History of inductively coupled plasma mass spectrometry-based immunoassays

Spectrochimica Acta Part B 76 (2012) 27–39

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Review

History of inductively coupled plasma mass spectrometry-based immunoassays☆ Charlotte Giesen a,⁎, Larissa Waentig b, Ulrich Panne b, c, Norbert Jakubowski b a b c

University of Zurich, Institute of Molecular Life Sciences, Winterthurerstrasse 190, 8057 Zurich, Switzerland BAM Federal Institute for Materials Research and Testing, Richard-Willstaetter-Strasse 11, 12489 Berlin, Germany Humboldt-Universität zu Berlin, Department of Chemistry, Brook-Taylor-Straße 2, 12489 Berlin, Germany

a r t i c l e

i n f o

Article history: Received 16 May 2012 Accepted 18 June 2012 Available online 26 June 2012 Keywords: ICP-MS LA-ICP-MS Immunoassay Elemental tagging Multiplexing

a b s t r a c t The analysis of biomolecules requires highly sensitive and selective detection methods capable of tolerating a complex, biological matrix. First applications of biomolecule detection by ICP-MS relied on the use of heteroelements as a label for quantification. However, the combination of immunoassays and ICP-MS facilitates multiparametric analyses through elemental tagging, and provides a powerful alternative to common bioanalytical methods. This approach extends the detection of biomarkers in clinical diagnosis, and has the potential to provide a deeper understanding of the investigated biological system. The results might lead to the detection of diseases at an early stage, or guide treatment plans. Immunoassays are well accepted and established for diagnostic purposes, albeit ICP-MS is scarcely applied for the detection of immune-based assays. However, the screening of biomarkers demands high throughput and multiplex/multiparametric techniques, considering the variety of analytes to be queried. Finally, quantitative information on the expression level of biomarkers is highly desirable to identify abnormalities in a given organism. Thus, it is the aim of this review to introduce the fundamentals, and to discuss the enormous strength of ICP-MS for the detection of different immunoassays on the basis of selected applications, with a special focus on LA‐ICP‐MS. © 2012 Elsevier B.V. All rights reserved.

Contents 1. 2. 3.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . Definition label and tag . . . . . . . . . . . . . . . . . . . . . Heteroelement detection of biomolecules by ICP-MS . . . . . . . . Heteroelement detection of biomolecules by LA‐ICP‐MS . . 4. Immunoassays . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Antibodies . . . . . . . . . . . . . . . . . . . . . . . 4.2. Tagging chemistry for ICP-MS applications . . . . . . . . 4.3. Strenghts of ICP-MS detection for immunoassay applications 5. Applications of immunoassays with ICP-MS detection . . . . . . 5.1. Immunoassay detection by solution nebulization ICP‐MS . . 5.1.1. One-parameter assays . . . . . . . . . . . . . . 5.1.2. Multi-parameter assays . . . . . . . . . . . . . 5.2. Immunoassay Detection by LA‐ICP‐MS . . . . . . . . . . 5.2.1. One-parameter assays . . . . . . . . . . . . . . 5.2.2. Multi-parameter assays . . . . . . . . . . . . . 6. Conclusions and Future Trends . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction ☆ This paper is dedicated to Gary M. Hieftje, on the occasion of his 70th birthday, in recognition of his boundless contributions to spectroscopy and analytical chemistry. ⁎ Corresponding author. E-mail address: [email protected] (C. Giesen). 0584-8547/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.sab.2012.06.009

The sequencing of the human genome with its 2.85 billion nucleotides [1], and the mapping of our 22 500 genes [2] has enabled the tracing of diseases to their molecular causes. However, the

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corresponding proteome [3] is highly dynamic since the protein expression patterns reflect current cell and environmental conditions. Consequently, proteins exhibit a diversity of posttranslational modifications and varying concentrations (10 2–10 6 copies per cell) [4]. Hence, their detection and identification is challenging, and is addressed by mass-spectrometry (MS) based proteomics, which was reviewed by Aebersold and Mann (see further references therein) [5]. The detection of proteins in situ is feasible by immunoassays, which are based on the specific binding of antibodies with their corresponding antigens. Usually, immunoglobulin G (IgG)‐type antibodies are employed for this purpose. The first description of an antibody carrying a tag for subsequent detection was published by Coons in 1941 [6]. He introduced the coupling of fluorescein by an isothiocyanate derivate. However, fluorescent tags suffered from quenching until long-lived fluorescence in rare earth elements was discovered, which was exploited for immunoassays many years later [7]. The first clinical immunoassay, making use of the specificity of antigen-antibody reactions, was developed for the determination of insulin in human blood by Yalow and Berson in 1960 [8]. The assay was performed in a competitive format: The antibodies bind to insulin, and by doing so, the binding of crystalline insulin–131I is inhibited competitively. In this assay, radioactive tags were applied for detection, but the accompanying hazards led to the development of enzymatic tags, and initiated the era of enzyme‐linked immunosorbent assays (ELISA) [9,10]. For this technique, enzymes like horseradish-peroxidase are employed as tags, and by addition of hydrogen peroxide as a substrate, and tetramethylbenzidine as a chromogen, a photometric detection becomes possible. Such conjugates have also been used histochemically to detect antigens in tissue sections [11]. The resulting immunohistochemical (IHC) staining allows an assessment of the expression level and the intracellular localization of a target protein directly in the tissue. An overview on different types of tags employed for bioassays is given in a review by Hempen and Karst [12]. Another benchmark in modern biochemistry is based on the separation of proteins in a complex sample by 2D‐gel electrophoresis (GE) [13,14]. After tryptic digestion of selected protein spots, the resulting peptides are usually separated by liquid chromatography (LC). They are subsequently analyzed by mass spectrometry, which became the main workhorse for protein identification with the introduction of electrospray ionization (ESI) [15] and matrix‐assisted laser desorption/ionization (MALDI) [16] for the ionization of peptides and proteins. In addition to protein identification, the determination of protein quantity or quantitative change in a complex sample is highly desirable. This knowledge provides new insights into the functionality of a living organism, facilitates the identification of disease markers, and contributes to the discovery of proteins as therapeutic targets [17]. The combination of LC‐MS/MS with stable isotope tags in synthetic amino acids treated cell culture (SILAC) [18], isotope‐ coded affinity tags (ICAT) [19], or isobaric tags for relative and absolute quantification (iTRAQ) [20], yields semi-quantitative data, and started the era of quantitative proteomics. Nevertheless, this approach still poses a challenge to methodologies for absolute quantification like PROTEIN‐AQUA (protein absolute quantification) [21], since standards need to be synthesized for any investigated peptide (upon tryptic digestion, a single protein generates 30–50 different peptides [22]). Although a number of promising quantification strategies have been proposed for the application of mass spectrometry as a quantitative tool, this field remains a topic of current research. On the contrary, inductively coupled plasma mass spectrometry (ICP-MS) is the preferred means to quantify the elemental composition of materials, especially their ultra-trace components. It has found acceptance in various applications including environmental (e.g., drinking, river, sea and waste water analyses) [23,24], geological (e.g., trace element patterning) [25], clinical (e.g., determination of trace metals in blood, serum and urine) [26], and industrial [27] analysis. With few exceptions, ICP-MS is a detection and diagnostic tool

not commonly applied in the biological or biochemical arena, although its high sensitivity which is independent of structure, wide linear dynamic range, and multielement capabilities provide promising features for bioanalytical applications [28]. Furthermore, methods based on ICP‐MS offer simple quantification concepts, low matrix effects compared to conventional bioanalytical techniques, and biologically relevant limits of detection (LODs) in the low pg g −1 range [29]. Since a variety of biological assays are conducted on solid surfaces, the application of direct sampling methods is advantageous for high throughput analyses. Concerning detection by atomic spectroscopy two approaches look very promising: 1) direct monochromatic imaging by glow discharge emission spectroscopy as pioneered by Gary Hieftje's group (see for instance Engelhard et al. [30]), and 2) laser ablation (LA)‐ICP‐MS. In this review, we will focus on ICP‐MS‐based techniques to discuss their advantages, shortcomings, and perspectives for the investigation of biological samples. 2. Definition label and tag A lot of confusion exists about the definition of labels and tags in the MS community since there is no consistent definition applied in the literature. Hence, both expressions are used synonymously. Therefore, we would like to propose a definition derived from the packaging industries to differentiate between those two expressions: The difference between a label and a tag is how they are attached to the material. A label, e.g. a sticker, is stuck onto the package, and a tag is tied or wired to the package. Thus, a label is nothing else than a modification of the atomic composition of the same biomolecule. The number of atoms stays constant, and the molecular weight is not changed significantly. This particularly holds true for isotopically modified biomolecules, where the biochemical activity or the chemistry of the biomolecule is not altered. In contrast, a tag is always chemically attached to a biomolecule by use of an adequate derivatization method, the so-called bioconjugation. Thus, it will change the chemical and biological properties significantly, such as the molecular weight, the solubility, the isoelectric point, etc. 3. Heteroelement detection of biomolecules by ICP-MS In our review we focus on those methods in which antibodies are applied for detection of a target molecule, i.e. the biomarker. However, the investigation of new matrices, e.g. thin tissue sections, was first conducted by heteroelement detection of biomolecules. Thus, we would like to point out first applications of biomolecule detection by ICP‐MS, which relied on the use of heteroelements as a label for quantification. Many quantification schemes in proteomics are based on such labels, and often isotopically enriched labels are employed for absolute quantification, as we have discussed in the previous chapter. Metal-containing proteins comprise one third of the whole proteome [31], and are detectable by ICP‐MS in a straightforward way. However, this technique features a structure-independent detection, and molecule specific MS is needed for protein identification in order to investigate the diverse functions of metalloproteins in living organisms. On the other hand, this limitation provides one of the main advantages of ICP‐MS: the structure‐independent detection offers low matrix effects in biological samples, and reduces the complexity of the sample. The benefits of combining molecular MS with elemental MS have been recently reviewed by Becker and Jakubowski [32]. In the past years, ICP‐MS has been applied more and more often for the relative and absolute quantification of biomolecules, and an overview on the use of heteroatoms, labels, and tags for quantitative MS analysis of biomolecules was given by Prange and Pröfrock [33]. The detection of proteins by, for example, sulphur (methionine or cysteine moieties), phosphorus (phosphoproteins), or metal heteroatoms has become an established method during the last ten years.

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Furthermore, the impact of heteroatom-based proteomics has been discussed by Alfredo Sanz-Medel [34]. A detailed review on organometallic derivatizing agents was presented by Bomke et al. [35]. The application of isotope dilution analysis enables absolute protein quantification, which was highlighted by Jörg Bettmer [36]. The above mentioned reviews mainly focus on solution nebulization ICP‐MS and thus, we add a short overview on the development of biomolecule detection by LA‐ICP‐MS. Heteroelement detection of biomolecules by LA‐ICP‐MS For a variety of applications it is important to sustain spatial information from a sample and hence, LA‐ICP‐MS techniques are employed. The sample is ablated in an air-tight chamber by a focused laser beam, and the generated aerosol is transported to the ICP by a carrier gas [37]. LA‐ICP‐MS‐based methods routinely offer a spatial resolution down to 5 μm [38]. In a recent work employing laser micro dissection ICP‐MS, a laser spot size of 1 μm was employed for two single line scans to analyze Cu in a brain standard slice [39]. However, the spatial resolution of an analysis is mainly determined by the sensitivity of the element of interest at a certain laser spot size. LA‐ICP‐MS has been increasingly utilized to gather information on biological samples. This technique can provide quantitative information, which is difficult to achieve by conventional bioanalytical approaches. In a recent review by Konz et al., the use of LA‐ICP‐MS for quantitiative biomedical applications is discussed in detail [40]. First biological applications of this technique focused mainly on SDS‐ PAGE-separated proteins and the detection of metals bound to those proteins. Furthermore, the analysis of thin tissue sections is performed by LA‐ICP‐MS. The potential of LA‐ICP‐MS to detect, quantify, and map biomolecules was first described by Neilsen et al. in electrophoresis gels [41]. In this work, human serum was enriched with Co, and the proteins were separated by gel electrophoresis. A distribution map of Co was obtained after LA‐ICP‐MS analysis, which enabled the identification of the main Co binding serum proteins by comparison to a parallel Coomassie Brilliant Blue stained gel. Furthermore, the possibility of using metal enriched gel standards for calibration was demonstrated by the authors. For the determination of the detection limit, gels were cast with Co enriched Milli‐Q water in a concentration range from 0.7 to 700 μg mL −1. The limit of detection was 0.29 ng Co [41]. One year later, the binding of lead to humic and fulvic acids by gel electrophoresis and LA‐ICP‐MS was studied by Evens and Villeneuve [42]. The detection and quantification of phosphorylated proteins by LA‐ ICP‐MS in the pmole range after gel electrophoresis and electroblotting was first described by Marshall et al., [43], and Wind et al. [44]. In 2003, LA‐ICP‐MS was implemented by Chéry et al. for the detection of selenoproteins in red blood cell extracts and in selenized yeast after 1D and 2D sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS‐PAGE), respectively [45]. A comparison with the most sensitive staining method for electrophoresis gels shows that LA‐ICP‐MS provides usually lower detection limits for metal binding proteins [46] than those achieved by silver staining, which typically amount to 1 ng of protein [47]. Further details on gel electrophoretic separation of metalloproteins, and the use of LA‐ICP‐MS as a detection tool are given in a review by Ma et al. [46], and more recently by Sussulini and Becker [48]. However, the application of SDS‐PAGE with LA‐ICP‐MS detection is still hampered due to the high risk of metal losses during the sample preparation, and separation procedure [49]. Metal losses are influenced by the affinity between metal and protein and thus, a careful optimization of the involved sample preparation steps is necessary, as pointed out by Sussulini and Becker [48]. The bioimaging of tissue sections is increasingly applied in modern medicine as it can be an important tool for the interpretation of diseases. For this reason, bioimaging analytical techniques have been developed in the last years to monitor elemental and molecular

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distributions in tissues, as has been recently reviewed [38]. Besides LA‐ICP‐MS [38,50], these include scanning electron microscopy with energy-dispersive X-ray analysis (SEM-EDX) [51], synchrotron X-ray fluorescence (SXRF) [38], proton-induced X-ray emission (PIXE) [52], secondary ion mass spectrometry (SIMS) [38], and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) [53]. Metals are usually heterogeneously distributed in tissues or cells and thus, LA‐ICP‐MS seems to be the most convenient alternative for elemental bioimaging in tissue sections, since it provides a virtual lack of sample preparation, multielemental detection with high sensitivity, and high spatial resolution [50]. Indeed, it has been widely used over the last decade for bioimaging of tissue samples [54,50]. ICP-MS excels by its exceptional accuracy and ease of calibration. However, elemental quantification in LA-ICP-MS cannot be applied in a straightforward way. Generally, calibration methods require the use of an internal standard to account for matrix dependence of the ablation process, variations in mass ablated, differences in transport efficiency, and instrumental drift [55]. The isotope 13C has been typically applied as an internal standard in elemental bioimaging applications, despite its difference in mass and first ionization potential to many analytes, the possibility of abundance sensitivity effects, and a poor signal to noise ratio [56]. Furthermore, it may be heterogeneously distributed in tissue samples owing to differences in the water content [57]. A recent study by Frick et al. demonstrated the difficulties in the use of 13C as an internal standard for biological applications. In contrast to the analytes of interest, which mainly localize in the particulate phase after laser ablation, a matrix dependent formation of a carbon gas phase was observed, which limits the quantification using non-matrix matched calibration standards [58]. Thus, the application of an alternative internal standard might be advantageous for thin tissue analysis. A new approach based on iodine as an internal standard for tissue sections was recently proposed by Giesen et al., [59]. A short incubation of 60 s with KI•I2 caused iodination of the tissue, and the resulting iodine signal in the ICP‐MS correlated with the tissue thickness. Only very few reference materials are available for the quantification of biological tissues by ICP-MS. Pressed pellets of TORT‐2 (lobster hepatopancreas), DOLT-2 (dogfish liver), and DORM-2 (dogfish muscle) were applied by Jackson et al. to perform a quantification of Cu, Zn, and Fe in rodent brains by LA‐ICP‐MS [60]. For most other cases, laboratory standards are produced to permit an improved matrix matching of standards to samples. Some authors have proposed quantification methods based on matrix-matched laboratory standards. Examples are spiked, frozen and embedded blood [61], spiked tissue homogenate [62], or solution-based calibration [62]. With these methods, detection limits in the low μg kg−1 range were achieved for Th and U in human brain samples [62], and for Pt in rat brain [61,63]. Furthermore, quantification using spiked thin polymer films was reported [64]. The detection of non-metals such as sulfur and phosphorus [65], and the distribution of metals such as Cu, Zn, Fe, Li, K, and Na in tissue sections [66] has been subject of many research projects. It was shown in the literature that tumor boundaries were clearly marked by imaging of 31P in lymph node biopsies [65]. The mapping of metallodrugs by LA‐ICP‐MS is also possible [61,67,63]. Becker et al. used matrix‐matched laboratory standards to receive information on the quantitative distribution of Cu, Zn, U, and Th in 20 μm thin tissue sections of human brain (hippocampus) [62]. Moreover, images of element distribution of metals (Zn, Cu, Fe, Mn, and Ti) in mouse heart tissue were compared to SIMS images of alkali metals and biomolecules [66]. In a recent study by Wu et al., laser microdissection ICP‐ MS was applied for imaging of heteroelements in brain tissue at laser spot sizes from 30 to 4 μm [39]. Furthermore, Giesen et al. presented a spatial resolution at the single cell level in an investigation of a liver biopsy tissue [59]. The cell nuclei of fibroblast cells and of a tissue were stained by iodine, which was analyzed by LA‐ICP‐MS.

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4. Immunoassays Although heteroelement detection is already well established for detection and quantification of biomolecules by ICP-MS, it does have an important limitation: heteroelements are often not specific for a target molecule, and the number of natural labels detectable by ICPMS is rather low. A technique which offers high target specificity and amplification of the tag looks promising for biomolecule detection. Immunoassays fulfil these needs and thus, are routinely employed in biochemistry and medical diagnostics due to their capability of fast identification and quantification of biomolecules [68]. The target specificity achieved is based on antibodies. These serum proteins are produced in a vertebrate's immune system by B‐lymphocytes and plasma cells as a response to foreign target molecules (‘antigens’).

4.1. Antibodies The efficiency of any immune reaction is highly dependent on the specificity, and the affinity of the antibody to its antigen. It needs to be kept in mind that the affinity of the antibody is the limiting factor in an immune reaction. The type of antibody used in immunoassays is immunoglobulin G (IgG), which accounts for 75% of all serum immunoglobulins [69]. It consists of two identical heavy chains (H) of 50 kDa, and two identical light chains (L) of 24 kDa [70]. Light and heavy chain, as well as their identical counterpart, are linked by non-covalent forces and disulfide bonds (see Fig. 1). The N‐terminal domains of each light and heavy chain are highly variable, and represent the two antigen-binding sites. The C‐terminal domains are constant in their amino acid sequence, and account for antibody binding to a cell surface. Upon papain digestion, the antibody dissociates into three parts: two Fab fragments (antigen binding), and one Fc fragment (crystallizable region). For antibody production, a substance triggering the immune response (‘immunogen’) is injected to a host animal. By means of the Fab fragment, antibodies recognize a specific steric pattern (‘epitope’) on the surface of

an antigen. Small molecules below 1500Da, which do not trigger an immune response (‘haptens’) [72], can be conjugated to a carrier protein for immunization [73]. Polyclonal antibodies are obtained from serum and therefore originate from different B‐lymphocytes, causing heterogeneous specificity for a variety of epitopes. The reproducible production of homogeneous, and thus highly specific monoclonal antibodies is feasible by the production of genetically identical hybridoma cell lines [72].

4.2. Tagging chemistry for ICP-MS applications Apart from the detection of heteroelements, any biomolecule of interest can be analyzed by the use of elemental tags. In ICP‐MS, identically tagged biomolecules provide equivalent sensitivity, which is a prerequisite for quantification [74]. The application of different metal tags enables the discrimination of various parameters within a single experiment and hence, the analysis of a complex biological system. Commonly used metal tags are nanoparticles or bifunctional ligands. The latter consist of a reactive group, a linker or spacer, and a chelate complex as it is shown schematically in Fig. 2. These bifunctional ligands employed for tagging often covalently bind to ε‐amino groups of lysine residues and to N-terminal α-amino groups at alkaline pH values. Isothiocyanatobenzyl residues (SCN) and N-hydroxysuccinimide ester (NHS) are frequently applied reactive groups for this kind of tagging chemistry. Alternatively, maleimidoethylacetamide (maleimido) residues are used for conjugation to sulfhydryl residues after selective reduction of the antibody's cysteine-based disulfide bridges. The latter reaction step is critical, as it might affect the antibody's binding efficiency. Concerning the chelating group, different linear or macrocyclic compounds based on polyaminocarboxylic acid are usually applied to bind the metal. These chelating groups are selected based on the charge state of the metal. Furthermore, they should improve water solubility, and should have a high complex constant in order to be resistant against metal exchange. The most important compounds in this group are: diethylene-triamine-tetraacetic acid (DTTA), diethylene-triamine-

Fig. 1. Scheme of immunoglobulin G (IgG). Light (light blue) and heavy (dark blue) chains are connected by disulfide bonds. Parts with variable amino acid sequence, which represent the antigen binding sites, are ruled. VH: variable heavy; VL: variable light. Upon papain digestion, the antibody dissociates into three parts: two Fab fragments (antigen binding), and one Fc fragment (crystallizable region). Figure adapted from reference [71].

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Fig. 2. Bifunctional ligand employed for antibody tagging. The chelate complex is attached to a reactive group, which enables conjugation to the antibody. A spacer is introduced in between the chelate and the reactive group to improve reacitivity. M3+ is typically a lanthanide ion.

pentaacetic acid (DTPA), and 1,4,7,10-tetraazacyclo-dodecane-1,4,7,10tetraacetic acid (DOTA). Historically, these chelating compounds were employed in combination with radioactive tracers. McDevitt et al. presented a method for binding 225Ac to a SCN-DOTA, and attached the complex to an IgG or other biomolecules [75]. The use of a NHS linker attached to DOTA was described by Lewis et al., who performed tagging with the radioactive isotopes 111In and 90Y [76]. To avoid health hazards caused by radioactive material, fluorescent lanthanides (Eu, Sm, Tb, Dy) were applied as central ions (AutoDELFIA), which offer multiparametric approaches based on time resolved fluorescence (TRF) [77]. The use of the ligand DOTA for MS-based applications was demonstrated by Whetstone et al. for the differential tagging of peptides [78]. The authors showed that this approach was suitable for reversed phase chromatography separation and subsequent MS/MS experiments. The high log K (DOTA) values for lanthanides result in particularly stable complexes [79]. This provides a variety of choices for different metal tags to be employed in multiparameter experiments. In 2005, Krause et al. applied for a patent for a DOTA-based reagent with a cysteine reactive maleimide group, and a biotin modification for purification and enrichment of tagged peptides via biotin–avidin affinity chromatography, named metal-coded affinity tag (MeCAT) [80]. In a study by Ahrends et al. reaction parameters were optimized, and MeCAT was used to analyze proteins of the Sus scrofa eye lens in a model system [81]. It was the first time that lanthanide tagging approaches for ICP‐MS detection were combined with a proteomic workflow for protein identification. The authors proved that the same differentially tagged peptides co-elute during LC‐ESI‐MS/MS, which is a prerequisite for multiparametric proteomics. Hence, the low matrix effects of biological samples in ICP‐MS, and the use of simple, non-species specific metal standards opened up new possibilities for absolute quantification of proteins and peptides, which can be identified by molecule specific MS in a parallel experiment. The limit of detection for Ho 3+-tagged bovine serum albumin was calculated as 110 amol. Furthermore, an investigation of variations in an Escherichia coli (E. coli) proteome due to different growth temperatures was conducted with this technique [82]. An implementation of MeCAT to absolute peptide, and standard protein quantification was published recently: Selected peptides were tagged with MeCAT‐Eu and quantified by isotope dilution ICP‐MS [83]. A peptide mix at different concentrations was separated by reversed‐phase (RP) chromatography before ICP‐MS detection and thus, a calibration was performed in one run. For quantitative protein identification, a lysozyme and a bovine serum albumin tryptic digest were tagged with MeCAT‐Eu. The resulting peptides were analyzed by parallel LC‐ICP‐ MS and LC‐(RP)‐ESI‐ion trap Fourier transform ion cyclotron resonance (IT‐FTICR) [83]. In the case of immunoassays analyzed by ICP-MS even better limits of detection can be expected with the polymer-based elemental tagging kit developed by Lou et al. [84],

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who used a polymer with various binding sites for DOTA to even increase the number of metal tags per binding site. Another possibility to increase sensitivity is the application of nanoparticles, which include thousands of atoms, at least. Nanoparticles are mainly applied for the visualization of proteins and oligonucleotids in electron microscopy, but also for their detection and quantification in combination with UV/Vis and fluorescence spectrometry, or electrochemical methods as bioassays [12]. The particle size can differ between smaller than 2 nm (gold cluster) to over 100 nm (colloidal gold), depending on the application. Especially gold nanoparticles are often employed as tagging reagents for antibodies and enzymes owing to their robustness and easy handling. Particles are coupled to reactive groups like maleimido or NHS residues for tagging strategies, as is also the case for bifunctional ligands. But there are some critical points to be kept in mind using Au nanoparticles as tagging reagents: Gold has a high affinity to surfaces and hence, good blocking and washing steps are required, and the nonspecific binding rate is required for analysis. Further, it is difficult to synthesize nanoparticles of uniform size, which is required for quantification strategies. In the paper discussed subsequently different kinds of gold nanoparticles and tagging strategies are presented. Some groups synthesized nanoparticles on their own (Zhang et al.) [85], others used commercially available gold tagged secondary antibodies (Quinn et al., Seuma et al.) [86,87]. Some groups additionally employed silver enhancement kit for further signal amplification (Seuma et al., [87], Liu et al., [88]). The different strategies are summarized in Tables 1 and 2. The tagging of peptides [89] and proteins with iodine has also been described in the literature. The binding is covalent and hence, this approach is applicable even in advance to electrophoresis and electro-blotting [90,91]. Radioactive iodine has been first used by Hunter and Greenwood [92] for tagging of hormones. Up to now, many other complex iodination agents have been applied [93–96], but the use of a saturated iodine solution in potassium iodide is less laborious and inexpensive. Direct tagging of proteins with iodine occurs at the two ortho-positions of tyrosine and at histidine residues. These electrophilic aromatic substitutions require reactive iodine species like triiodide, which is formed in aqueous solution from iodide and elemental iodine. The iodination of proteins, whole proteomes, and antibodies for analysis by LA‐ICP‐MS of Western Blot membranes was examined by Waentig et al. [90]. For all tagging strategies described above, the conjugation chemistry is rather delicate. On the one hand the antibody's binding capability to its antigen should not be affected, and on the other hand other properties such as charge, solubility, pI, and pH stability should not deviate too much from the untagged species. Thus, the tagging conditions have severe consequences for the analytical figures of merit of the immunoassay. Owing to the different types of chemistry, the number of reaction sites might vary. Generally, the number of metals which can be attached to the antibody limits the assay sensitivity. 4.3. Strenghts of ICP-MS detection for immunoassay applications As has been mentioned before, tagging reagents historically consisted of radioactive tags, fluorophores, enzymes, or organic dyes. Although these tags are highly sensitive, they are accompanied by several limitations, which can be overcome by ICP‐MS detection. The application of radioactive tags for immunoassays has almost vanished from the literature nowadays due to the accompanying hazards, and high expenses in radioactive waste disposal. On the other hand, the use of organic dyes as a direct tag, or created by an enzymatic reaction, is commonly employed for diagnostic purposes owing to low assay costs. The downside is that multiparametric studies are difficult to realize due to a spectral overlap of available dyes. The simultaneous analysis of more than 10 parameters is feasible by fluorescent detection [97]. However, this application is declined by

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Table 1 Solution nebulization ICP-MS-based immunoassays. Year

Application

One-parameter assays 2001 Determination of thyroid hormones in human serum in a sandwich-type immunoreaction 2002 Indirect measurement of rabbit-anti-human IgG 2002 Determination of proteins

2002 Monitoring of cell line transfection 2002 Determination of thyroxin (T4) in human serum in a competitive immunoassay 2007 Determination of peanut allergens

2010 Determination of Ochratoxin A (OTA) in wine 2010 Determination of E. coli O157:H7

2011 Determination of human carcinoembryonic antigen 2012 Determination of progesterone in cow milk

Multi-parameter assays 2002 Determination of endogenous proteins Smad2 and Smad4 in mouse skeletal muscle C2C12 cell line 2004 Detection of tumor markers in human serum in a dual sandwich immunoassay 2004 Analysis of prostate cancer markers in human serum in a dual sandwich immunoassay 2006 Identification of four antigens on MBA-4 cells 2006 Detection of mRNA in MBA-1 cells

Tag

Detection limit

Reference

Biotinylated antibodies and Eu3+-tagged streptavidin

0.5 mIU L−1, linear up to 170 mIU L−1

[102]

Colloidal Au goat-anti-rabbit IgG: 0.008ngmL−1 Immunoassay: 0.4ngmL−1 of rabbit-anti-human IgG, linear between 0.8 and 50ngmL−1 Eu-based AutoDELFIA assay Target proteins: 0.1–0.5 ngmL−1 alpha-fetoprotein: 0.22 ngmL−1; sample volume: 200 μL ➔ 44 pg NANOGOLD goat anti-human Fab′ conjugate (Fab′-nanoAu), Fab′-nanoAu: 0.1 fmol mL−1 gold nanoparticle diameter approximately 1.4nm Fab′-nanoAu – DTTA-Eu3+-tagged BSA, conjugated to T4 7.4 ng mL−1 thyroxin in 25 μL ➔185 pg ➔ 0.24 pmol Eu3+-tagged anti-mouse antibody (AutoDELFIA) Free antibody: 0.1 ng mL−1, linear from 2 ng mL−1 until 100 μg mL−1 raw peanut protein: 1.5 ng mL−1 in cereal matrix: 2 mg kg−1 peanuts Gold nanoparticle (diameter approximately 40 nm) tagged 3 pg mL−1 OTA in 50 μL ➔ 150 fg secondary antibodies ➔ 370 amol Detection limit: 500 E. coli cells per mL, linear Gold nanoparticles (diameter approximately 10 nm) conjugated with mouse monoclonal antibodies against E. dynamic range between 500 and 5×105 E. coli coli O157:H7 cells per mL Gold nanoparticles with silver enhancement 0.03 ngmL−1 (0.15 pM), linear dynamic range 0.07–1000 ng mL−1 CdSe/ZnS quantum dots High analyte concentration: 0.32 ngmL−1, linear range 1.7–31 ng mL−1 Low analyte concentration: 0.028 ng mL−1, linear range 0.07–1.43 ngmL−1 Goat-anti-rabbit IgG, tagged with colloidal gold nanoparticles (diameter approx. 15 nm)

[85]

[74]

[105] [103]

[112]

[106]

[107]

[88] [108]

Eu3+-tagged antibody (AutoDELFIA), Fab′-nanoAu

Target proteins detectable between 2 and 100 ngmL−1, sample size: 0.5 mL

[86]

SCN-DTTA-Eu3+ and SCN-DTTA-Sm3+ tagged antibodies

Injected volume: 200 μl; LODs: 1.2 μgL−1 for AFP; 1.7 μg L−1 for hCGβ Injected sample volume: 120 μl; LODs: 0.01 μgL−1 (0.22 ngL−1 Eu) for fPSA; 0.02 μgL−1 (0.11 ngL−1 Sm) for tPSA –

[109]

Eu3+ and Sm3+ tagged antibodies (AutoDELFIA)

Lanthanide-tagged (Eu, Tb, Sm)3+ affinity reagents (AutoDELFIA), Fab′-nanoAu Gold tagged anti-mouse-Au (NANOGOLD), Eu3+-tagged anti-rabbit antibody (AutoDELFIA), Tb3+-tagged streptavidin (AutoDELFIA) DOTA (Pr, Eu, Gd, Ho, Tb)3+-tagged antibodies

2009 Detection of five cancer biomarker proteins in human serum Lanthanide-tagged polymertags (DVS Sciences) 2011 Measurement of 34 parameters simultaneously in single cells of human bone marrow 2012 Quantification of 18816 phosphorylation Lanthanide-tagged polymertags (DVS Sciences) sites in single PBMCs

the compensation needed for a correction of spectral overlap [98], which is virtually absent for ICP‐MS‐based detection. Furthermore, fluorescence is affected by the vicinity of the probe, which hampers quantification. On the contrary, elemental tags are stable, and the ICP signal is independent of the tag's vicinity. Additionally, there is no equivalent in ICP‐MS to the phenomenon of autofluorescence, which produces a constant background signal in fluorescent detection. Combined with the considerably higher number of parameters, which are simultaneously accessible, ICP‐MS is a technique with great potential for our comprehension of biological systems. Briefly, a sample, most commonly an aerosol produced by nebulization or by laser ablation (LA), is injected into a high temperature plasma where the sample is promptly vaporized, atomized and

[29]

[116]

–

[111]

CA125 antigen: 12–24 IU mL−1

[113]

–

[98]

–

[114]

ionized. Mass analysis provides a “mass fingerprint” that identifies the isotopes, in our case the antibody tags contained in the sample. Relative to existing analytical methods, the ICP-MS approach provides several important advantages for affinity-based assays: • The use of metal tags in combination with ICP‐MS provides equivalent sensitivity to identically tagged biomolecules [74], which is a prerequisite for quantification. ICP‐MS methods offer a promising quantification concept based on simple, nonspecies specific metal standards, which opens up new possibilities for absolute biomolecule quantification with biologically relevant limits of detection (LODs) in the low pg g −1 range. However, it should be kept in mind that sample preparation

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Table 2 LA‐ICP‐MS-based immunoassays. Year

Application

One-parameter assays 2005 Analysis of Mre11 protein in crude lysates of CHO-K1 fibroblasts 2005 Detection and imaging of betaamyloid protein in immunohistochemical sections from mouse brain 2008 Imaging of cancer biomarkers in tissue sections and tissue microarrays 2011 Imaging of triiodide-tagged antibodies 2012 Determination of holoceruloplasmin in human serum. Antibodies were immobilized on microarrays. Multi-parameter assays 2007 Multiple protein detection on immuno-microarray 2008 2009 2011

2011

• • •

•

•

•

•

•

Detection limit

Goat-anti-rabbit secondary antibody coupled to gold cluster (diameter approximately 10 nm, Auroprobe, Amersham) Biotinylated secondary antibody, Eu3+-tagged streptavidin

Gold standard: 0.449 fmol gold cluster tagged antibody: 100 μm [117] 10.66 fmol➔0.200 amol difference caused by relative high background of gold on blot membrane – 40 or [118] 100 μm

Gold nanoparticle tagged secondary antibody (diameter approximately 5 nm) plus silver enhancement solution Iodine

LODs calculated from a tissue section spiked with colloidal Au and Ag dilution series: 0.01 ng Au; 0.005 ng Ag BSA: 0.75 pmol Beta-casein: 2 pmol Cu: 46.7 ngmL−1 ➔126 nmol L−1, sample volume 126 pL➔15 amol holoceruloplasmin

Copper (heteroelement of holoceruloplasmin)

Sm3+-tagged anti-alpha-fetoprotein antibody, Eu3+-tagged carcinoembryonic antigen antibody, Au nanoparticle tagged goat-anti-human IgG Western blot assay for Cytochrome Iodinated anti-CYP2E1, SCN-DOTA (Eu3+) tagged P450 in rat liver microsomal protein anti-CYP1A1 Western blot assay of BSA, lysoAntibodies tagged with DOTA (Eu, Tb, Tm, Ho)3+ zyme, casein Multiparameter Western blot assay Antibodies tagged with SCN-DOTA (Tb, Tm, Ho, Eu, Lu)3+ for Cytochrome P450 in rat liver microsomal protein Simultaneous detection of tumor Antibodies tagged with DOTA (Tb, Tm, Ho)3+ markers in breast cancer tissue

recoveries need to be identical for different biomolecules to prevent a bias in quantification. Matrix effects of biological samples in ICP‐MS are low compared to conventional bioanalytical techniques. The matrix and substance independent calibration provides high precision and accuracy of measurements. Generally, ICP‐MS offers a large linear dynamic range between 9 and 12 orders of magnitude, depending on the manufacturer and thus, MS detection is less prone to saturation effects. Furthermore, the reagents are stable against time, temperature, and light (the isotopic masses do not change, bleach, or degrade). Lower background and blank levels are observed if lanthanides or rare earth elements are used as elemental tags, for which lowest limits of detection can be achieved in ICP-MS due to two reasons: First, the very low first ionization potential guarantees 100% ionization efficiencies. Second, the blank values of these elements are low in the environment, in biological and medical samples, and in the biomolecules of interest. The background is not affected by materials used for production of the containers, cuvettes, wells or the sample composition as it is the case for instance from plastic containers and plates in case of fluorescence or photometric detection. Reduction of non-specific background: In ICP‐MS, there is no equivalent to autofluorescence. However, sample preparation steps need to be carefully optimized to reduce non-specific background from tagged antibodies. Independence of analytical response from incubation or storage times (as protein degradation does not affect analysis of an elemental tag). Larger multiparametric and multiplexing potential. Mass cytometry might allow the simultaneous detection of around 100 parameters that can be analyzed by distinguishable single (enriched) isotope tags [4,98].

Laser spot size

Reference

Tag

[87] 100, 20, 5 μm 500 μm [90] 200 μm [100]

0.2, 0.14, 0.012 ng mL−1, respectively

30 or 90 μm

140 fmol CYP1A1, 70 fmol CYP2E1

500 μm [119]

ng levels of antigen➔sup mol range

500 μm [120]

–

500 μm [121]

–

200 μm [122]

[99]

• Better spectral resolution depending only on the abundance sensitivity of the selected mass spectrometer and the isotope/element selected for tagging. • Simple signal amplification properties by attaching more than one tag per biomolecule. As mentioned before, the low elemental detection limits in the pgg−1 range, the possibility for absolute quantification, and the multi element capabilities make ICP‐MS an ideal detection method for bioanalytical techniques like immunoassays, facilitating a high throughput format. Furthermore, the application of laser ablation (LA)‐ICP‐MS is advantageous for the analysis of solid samples such as tissues [38] or microarrays [99,100] since it provides high lateral resolution in the low μm range [101], combined with the virtual lack of sample preparation. In the life sciences, this is of high benefit for a convenient analysis of a variety of different biomarkers, antigens, and metalloproteins. 5. Applications of immunoassays with ICP-MS detection In this section, we focus on two different detection methods for ICP‐MS‐based assays: Detection by solution nebulization ICP‐MS, and detection by LA‐ICP‐MS. For the former detection mode, the assay is usually conducted in solution, on a cell surface, or on a microtiter plate. For the latter, one of the binding partners (antibody or antigen) is immobilized on the surface of a substrate (microarray or Western Blot membrane). Furthermore, thin cuts of tissue samples are investigated by laser ablation as a sample introduction system for immuno-imaging. All formats will be discussed in more detail in the following chapters. From the discussion in the previous paragraph it can be concluded that the analytical figures of merit of an ICP‐MS‐based immunoassay are directly related to the antibody affinity, the number of tags attached to the antibody, the element background in the sample, and the number of antigens, i.e. antibodies, being present in the assay.

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Fig. 3. This scheme illustrates an immunoassay applied for the multiparametric detection of four antigens on a cell surface. The antibodies are tagged with lanthanides and specifically bind to their antigens (in this exemplary case different cell surface markers). The sample is introduced into the ICP, combusted, and detected. The resulting spectrum reflects the antigen abundance on the cell surface (1:1:1:1).

5.1. Immunoassay detection by solution nebulization ICP‐MS In this assay type the antibody tag is usually dissolved by acids, and the resulting solution is analyzed by conventional pneumatic nebulization. From an analytical point of view, dissolution is inherently connected with a dilution process and thus, the limit of detection of this type of assay might be degraded compared to a direct analysis. However, this type of assay can easily be quantified by liquid standard solutions. A typical ICP‐MS‐based multiparameter assay is depicted in Fig. 3. Lanthanide tagged antibodies specifically bind to their antigens, in this exemplary case cell surface markers. The sample is combusted in the ICP, and detected subsequently. The resulting spectrum reflects the antigen abundance on the cell surface. 5.1.1. One-parameter assays The first element-tagged immunoassay was described in 2001 by Zhang et al. for the determination of thyroid hormones in human serum by means of a sandwich‐type immune reaction [102]. The authors immobilized anti-thyroid hormone antibody on a well surface, incubated with the hormone, and employed biotinylated secondary antibodies plus Eu 3+‐tagged streptavidin for detection. A 1% HNO3 solution was used to dissociate Eu from the immobilized complex. The liquid extract was subsequently analyzed by ICP‐MS, and the results were in agreement with a radioimmunoassay performed for comparison. In 2002, the same group described the determination of thyroxin in a competitive immunoassay [103]. The concentration of thyroxin was determined by its ability to inhibit the binding of Eu-tagged thyroxin to the corresponding monoclonal antibody. Owing to the enrichment of antigen on a microwell surface, a detection limit of 7.4 ng mL −1 was achieved in 25 μL sample, with a working range up until 233 ng mL −1. This result is comparable to an enzyme immunoassay for thyroxin with photometric detection, which provides a working range of 10–240 ng mL −1 [104]. Apart from lanthanides, gold nanoparticle tags were applied for tagging as well. Colloidal gold nanoparticles conjugated with secondary antibodies were employed by Zhang et al. for a sandwich‐type immune reaction [85]. The detection limit for colloidal Au goat-anti-rabbit IgG antibody was 0.008 ngmL−1, whereas the detection limit of the immunoassay for rabbit-anti-human IgG antibody was reported as 0.4 ng mL −1 and thus, about two orders of magnitude higher. Beyond that, the authors conducted a study of nanoparticle atomization, and showed that the ICP‐MS signal is not influenced by the organic matrix [85]. Furthermore, Baranov et al. implemented a gel‐filtration‐based ICP‐MS immunoassay to separate antibodies conjugated to Fab′‐nanoAu or (Fab′)2‐nanoAu. The Fab′-nanoAu antibodies were applied for the monitoring of cell line transfection by ICP‐MS [105]. An ICP‐MS‐based immunoassay for ochratoxin A (OTA) determination in wine was developed by Giesen et al. employing gold nanoparticle tagged secondary antibodies. The

limit of detection for this indirect, competitive assay was 3 pgmL−1 for OTA in wine [106]. Gold nanoparticle tagging was also employed for the sensitive detection of E. coli O157:H7 by ICP‐MS. For this application, monoclonal antiE.coli antibodies were conjugated with gold nanoparticles, and incubated with cells containing E. coli. After acid digestion, the samples were analyzed by ICP‐MS. A detection limit of 500CFUmL−1 (colony-forming-unit per mL)) was obtained for E. coli O157:H7 cells, and the dynamic range was between 500 and 5×105 CFUmL−1. Furthermore, the authors compared the application of directly tagged antibodies with those obtained by tagging via streptavidin–biotin-linkage. The latter approach was less sensitive with a detection limit of 5×104 CFUmL−1 [107]. The determination of carcinoembryonic antigen was achieved by Liu et al. with a limit of detection of 0.03ngmL−1 by use of silver enhanced Au nanoparticles as tagging reagent [88]. Furthermore, quantum dots can be applied as tagging reagents for immunoassays. A recent paper by Montoro-Bustos et al. describes the determination of progesterone in cow milk in a comparison study between fluorimetric and ICP‐MS detection. The latter technique provided a better limit of detection (0.028 ng mL −1 versus 0.11 ng mL −1 for fluorimetric detection) at low analyte concentration [108]. A chronological overview on ICP‐MS-based immunoassays is given in Table 1. 5.1.2. Multi-parameter assays The multiparametric determination of proteins using element-tagged immunoassays coupled with ICP-MS detection was pioneered by Tanner and co-workers [86]. They implemented the Wallac AutoDELFIA reagents, which were originally designed for the time-resolved fluorescence (TRF) detection of enzyme-linked immunosorbent assays (ELISAs). The limit of detection achieved by these reagents was 0.1–0.5ngmL−1 of target proteins, which was about one order of magnitude inferior to the AutoDELFIA™ method [74]. In the first multiparameter assay for the determination of endogenous proteins Smad2 and Smad4 in mouse skeletal muscle C2C12 cell line, one antibody was tagged with Eu (Wallac AutoDELFIA™), and the second antibody was tagged with 1.4 nm nanogold clusters (NANOGOLD®) [86]. Furthermore, Zhang et al. designed a dual sandwich immunoassay method for the simultaneous determination of two tumor markers (alpha-fetoprotein (AFP) and free beta-human chorionic gonadotropin (hCGß)) in human serum [109]. First, the tumor markers were captured by antibodies immobilized on microtiter plates. Second, Eu3+-tagged anti-AFP and Sm 3+-tagged anti-hCGß monoclonal antibodies were applied for detection. The antibodies were conjugated with N-[p-Isothiocyanatobenzyl]-diethylene-triamine-N′,N′′,N′′′, N′′′-tetraacetate (SCN-DTTA-Eu 3+ or SCN-DTTA-Sm 3+). As a last step, Eu 3+ and Sm 3+ were dissociated from the immunocomplex by means of HNO3, and analyzed by ICP‐MS. The measurable ranges of AFP and hCGß were 4.6–500 μg L −1 and 5.0–170 μg L −1 with detection limits of

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1.2 μg L−1 and 1.7μg L −1, respectively. The authors stated that the ratio of tagged lanthanide to the corresponding antibody is crucial. Since signal intensity in ICP‐MS is linearly proportional to the number of atoms provided by the tags, the sensitivity of ICP-MS-linked immunoassays is directly affected. Another dual immunoassay for the simultaneous detection of two prostate cancer markers via flow injection ICP-MS was published by Hutchinson et al. in 2004 [29]. A commercial sandwich immunoassay kit for TRF (AutoDELFIA) was used, employing Eu3+- and Sm 3+-tagged antibodies for detection of free prostate specific antigen (fPSA) and total prostate specific antigen (tPSA) in human serum. After finishing TRF analysis the same samples were analyzed by ICP-MS. Detection limits were 0.01 μg L −1 (0.22ngL−1 Eu) for fPSA and 0.02 μg L−1 (0.11 ngL −-1 Sm) for tPSA. The FI‐ICP‐MS analysis was competitive with TRF in terms of sensitivity and reliability. However, in case of a dual immunoassay, ICP-MS detection is more time consuming than TRF. A typical 96 well plate analysis took approximately 4 hours in this study compared to 30 minutes by TRF. However, the AutoDELFIA kit is limited to four tags which could be detected in one run by TRF. On the contrary, ICP-MS offers the capability of highly multi-analyte assays. In the process of further development of multiparameter ICP-MSbased immunoassays, Ornatsky et al. presented a 4-parameter assay in 2006. In this work, a human leukaemia cell line model was studied by using Sm, Eu, and Tb tags (Wallac AutoDELFIA™) in combination with a NANOGOLD® reagent [110]. The same procedure was also applied for mRNA detection [111]. The AutoDELFIA reagents were also employed by Careri et al. for the detection of hidden peanut allergens in foods implementing Eu3+-tags [112]. The limit of detection calculated for raw peanut protein extracts was 1.5ngmL−1. In a cereal matrix, approximately 2mgkg−1 of peanuts were detectable. Moreover, the authors illustrated that the detection limit is influenced by antibody binding efficiency and non-specific binding, since the ICP‐MS instrumental detection limit of Eu‐tagged antibody was as low as 0.1ngmL−1 [112]. A clinical application for a multiparametric ICP-MS determination of cancer biomarkers in serum and tissue lysates was investigated by Terenghi et al. [113]. The authors employed size exclusion chromatography (SEC) to separate the antibody-antigen complex from unbound species. With this method, a discrimination of ovary and uterus tumor tissue and control samples was achieved. For signal amplification in ICP‐MS, Tanner and co-workers developed a maleimide-functionalized polymer tag, which significantly increases assay sensitivity [84]. These tags enable the detection of proteins at different abundances in a single assay. The authors presented the detection of two cell surface markers, which differ by a factor of around 500. In this work, a 5‐parameter assay was presented, demonstrating the multiparameter capabilities of their polymer tags. This opened up new possibilities for larger multiplex assays, i.e. the multiparametric investigation of different systems states. A landmark publication to achieve this goal was the presentation of a new mass cytometer in 2009. Bandura et al. coupled flow cytometry to a high spectrum generation frequency ICP-time-of-flight (TOF)-MS, which is commercially available as the CyTOF (DVS Sciences Inc., Markham, Ontario, Canada) [4]. Such an instrument was employed for the simultaneous detection of 20 antigens on the surface of single cells of leukemia cell lines and leukemia patient samples, which is hard to achieve by quadrupole-based ICP‐MS instruments. The differential tagging was accomplished by polymer tags, which were chelated with a variety of lanthanide isotopes. These findings formed a basis for highly multiplexed analyses enabling a fingerprint detection of individual types of cell lines, which was realized by Bendall et al. [98]. The authors reported on the simultaneous detection of a multiplex assay with 34 cellular parameters in the primary human hematopoietic system, revealing unexpected cell-type specific signaling. Recently, Bodenmiller et al. compared the signaling response of human peripheral blood mononuclear cells (PBMCs) in eight different donor samples to characterize their signaling dynamics, and to investigate the influence of 27

35

small molecule inhibitors. For each compound, 14 phosphorylation states were measured per cell in 14 PBMC types under 96 conditions, resulting in 18816 quantified phosphorylation measurements from a single sample [114]. This work enabled an inhibitor analysis across cell type in a high-dimensional level, which was previously unknown. These recent publications clearly demonstrate that mass cytometry is a powerful tool for cellular fingerprint analyses, helping to understand drug action, cellular dysfunction, or cancer. Since the ICP signal is proportional to the number of tags attached, a further signal enhancement might be possible by use of lanthanidecoded particles, which in the past could not be applied for tagging in a straightforward way due to difficulties in biofunctionalization. Winnik et al. proposed a solution by growing a functional polymer shell onto the particle surface, which subsequently was biofunctionalized with NeutrAvidin [115]. 5.2. Immunoassay Detection by LA‐ICP‐MS Since a variety of immunoassays are conducted on solid surfaces like blot membranes or microarrays, the application of direct sampling methods is advantageous for detection, and provides a possibility for high throughput analyses. Furthermore, the immunohistochemical localization of a biomarker directly in a tissue is feasible by LA‐ICP‐MS of thin tissue sections. Similar to solution nebulization ICP‐MS, both nanoparticles and chelated lanthanides were employed for tagging and detection. A chronological overview on LA-ICP-MS-based immunoassays is given in Table 2, which differentiates one-parameter and multi-parameter assays. 5.2.1. One-parameter assays Müller et al. employed LA‐ICP‐MS of Western Blot membranes for the first time, employing gold‐cluster‐tagged antibodies for analysis of the Mre11 protein in crude lysates of CHO‐K1 fibroblasts [117]. A detection limit of 0.20 amol was achieved due to the metal cluster, which enhanced sensitivity in correlation to the number of cluster atoms [117]. The direct detection of biomarkers in tissue sections is facilitated by the tagging of antibodies. Hutchinson et al. applied Eu- and Ni‐ coupled secondary antibodies for LA-ICP-MS imaging of beta‐amyloid deposits in mouse brain tissue to study Alzheimer´s plaques [118]. The methodology based on single-spot ablation, single line rastering and 2D-rastering revealed the distribution of tagged proteins in histological sections of brain tissue. Ni displayed a higher background than Eu and thus was distributed in the whole tissue. However, Ni signals in the LA‐ICP‐MS image correlated with a Diaminobenzidine–Ni stain highlighting amyloid plaque deposition. When Eu-coupled antibodies were employed, the 153Eu map highlighted the plaque regions as well. Moreover, the mapping of different trace elements in the tissue sections such as Cu was possible at the same time by LA-ICP-MS. Furthermore, the distribution of mucin 1 (MUC 1) or human epidermal growth factor receptor 2 (Her 2) in breast cancer tissue sections and on microarrays were studied by employing gold nanoparticle tagged secondary antibodies and silver enhancement for signal amplification [87]. For tissue microarrays cores of 2 mm diameter were taken from 28 FFPE breast cancer tissue blocks and were incubated first with a primary antibody against Her 2, and after some washing steps a gold tagged secondary antibody followed. The array was analysed by single spot ablation (beam diameter 515 μm). For quantitative assessment a commercial available standard test was analysed, which consisted of negative controls and samples of different expression levels (0, 1+, 3+). The wide linear dynamic range of ICP-MS enabled the detection of Her 2 even in the low expression level standards (0), which were below the range for a typical visual microscopic evaluation. Additionally, Seuma et al. investigated the distribution of MUC 1 or Her 2 in formalin-fixed and paraffin-embedded (FFPE) breast cancer tissue sections of 4 μm thickness via Au/Ag tagged secondary antibodies. The

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conditions for LA-ICP-MS analysis of tissue sections were optimized and good sensitivity and spatial resolution down to 5 μm were achieved. However, spatial resolution has been inferior to light microscopy due to a loss of sensitivity when using small laser spot sizes. Therefore, important information on tissue morphology is lost, as compared to hematoxylin and eosin (HE) staining in conventional histology. Recently, Joo and Lim presented the determination of holoceruloplasmin (holo-CP) in human serum by LA‐ICP‐MS with a sample consumption as low as 126 pL [100]. The serum was deposited on microarray chips with 150μm diameter pillars, on which ceruloplasmin antibodies had been immobilized. Holo-CP was detected by LA-ICP-MS via its Cu ions in single shot mode. The limit of detection achieved for Cu was 46.7ngmL−1, which equates to 126 nM of holo-CP. Since the sample volume amounted to 126 pL, the absolute number of detected holo-CP was reported as 1.54×10−17 mole [100]. 5.2.2. Multi-parameter assays The first LA‐ICP‐MS‐based multiparameter approach was demonstrated by Hu et al. [99]. The authors applied antibodies conjugated with different lanthanide ions or nanoparticles in a sandwich immunoassay and reported on the detection of three proteins on a single microarray spot by means of LA-ICP-MS. Therefore, a mixture of three different antibodies was printed on a microarray in one spot and react first with the antigens of unknown concentration. Then, tagged antibodies were added which coupled to a second binding site of the antigens. Single laser pulses were used for analysing the microarray spots. The detection limits were 0.20, 0.14, and 0.012 ng mL −1 employing Sm-tagged, Eu-tagged, and Au nanoparticle-tagged antibodies, respectively. The latter limit of detection reflects the nanoparticle, which provides a higher sensitivity per tag in ICP‐MS, than simple Sm 3+ or Eu 3+ tags. The detection of electrophoretically separated and electroblotted cytochromes P450 (CYP) by europium and iodine tagged monoclonal antibodies via LA-ICP-MS was described by Roos et al. [119]. The authors showed that two different cytochromes P450 with a difference in their molecular weight of 2.8 kDa can be detected simultaneously in one SDS‐PAGE separation via differentially tagged monoclonal antibodies specific for each CYP. Furthermore, the multi element capability of LA‐ICP‐MS directly on a Western blot membrane has been investigated with detection limits in the sub pmol range. In this study, antibodies for bovine serum albumin, lysozyme, and casein were tagged with a lanthanide element via SCN‐DOTA. After specific binding to their corresponding antigens, the elemental profile on the Western blot membrane was analyzed by LA-ICP-MS [120]. The principle of this method is illustrated in Fig. 4. In 2011 Waentig et al. extended the number of simultaneously analysed CYP up to five, using SCN-DOTA loaded with different lanthanides as tagging reagent [121]. Competition of antibody binding could be neglected as the quantitative data obtained by single immunoassays were comparable to the multiparameter assay. The detection limits achieved in the medium were in the fmol range. Concerning the analysis of multiple parameters on tissue sections, this approach was hampered in the past by the use of secondary, tagged antibodies. However, in conventional immunohistochemistry assays usually several tissue sections need to be stained subsequently to receive information on the tumor marker profiles. Thus, the number of simultaneously detected antigens should be increased. This issue was addressed in a recent publication by Giesen et al. The authors introduced the first multiparametric immunohistochemical approach for the simultaneous detection of three tumor markers in a single cancerous tissue section [122]. In this case, primary antibodies against Her 2, MUC 1, and Cytokeratin 7 (CK7) were tagged with holmium, terbium and thulium via SCN-DOTA. A breast cancer tissue section of 5 μm thickness was incubated with the lanthanide tagged antibody pool. Here, no secondary antibodies were needed. The expression levels of the tumor markers could be directly compared on

Fig. 4. LA‐ICP‐MS of lanthanide tagged primary antibodies on a Western blot membrane. A mixture of bovine serum albumin, lysozyme, and casein was separated by SDS‐PAGE, and transferred to a nitrocellulose membrane by electroblotting. After the immune-reactions of the lanthanide tagged antibodies, the elemental profile is directly analyzed on the Western blot membrane [120].

the same section which is of great benefit for standardization of the results in immunohistochemistry. Moreover, the LA-ICP-MS analysis indicated different expression levels for Her 2, MUC 1, and CK 7, which was not obvious from the conventionally stained sections. To summarize, LA‐ICP‐MS‐based immunoassays provide insight into the spatial distribution of biomarkers, which are of special interest in biochemical or medical applications. However, spatial resolution is limited by the amplification of the label, or tag employed. This trend is reflected in Table 2, where an increased spatial resolution is related to an amplifying tag, e.g. a nanoparticle. Further options towards single molecule imaging by LA‐ICP‐MS are discussed in the following section. Although absolute quantification is in theory applicable to LA‐ICP‐MS, it remains challenging concerning biological samples due to difficulties in the design of suitable matrix-matched standards, which usually need to be synthesized for any special application.

6. Conclusions and Future Trends In this review we have shown that ICP‐MS is a new and very promising analytical tool in various applications of different immunoassays. It excels by its high sensitivity, wide linear dynamic range, independency of the organic matrix compared to other bioanalytical techniques, and in particular by its multielement capability. The analysis of biomolecules via ICP‐MS detection is commonly achieved by (i) labels, or (ii) tags. The advantages of ICP‐MS for the life sciences lie in the potential of multiplexed and sensitive quantitative analyses at high linear dynamic range. With this technique, a variety of modifications can be queried in a single experiment. Summarizing so far, we can say that ICP-MS is still a sleeping beauty in many life science applications such as biochemistry, proteomics, medical diagnostics, treatment control, toxicology, or cancer research. At the end of this review a very personal view will be presented where the authors see room for further improvements and novel applications. Compared to conventional immunoassays the following features look most promising and can be used to improve or develop new assays:

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1. The outstanding sensitivity, which already guarantees sub fmol detection limits sufficient to detect the more abundant biomolecules. But it has to be kept in mind that the analysis of low abundant proteins still poses a challenge to ICP‐MS detection, since only a few labels or tags are conjugated with the biomarker of interest. Hence, the amplification of the tag either by binding it to a polymer, or by application of nanoparticles has been of interest recently. Considering the use of nanoparticles, new strategies for multiparametric analyses need to be developed, which are comparable to the possibilities provided by lanthanide tags. However, nonspecific binding on the sample surface lowers signal‐to‐background ratios and thus, impedes the application of these signal‐amplifying tags unless suitable blocking conditions are elaborated. Although it sounds very enthusiastic, the signal amplification or better to say the gain which can be achieved by use of nanoparticles is even higher than 106. Similar gain factors have been recently achieved by the discovery of the polymerase chain reaction which has revolutionized genomics. Without any prophecy we want to claim that we now have the tools to initiate a similar revolution in proteomics 2. The excellent lateral resolution of laser ablation already opens a door for immuno-imaging of tissue samples with single cell resolution capability, but this technique is still inferior to light microscopy. However, Giesen et al. presented a spatial resolution at the single cell level in an investigation of a liver biopsy tissue by LA‐ICP‐MS after iodination of the cell, and even the nucleus became visible [59]. We are not at the end, but at the beginning of quite new capabilities. If amplifying tags are employed in future analyses of tissue sections, sensitivity will be improved significantly and thus, will hopefully enable a reduction of the laser spot size down to a μm, or even less. Single cell detection by the CyTOF is already routinely applied; immuno-imaging of single cells is still challenging, because most commercial laser ablation systems are not very well suited for this purpose, and they urgently need to be improved. To further increase the spatial resolution, near-field (NF) techniques have been applied to deliver the laser radiation via the tip of a thin silver needle onto the sample surface. As a function of the tip diameter and the tip-to-sample distance, laser craters in the range from 200 nm to 2 μm were produced [123]. The detection efficiency of NF‐ LA‐ICP‐MS was calculated to 2.7×10 −5 cps per ablated atom based on a typical laser crater of 600 nm on a Au-covered Si material [124]. Hence, the technique should be suitable for single molecule imaging if a 3D-movement of the Ag needle is realized. So far, the analysis of major sample constituents in single-shot measurements was feasible by this technique. However, the quantification of minor constituents will require an increase in ICP‐MS sensitivity by at least two or three orders of magnitude [125]. 3. The ability to develop simple calibration and quantifications schemes is another important feature of ICP-MS, because most conventional assays are difficult to calibrate. Additionally, most of the assays previously described are not yet standardized which makes a comparison difficult. For development of quantification schemes, the stoichiometry of the tagged antibody is required for the quantification, and reliable standards are still missing. This becomes even more challenging for tissue samples, or better to say for immuno-imaging of tissues where we need to improve our internal standards to correct for the thickness or density of the tissue. Although first results of Giesen et al. look promising more fundamental research is needed in this direction [59]. To conclude, ICP‐MS is capable of the detection and quantification of proteins or protein modifications, but other mass spectrometry techniques like ESI or MALDI are often needed for their identification.

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