO 2  substrate were used. Standard BSA digest was spotted at 500 attomole in 0.25 mg/ml CHCN matrix. The following results showed nanostructured Au/Si/SiO 2  substrate provided better results.