Anal Bioanal Chem (2012) 402:601–623 DOI 10.1007/s00216-011-5120-2

REVIEW

Gold nanomaterials as a new tool for bioanalytical applications of laser desorption ionization mass spectrometry Rosa Pilolli & Francesco Palmisano & Nicola Cioffi

Received: 30 March 2011 / Revised: 13 May 2011 / Accepted: 17 May 2011 / Published online: 5 June 2011 # Springer-Verlag 2011

Abstract Nanomaterials have emerging importance in laser desorption ionization mass spectrometry (LDI–MS) with the ultimate objective being to overcome some of the most important limitations intrinsically related to the use of conventional organic matrices in matrix-assisted (MA) LDI–MS. This review provides a critical overview of the most recent literature on the use of gold nanomaterials as non-conventional desorption ionization promoters in LDI– MS, with particular emphasis on bioanalytical applications. Old seminal papers will also be discussed to provide a timeline of the most significant achievements in the field. Future prospects and research needs are also briefly discussed. Keywords Laser desorption ionization . Mass spectrometry . Gold nanomaterial . SALDI . SELDI . NALDI

Introduction Matrix-assisted laser desorption ionization mass spectrometry (MALDI–MS) is a high-throughput analytical tool for the detection of a wide range of compounds, above all biological macromolecules [1, 2]. Enabling rapid desorpR. Pilolli : F. Palmisano : N. Cioffi (*) Dipartimento di Chimica, Università degli Studi di Bari “Aldo Moro”, Via Orabona, 4, 70126 Bari, Italy e-mail: [email protected] F. Palmisano : N. Cioffi Centro Interdipartimentale di Ricerca S.M.A.R.T., Università degli Studi di Bari “Aldo Moro”, Via Orabona, 4, 70126 Bari, Italy

tion of high-molecular-weight molecules, with either limited or no fragmentation, remarkable detection accuracy, and sensitivity over a wide mass range, MALDI–MS is an ideal choice for direct identification of biomolecules, even in complex samples. Typical sample preparation involves mixing the analyte with an organic UV-absorbing matrix. The latter is crucially important in this technique because it absorbs the laser energy and promotes desorption and ionization of the analyte into the gas phase. Matrix selection and optimization in the sample-preparation procedure are important stages of the analysis although, unfortunately, they are typically based on an empirical approach, thus limiting the usability of the technique. In fact, the main drawbacks of MALDI–MS are directly ascribable to the organic matrices themselves. Remarkable spectral interferences from matrixrelated signals make the spectral range below 800 m/z, scarcely accessible. Moreover, the non-uniform matrix crystallization or matrix–analyte co-crystallization steps, severely affect the efficiency of LDI processes, and the resulting signal quality in mass spectra. Consequently, limited shot-to-shot and sample-to-sample reproducibility prevents the challenge of quantitative analysis from being fully addressed [3]. Several alternative approaches have been proposed for analysis of low-molecular-weight (LMW) compounds; these have been based on the use of high-molecularweight organic matrices, silicon substrates and nanoporous surfaces, sol–gel materials, polymers, and carbon-based micro- or nanostructures; most of these have already been reviewed elsewhere [3–7]. More recently, replacing conventional organic matrices with metal nanoparticles (NPs) and nanostructured metal surfaces is a further promising alternative [8–12]. Indeed these nanomaterials, because of their unique properties, can

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meet the same requirements that conventional organic matrices fulfil, i.e. they can: 1. absorb the incoming laser radiation; 2. transfer the absorbed energy to the analyte, promoting its desorption; and 3. provide a source of ionization (mainly by cationization processes). Furthermore, the nanostructure surface is expected to efficiently interact with analyte molecules, thus preventing their detrimental aggregation. Unlike organic matrices, further advantages can potentially be achieved, namely, reduction of spectral interferences in the low m/z range, and simplification of the sample-preparation procedure, thus improving the reproducibility of the mass spectra. Early work by Tanaka et al. demonstrated the ability of NP dispersions in glycerol to desorb/ionize large molecules, for example proteins and polymers, in the “two-phase MALDI” approach [13]. The basic features supporting the application of metal nanoparticles in MALDI–MS were soon recognized—high photo-absorption, low heat capacity, and extremely large surface area per unit volume [13]. However, the contemporary introduction of organic matrix by Hillenkamp et al. [14] obscured the role of NPs. Only after the introduction of DIOS (desorption ionization on silicon) [15], the proved correlation between LDI enhancement and nanostructured surfaces generated renewed interest in the use of nanomaterials; recent and straightforward advances in nanotechnology have, moreover, provided the necessary tools to develop a new generation of high-throughput devices. Since then, several metal nanostructure morphologies and functionalization approaches have been proposed, in order to improve LDI–MS performance in terms of selectivity, sensitivity, reproducibility, and suitability for the detection of LMW analytes. Some review papers on the application of metal nanomaterials (and particularly nanoparticles) in this field have recently been published [8–11]. The objective in this paper is to provide a comprehensive critical overview of the use of gold-based nanomaterials (thin metallic films, nanorough surfaces, spherical and rodlike particles, functionalized NPs, nanofractals, nanowires, etc.) in LDI–MS bioanalysis. In the last two decades, gold nanomaterials have been the preferred choice for NP-based LDI–MS applications, because of a huge number of wellestablished synthetic routes able to provide high dimensional control. Moreover, the ease of surface design, mainly based on Au–S linkage chemistry, has enabled fully customizable nanomaterials which can address applicationrelated requirements. This paper is organized in three sections dealing with specific aspects of the use of gold nanomaterials in LDI–

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MS. First, their use as extraction and concentration probes in conventional MALDI–MS analysis is discussed. The second section focuses on thin gold films (from 10 to 120 nm thick) as active promoters of LDI processes. The third section, devoted to the application of different gold nanostructures, is organised into sub-sections according to the wavelength of the incident laser irradiation, and the kind of nanomaterials used (as-synthesized nanomaterials, engineered nanomaterials for enhanced ionization or selectivity, and implanted nanomaterials for tissue imaging).

AuNPs as extraction and concentration probes in conventional MALDI–MS analysis Sample-preparation procedures are crucial steps in determining the quality of the mass spectra generated; therefore, a wide variety of approaches has been developed to maximize the information content provided by MALDI– MS. Extraction and concentration methods can be roughly grouped into two classes: on-probe (pre-concentration steps directly performed on the MALDI target) and off-probe procedures (purification performed by traditional chromatographic separation techniques and/or solid-phase extraction methods, for example Ziptip [16]). The first approach enables reduction of sample contamination and analyte losses by minimizing sample handling and is used either for desalting [17–19] or for bio-affinity-based purification in surface-enhanced laser-desorption ionization (SELDI) MS methods [20–22]. The off-probe approach basically decouples the extraction and MALDI analysis steps so they can be independently optimized; implying more laborious sample manipulation, the latter approach increases the potential for contamination and sample loss. An alternative strategy, combining some advantages of both the on and off-probe methods, consists in the use of discrete particles (or beads) to extract and concentrate analytes followed by their direct MALDI–MS analysis. In this technique nanoparticles are extremely important, offering many desirable attributes for the development of selective extraction and pre-concentration agents. Compared with the micron-sized particles commonly used in most solid-phase extraction methods, NPs have much higher surface area-to-volume ratios, thus leading to a greater extraction capacity. Gold-based nanoparticles (AuNPs), in particular, have well established biocompatibility which facilitates their in-vitro interaction with biological samples, including tissues and cells (Ref. [8] and references cited therein). Some bare metal and metal oxide NPs have intrinsic affinity for some functional groups; they can, therefore, be used to selectively extract and enrich specific classes of analytes (e.g. TiO2 NPs for phosphopeptides [23], or

Gold nanomaterials as a new tool for LDI-MS applications

AuNPs for thio compounds [24]). Furthermore, design of the NP surface provides customizable interactions with biomolecules such as cell membrane lipids, proteins, and nucleic acids, thus leading to new means of improving mass spectrometric performance. The use of NPs for selective extraction of specific analytes has been designed for both non-covalent (either non-specific [25–28] or bio-specific [29–31]) and covalent extraction [32–36]. Non-covalent, non-specific extractions are mainly based on electrostatic and/or hydrophobic interactions [25–28]. Charged surface ligands (i.e. anionic or cationic stabilizers) encapsulating AuNPs prevent their aggregation in solution, enabling electrostatic interaction with oppositely charged species from the sample solution. This approach has been typically used to develop nanoparticle-based probes that selectively trap and concentrate target species in sample solutions with efficiencies that depend on the peptide isoelectric point (pI) and solution pH [25–27]. In 2004, AuNPs–magnetic particle conjugates were evaluated as selective and concentrating probes for the detection of small amounts of peptide residues from the tryptic digest products of 100 nmol L−1 cytochrome c [25]. Similarly, in 2006, mixed monolayer protected AuNPs with a mean core diameter of 2 nm were used to efficiently preconcentrate several peptides, providing detection limits as low as 500 pmol L−1 for bradykinin [26]. Noteworthy, practical advantages have been achieved, minimizing sample manipulation by direct MALDI–MS analysis, leading to reduced analysis time without substantial sample losses and contamination [26]. In 2005, Sudhir et al. first introduced gold nanoparticles into single-drop micro-extraction (SDME) [27]. Tetraalkylammonium bromide-capped AuNPs prepared in toluene were used as electrostatic probes for rapid analysis and signal enhancement of peptide mixtures in atmosphericpressure MALDI–MS. Conditions affecting the extraction yield, for example solvent selection, extraction time, agitation rate, and pH were investigated in depth, enabling detection of Met-enkephalin and Leu-enkephalin at concentrations of 200 and 170 nmol L−1, respectively [27]. Non-covalent, biospecific interactions have also been achieved [29–31]. Evans-Nguyen et al. reported the use of custom-patterned nanostructured gold substrate for affinity extraction of proteins. Patterning a super-hydrophobic substrate with hydrophilic spots created a strong wettability contrast, providing a highly effective microarray for protein detection [31]. A gold NPs assembly entrapping enzymes was designed to build up a microchip reactor for efficient on-line proteolysis of low-abundance proteins and complex extracts [29]. The nanostructured network containing trypsin was assembled via layer-by-layer electrostatic deposition of

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poly(diallyldimethylammonium chloride) (PDDA) and AuNPs on poly(ethylene terephthalate). The specific features of the PDDA/AuNPs network provided high affinity for immobilization of the enzyme and also hosted the enzymes in a favourable microenvironment. Trace amounts of proteins (e.g., 16 fmol cytochrome c), were digested by use of the microchip reactor, more quickly than in insolution digestion; the resulting peptides were then successfully analysed by MALDI–MS [29]. The applicability of MALDI–MS to the analysis of molecules such as DNA, characterized by poor ionization efficiency and facile fragmentation, can be severely restricted [37]. To overcome this limitation, the translation of DNA hybridization events into peptide identification by MALDI–MS has recently been proposed [30]. As illustrated in Fig. 1a, the plasmid-encoded peptide tags act as surrogate molecules for mass spectrometric identification of the target DNA (a possible disadvantage is the additional steps required to obtain the surrogate peptide). However, the potential of this DNA-detection strategy was fully supported by both the detection capability achieved under non-optimized conditions (4 pmol L−1, Fig. 1g), the absence of false positive results, and the possible application in parallel multiplexed analyses at reasonable cost [30]. Finally, covalent extractions have been achieved by forming covalent bonds between the target analytes and either the functional groups on the AuNP surface [32–34, 36] or the metal surface itself [35]. Covalent bonding enables non-specifically bound analytes to be more effectively removed via an extensive washing procedure. Selective enrichment of glycopeptides using 4mercaptophenylboronic acid (4-MPBA)-modified colloidal [32] and supported [33] gold nanoparticles has been proposed on the basis of the well known ability of the boronic acid group to form strong but reversible covalent bonds with glycopeptides containing cis-1,2-diol groups. In the first example, 4-MPBA-functionalized NP suspensions were mixed directly with glycoprotein solutions and, after incubation, recovered by centrifugation [32]. In the second example, the same nanoparticles were spotted and sintered on a stainless steel plate, providing a porous substrate for on-plate selective enrichment [33]. Good enrichment factors were obtained for both standard solutions and complex samples, for example glycopeptide residues from tryptic digestion and peptide mixtures at picomolar [32] and nanomolar [33] concentrations, respectively. Two major advantages were provided by the supported designed AuNPs compared with the colloidal analogues: the in-situ enrichment of glycopeptides on MALDI target plate spots (which acted as both solid-phase extractor and support of the matrix for the LDI processes), and the reusability of these functionalized target plates after regeneration in acidic solution [33]. Control experiments were performed to

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Fig. 1 Schematic representation of: (a) AuNP probe design and fabrication via functionalization of citrate-stabilized AuNPs, (b) DNA detection by MALDI–TOF MS identification of the surrogate peptide (SP) expressed by a plasmid. Single-target DNA detection: (c) MALDI–TOF spectrum in the absence of the target; (d) MALDI– TOF spectrum of an independently synthesized peptide with the same

sequence of SP; MALDI–TOF spectrum in the presence of the target at a concentration of 40 μmol L−1 (e), 40 nmol L−1 (f), 40 pmol L−1 (g), 4 pmol L−1 (h) (1484 Th, [SP + H]+, 1522 Th, [SP + K]+). (Reprinted with permission from Ref. [30]. Copyright (2010) American Chemical Society)

confirm the absence of memory effects after plate regeneration; the enrichment capability was fully preserved after two regeneration cycles. Aminoxy-functionalized AuNPs have been used to efficiently capture glycosphingolipids (GSLs) [34]. Figure 2 illustrates the general strategy for selective enrichment of cellular GSLs on to the AuNPs surface by glycoblotting, a method based on chemoselective oxime bond formation between aldehyde and aminoxy functional groups. GSLs were extracted from melanoma cells or mouse brain samples and converted into aldehydes by selective ozonolysis of carbon–carbon double bonds. The GSLs captured by glycoblotting were then directly analysed by MALDI– MS [34]. It is worthy of note that this contribution was the first comprehensive (structural and functional) glycosphingolipidomics approach based on the glycoblotting method, with a widespread utility for identifying and characterizing whole GSLs present in living cell membranes. Recently, a simple method for identification of protein Smodification sites was proposed in which AuNPs were used simultaneously as isolating and enriching probe for thiolcontaining peptide residuals from digestion [35]. The MALDI–MS analysis of these AuNP-bound peptides resulted in significant enrichment of free thiol-containing

species, and S-nitrosylated, S-glutathionylated, and Salkylated peptides [35].

Thin gold film-assisted LDI–MS analysis The use of thin gold films to assist the direct LDI–MS analysis of low-molecular-weight species can be traced back to the early nineties. In 1990, Li et al. investigated DI mechanisms of small peptides from 100 nm to 500 μm thick gold films. Analytes could be detected in negative-ion mode and very simple spectra were obtained, with limited fragmentation. The occurrence of either thermal or nonthermal desorption processes was shown to be a function of the wavelength of the incoming laser radiation. Thin Au films, reaching a high surface temperature much more easily than thick films, had lower threshold energy values for the LDI of peptides. A mechanism involving excitation of Au electrons, followed by hot electron transfer to the adsorbate was hypothesized to occur at shorter excitation wavelengths, for example 248 nm [38]. The acronym TGFA-LDI (thin gold film-assisted laser desorption/ionization) was used few years later by Wahl et al. [39], who investigated processes involving gold films

Gold nanomaterials as a new tool for LDI-MS applications

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success [39]. It is worthy of note that the detection limit for gramicidin S was 100 fmol per laser shot; this yielded an FT-ICR mass spectrum with signal-to-noise ratio of ca. 5 [39]. Thin Au film-coated nanoporous alumina substrates have also been proposed as active surfaces for direct LDI– MS [40–43]. The effects of several structural properties, for example Au layer thickness [41], alumina pore size (i.e. depth and width) [41], and geometry (i.e. order and density) [42] were independently investigated (Fig. 3). Several authors agreed the nanoporous structure was crucially

Fig. 2 (a) A general strategy for selective enrichment of cellular glycosphingolipids (GSLs) on an aminoxy-functionalized AuNPs (aoAuNPs) surface by glycoblotting. (b) Oxime bonds formed between GSL aldehydes and aoAuNPs are disrupted by laser irradiation in the MALDI process to afford highly sensitive imino alcohol ions. (Reprinted with permission from Ref. [34]. Copyright (2009) American Chemical Society)

much thinner than those previously proposed [38]. A film 10 nm thick, evaporated on a glass slide, was successfully used at the fundamental wavelength of a Nd:YAG laser (near-IR, 1064 nm). The gold film acted as vehicle for analyte desorption into the gas phase, whereas ionization was achieved by adduct formation with added traces of alkali ions. The film thickness was tuned in order to match its maximum absorption wavelength to the excitation laser wavelength. A tentative explanation of the observed phenomena was provided: the rapid heating caused by laser irradiation supplied an adequate amount of energy to vaporize the thin gold film and to bring single or clustered metal atoms into the gas phase with the analyte, without appreciable fragmentation. As a proof of concept of the feasibility of this new approach, several classes of analyte, for example peptides, nucleotides, saccharides, and phospholipids, were tested, although with different degrees of

Fig. 3 (a) Scanning electron microscopy images of porous alumina surface showing the pore-widening effect of etching with phosphoric acid solution. (b) Variation of LDI signal intensity with different pore width. (c) Variation of LDI signal intensity with different thickness of the gold layer (Al thicknesses, 500 and 700 nm; analyte angiotensin II 40 pmol). Data labelled “degraded sample” are from repeated laser shots on the thinnest gold layer after the initial 100 shots used to plot the upper point on the graph. (Reprinted with permission from Ref. [41]. Copyright (2007) American Chemical Society)

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important to the improved ion yield. Nevertheless alumina was not directly involved in the laser energy adsorption and transfer; the laser beam, in fact, was hypothesized to interact with the free electrons at the surface of the thin Au layer thereby enabling rapid temperature increase by a surface plasmon resonance effect. The porous alumina layer was hypothesized to act only as a thermal insulator, the energy confinement being a function of pore depth [41–43]. The versatility of this technique was assessed for a variety of analytes with a relatively wide range of molecular weights (from 260 to 5800 Da; Table 1). When systematic comparison with conventional MALDI– MS analyses was carried out, results were similar in terms of signal intensity even if at slightly higher laser power [41]. Au-coated porous alumina surfaces are, therefore, a useful low-cost substitute for conventional MALDI matrices, without the variability arising from sample preparation (e.g. co-crystallization) and with reduced background in the low-m/z region. Furthermore, it is also worthy of note that coating the surface with noble metal conferred the additional advantage of extending the plate shelf-life: ionization performance of 10 fmol angiotensin I remained unchanged over several months exposure to air [42]. As a further confirmation of the reliability of this approach, a nanoporous calcined silicate film was recently

fabricated on a gold substrate and successfully used for matrix-free LDI–MS analysis of biomolecules [44, 45]. Although with an inverted configuration, the twocomponent-based operating system was reconfirmed: the highly absorbing and conductive metal collects the incident laser energy whereas the nanoporous layer confines the thermal energy, thus maximizing the local temperature peak [44, 45]. Direct comparison with conventional MALDI was performed. Several aspects, for example ease of preparation and modification of the surface, reproducibility, cost, compatibility with common organic solvents, wettability, need for specific additive, shelf life, and reusability of the surfaces were evaluated. The efficiency of the film in LDI–MS was outstanding, with extremely low background noise in the low-m/z region; the useful mass range was large enough to include both amino acids and small proteins (Fig. 4 and Table 1). The detection limit with assistance of citric acid was at the sub-picomole level (i.e. 100 fmol [Sar1, Thr8]-angiotensin II gave a S/N ratio of approximately 38) [44], and could be further improved by chemical functionalization of the calcined upper layer with a hydrophobic octadecyltrichlorosilane (OTS) monolayer [45]. OTS-calcined nanofilms guaranteed sensitivity enhancement (low femtomolar range) by eliminating interferences from contaminants, for example salts and surfactants, which arose from a

Table 1 Analytical conditions used for gold thin-film-assisted laser desorption ionization MS Gold thin film (thickness)

Substrate

Laser source/ analyser

Additives

Analyte

Ref.

Thermally evaporated (10 nm)

Glass

Nd:YAG laser (1064 nm)/FTICR N2 laser (337 nm)/ToF N2 laser (337 nm)/ToF

Potassium bromide 5 mmol L−1

Gramicidin S, leucine enkephalin, gramicidin D, guanosine

[39]

Citrate buffer

[43]

N2 laser (337 nm)/ToF N2 laser (337 nm)/ToF

Citric acid 40 mmol L−1

Tryptic digest of BSA, tryptic digest of catalase, tryptic digest of lactoperoxidase Angiotensin I, insulin, reserpine Triton X, poly(propylene glycol), poly(1,4butylene adipate) Angiotensin II, bradykinin, tryptic digest of BSA, insulin peptide calibration mixture Angiotensin I

N2 laser (337 nm)/ToF

Citric acid 10 mmol L−1

Peptide mix, amino acid mix, insulin chain B, cytochrome C

[44]

N2 laser (337 nm)/ToF

None NaCl 1 mmol L−1 / NaAc 200 mmol L−1 / 8 mol L−1 urea None NH4HCO3 50 mmol L−1 800 mmol L−1 urea

Peptides mix Peptides mix

[45]

Sputter coated Nanoporous (120 nm) alumina Sputter coated (50 nm) Submicroporous alumina Sputter coated Nanoporous (120 nm) alumina Sputter coated (50 nm) Ordered nanoporous alumina e-Beam sputtered Stainless steel nanoscale calcined tape /glass (46 nm) e-Beam sputtered Stainless steel nanoscale calcined tape /glass (46 nm)

Trifluoroacetic acid 0.1% Sodium iodide 1 mg mL−1

Trifluoroacetic acid 0.1%

Abbreviations: FT-ICR, Fourier transform ion cyclotron resonance; TOF, time of flight

Peptides mix Tryptic digest of cytochrome C Tryptic digest of BSA

[40]

[41] [42]

Gold nanomaterials as a new tool for LDI-MS applications

Fig. 4 Mass spectra obtained from a peptide mixture ([Sar1, Thr8]angiotensin II and neurotensin, 20 pmol each with 10 mmol L−1 citric acid) on a calcined glass surface (a) and with conventional CHCA matrix (b). Mass spectra obtained from an amino acid mixture (Lys, His, and Arg, 60 pmol each with 10 mmol L−1 citric acid) on a calcined glass surface (c) and with conventional CHCA matrix (d).

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Mass spectra obtained from insulin chain b, 20 pmol (e) and cytochrome c from bovine heart, 40 pmol (f) with 10 mmol L−1 citric acid. Inset: spectrum obtained with CHCA matrix on a steel MALDI plate under the same conditions. (Reprinted with permission from Ref. [44]. Copyright (2010) American Chemical Society)

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simple on-plate desalting step. Three main features accounted for this improved performance: & & &

the “buffer” effect of the OTS layer, reducing the thermal conductivity of the film; the increased stability of the latter to the laser irradiation; and the reduction of the sample spot size, owing to the surface hydrophobicity [45].

In addition to widening the mass range, further advantages are worthy of mention, for example high reproducibility, low cost, excellent long-term durability, and reusability up to 10 cycles [44]. The most recent relevant application of thin Au films was in MS imaging: coating a tissue section (Miscanthus x giganteus) with a thin layer of metal was found to be effective in reducing surface charging and enhancing MS imaging quality [46]. However, when direct, conventional matrix-assisted and Au film-assisted LDI experiments were compared it was found that no single sample-preparation method provided complete coverage of the ions of interest. Therefore, for this specific application, a combination of different matrices was needed to maximize the chemical information obtained while minimizing matrix interference [46].

Gold nanomaterial-assisted LDI–MS analysis As-synthesized nanomaterials Focusing on the use of gold nanomaterials for direct LDI– MS analysis of biomolecules without any other organic adjuvant, the investigation performed by Russell and coworkers in 2005 is a milestone [47]. For the first time, sizeselected AuNPs (2, 5, and 10 nm in diameter) were proposed as low concentration and selective DI promoters for peptides and small proteins. The conventional matrixto-analyte (M/A) ratio of 104:1 required for organic UVabsorbing molecules, was reversed to 1:107–109, confirming results previously obtained from samples implanted with massive gold clusters (i.e., Au4004+) [48]. AuNPmediated analyte ionization, as performed in both positive and negative modes, with different success. Analyte cation adducts (i.e. [M + H]+, [M + Na]+, and [M + K]+) and deprotonated molecular ions were observed in positive and negative-ion modes, respectively, and Au cluster-related ions were observed in both modes (Fig. 5). In general, spotto-spot precision, defined as relative standard deviation (RSD), in signal intensity was below 10%, with limits of detection of approximately 100 fmol [47]. Although the latter LOD value was not dramatically lower than those obtained by use of other methods, it is worth noting that

they could be obtained under unusual experimental conditions, resulting in significant spectral advantages at low m/z values. Moreover, AuNP LDI promoters were shown to afford selectivity, typically not observed with organic matrices, leading to the preferential ionization of phosphotyrosine (pTyr) over phosphoserine (pSer) or phosphothreonine (pThr)-containing peptides [47]. A tentative explanation of the energy-transfer processes driving the AuNP-assisted LDI, was provided on the basis of the same thermal mechanism proposed by Tanaka et al. [13]. For all the size distributions used, the calculated heat diffusion length (ddiff) is ca 3 orders of magnitude higher than the NP diameter, indicating that the entire NP volume reached the same temperature (with a calculated maximum surface temperature of 104 K). This model could account for the mass spectra obtained using 5 and 10 nm AuNPs but could not explain the different ion yields observed for 2 nm AuNPs. Quantum confinement effects arising from the latter were hypothesized to result in electronic excitationbased energy transfer and analyte ionization [47]. Starting from the bases provided by Russell and coworkers, several papers were published with the objective of addressing specific open issues of this innovative application [49–64]. A few fundamental studies have been carried out, providing detailed information on the appropriate choice of metal NPs [56] and their surface chemistry [57], both of which significantly affect the efficiency of LDI processes. Stabilizer-free bare nanoparticles of Ag, Au, Cu, and Pt were prepared by laser ablation and their LDI performance for detection of peptides, surfactants, and synthetic polymers was compared. It was demonstrated, for the first time, that MS performance was largely dependent on the NP species and that gold and platinum NPs outperform silver and copper NPs in the LDI–MS of peptides (at least when citrate buffer was added as a proton source) [56]. It was hypothesised that these differences were because of the different laser-induced temperature increases achieved under irradiation, arising from specific physical properties of each metal, i.e. absorbance at the working wavelength, heat capacity, melting temperature, and heat conductivity. Moreover, the effects of different salts on the physical properties of AuNPs and on the resulting LDI ion yields were evaluated, revealing significant sensitivity to the presence of passivating impurities. Three key issues regarding the surface chemistry were emphasized: 1. species (for example fluoride and chloride) that did not interact (chemically and physically) or that were chemically unreactive towards the AuNP, did not affect the LDI processes; 2. reactive species (for example iodide) led to dramatic changes in the LDI ion yields; and

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Fig. 5 LDI-TOF mass spectra of substance P obtained by using AuNPs: (A) 2 nm, (B) 5 nm, and (C) 10 nm (337 nm, laser energies of ca. 120 and 180 μJ pulse−1 for positive and negative mode LDI,

respectively). (Reprinted with permission from Ref. [47]. Copyright (2005) American Chemical Society)

3. modification of the AuNP surface with organic ligands minimized or eliminated such effects, protecting and stabilizing the NPs [57].

are more tolerant of high salt concentrations, enabling direct analysis of untreated complex biological sample, for example urine (Fig. 6) [51]. Another potential application of both bare and citratecapped AuNPs which has attracted increasing attention is the detection of aminothiols, for example glutathione (GSH), cysteine (Cys), and homocysteine (Hcys), owing to their important biological functions [50, 53–55]. Because an increase in the concentrations of these thio compounds can be correlated with several diseases, many separation techniques typically coupled to MS and laser-induced fluorescence detection [53] have been developed for their analysis in biological samples; however expensive and time-consuming sample pretreatment procedures are usually required. Clearly, in these circumstances, use of AuNPs as selective probes and promoters for direct LDI–MS detection makes them important analytical tools. To provide a valid approach for the analysis of aminothiols, Chang and co-workers recently proposed the use of a mixture of two AuNPs of different sizes (average diameters 3.5 and 14 nm) [53]. Several conditions were investigated—the molar ratio of the AuNPs, buffer concentration, sample pH, and the effect of salt—to maximize detection sensitivity. Using the 3.5 and 14-nm AuNPs as probe and promoter, respectively, in the presence of 0.5 mmol L−1 ammonium citrate, quantitative LDI–MS analysis afforded limits of detection of 2 fmol for GSH, 20 fmol for Cys, and 44 fmol for Hcys, with good quality calibration plots [53]. The improved sample-preparation procedure and spot homogeneity resulted in significantly higher spot-to-spot reproducibility; the signal intensities varied by less than 20% for AuNPassisted LDI–MS analyses recorded over 50 sample spots,

Among the most attractive LMW biomolecules, neutral carbohydrates are a challenge for direct MALDI analysis, because of the absence of strong basic or acidic groups in their structure. Bare AuNPs, although less chemically stable than capped AuNPs, have been proved to efficiently capture different small neutral carbohydrates (including cyclic oligosaccharides) on their surface, thus efficiently promoting their desorption and gas-phase cationization [49–52]. The minimum detectable amount for nonderivatised ribose, glucose, cellobiose, maltose, and cyclic oligosaccharides (α, β, and γ-cyclodextrin) at a S/N ratio of 3 was in the fmol range (Table 2) [49, 50]. In comparison with conventional organic matrixes (e.g., 2,5-dihydroxybenzoic acid), bare AuNPs have many advantages, for example: 1. ease of sample preparation; 2. no requirement for derivatization/additives; 3. high ionization efficiency for neutral carbohydrates; and 4. high shot-to-shot reproducibility [49]. As already mentioned, the main disadvantage of stabilizer-free NPs is their lack of chemical stability; in our opinion this may severely limit overall method reproducibility. Even if bare have AuNPs afforded, so far, the highest carbohydrate detection sensitivity, citrate-capped AuNPs

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Table 2 Analytical conditions for laser desorption ionization MS assisted by as-synthesized nanostructures DI promoters (size)

Synthesis

Additives

Analyte

Minimun detected amount

Ref.

AuNPs (2, 5, 10 nm)

Chemical reduction by sodium citrate

None

Chemical reduction by sodium borohydride

None

AuNPs film by evaporation driven vertical colloidal deposition

Chemical reduction by sodium citrate

TFAA 0.1%

NA 100 fmol 41 fmol 82 fmol 144 fmol 151 fmol 10 pmol 10 pmol 10 pmol 10 pmol 10 pmol

[47]

AuNPs (12.0±1.9 nm)

AuNPs (12.0±1.9 nm)

Chemical reduction by sodium borohydride

None

Bovin insulin Substance P glucose ribose cellobiose maltose β-cyclodextrin Leucine-enkephalin bradykinin angiotensin I substance P Cyclic oligosaccharides mix carbohydrates mix steroids mix aminothiols mix substance P insulin Angiotensin I insulin cytochrome C Angiotensin I Val4-Angiotensin II

Angiotensin I peptides mix Glutathione cysteine homocysteine Steroids mix carbohydrates mix indolamine mix monosialoganglioside angiotensin I untreated urine sample Gentamicin kanamycin A apramycin neomycin paromomycin Arginine leucine Enkephaline Glutathione cysteine homocysteine glutathione in blood lysates glutathione in cell lysates Glutathione angiotensin I

500 fmol

[56]

2 fmol 20 fmol 44 fmol

[53]

Layer by Layer self-Assembled multilayer films of AuNPs

Chemical reduction by sodium citrate

AuNPs (2, 5, 10 nm)

Chemical reduction by sodium citrate

AuNPs (2–30 nm)

Laser ablation

PFOS 0.5 mmol L−1 NaX and NH4X (X: F-, Cl-, Br-, I -, NO3−, -SO42−) Citrate buffer

AuNPs (mixture of two different sizes 3.5 and 14 nm)

Chemical reduction by sodium borohydride and sodium citrate

Ammonium citrate 5 mM

AuNPs (13.2±1.2 nm)

Chemical reduction by sodium citrate

NaCl 100 mmol L−1

Citrate buffer

Silver coated AuNPs (39±5 nm)

Chemical reduction by sodium citrate in presence of tannic acid + seed Ag growth with hydroquinone

Phosphate buffer 10 mM

AuNWs (350 nm×40 nm×1 cm) AuNPs (14 nm)

Electrochemical deposition

NaCl 0.1 mM

Chemical reduction by sodium citrate

Ammonium citrate 0.5 mM (with internal standard)

Chemical reduction by sodium citrate

Ammonium citrate 0.5-50 mM

AuNPs (14±2 nm)

[49]

[58]

[50]

10 pmol 100 pmol 10 fmol 5 fmol 5 fmol 700 amol 10-50 fmol

[59]

[57]

[51]

1648.4 fmol 5115.7 fmol 30 fmol 25 fmol 38 fmol 15 fmol 3 fmol 100 pmol 100 pmol 100 fmol 2 pmol 500 fmol

140 fmol 810 fmol

[64]

[61] [54]

[55]

Gold nanomaterials as a new tool for LDI-MS applications

611

Table 2 (continued) DI promoters (size)

AuNFs

Synthesis

Electrochemical etching

Additives

Citrate buffer

Analyte

Minimun detected amount

insulin 7.7 pmol Calibration mix angiotensin I 8 fmol ornithine 1.5 pmol Standard protein digests of bovine serum albumin, bovine catalase and bovine lactoperoxidase

Ref.

[63]

Abbreviations: AuNPs, gold nanoparticles; AuNWs, gold nanowires; AuNFs, gold nanofractals; TFAA, trifluoroacetic acid; PFOS, heptadecafluorooctanesulfonic acid; CD, cyclodextrin

Fig. 6 Mass spectra of a urine sample obtained using (a) 20.0 mg mL−1 DHB and (b, c) 13.0 nmol L−1 AuNPs. Unidentified peaks are marked by arrows. (Reprinted from J Am Soc Mass Spectrom, vol. 20, Wu HP, Yu CJ, Lin CY, Lin YH and Tseng WL “Gold Nanoparticles as Assisted Matrices for the Detection of Biomolecules in a High-Salt Solution through Laser Desorption/Ionization Mass Spectrometry” 875–882, Copyright (2009), with permission from Elsevier)

whereas it could be as much as 60% when using a conventional matrix (2,5-dihydroxybenzoic acid) [53]. In addition, the same mixture of NPs was proficiently applied to the analysis of spiked cell lysates and human plasma samples. With the objective of improving the accuracy of quantification of aminothiols in cells, in 2010 the same group developed a simple internal standard-based method [54]. The target thiols were first captured using the unmodified AuNPs; N-2-mercaptopropionylglycine (MPG) modified AuNPs were then added as internal standard, and, finally, the sample was analysed (upper panel of Fig. 7). After optimization of conditions such as surface density of the internal standard, solution composition, and salt concentration, the technique was validated by quantitative analysis of GSH in the lysates of human red blood cells and MCF-7 cancer breast cells in the presence and absence of the anti-inflammatory drug sulfasalazine [54]. Good response linearity and further improved reproducibility were obtained for Cys, Hcys, and GSH. Inter-spot RSD less than 10% (n=15) was obtained in the presence of MPG-AuNPs (lower panel of Fig. 7) [54], definitely better than that (ca 20%) obtained with the mixture of AuNPs of two sizes [53]. In addition, within and between-day accuracy was less than 8.5 and 12.5%, respectively, and within and betweenday precision, as RSD, was less than 7.8 and 10.4%, respectively [54]. This internal-standard LDI–MS was therefore proved to be a simple, accurate, and precise approach for determination of GSH in cells under drug invasion, resulting in a promising potential application of LDI–MS for quantitative analysis of a variety of analytes. Since 2005, great improvements have been achieved in direct AuNPs-assisted detection of peptides and small proteins [50, 51, 55–62]. Supported gold nanostructures [58, 61], AuNPs embedded into polymeric films [59, 60, 63], and patterned nanostructured gold thin films [62], among others, have been proposed. Generally referred to as matrix-free LDI surfaces, they rely on the same basic concept of ionization

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ƒFig.

7 Schematic representation of determination of aminothiols using N-2-mercaptopropionylglycine (MPG)–AuNPs as the internal standard and AuNPs as the matrix. Relative standard deviation of [aminothiol + Na]+ intensities for (a) glutathione, (b) cysteine, and (c) homocysteine, 20 μmol L−1 each, captured by AuNPs in the absence (empty squares) or presence (filled squares) of MPG–AuNPs from 15 different sample spots. (Reprinted from Nanomed: Nanotechnol, Biol Med, vol 6, Chiang CK, Lin YW, Chen WT and Chang HT “Accurate quantitation of glutathione in cell lysates through surface-assisted laser desorption/ionization mass spectrometry using gold nanoparticles” 530–537, Copyright (2010), with permission from Elsevier)

from a functional (nanostructured) surface. These options have resulted in the best performance in terms of both sensitivity and accessible mass range. Arakawa and co-workers used polymer films to immobilize AuNPs, in two different configurations. One used ammonium citrate-capped AuNPs on a silicon substrate [60]; the second used a layer-by-layer (LbL) approach to assemble negatively charged AuNPs with an oppositely charged poly(allylamine) hydrochloride polymer on a silicon oxide substrate [59]. In the first approach, the dependence of ionization efficiency on on-plate nanoparticle distribution was evaluated; clustering of AuNPs by polymer micelles resulted in greater absorption of the incident UV light, leading to rapid heating of the AuNPs and enhanced DI of peptides; controlling the aggregation and/or the density of the AuNPs on the plate was therefore indicated to be a key factor for tuning LDI performance [60]. In the second approach, five polymer/AuNP overlayers were used to obtain an LDI-active substrate [59]. Use of LBL multilayer films containing nanoparticles had important advantages, mainly arising from the possibility of tuning the density and the aggregation of the AuNPs by changing experimental conditions such as dipping time and number of LBL deposition cycles. The useful analyte mass range was widened by increasing the number of layers; when five polymer/AuNP layers were used it became possible to detect small proteins, for example 5.8 kDa insulin (5 pmol) and 12.4 kDa cytochrome c (5 pmol). The detection limit for angiotensin I (1.3 kDa) was as low as 10 fmol in the presence of citrate buffer as proton donor, and it was further reduced to 0.7 fmol in the presence of heptadecafluorooctanesulfonic acid [59]. However, the excellent sensitivity was achieved at the expense of a timeconsuming preparation procedure (deposition time being of the order of 68 h for each AuNPs layer and 30 min for each polymer layer), with critical washing steps that could significantly degrade the MS signal. Indeed, signal reproducibility was found to be strongly dependent on the washing step between the deposition of each layer [59]. Recently, a novel matrix-free LDI–MS technique using patterned nanostructured gold thin film, was proposed [62]. The versatility of such a surface was assessed by performing peptide mass fingerprinting of protein digests obtained by

Gold nanomaterials as a new tool for LDI-MS applications

use of different enzymes and by analysis of LMW compounds. Protein digestion using multiple proteases (trypsin and Glu-C) improved the sequence coverage, thus improving protein identification. Compared with the aggregated AuNPs as reported by Arakawa and co-workers [59, 60], the fractal-like morphology of the nanostructured gold surface provided a highly enhanced gold surface area, together with a shorter fabrication time [62]. Engineered nanomaterials for improved selectivity Engineered nanomaterials are a valid alternative to assynthesized nanostructures, in those cases where introducing surface functionality can afford novel or improved interaction with biomolecules. The high control of surface chemistry on gold materials, mainly based on the Au–S chemistry, has greatly assisted advances in this field. In many cases, self-assembled monolayers (SAMs) of alkanethiols chemisorbed on to gold surfaces can be cleaved and directly ionized upon laser irradiation [65]. On the basis of this observation, the nanoparticles themselves can often be analysed, providing information about the composition of surface functionality and atom numbers in the NP core [66–68]. Although this specific application is beyond the scope of this review, introducing monolayers on to NPs surfaces enables similar “read out” to be performed by MS. In effect, these surface ligands can then act as mass “barcodes” for the NP. This approach has been used in several recent biological studies under different experimental configurations [69–72]. Nagahori and Nishimura used AuNPs as a scaffold to immobilize carbohydrate functionalized alkanethiol monolayers (i.e. N-acetylglucosamine, GlcNAc) and to display acceptor substrates for glycosyltransferases. Once treated with the proper glycosyltransferase enzyme, the mass change of alkanethiol ligands on glycosylation was monitored by LDI–MS, affording very rapid and direct detection of the enzymatic reaction, even in presence of contaminants such as proteins and salts [69]. This simple assay was then successfully applied to detection of the enzymatic activity of crude cell extracts from Escherichia coli, which required purifying pretreatments [69]. Zhu et al. have recently developed an LDI–MS technique for simultaneous identification and quantification of cellular uptake of multiple AuNPs [70]. AuNPs were tagged with readily ionisable cationic or neutral surface functionalities, and used as mass barcodes. On laser irradiation, the surface functionalities were simply read out by LDI–MS, providing characteristic peaks suitable for identifying the AuNPs. The key advantage of this approach is direct control of the cellular uptake of the NPs, on the basis of NP surface functionality, suggesting that different cellular uptake and specific cell targeting might be possible if the NPs are

613

appropriately designed. Moreover, different NPs could be simultaneously identified and quantified at levels as low as 30 pmol, enabling multiplexed screening [70]. Mass barcodes on AuNPs have also been used to report and amplify the signal associated with DNA hybridization; as already mentioned, MS detection of molecules with low ionization efficiency and facile fragmentation, for example DNA, is still challenging. Qiu et al. and Yang et al. have proposed a three-component sandwich assay based on barcoded AuNPs for ultrasensitive detection of DNA [71] and of single nucleotide polymorphism (SNP) [72]. The high selectivity was based on two sequential oligonucleotide hybridizations: the first is capture of the target strand by specific probes immobilized on to either chip [71] or magnetic beads [72]; the second is recognition by gold nanoparticles functionalized with complementary oligonucleotides. First, gold nanoparticles were functionalized with modified complementary oligonucleotide structures; mass-tag molecules (for example, a symmetric disulfide featuring an ethylene glycol oligomeric unit) were incorporated as barcode. The surrogate alkanethiol monolayer interacted preferentially with the AuNPs surface—rather than the DNA strands—and could be directly identified by LDI– MS, thereby providing an amplified signal of the target DNA strand hybridization [71, 72]. In the initial proof-of-concept demonstration, on-chip detection of the oligonucleotide sequence associated with the anthrax lethal factor was chosen as a test target; sensitivity was 100 pmol L−1 (equivalent to a minimum detected amount of 3 fmol) [71]. Single nucleotide polymorphism genotyping without the need for primer-mediated enzymatic amplification was achieved successfully [72]. The most frequent SNPs in the MDR1 (multiple drug resistance) gene, considered to contribute to multidrug resistance during cancer chemotherapy and to drug disposition, were detected and the sensitivity of this new method was reduced to the 0.1 fmol L−1 range, thus approaching the PCR (polymerase chain reaction) performance level [72]. Alternatively to the mass barcode approach, functionalized AuNPs have also been applied to the direct desorption/ ionization of biomolecules such as peptides and proteins [73, 74], thiols [24, 74], and nucleotides [75]. Chen et al. proposed the use of carbohydrate-encapsulated AuNPs as affinity probes for the separation and enrichment of target proteins, and their epitope mapping by on-probe digestion, followed by LDI–MS detection after removal of unbound peptides [73]. Worthy of note is that in the work discussed in this paper detection of up to 78 fmol of Pseudomonas aeruginosa lectin I protein was achieved, thus extending the upper limit of the molecular weight (13 kDa) approachable with NP-promoted LDI–MS analysis [73]. Moreover, Nile red-absorbed [24] and aptamer-modified [75] AuNPs have been shown to promote selective

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detection, at the nmol L−1 level, of aminothiols, (e.g. glutathione, cysteine, and homocysteine) and adenosine triphosphate, respectively (Table 3). Both the approaches have been validated on real samples, for example lysed human red blood cells and blood plasma. The same research group recently developed an internal standard method for accurate and precise detection of captopril in urine (inter-day RSD <9%), using mercaptobenzoic acid-capped AuNPs both as DI promoter and internal standard [74]. Finally, a binary matrix composed of a mixture of a conventional organic matrix and multifunctional nanoprobes coupled with SDME has been shown to substantially

outperform the single-matrix approaches (organic matrix or AuNP) in the detection of peptides and proteins. For instance, significant signal enhancement (up to 35-fold) was achieved for the hydrophobic peptide gramicidin D [76]. Engineered nanomaterials for enhanced ionization Notwithstanding the increasing research activity of several groups into AuNP-promoted DI processes, a universal method affording efficient ionization and energy-transfer for different classes of analyte and real samples is still not available. DI efficiency seems to be strongly analyte-

Table 3 Analytical conditions for laser desorption ionization MS assisted by engineered nanostructures for improved selectivity DI promoters (size)

Capping agents

AuNPs (4±1 nm)

Two carbohydrates-galactose (linker: ethylene None glycol treated with thioacetic acid); Pk antigen (linker cysteamine)

AuNPs (3–8 nm)

None

N-Acetylglucosamine (linker: ethylene glycol polymer) AuNPs (14, 32, 56 nm) Nile red

Additives

0.5 mmol L−1 ammonium citrate

AuNPs (13.3±1.2 nm)

Aptamer

0.5 mmol L−1 ammonium citrate

AuNPs (ca 2 nm)

Tetraalkylammonium (linker:ethylene glycol thiolate)

None

AuNPs mass barcoded (13 nm)

Thiol-terminated oligonucleotides+disulfide ethylene glycol oligomer

None

AuNPs (13.3±1.2 nm)

Mercaptobenzoic acid (as internal standard)

AuNPs+α-CHCA

(4-mercaptophenylimino methyl)-2methoxyphenol

0.5 mmol L−1 ammonium citrate None

AuNPs mass barcoded (13 nm)

Analytes

Minimum detected amount

Ref.

Pseudomonas aeruginosa lectin I Pseudomonas aeruginosa lectin I digested peptides (chymotrypsin) Reaction products of glycotransferase Glutathione cysteine

78 fmol

[73]

[69] 25 fmol 54 fmol

homocysteine 34 fmol analysis of glutathione in red blood cells and of cysteine in plasma Adenosine triphosphate 480 fmol glutathione + adenosine triphosphate in cell extract Multiplexed analysis of cellular uptake of functionalized AuNPs (30 pmol) Oligonucleotide sequence 3 fmol associated with anthrax lethal factor Captopril 1 pmol

[24]

[75]

[70]

[71]

[74]

Insulin 17.4 fmol [76] ubiquitin 23.3 fmol gramicidin D 53.1 fmol HW6 450 fmol cytochrome C 40 fmol myoglobin 35.3 fmol lysozyme 13.2 fmol milk samples: proteins detected proteoso pep-PP81, 3-casein, lactoalbumin and lactoglobulin Thiol-terminated oligonucleotides + disulfide 0.1% trifluoroacetic Multiple drug resistance gene 100 amol L−1 [72] ethylene glycol oligomer acid

Abbreviations: AuNPs, gold nanoparticles; α-CHCA, α-cyano-4-hydroxycinnamic acid

Gold nanomaterials as a new tool for LDI-MS applications

dependent. Furthermore, the positive ion mass spectra obtained by using AuNPs and their derivatives as DI promoters often results in analyte fragmentation and/or in the massive presence of alkali metal ion adducts. Previous studies have shown that the presence of a proton donor is crucial for ionization of polar biomolecules [13]; 4-aminothiophenol (4-ATP) and 4-mercaptobenzoic acid (4-MBA)-functionalized AuNPs were designed and investigated by Russell and co-workers as proton sources [77, 78]. The rationale was that capping AuNPs with an organic monolayer acting as efficient proton donor would enhance ionization efficiency while maintaining the advantages provided by the nanomaterials. Screening of a wide range of peptides and small proteins revealed the intensity of quasi-molecular ions was significantly increased by using either 4-ATP or 4-MBA-capped AuNPs compared with as synthesized citrate-capped AuNPs [77, 78] (Table 4). Furthermore, simplified mass spectra were obtained, because of the drastic reduction of: 1. alkali metal adducts of analyte molecules; 2. analyte fragments; and 3. interfering ions ascribable to gold clusters. The tentative explanation for this observation was that the presence of either 4-ATP or 4-MBA at the surface of the nanoparticle reduced analyte fragmentation by decoupling the analyte from the nanoparticle itself. The chemisorbed monolayer inhibited direct adsorption of the analyte molecules by the nanoparticle surface, thus reducing the energy required for analyte desorption and enabling softer desorption/ionization (ions with less internal energy). Another advantage of replacing the citrate capping agent with such an organic monolayer was an increase of the useful analyte mass range for LDI–MS; as an example, analyses could be carried out on a relatively high-molecular weight analyte such as cytochrome c (12,400 Da). The latter evidence was interpreted in terms of increased production and survival of analyte ions. For 4-MBA capped AuNPs, a mixed monolayer-capping agent was developed, to behave both as analyte capture probe

615

and ionization enhancer (Fig. 8) [78]. Selective capture and direct desorption/ionization of bradykinin (1–7) from a twopeptide mixture was achieved by using β-cyclodextrin functionalized AuNPs, thus confirming that controlled surface chemistry is of key importance in designing the properties of nanoparticles for MS applications [78]. Recently, AuNPs functionalized by α-cyano-3-hydroxycinnamic acid (CHCA) have been proposed as an improved DI promoter compared with the CHCA matrix itself. Gold nanophases were capped by means of an organic hybrid shell containing, first, a cysteamine selfassembled monolayer that acted as a linker for subsequent covalent bonding of CHCA. By use of these NPs, direct ionization of peptides such as [Sar1, Thr8]-angiotensin II and neurotensin was achieved [79]. Covalent linking of this conventional organic matrix to AuNPs resulted in marked improvement of peptide ionization compared with standard citrate-capped nanoparticles: 1. the NP coating effectively suppressed the formation of Au cluster ions and analyte fragment ions, leading to cleaner mass spectra; 2. nearly twofold higher analyte ion responses and better signal-to-noise ratios were obtained; and 3. protonated analyte ions were obtained in mass spectra (in contrast with other NPs, which mainly produce adducts such as alkali ion–neutral analyte species [79]). However, these capped AuNPs tended to aggregate and precipitate at the edge of the sample spot, resulting in inhomogeneous spot composition. To further enhance ionization efficiency, a variety of additives, including glycerol, organic acids, and ammonium salts were systematically investigated [79]. Glycerol addition resulted in more uniform spots (with enhanced AuNPs dispersion), and contributed to an increase in the extent of sample ionization, thus improving the quality and reproducibility of spectra. Moreover, addition of citric acid as an external proton donor resulted in high-intensity spectra, dominated by the protonated analyte ion and with reduced peptide fragmentation [79]. To assess the applicability of the method to proteomics, a tryptic digest of cytochrome c

Table 4 Analytical conditions for laser desorption ionization MS assisted by engineered nanostructures for enhanced ionization DI promoters (size)

Capping agents

Additive

Analytes

Ref.

AuNPs (5 nm) AuNPs (10 nm)

4-Aminothiophenol 4-Mercaptobenzoic acid cyclodextrin α-Cyano-3-hydroxycinnamic acid

0.1% Trifluoroacetic acid 0.1% Trifluoroacetic acid

ACTH cytochrome C insulin Angiotensin II mixture: bradykinin + c-telopeptide bradykinin (extracted from two peptides mixture) Mixture of [Sar1, Thr8]-angiotenisn II and neurotensin (20 pmol) tryptic digest of cytochrome C

[77] [78]

AuNPs (21.5±0.4 nm)

Abbreviations: AuNPs, gold nanoparticles

5% Glycerol + 20 mmol L−1 citric acid

[79]

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R. Pilolli et al.

was analysed by means of CHCA-capped AuNPs, although with limited success only. Indeed, the ionization efficiency was still found to be poorer than that for CHCA, because a ca. fivefold excess of digest was required to obtain results similar to those provided by the organic matrix [75]. Presumably, the efficiency of this hybrid nanosystem is still limited when applied to the analysis of complex samples. Moreover, in our opinion, this method strongly resembles the “two-phase” MALDI Tanaka et al. approach [13], rather than being exclusively based on nanophases. Matrix-implanted LDI–MS analysis and imaging MS

Fig. 8 (a) Schematic representation of a hybrid nanoparticle capped with both ionization enhancing (mercaptobenzoic acid, MBA) and analyte-capture ligands (β-cyclodextrin). LDI–TOF mass spectra of angiotensin II using citrate-capped AuNPs (b) and 4-MBA-capped AuNPs (c). (Reprinted from J Assoc Lab Autom, vol. 13, Castellana ET, Sherrod SD, and Russell DH “Nanoparticles for Selective Laser Desorption/Ionization in Mass Spectrometry” 330–334, Copyright (2008), with permission from Elsevier)

Tissue profiling and imaging by MALDI–MS enable the direct analysis and location of biomolecules, preserving the anatomical information about the tissue. In this method, the UV-absorbing matrix is deposited directly on to the surface of the biological tissue section, which is then analysed with no further preparation. The uniform incorporation of organic matrix and perturbation of the spatial distribution of the analyte by matrix solution deposition are severe limitations of such applications. To overcome this problem, an alternative method for homogeneous non-destructive incorporation of matrix metal clusters into a bioorganic solid material was developed in 2004 by Novikov et al. [48] A liquid metal ion source (LMIS) was used to implant gold clusters (Au4004+) into the top layers of biological sections with the objective of providing a matrix-like effect in UV laser desorption mass spectrometry [48]. Clearly, this study is the first proof of concept of the feasibility of replacing conventional organic matrices with AuNPs. In these experiments, the so-called matrix-implanted laser desorption/ionization (MILDI) approach was applied to a variety of samples (dynorphin 1–7, thymic factor, bovine insulin B chain, and slices of rat brain tissue) and excellent homogeneity of ion emission from the entire implanted surface in response to laser irradiation was achieved [48]. Owing to the in-situ nature of this method, preliminary purification and separation steps are not possible, because information about the spatial location of the bioanalytes must be preserved. Therefore, the acquired mass spectra can be extremely complex, particularly at m/z values below 2,000, where lipid, peptide, and matrix signals overlap. MALDI–ion mobility (IM)–orthogonal time-of-flight mass spectrometry (oTOFMS) provided a partial solution to this issue by initially separating different classes of biomolecule by their IM drift times before mass analysis [80, 81]. AuNPs were used to detect and map neutral cerebrosides in rat brain sections. It is worthy of note that cerebrosides, although occurring at high concentration, were usually undetected in conventional MALDI–MS because of suppression effects [81]. Figure 9c and b illustrate, respectively, an example of a MALDI–IM–oTOFMS image obtained

Gold nanomaterials as a new tool for LDI-MS applications

Fig. 9 (a) MALDI-ion mobility 2D plot obtained from a rat brain tissue section with DHB matrix in positive-ion mode. (b) 1D mass spectrum from the tissue section recorded by using AuNPs (5.5 nm) in positive-ion mode. (c) Image of sodiated cerebroside 24:0 h at m/z 850.7 (40×40 pixel). The adjacent frame is a photograph of the rat brain section (Cx, cortex; fmi, forceps minor of the corpus callosum; Cpu, caudate putamen (striatum); Acb, nucleus accumbens; ac, anterior commissure; lo, lateral olfactory tract). (Reprinted with permission from Ref. [81]. Copyright (2007) John Wiley and Sons)

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from a rat brain cerebrum coronal section in positive-ion mode and the total 1D mass spectrum obtained by summing all the ions within this IM–m/z region of interest [81]. Mapping of glycosphingolipids (GSLs) in mouse brain sections using AuNPs was reported in 2010 by Goto-Inoue and co-workers who demonstrated twentyfold-enhanced detection sensitivity compared with the conventional 2,5dihydroxybenzoic acid matrix [82]. Ion images were acquired from 14 sulfatides and 10 gangliosides, thus also exceeding the performance of DHB in terms of number of detectable components (Fig. 10) [82]. A preliminary but interesting application of AuNPassisted imaging mass spectrometry in forensic science is the detection of latent fingerprints (LFP) [83]. Most achievements in this field have been focused on visualization of the physical pattern but were lacking molecular information. By integrating distinctive properties of gold nanoparticles with imaging MS, both these objectives can be achieved. Tang et al. discovered that sputtered AuNPs tended to aggregate differently on the ridges and grooves of LFPs, affording two contrasting colours [83]. Consequently, the physical pattern of LFPs on different substrates, for example plastic, glass, and paper, was clearly visible to the naked eye, enabling individual identification. Moreover, AuNPs were an effective medium for MS detection and imaging of endogenous (e.g. fatty acids) and exogenous (e.g. verapimil) compounds embedded in the LFPs, also relevant for identification of the age and sex of individuals. The “molecular images” obtained led also to the discrimination of two overlapped fingerprints relying on the presence of specific compounds [83]. Coating the LFPs with sputtered AuNPs enabled their preservation for more than a month and also direct analysis by scanning electron microscopy, thus achieving, with a one-step sample-preparation procedure, three levels of characterization—macroscopic, microscopic, and molecular —furnishing more important evidence [83]. Further development of this method is needed for on-site and real sample analysis, because current LDI–MS instrumentation is not usually suitable for fingerprint samples. The most recent application of gold nanoparticles in forensic science, proposed by the same group, was direct chemical analysis and molecular imaging of documents of questionable validity [84]. A thin layer of AuNPs was applied, by ion sputtering, to banknotes and cheques in a dry state. Such a layer provided combined information about the spatial distribution and chemical composition of visible and fluorescent inks and their order of printing, thus differentiating real banknotes from fakes [84]. The method was also used to identify forged parts of documents, for example number/writing alteration on a cheque, by tracing different writing patterns that come from different pens [84].

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Fig. 10 Negative-ion mode MALDI imaging mass spectrum acquired from mouse brain tissue with DHB (a) and with AuNPs (b). (arrow head indicates peak derived from AuNPs). The ion images of ten kinds of sulfatide molecular species with DHB (c) and with AuNPs (d). (Reprinted from J Am Soc Mass Spectrom, vol. 21, Goto-Inoue

N, Hayasaka T, Zaima N, Kashiwagi Y, Yamamoto M, Nakamoto M and Setou M “The detection of glycosphingolipids in brain tissue sections by imaging mass spectrometry using gold nanoparticles” 1940–1943, Copyright (2010), with permission from Elsevier)

Gold nanomaterial-mediated visible/near-infrared LDI–MS analysis

number of suitable matrices capable of operating in this spectroscopic range. Early investigations on Vis-MALDI were conducted by Tang et al. using rhodamine dyes as matrices [85], followed by a series of approaches based on binary matrix (rhodamine + liquid matrix) [86, 87], and neutral red [88], to cite just a few. Despite the high optical absorption of the organic dyes, the ionization performance was not as efficient as that of the UV-MALDI matrices. As an alternative to organic matrices, Schurenberg et al. proposed titanium nitride nanoparticles suspended in

Visible-MALDI (Vis-MALDI) may have advantages over traditional UV-MALDI for organic molecules absorbing in the UV region that are susceptible to fragmentation under UV laser irradiation. Most small organic molecules do not absorb in the visible wavelength range and are, therefore, less prone to fragmentation by direct absorption of incident laser radiation. However, there are relatively few reports on Vis-MALDI mass spectrometry, because of the restricted

Gold nanomaterials as a new tool for LDI-MS applications

glycerol, thus leading toward use of inorganic nanostructures as matrices in visible and near infrared LDI–MS [89]. For metals such as gold, surface plasmon resonance occurs under visible light, leading to a huge concentration of optical near-field in a small volume, usually called a “hot spot”, with consequent local heating. Besides the plasmonic heating, non-thermal excitation can also occur on the nanostructured surface and be relevant to LDI processes [90–97]. Gold colloids, in particular, absorb strongly at approximately 520 nm, which can be drastically shifted by size or aggregation effects. To keep this variable under control, the first investigations on this topic were mainly based on the use of supported gold nanostructures. Hori and co-workers demonstrated visible laser desorption/ionization on gold nanorod arrays [93] and gold thin films [90]. The nanostructures were irradiated with a Nd:YAG laser and desorption/ionization was found to be favoured by use of a 532 nm visible laser, which is in the range of the localized surface plasmon resonance. Detection performance was assessed for peptides over a relatively wide mass range (from ca 200 to 6000 Da), with suitable detection sensitivity at the upper approachable mass range limit (Table 5) [93]. It is worthy of note that, because of the inert nature of gold, the nanorod array substrate could be stored under atmospheric conditions and reused after proper cleaning, without significant loss of performance. Spencer et al. used a tuneable dye laser to scan the visible plasmonic band of gold nanospheres suspended in aerosols (from 440 to 680 nm), thus performing LDI–MS detection of individual aerosol particles containing ~50 attomoles of a small peptide [97]. The peculiar experimental conditions proposed in this study were quite different from those used in conventional LDI–MS analysis, because an unusually high laser power (~1 mJ per pulse) and a dualpolarity time-of-flight mass spectrometer were used to obtain both positive and negative-ion mass spectra simultaneously from individual particles. Therefore, direct comparison of these results with those in other papers would not be completely appropriate. Interestingly, the laser wavelength coincident with the surface plasmon resonance absorption (λ=500–540 nm) generated the most intense ion species ([M + Na]+ and [M−H]−) with less fragmentation than those obtained by excitation under off-resonant conditions [97]. Recently, more in-depth understanding of the mechanism of ionization in surface plasmon enhanced LDI has been obtained by starting from the idea that it is unlikely the rapid thermal energy supply is the only process promoting LDI [91, 92]. Surface effects resulting from either local enhancement of the electromagnetic (EM) field or chargetransfer (CT) effects could also occur. CT effects would act

619

as a direct supply of energy from the metal surface to individual adsorbed molecules. Nagoshi and co-workers and Shibamoto and co-workers ascribed the ultra-highsensitivity detection in AuNPs-based Vis-LDI–MS to the CT effect [91, 92]. A clear correlation between surface plasmon excitation efficiency at 532 nm (in turn correlated with the diameters of gold nanoparticles) and analyte (i.e. dewatered N-acetyltetraose, N-AT) ion intensity is clearly apparent from Fig. 11 [92]. The dependence of the ion intensity obtained by use of 50-nm gold nanoparticles on the concentration of N-AT in the sample was also measured. The minimum detected amount of N-AT was 10 amol. The ion intensity increased proportionally with concentration from the 10 amol sample to the 1 fmol sample, i.e. until N-AT molecules completely covered the gold nanoparticle surface with a monolayer; after this concentration limit, the ion intensity became inversely proportional to analyte concentration (Fig. 11b) [92]. This trend was not explained solely by the contribution of EM effects, because they would have affected all piled analyte molecules with the same proportionality. More likely, charge interactions occurred at the same time and acted mainly on the first adsorbed monolayer. The different relative contribution of each of these two processes accounted for the final trend of the ion intensity as the concentration decreased [92]. The most recent research trend is extending the usable excitation wavelength for gold nanostructures towards the near-infrared [95, 96]. Russell and co-workers proposed a strategy for analyte ionization based on chemical derivatization of gold nanorods. A self-assembled monolayer was required for ionization of non-preformed ion analytes, assisting the transfer of a proton and/or sequestering the analyte in close proximity to the nanorod surface [96]. A systematic study of the wavelength dependence of LDI performance delivered by an Nd:YAG laser (266, 355, 532, and 1064 nm) has been carried out with the objective of providing further information about the dominant processes involved [95]. Gold nanosphere, nanorod, and nanostarassisted LDI was investigated, to reveal the laser fluence thresholds for the appearance of cationized analyte adducts. A close correlation was found between the optical absorbance of the nanoparticles and the laser fluence thresholds. For nanospheres, plasmonic excitation in the visible range seemed to be more efficient than non-plasmonic excitation at shorter UV wavelengths (valence electron excitation), confirming previous evidence [95]. The thermal mechanism in these experiments was assumed to be relevant, even if not dominant. For gold nanorods and nanostars, the induced LDI mechanism was likely to feature photothermal character, and it was therefore hypothesized it would not depend strongly on the type of electronic excitation (valence electron or plasmonic); the contribution of heating was found to be

Sputter coated

Chemical reduction by None tetrakis(hydroxyl-methyl) phosphonium chloride Argon ion sputtered None

Gold film (10–15 nm-AuNPs layer) on porous silicon

5 nm AuNPs aerosol

NA

Seed-mediated growth

50 nm AuNPs

AuNRs

Analytes

Nd:YAG (532 nm) incident light 45° Nd:YAG (532 nm) incident light 45°

Bradykin angiotensin I thermometer molecule ( 4-chlorobenzylpyridinium chloride) None Rhodamine B, malachite green, crystal violet None N-acetyltetraose crystal violet angiotensin II 0.1 % TFAA Angiotensin II benzyldimethylhexadecylamm onium

Nd:YAG (532 nm) incident None light 60°

4-Aminothiophenol Nd:YAG (1064 nm) CTAB

None

None

None

Additive

Nd:YAG (532 nm) incident NaCl 10 ppm Lactose light 60° melittin none Lys-Lys bradykinin citric buffer bradykinin bovine insulin Nd:YAG (532 nm) incident None Angiotensin I light 60° pentalysine lactose Bradykinin pentalysine lactose Tunable wavelength laser None WGG peptide, D-ribose, from 400 to 680 nm L-arabinose

Laser source

Abbreviations: AuNRs, gold nanorods; AuNPs, gold nanoparticles; CTAB, cetyl trimethylammonium bromide; TFAA, trifluoroacetic acid

NA

60 nm AuNPs

10-50 nm AuNPs by in-situ laser ablation of gold thin film

Electrodeposition of gold into alumina porous template

AuNRs array (~15 in diameter ~100 nm in length)

None

Electrodeposition of gold into alumina porous template

AuNRs array (~15 in diameter ~100 nm in length)

Capping agents

Synthesis

DI promoters (size)

Table 5 Analytical conditions for gold nanostructure-mediated visible/near-infrared LDI–MS

10 amol 500 zmol Several fmol NA 5000 pmol

NA

NA

10 pmol 5 pmol 6 pmol 5 pmol 650 fmol 2 pmol NA 5 pmol 5 pmol 5 pmol 5 pmol 5 pmol 50 amol / aerosol particle

[96]

[92]

[91]

[94]

[97]

[90]

[93]

Minimum detected amount Ref

620 R. Pilolli et al.

Gold nanomaterials as a new tool for LDI-MS applications

621

Conclusions

Fig. 11 (a) Dependence of de-watered biose ion intensity and SP excitation efficiency on the diameter of the gold nanoparticle (triangles, right axis; black dots, left axis). (b) LDI mass spectra obtained from different amounts of the N-acetyltetraose sample with 50 nm AuNPs (4.5×105 particles). (Reprinted with permission from Ref. [92]. Copyright (2009) American Chemical Society)

approximately constant at the four wavelengths investigated. The mechanism difference between these two categories was ascribed to the different capping agents of the specific materials under investigation. Light citrate coating of nanospheres resulted in enhanced local field and charge effects associated with plasmon excitation which affected the analyte. In contrast, because the bulky polymers used for stabilization of nanorods and nanostars prevented the direct interaction of the analyte with the nanostructure metal core, the thermal mechanism only was assumed to be effective in determining LDI processes [95].

Gold nanomaterials are an emerging and powerful tool for bioanalytical applications of LDI–MS. They improve the performance of conventional matrix-assisted LDI–MS in terms of approachable mass range (especially in the low-m/z region), sensitivity, reproducibility, and ease of sample preparation, thus overcoming some of the limitations arising from the use of conventional organic matrices. So far, the viability of this approach has been demonstrated over a large mass range (from ~123 Da for cysteine to ~18.5 kDa for lactoglobulin), with good sensitivity. The main disadvantage is that the efficiency of energy transfer, desorption, and ionization processes seems to be strongly dependent on the particular molecule detected, and thus changes as a function of the specific nanomaterial–analyte combination. Moreover, the use of additives, for example salts, buffers, and proton donors, is often an essential requirement in order to achieve satisfactory ionization yield. This problem is a critical limitation of most matrixfree approaches, suggesting that the nanomaterial itself is probably involved in the desorption process, but that an “external” additional ionization source is also required. Several classes of analyte have been investigated to assess the versatility and reliability of application of AuNPs to LDI–MS; detection sensitivity generally ranges from low femtomoles (thio compounds, amino acids, peptides, small proteins, antibiotics) to sub-picomoles (neutral carbohydrates, steroids). To validate the approach, complex real samples, for example untreated urine, cell extracts, human lysed blood cells, lysed blood plasma, and milk have been analysed. With regard to reproducibility, an internal standard-based approach resulted in spot-to-spot relative standard deviation below 10% for aminothiols, leading to the possibility of quantitative analysis of some target molecules. Moreover, supported gold nanostructures and/or nanostructured surfaces (i.e. gold thin films) addressed the sample preparation challenge, avoiding the matrix–analyte co-crystallization step, and resulting restrictions, and sometimes enabling simple on-plate desalting and direct LDI analysis. Despite efforts devoted to rapid development of these applications, lack of systematic fundamental investigation of the mechanisms and energetics involved is still limiting widespread application of the technique. A full understanding of surface effects promoting DI mechanisms would speed nanomaterial design and optimization steps leading to improved analytical performance. Finally, a critical comparison of gold nanostructures assisted LDI with other matrix-free approaches, would be useful to emphasize the possible advantages of gold

622

compared, for example, with other commercially available materials, for example silicon oxides, metal oxides, and other inorganic nanostructures. Acknowledgements The authors acknowledge financial support from the Italian Project “Nanomaterials and laser ionization mass spectrometry: a new bio-analytical approach” FIRB Futuro in Ricerca 2008, funded by the Ministero dell’Istruzione, dell’Università e della Ricerca.

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