Ferrocene-based derivatization in analytical chemistry

Anal Bioanal Chem (2008) 390:181–200 DOI 10.1007/s00216-007-1639-7 REVIEW Ferrocene-based derivatization in analytical chemistry Bettina Seiwert & Uwe Karst Received: 2 August 2007 / Revised: 14 September 2007 / Accepted: 17 September 2007 / Published online: 13 October 2007 # Springer-Verlag 2007 Abstract Ferrocene-based derivatization has raised considerable interest in many fields of analytical chemistry. This is due to the well-established chemistry of ferrocenes, which allows rapid and easy access to a large number of reagents and derivatives. Furthermore, the electrochemical properties of ferrocenes are attractive with respect to their detection. This paper summarizes the available reagents, the reaction conditions and the different approaches for detection. While electrochemical detection is still most widely used to detect ferrocene derivatives, e.g., in the field of DNA analysis, the emerging combination of analytical separation methods with electrochemistry, mass spectrometry and atomic spectroscopy allows ferrocenes to be applied more universally and in novel applications where strongly improved selectivity and limits of detection are required. Keywords Ferrocene . Derivatization . Labeling . Electrochemistry . Mass spectrometry Abbreviations AAS atomic absorption spectroscopy AED atomic emission detection APCI atmospheric pressure chemical ionization CGE capillary gel electrophoresis CV cyclic voltammetry DPV differential pulse voltammetry DRC dynamic reaction cell EC electrochemistry B. Seiwert : U. Karst (*) Institute of Inorganic and Analytical Chemistry, University of Münster, Corrensstr. 30, 48149 Münster, Germany e-mail: [email protected] ECD ECL ESI FBA FCA FCC FEM FMEA GC HPLC ICP LC MALDI MS OES SWV TOF electrochemical detection electrochemiluminescence electrospray ionization ferroceneboronic acid ferrocenecarboxylic acid ferrocenecarboxylic acid chloride N-(2-ferroceneethyl)maleimide ferrocenecarboxylic acid-(2-maleimidoyl) ethylamide gas chromatography high-performance liquid chromatography inductively coupled plasma liquid chromatography matrix-assisted laser desorption/ionization mass spectrometry optical emission spectroscopy square wave voltammetry time-of-flight Introduction Since the discovery of ferrocene in 1951, many of its derivatives have been synthesized and characterized. They are of considerable interest in various areas of research and application, like asymmetric catalysis [1], electrochemistry (EC) [2], functional biomaterials [3] and even for medical purposes [4, 5]. A large variety of attractive analytical applications of ferrocene and its derivatives have been reported as well. The present review article highlights the detection-oriented derivatization with ferrocenes in analytical chemistry. The review mainly focuses on the direct covalent derivatization of various functional groups by ferrocenes as well as on the 182 Anal Bioanal Chem (2008) 390:181–200 respective separation and detection techniques. Other important analytical applications of ferrocenes, in which no covalent derivatization is carried out, are only mentioned in those cases where they help to understand general reaction mechanisms or detection strategies. This is particularly valid for the use of ferrocenes as electron-transfer reagents ("mediators") in electrochemical biosensors, which has already been summarized in reviews by Ali et al. [6], Hill [7] and Frew and Hill [8]. The mediators serve to facilitate the electron transfer between an enzyme and the working electrode of an amperometric transducer, but are not involved in any covalent bond formation or cleavage and therefore do not fit into the focus of this article. The same is valid for selected approaches for electrochemical DNA detection, which have also been the subject of a recent review [9]. Ferrocene and its derivatives mostly are neutral compounds and, unlike many other organometallics, are stable in the presence of water and air. However, they are preionic compounds, because they are highly reversible redox systems which may readily be switched electrochemically between the ferrocene and the respective ferrocinium cation at low potentials. A typical cyclic voltammogram of ferrocene derivatives, in this case of the ethyl isocyanante derivative of ferrocenoyl piperazide [10], is shown in Fig. 1, which demonstrates the excellent reversibility of the oxidation of ferrocene to the ferrocinium cation. N Fe II + O O N - H - 1e N + 1e- N Fe III H N N O O Furthermore, the chemistry of ferrocenes is well explored and a large variety of ferrocene derivatives is easily accessible via established synthetic routes. The redox behavior of ferrocene is sensitive to its covalent or noncovalent binding to other molecules. A unique property of metallocenes is the possibility of introducing substituents on one or both of the cyclopentadienyl rings while retaining the properties of a simple one-electron redox couple. The electrochemical oxidation potential is tunable by changing the nature of the substituents. Therefore, ferrocenes allow the use of a large variety of electrochemical detection (ECD) techniques, including amperometry or voltammetry [11]. Other detection techniques are possible as well. UV/vis absorption detection is an option owing to the intense color of the ferrocenes. It is well known from the literature that the color of the ferrocenes strongly changes upon oxidation, thus allowing spectroelectrochemical measurements [12]. Iron as a central ion furthermore enables the use of atomic spectroscopy for ferrocene detection [12]. Atomic absorption spectroscopy (AAS) as well as inductively coupled plasma (ICP) excitation with optical emission spectroscopy (OES) or mass spectrometry (MS) have been used to detect ferrocene derivatives [13–18]. Naturally occurring iron consists of four isotopes: 5.8% of 54Fe, 91.7% of 56Fe, 2.2% of 57Fe and 0.3% of 58Fe [19]. The resulting characteristic isotopic pattern of ferrocene allows the identification of an iron-containing compound in complex mixtures when using “organic” MS, e.g., electron impact or electrospray ionization (ESI) MS. The measured as well as the calculated isotopic pattern of the cysteine derivative of N-(2-ferroceneethyl)maleimide (FEM) are presented in Fig. 2. ∆V= 58 mV 80 60 430.2 430.2 20 0 -20 431.3 432.2 428.2 Intensity [-] Current [µA] 40 O OH O 431.3 -40 -0.5 0.0 0.5 428.2 1.0 Potential [V] vs. Ag/AgCl Fig. 1 Cyclic voltammogram of the ferrocenoyl piperazide derivative of ethyl isocyanate dissolved in a mixture of 10 mL ammonium formate buffer (0.1 M) and 10 mL acetonitrile to form a 0.3 mM solution. As a three-electrode assembly, a platinum electrode as the working electrode, a Ag/AgCl reference electrode and a glassy carbon counter electrode were used. The cyclic voltammogram was recorded in a potential range from -1,000 to 1,000 mV with a scan rate of 50 mV/s NH2 O Fe -60 -1.0 S N 432.2 433.2 420 425 430 435 440 445 m/z Fig. 2 Electrospray ionization (ESI)/mass spectrometry spectrum of the cysteine derivative of N-(2-ferroceneethyl)maleimide (FEM). The inserts show the chemical structure of the analyte as well as the calculated isotopic pattern Anal Bioanal Chem (2008) 390:181–200 Ferrocene-based derivatizing agents Despite some laborious work protocols and their demand for additional analysis time, derivatization reactions are powerful tools in analytical chemistry. In many cases where direct analysis is not possible because chromophoric, fluorophoric or electroactive groups are missing or the analytes are chemically instable, derivatization is the best option. A suitable derivatization technique may: – – – Convert a chemically labile compound into a stable product, Enable or improve the use of more selective or more sensitive detectors, Modify the polarity of a compound to improve its chromatographic separation. In the past, a large variety of ferrocene-based derivatizing agents were developed for coupling to several functional groups. A selection of these reagents, including reaction conditions and detection technique used, are summarized in Table 1. Solid-phase synthesis approaches and low-yield methods, which are not suitable for quantitative analysis, are not considered. From Table 1, it is obvious that the largest number of the known ferrocenebased derivatizing agents are dedicated to the analysis of amine functionalities, including small organic molecules and amino acids as well as peptides or proteins. This correlates well with the strong demand for powerful analytical methods for proteins, peptides and amino acids. Alcohols, diols, thiols, isocyanates, carboxylic acids and unsaturated hydrocarbons are other important functional groups which have successfully been derivatized with ferrocene-based reagents. For all reagents, the most important reaction conditions have been added as well as the preferred analytical technique, mostly based on a chromatographic separation with selective detection. As the chemical reactions are mostly established in organic chemistry, the following parts of this review have been sorted by the detection techniques used. Analytical methods used in conjunction with ferrocene-based derivatization Electrochemical detection General aspects of electrochemical detection The ferrocene/ferrocinium couple shows a simple oneelectron redox behavior; hence, it is used as a reference redox system for organic and, with some limitations, for aqueous systems [84]. The derivatization with ferrocenes yields electroactive products even from nonelectroactive analytes. 183 The ferrocene group retains its speed and reversibility of electron transfer in more complex molecules, too. Therefore, ferrocene, many of its derivatives and even ferrocene-labeled proteins [16, 44, 56, 85] are frequently used as mediators for electrochemical measurements with particular focus on amperometric biosensors. As this aspect was summarized by Frew and Hill [8], it will not be discussed in more detail here. This part of the review therefore focuses on those analytical methods where the electrochemical signal is based on the attachment, steric shielding or dissociation of ferrocene-based derivatizing agents. These include the ECD of changes in diffusion coefficients and changes upon chemical reaction of the derivatives. Furthermore, this part includes the ECD based on the electrochemical cycling of the redox couple. ECD of attached ferrocene derivatives The derivatization with ferrocenes enables the detection of originally nonelectroactive analytes by electrochemical methods like cyclic voltammetry (CV), differential pulse voltammetry (DPV) and square wave voltammetry (SWV). The detected current is proportional to the concentration of the analyte at the electrode. The attachment method was used to identify derivatized biomolecules. Di Gleria et al. [86] used the attachment of ferrocenes to monitor cysteinecontaining biomolecules by CV measurements. Ferrocenecarboxylic acid (FCA) was coupled by carbodiimide to enzymes with the goal of altering the electron transfer with the electrodes [87]. Several ferrocene-labeled linkers for amino functionalities in proteins were evaluated by Tranchant et al. [58] with respect to the ECD by CV. The attachment of ferrocene was used to monitor enzymatic reactions, for example, those catalyzed by tyrosinase [88]. Tyrosinase catalyzes the oxidation of tyrosine (or other phenols) to L-dopa (or other respective catechol derivatives). The diol functionalities formed were derivatized by ferroceneboronic acid (FBA) and this derivatization was monitored by DPV. Furthermore, the ferrocene derivatization has been applied to monitor immunological reactions. Biotinylated antigens were immobilized on a streptavidin-modified gold electrode and the antibody labeled with ferrocene moieties binds in a competitive immunoreaction to the immobilized antigen (Fig. 3) The signal was detected by DPV [59]. In a sandwich electrochemical immunoassay [57], a capture antibody connected to the electrode reacts with the antigen and, in a second step, the antigen binds to the signal antibody containing the ferrocene unit as shown in Fig. 4. In this case, human chorionic gonadotropin in buffer or serum samples was incubated with the capture antibody, afterwards immobilized on the electrode and, after washing, 184 incubated with a FCA-conjugated signal antibody and detected by CV. The “sandwich assay” is the most common design for an electrochemical DNA sensor as well (Fig. 5). The assay consists of three individual DNA components: An immobilized capture strand, which is immobilized on a gold electrode, a target strand and a probe strand containing the ferrocene reporter group. If all components are combined and hybridized together, an electrochemical response at the Anal Bioanal Chem (2008) 390:181–200 electrode is detected because the ferrocene unit is close to the electrode. If a non-target DNA strand is used, this strand is combined with the probe strand, but no hybridization takes place with the capture strand and no signal is detected [60, 89]. A further development of this assay was achieved by using a two-piece reagentless assay for DNA. In this case, the capture strand is connected to the probe strand via a linker [89]. Dehybridization from the surface-bound strand will Table 1 Ferrocene-based derivatizing agents for different functional groups with appropriate reaction conditions, detection methods and references Anal Bioanal Chem (2008) 390:181–200 Table 1 (continued) 185 186 Table 1 (continued) Anal Bioanal Chem (2008) 390:181–200 Anal Bioanal Chem (2008) 390:181–200 187 Table 1 (continued) AAS atomic absorption spectroscopy, AED atomic emission detection, CGE capillary gel electrophoresis, CV cyclic voltammetry, DMAP 4dimethylaminopyridine, DPV differential pulse voltammetry, EC electrochemistry, ECD electrochemical detection, EDC N-ethyl-N(dimethylaminopropyl)carbodiimide, ESI electrospray ionization, Fc ferrocenyl, GC gas chromatography, HPLC high-performance liquid chromatography, ICP inductively coupled plasma, LC liquid chromatography, MALDI matrix-assisted laser desorption/ionization, MS mass spectrometry, RT room temperature, SWV square wave voltammetry 188 Anal Bioanal Chem (2008) 390:181–200 Fc Fig. 3 A competitive immunoassay with electrochemical detection [59] Fc Au electrode Fc Au electrode Fc Au electrode streptavidin biotinylated antigen occur at temperatures at which a fully matched strand remains hybridized to the surface-bound strand. Using this strategy, one can detect single nucleotide mismatches [89]; however, the background current is very high, thus hampering the limit of detection for these measurements. Bioelectrocatalytic reactions were used to amplify the detection of ferrocene connected to DNA after hybridization [90]. For this purpose, the redox-active DNA replica is coupled to the glucose oxidase mediated oxidation of glucose. Furthermore, an amplification of the voltammetric signal may be obtained by the use of labels containing more than one ferrocene unit, like triferrocenyl-tris(hydroxymethyl)aminomethane [23, 24], ferrocene-derivatized diblock and triblock copolymers [91] or ferrocene-coated gold nanoparticle/ streptavidin conjugates [92–94]. The remarkable sensitivity of the ferrocene-coated nanoparticles may be attributed to the large number of ferrocenes present at the surface (127 per gold nanoparticle) and the close proximity with respect to the underlying electrode. The method was used for the determination of sulfhydryl groups in surface-bound proteins or peptides [94] and for DNA hybridization detection [92, 93] as shown in Fig. 6. The block copolymer conjugates were utilized as DNA probes in a three-component sandwich-type ECD strategy. The single-polymer-based DNA probe contains an average of approximately ten ferrocenyl groups. The formation of multiple layers of DNA hybrids on polymers (“multilayering”) is possible as well. This leads to further enhanced signals [91]. antibody ferrocene-labeled antibody ECD of the disappearance of the electrochemical signal by steric hindrance In another approach, steric effects cause the disappearance of the electrochemical signal. One example is the change in flexibility and electron-transfer tunneling distance of the electroactive tags connected to the electrode. A ferrocenelabeled probe [95, 96] or labeled stem-loop oligonucleotide [97] that alters its flexibility by hybridization may be used to detect complementary target DNA. This approach was applied to immunosensors as well: The reaction, biocatalyzed by the antigen bound to the electrode surface, of ferrocene-labeled glucose oxidase disappeared owing to steric shielding of the surface when an antibody is bound to the antigen as shown in Fig. 7 [45, 46]. An electroactive double-conjugated protein, ferrocene-bovine serum albumin-digoxin [48, 50], may be used for the same purpose with improved sensitivity. The amplification is due to the combined effect of enzymatic amplification and electrochemical amplification by multilabeled ferrocene. The determination of thrombin is another application of this method. A bifunctional derivative of a thrombin-binding aptamer with a redox-active ferrocene moiety is used for this purpose. An aptamer is an oligonucleotide selected from combinatorial libraries by systematic evolution of ligands by exponential enrichment, which selectively binds with target antibodies. Aptamers against many different targets have been produced in recent years. The signal in DPV is Fig. 4 Setup of a sandwich electrochemical immunoassay [59] glassy carbon electrode immunoelectrode preparation glassy carbon electrode Fc Fc antigen Fc Fc signal antibody capture antibody 189 Au electrode Anal Bioanal Chem (2008) 390:181–200 1) immobilized capture strand 1) 2) 2) Fe probe strand immobilized capture strand target DNA probe strand e- Fe non-target DNA Au electrode Au electrode target DNA Fe probe strand immobilized capture strand non-target DNA probe strand Fe Fig. 5 Principle of the “sandwich assay” applied in electrochemical DNA sensors [60, 89] enhanced by the change of the aptamer conformation at the electrode from the uncomplexed coil-like form to a quadruplex upon association of the aptamer with thrombin [98]. ECD of the change in redox response by dissociation of the ferrocene label Changes in redox response upon dissociation of the ferrocene label may be used for analytical purposes as well: Ferrocene-labeled helical peptides with a specific sequence are attached to an electrode. In the presence of matrix metalloproteinases, the signal decreases because the part of the peptide with the ferrocene attached is cleaved and the ferrocene as a sensing element is not connected to the electrode surface anymore [99]. This is a sensitive approach to detect the matrix metalloproteinase activity, and was also applied to the detection of complementary oligonucleotides by hybridization with an electrochemically active oligonucleotide probe and subsequent dissociation of the terminally labeled 5′ nucleotide by T7 exonuclease [100]. Ligase activity may be monitored by using tethered and ferrocene-terminated DNA hairpins. These molecules are made of a single strand of DNA, which carries sequences of bases that are complementary to each other in each of its two terminal regions. As a result, when the base pairs of these two sequences are formed, the molecule takes the shape of a hairpin. The exposure to DNA ligase is followed by conditions that denature the hairpin and the corresponding dissociation of the ferrocene label is monitored by CV [101]. Another interesting example is the sugar-induced disintegration of a ConA-ferrocene-modified glycogen multilayer film, which depends on the type of sugar and its concentration. The CVof such an electrode can be used for the determination of sugars at a millimolar level [66]. Electrochemical monitoring of changes in oxidation potential The tunability of the electrochemical properties of ferrocene derivatives can be used to design systems for monitoring several reactions and to investigate catalysts or/and activity of enzymes. This is achieved by monitoring reaction products that have different electrochemical properties compared with the educts. An example is an amperometric 190 Anal Bioanal Chem (2008) 390:181–200 1) thiolated ODN probe hexanethiol 2) Au electrode Au electrode biotinylated ODN target biotinylated ODN target streptavidin Fc Fc Fc Fc Fc Fc Fc Fc Au Au Fc thiolated ODN probe Fc Fc Fc thiolated ODN probe Fc Au Au Fc Fc Fc Fc Fc Fc Fc hexanethiol hexanethiol Au electrode Au electrode Fig. 6 The enhanced voltammetric detection of a biotinylated oligodeoxynucleotides (ODN) target [92, 93] sample may be analyzed by SWV within one measurement as shown for RNA and DNA [37]. assay for aldolase activity, where ferrocenylethylamine and the retro-aldol retro-Michael substrate are monitored at different potentials [102]. Ferrocene-based derivatives having different redox potentials were measured competitively on the electrode. With use of ferrocenyl carbodiimide derivatives based on FCA and ferrocenepropionic acid, the standard and the test Fig. 7 Amperometric analysis of an antibody by an antigenmonolayer electrode using a bioelectrocatalyst as a redox probe [45, 46] e- ECD based on the reversibility of the oxidation/reduction The reversibility of oxidation/reduction of ferrocene can be used to detect ferrocene derivatives by a recycling bienzyme Fc glucose e- e- GOx GOx gluconic acid antigen Fc glucose oxidase antigen antibody Anal Bioanal Chem (2008) 390:181–200 electrode. The electrode consists of a laccase-glucose dehydrogenase bienzyme membrane coupled with an oxygen electrode and can be used for the detection in immunoassays [103]. ECD based on changes in diffusion coefficient An affinity assay that is based on the modulation of the diffusion coefficient of an electroactive ferrocene label upon complementary recognition, thus leading to an increase of the molecular weight, was developed by Mosbach and Schuhmann [51]. With use of an electroanalytical technique, which is correlated to the diffusion coefficient of the redox species, e.g., CV, the decrease of the diffusion coefficient can be monitored as a decrease of the diffusion-limited current. The signal is intensified by redox cycling [104]. ECD coupled to separations High-performance liquid chromatography with ECD High-performance liquid chromatography (HPLC) with ECD is a useful method for the determination of trace components in complex matrices because of its excellent selectivity and sensitivity as shown in an earlier review on this topic [105]. The oxidation potential ranges from 0.2 to 0.6 mV vs. Ag/ AgCl for most ferrocene derivatives and depends on the substituents on the cyclopentadienyl ring. However, the electroactivity of ferrocene is generally even higher than that of catechols, whose oxidation to the o-quinone at 600 mV vs. Ag/AgCl has been used for routine analysis in HPLC/ECD. Ferrocenes can be selectively detected even in the presence of other electroactive aromatic compounds. The ferrocene derivative undergoes facile oxidation and the product can be readily reduced. The subsequent oxidation and reduction signals at the electrodes were used to enhance selectivity. The collection efficiency and thus the reversibility (ratio of the current at the downstream detector to that at the upstream detector) of the ferrocenyl group is even higher than that of the catechol group [106]. The use of the ferrocenyl group as an electrophore for HPLC/ECD measurements was first reported by Tanaka et al. [106]. N-Succinimidyl-3-ferrocenylpropionate and later ferrocenylisothiocyanate [47] were used as derivatizing agents for amines. The same authors later introduced derivatizing agents for alcohols (ferrocenoyl azide and 3ferrocenylpropionyl azide) as well [21]. The maximum sensitivity was obtained for the three derivatizing agents at 0.4 mV vs. Ag/AgCl with a detection limit of 3×10-8 M and 5×10-8 M, respectively. For thiols, N-(ferrocenyl)maleimide and FEM [76], for fatty acids, 3-bromoacetyl-1,1′-dimethylferrocene [61] and ferrocenylethylamine [62, 64, 65] were used with comparable limits of detection. The 191 ferrocenylethylamine derivatives of retinoic acid were detected by coulometric reduction (-100 mV) after on-line coulometric oxidation (+400 mV) with a detection limit of 2×10-8 M. Microcystin-LR was derivatized with 6-ferrocenylhexanethiol at its α,β-unsaturated carbonyl group and was selectively detected by HPLC/ECD [83]. Gamoh et al. [67] used FBA to derivatize diols prior to HPLC/ECD. Kubab et al. [20] introduced ferrocenecarboxy hydrazide for aldehyde detection by HPLC/ECD. Other groups reported HPLC/ECD methods for the analysis of peptides and proteins. In detailed studies by Eckert and Koller [25, 38] and Cox et al. [26], several ferrocene-based derivatizing agents were investigated for their usefulness in peptide and protein analysis. Recently, Williams and Koppang [27] reported on a selective analysis method for secondary amines. The method uses the selectivity of o-phthalaldehyde to "mask" primary amines prior to derivatization of the secondary amines by ferrocenecarboxylic acid chloride (FCC). Dual electrode detection (E1=0.7 V and E2=0.2 V vs. Ag/AgCl) was used to selectively detect FCC derivatives showing a complementary oxidation and reduction peak, whereas o-phthalaldehyde derivatives show only an oxidation signal at the upstream electrode. The coupling of HPLC and ECD can furthermore be used to detect the hybridization of a target nucleotide with a complementary oligonucleotide carrying an electrochemically active ferrocene group [107, 108]. The introduction of a ferrocene-labeled terminator by polymerase was measured by HPLC with coulometric detection, too [109]. The method is rapid and is characterized by a detection limit in the femtomolar range. The sensitivity of the method depends on the stability of the hybrid complex as measured as its melting temperature. However, a higher melting temperature implies decreased mismatch sensitivity. By incorporation of polymerase chain reaction, the sensitivity of the HPLC/ECD method can be enhanced at least 1,000fold [110, 111] and quantification is possible since DNA amplification proceeds exponentially under low cycles of polymerase chain reaction. A drawback of HPLC/ECD is the limitation with respect to isocratic elution. Only if special electrochemical methods that reduce the slope of the baseline caused by electrochemical reactions of background electrolyte and solvents are used, e.g., with the use of a coulometric array detector, gradient elution may be carried out. HPLC/ECD with differential labeling with two ferrocene-based derivatizing agents is possible to enhance the selectivity. Electrophoresis with ECD In principle, the number of theoretical plates and, therefore, the separation power of capillary electrophoresis is higher compared with HPLC. 192 Capillary gel electrophoresis (CGE) is the method of choice for DNA sequencing and fingerprinting, because it offers short run times, high efficiencies, low sample consumption, and it is compatible with automated sample injection [112]. The chip-based approach adds the ability to multiplex the analysis. Laser-induced fluorescence is usually applied for detection. Although the method is very sensitive, it is relatively difficult to miniaturize compared with ECD systems and expensive owing to the use of lasers and specialized optical components. Advantages of electrochemical methods are sensitivity, ease of microfabrication, simplicity and inexpensive instrumentation. As for the fluorescence approach, derivatization is mostly needed to enhance the general applicability of the method [113]. Ferrocene-based electrochemically active labels are particularly useful owing to their diversity and tunable potential, which enable the simultaneous detection of different analytes as well. Applying sinusoidal voltammetry, one can selectively monitor minimal differences in redox potentials. This technique coupled with CGE separation was applied to low resolution of four-color DNA sequencing and has been pioneered by Brazill et al. [39]. Discrimination between one tag and all others is accomplished through a “phase-nulling” technique. The signal for each tag is selectively eliminated, while the other three responses remain virtually unchanged. This analysis scheme allows the selective identification of differently tagged oligonucleotides. The same group demonstrated the applicability of the method to single nucleotide polymorphism screening of DNA [40, 114, 115]. The electroactive label is introduced by a primer with ferrocene acetate covalently attached to its 5′ end [40] or by a chain terminator like ferrocene-acycloATP [114, 115], which is incorporated by a thermostable polymerase. The extension product is separated by CGE from any excess terminator or the unextended primer and finally detected by sinusoidal voltammetry. The first electrochemical capillary flow immunoassay was developed by Lim et al. [116, 117]. The separation is based on differences in the isoelectric points of the antigenantibody complex and the unreacted antibody. Cationexchange capillary columns were applied to selectively trap the free ferrocene-labeled antibody, whereas the antibody-antigen complex with human choronic gonadotropin passes through the column and can be detected by a three-electrode flow-through cell [53]. The approach was later transferred onto a chip [41]. The multichanneled matrix column was functionally coated with cation-exchange resin to detect histamine in blood. Wang et al. [54] later developed a chip-based electrochemical immunoassay with two reaction formats: A direct and a competitive mode of operation. The reagents were mixed in a precolumn, followed by an immunochemical reaction. For the direct Anal Bioanal Chem (2008) 390:181–200 protocol (Fig. 8a), the free labeled antibody and the labeled antigen-antibody complex were separated by electrophoresis and detected downstream by a gold-coated carbon screen-printed anode. For the competitive immunoassay, the labeled antigen bound to the antibody was separated from the free labeled antigen and the internal standard (Fig. 8b). Recently, the electrophoretic immunoassay was performed on-chip using a column packed with boronateactivated agarose beads [118]. Separation based on the ionic and preionic character of ferrocene derivatives Selective concentration of positively charged compounds and repulsion of negatively charged compounds at a negatively charged electrode is a possibility to separate differently charged analytes. The activity of protein kinase A can be monitored using this approach [81]. A gold electrode modified with the anionic thioctic acid selectively accumulates and detects the positively charged ferrocene-labeled substrate. Protein kinase A introduces a phosphate group, changing the net charge to negative, and repulsion takes place. The polyanionic perfluorosulfonated Nafion polymer is known for its ability to exchange and accumulate cationic or precationic species and to repel anionic compounds. The preionic character enables the enrichment of ferrocene derivatives such as ferrocinium salt in Nafion by applying an anodic potential. With cathodic stripping, the accumulated ferrocene derivatives are released from the film in its neutral form [52]. By using these electrodes, one can discriminate between species that are oxidized at the same potential. Electrochemical enzyme assays for the detection of alkaline phosphatase [119, 120] were developed based on this method. One of them applies ferrocenylethyl phosphate as substrate. The Nafion film acts as an electrostatic barrier against the ester phosphate (Donnan exclusion) and the enzyme-generated ferroceneethanol is preconcentrated and detected [120]. A Nafion-loaded carbon paste electrode was used to amplify immunoassay signals by a preconcentration step of the derivatives labeled with ferrocene ammonium salts [121]. Mass-spectrometric detection General considerations for mass-spectrometric detection The derivatization with ferrocene increases the mass of the analyte. Furthermore, an iron atom, an electroactive group and a predetermined fragmentation site is thus introduced into the analyte. These properties facilitate the analysis of many compounds by MS. Mass-spectrometric detection may be targeted to either the iron atom (“inorganic” MS) or the derivatized analyte (“organic” MS). Anal Bioanal Chem (2008) 390:181–200 Fig. 8 The on-chip a direct and b competitive electrochemical immunoassay protocols [54] 193 a b running buffer running buffer antibody ferrocene-labeled antibody Fc antigen Fc Fc Fc Fc antigen + ferrocene-labeled antigen Fc Fc Fc internal standard working electrode The mass increase and the amount of iron correlate with the number of ferrocene labels per analyte. If quantitative derivatization takes place, the number of functional groups will correlate with the number of ferrocenes. Therefore, mass-spectrometric approaches such as ICP-MS [14, 92] or electron impact, ESI, atmospheric pressure chemical ionization (APCI) or matrix-assisted laser desorption/ionization MS may be applied. Of these, ICP-MS is most suitable for quantitative experiments, while the other techniques can provide valuable structural information. Inductively coupled plasma mass spectrometry For the ICP-MS approach, the concentration of the analyte has to be known to determine the number of labels [92] bound to each analyte molecule. The detection of iron by unit-resolution ICP-MS may lead to erroneous results owing to various polyatomic interferences such as 38 Ar16O+, 40Ar14N+ on the peak of 54Fe+, and 40Ar16O+ on 56Fe+ and 40Ar18O+ on 58Fe+. An effective method for the reduction of spectroscopic interferences is the use of a dynamic reaction cell (DRC) and/or collision cell technique or the application of high-resolution ICP-MS with a doublefocusing sector field or a time-of-flight (TOF) mass analyzer. Ammonia as reaction gas is used to eliminate argon-based spectral interferences for the analysis of iron [31]. Under the conditions used by Deng et al. [14], the detection limit was 3.2 ng/L for Fe. Furthermore, ICP-MS may be used for the quantification of ferrocene-labeled analytes. Ferrocene-tethered hydroxysuccinimide ester was coupled either directly or via horseradish peroxidase as a bridge to a monoclonal antibody for 2,4-dichlorophenoxyacetic acid. Competitive immunoreactions were carried out on a microplate. The bound conjugate was dissolved in 1% working electrode (v/v) nitric acid with In as an internal standard and the signal of 56Fe was detected by DRC-ICP-MS [14]. Matrix-assisted laser desorption/ionization mass spectrometry The mass of a ferrocene-based mass tag typically exceeds 200 Da. The mass tag can therefore easily be determined even with low-resolution mass spectrometers. Especially MALDI-TOFMS may be used for that purpose, leading to the singly protonated molecule even for proteins and other large molecules. The technique was used to verify the performance of labeling methods for proteins [38], DNA [40, 60, 75, 102, 114, 115, 122] and for the detection of single base extension products utilizing a 5′ ferrocene labeled primer [40]. Electrospray ionization mass spectrometry Peptides, proteins and other large molecules are typically detected with a charge state distribution in ESI-MS. By deconvolution, the mass of the analyte may be calculated using appropriate software tools. ESI-MS was applied to determine the number of thiol and disulfide groups in proteins. In the first case, the disulfide groups are first reduced to thiols, and all free and released thiol groups are then derivatized by FEM [79]. In a second approach, the subsequent use of two different reagents, FEM and ferrocenecarboxylic acid-(2-maleimidoyl)ethylamide (FMEA) allows the individual determination of both free and disulfide-bound thiols [78]. However, the detection of the mass increase is not the main reason why derivatization by ferrocenes is performed for MS measurements. ESI has been reported to show excellent results for the determination of ionic and polar 194 analytes, since they are either already ionized or can easily be ionized under comparably soft conditions. Analytes of lower polarity are less accessible for protonation or deprotonation, resulting in losses of sensitivity. Although being nonpolar, ferrocenes are preionic molecules: Owing to the electroactivity at relative low potential, the ESI-MS spectrum of ferrocene is dominated by the intact molecular cation [M]+. A one-electron oxidation mechanism, which takes place in the electrospray interface, is responsible for this effect [123]. The ferrocene-based chemical derivatization for ESI-MS was introduced by Van Berkel et al. [22]. This method has proven to be an effective tool for transforming analytes that are neither ionic nor readily ionized in solution into products, which are amenable for ESI-MS detection. In general, signal levels for ferrocenes in ESI-MS are significantly improved using this approach. This expands the applicability of ESI-MS to a broader range of (nonpolar) compounds and adds detection selectivity as well, because only one specific functional group is derivatized. The one-electron oxidation is performed with excellent results using either a specially designed interface [124] or an electrochemical cell positioned in front of the interface [32]. A further advantage of the electrochemical cell approach is that the potential can be precisely adjusted to allow selective oxidation. The electrochemical oxidation mechanism is proven by using a heated nebulizer as the interface, for which a commercial APCI interface with the corona discharge switched off may be used most conveniently [32]. By this method, only preionized substances were detected and oxidation of ferrocenes by the ESI voltage cannot occur. The spectra of most derivatives are very simple, usually consisting of the molecular cation with no or only little fragmentation. The applicability of the method was demonstrated for alcohols and phenols [32], alkenes [125], isocyanates [10], neutral steroids [22] and 1-α-hydroxyvitamin D [73, 74]. The detection limits are in the nanomolar range, but strongly differ for different compounds. The ferrocene derivatives exhibit a characteristic isotopic pattern due to the natural isotopic distribution of iron. This facilitates the determination of ferrocenes in complex mixtures. Furthermore, derivatization has been used to direct the fragmentation. Ferrocene derivatization of carbohydrates was employed to obtain more information from the tandem MS spectra, which are dominated by the loss of water molecules for the underivatized carbohydrates. The distinction between the diasteromers of isomeric low molecular weight monosaccharides and disaccharides may be achieved this way [71, 72, 126]. Each epimer shows the diagnostic ions derived from the cyclic boronate at m/z 254 (Fig. 9). The other fragment ions represent the successive loss of formaldehyde (30 Da) and water (18 Da). Most ferrocene-based derivatizing agents undergo a characteristic fragmentation pathway in ESI-MS. A frag- Anal Bioanal Chem (2008) 390:181–200 H OH H O + HO + O H H H O OH B B O Fe O Fe m/z 254 Fig. 9 Fragmentation scheme for ferroceneboronic acid derivatives with ESI as the ionization method [126] ment corresponding to a loss of 65 Da, corresponding to the cylcopentadienyl ligand (leaving as a radical), is the most abundant fragment of ferrocene in ESI-MS experiments. This fragment occurs as well for ferrocene derivatives which are directly connected to an alkyl chain. It was observed for amine derivatives of 3-ferrocenyl-succinimidylpropionate and thiol derivatives of FEM (Fig. 10). The detection selectivity for these derivatives is enhanced by using the neutral loss mode. Furthermore, several fragment ions yield high intensities, which enable their sensitive detection in the multiple reaction monitoring mode. Van Berkel et al. investigated the fragmentation pathways for ferrocenoyl azide derivatives of phenols and primary, secondary and tertiary alcohols. By using tandem MS, including both precursor ion scan (m/z 201, m/z 227, m/z 245) and neutral loss scan mode ([M-44]+) (Fig. 11), one can distinguish between these groups of compounds [22, 35]. Furthermore, the use of energy resolved product ion spectra leads to information about the molecular structure, including positional isomers [36]. The differentiation between alcohols and phenols is possible for FCC derivatives by the presence or absence of m/z 213 and m/z 230. The fragment ions m/z 213 and m/z 185 are also observed for FMEA derivatives of thiols [127] and iscocyanates derivatized by ferrocenoyl piperazide [10] (Fig. 12). The 4-ferrocenylmethyl-1,2,4-triazoline-3,5-dione derivative of vitamin D shows m/z 199 as the main fragment [73] (Fig. 13). The transition was used for the sensitive monitoring of the analyte. The multiple reaction monitoring mode was comparably insensitive for the underivatized analyte because the fragment ions, produced by the neutral loss of water molecules, only show low abundance. LC/MS and LC/EC/MS Analytes of low polarity are best suited for reversed-phase HPLC separations. Ferrocenes add a relatively large nonpolar group to the analyte and thus improve the reversed-phase Anal Bioanal Chem (2008) 390:181–200 195 Fig. 10 Fragmentation scheme for FEM and 3-ferrocenylsuccinimidylpropionate derivatives with ESI as the ionization method Fe O HPLC separation. Because of the preionic character of ferrocenes, they can easily be oxidized after separation in an electrochemical cell and detected by MS very sensitively. This combination was introduced by Diehl et al. [32–34] for the determination of phenols and alcohols, and was later applied for the analysis of isocyanates as well [10]. The degree of polarity of derivatizing agents can further be tuned by functional groups at the alkyl side chains. This possibility was used for the development of FMEA. It is slightly more polar than FEM and the separation of the derivatives of both reagents can easily be performed. The method was applied to the differential labeling of disulfides and thiols. While the FMEA derivatives are eluted first, the FEM derivatives are eluted later [127]. Precursor ion scan and neutral loss scan may, in addition to the discrimination by retention time, be used to distinguish between both groups of derivatives. The combination with EC enables the selective detection of ferrocene-labeled analytes. The charge state distribution in ESI-MS spectra of multiply charged analytes is broadened and shifted to higher charges. In many cases, the signal intensity will be increased if the electrochemical potential is applied owing to oxidation of the ferrocenes to ferrocinium ions. The performance of the method was shown for a tryptic m/z 227 NH . + O Fe O Fe H R N O .+ O [M-44]+ main fragment for benzylalcohols and methanol main fragment for sterols and benzylalcohols R2 . H N R3 + m/z 245 H R1 O N R Fe O R3 H R1 Fe H .+ R O O R2 main fragment for secondary and tertiary alcohols m/z 201 main fragment for primary alcohols Fig. 11 Fragmentation scheme for ferrocenoyl azide derivatives with ESI as the ionization method [22, 35] .+ O N [M-65]+ R .+ O N Fe S R R H [M-65]+ peptide mixture with the goal to selectively determine the cysteine-containing peptides [79]. Gas chromatography/mass spectrometry Although they readily sublime at temperatures exceeding 100 °C, ferrocenes are remarkably stable, thus allowing their separation by gas chromatography (GC) as well. Peaks of secondary alcohols tend to broaden and tail on different capillary columns, which makes their direct analysis difficult and inaccurate. As a derivatizing agent for the GC/MS analysis of phenols and alcohols, FCC was used [28]. The volatility of FCC esters is very high, and with a temperature program, alcohols up to a chain length of 34 carbon atoms were eluted. Electron impact ionization MS may be used to assign alcohols and phenols. The mass spectra of alcohols show a fragment of m/z 230 corresponding to the FCA cation. Phenol ethers show a base peak of m/z 213 corresponding to the ferrocenecarbonyl cation, and m/z 230 is absent. Thus, alcohol- and phenol-selective chromatograms can be recorded. In all cases, [M]+, which may be used to identify the compound, is present in the mass spectra as well [28]. GC/MS enables the separation and identification of structural isomers owing to its high resolution. In LC/MS, the chromatographic resolution is lower, thus leading to a more difficult separation of the lower alkylphenol derivatives. For the higher molecular weight derivatives, both GC/MS and LC/MS do not allow to completely separate the very large number of possible structural isomers. The LC/MS method, however, is in principle applicable to alkylphenols with higher masses. The derivatization by FBA and thus the large increase in mass and in gas-chromatographic retention time is an advantage for analyzing low molecular mass diols [69, 70]. The mass spectra of electron impact ionization are dominated by molecular ions [M]+. The ferrocenyl group largely inhibits fragmentation modes within the substrate moiety. The characteristic isotopic pattern reflects the presence of 10B, 54Fe and 57Fe as minor natural isotopes and gives rise to distinctive isotopic patterns. Electron impact spectra show several reagent-derived ions (e.g., m/z 230 [FcB(OH)2]+, m/z 212 [FcBO]+, m/z 186 [FcH]+ and m/ z 121 [C5H5Fe]+). Thus, the selected ion monitoring mode enables the detection of individual boronates. All spectra of the derivatives contain a strong and characteristic ion at 196 Fig. 12 Fragmentation scheme for ferrocenecarboxylic acid chloride, ferrocenecarboxylic acid-(2-maleimidoyl)ethylamide and ferrocenoyl piperazide derivatives with ESI or atmospheric pressure chemical ionization as ionization methods [10, 79] Anal Bioanal Chem (2008) 390:181–200 m/z 213 m/z 230 O ferrocenecarboxylic acid chloride derivative m/z 185 + . H O C O R O H Fe Fe alcohol .+ H C H R benzyl alcohol m/z 213 ferrocenecarboxylic acid-(2-maleimidoyl) ethylamide derivative . + m/z 185 O O Fe S N N R O H m/z 213 m/z 185 O ferrocenoyl piperazide derivative . + H N Fe N N R O m/z 212 [69]. Furthermore, it is possible to distinguish between isomers such as pinacol and 2-methylpentane-2, 4-diol by their characteristic fragments. Atomic spectroscopy methods General considerations for atomic spectroscopy The ferrocene label contains an iron atom, which enables the determination of analytes by spectroscopic methods. Atomic spectroscopy is a powerful tool, because the calibration is in principle only dependent on the element to be determined, but not on the individual compound. Atomic absorption spectroscopy The average number of ferrocene moieties bound to an analyte can be determined by using atomic absorption spectroscopy (AAS) [15]. The iron in the ferrocene derivatives is analyzed after direct aspiration of the aqueous solution using an air-acetylene flame and detection of the Fig. 13 Fragmentation scheme for 4-ferrocenylmethyl-1,2,4triazoline-3,5-dione derivatives with ESI as the ionization method [73] m/z 199 m/z 241 O N Fe O N R1 N R3 R2 .+ absorption at 248.3 nm. The detection limit observed is 0.06 mg/L for iron [15]. Two concentrations have to be measured to determine the number of protein labels: The protein concentration is determined by a bicinchoninic acid protein assay [128], which is, however, associated with the same large uncertainty as many other methods for protein quantification. The iron content is determined after digestion with nitric acid at 100 °C [16, 18]. With these two concentrations, the average number of labels is determined. An immunoassay based on the labeling with organometallic compounds was described by using the quantitative measurements of the ferrocene-labeled antigens by AAS [17]. The antibody was bound to a water-insoluble polymer. Upon centrifugation, the antibody was precipitated, carrying with it the antibody-ferrocene-metallohapten complex. Aliquots of the supernatant containing the residual ferrocene-labeled antigens were injected into a graphite furnace atomic absorption spectrometer [13]. Atomic emission detection The idea to use GC analysis coupled with atomic spectroscopy after derivatization of chemical functionalities with element tags was introduced by Hagen et al. [129]. Iron has excellent detection characteristics in atomic emission detection (AED): It may be detected at 302 nm down to 50 fg/s and exhibits a selectivity versus carbon of 4.6×106 [130]. Because volatile iron compounds are not naturally present, underivatized matrix compounds do not Anal Bioanal Chem (2008) 390:181–200 197 interfere. Thus, a simple and sensitive determination of alkylphenols after derivatization by FCC was developed by Rolfes and Andersson [29]. The method was shown to be useful for the quantification of 20 C0-C3 alkylpenols in crude oil [30] and o-phenylphenol in citrus fruits [28]. Furthermore, atomic emission spectroscopy was used to quantify ferroceneboronate derivatives of 1,2-diols and 1,3diols [68]. For FBA derivatives, boron can be detected at 249 nm in addition to iron at 259 nm. GC/AED is limited to volatile samples. The detection limit is much better than that of GC/MS. However, no information about possible coelution of different labeled analytes and about the mass and nature (phenol or alcohol) of the analyte is available. Therefore, GC/MS and GC/AED may be considered as excellent complementary tools for ferrocene-based derivatization approaches with volatile analytes. Electrochemiluminescence Electrochemiluminescence (ECL) is a form of chemiluminescence in which the chemiluminescence reaction is preceded by an electrochemical redox reaction. The oxidation of luminol by hydrogen peroxide as a classic chemiluminescence reaction is catalyzed by Fe(III), ferricyanide, horseradish peroxidase and other iron-containing compounds. Ferrocene and its derivatives catalyze the abovementioned system as well [131]. Owing to this reaction, ferrocinium as well as oxidized ferrocene-labeled proteins can be detected down to subnanomolar amounts by R Fe II NH2 O e- NH NH R Fe III O H2O2/OH- H2O/N2 hν NH2 O O O O Fig. 14 Electrochemiluminescence reaction of luminol with catalytic effect of the ferrocinium cation [131] a simple fluorimeter [18]. The ferrocinium cation generated at the electrode reacts with luminol to form an excited species, which is deactivated by light emission (Fig. 14). The intensity of the light may be further enhanced by suitable substituents at the cyclopentadienyl ring [132]. This detection method can be applied to immunoassays. As long as the labeled antigen is free, luminol oxidation is catalyzed and chemiluminescence is observed. The ferrocene loses its catalytic activity when embedded into an antibody, thus resulting in a signal reduction [133]. The selectivity of the method will be limited depending on the presence of other catalysts and on the stability of the ferrocinium cation. In another approach, ferrocene is used to quench ECL. Tris(2,2′-bipyridine)ruthenium(II) shows ECL in the presence of tripropylamine. The ferrocinium cation quenches the reaction, because it oxidizes tris(2,2′-bipyridine)ruthenium(I) to tris(2,2′-bipyridine)ruthenium(II). With use of ferrocene as a quencher label on a complementary DNA sequence, an intramolecular ECL quenching in hybridized oligonucleotide strands has been realized [75]. The major advantage of the chemiluminescence approaches is the relatively simple assembly compared with other optical methods, because no light source is needed. A combination of a chemiluminescence detector with an electrochemical cell could thus lead to a new simple, but sensitive detection method for ferrocenes in HPLC. UV/vis absorption Owing to the low molar absorptivity of ferrocene, a sensitive detection of its derivatives by UV/vis absorption is not possible; thus, methods using UV detection are not particularly promising for trace analysis. The ferrocene/ ferricinium redox couple has significantly different UV/vis absorption spectra. In ethanol, ferrocene is yellow and exhibits two absorption bands at 325 and 440 nm, whereas ferrocinium solutions exhibit a characteristic absorption peak at 620 nm [12]. Thus, ferrocene was used as a colorimetric dye for determining concentrations of glucose, glutamate, lactate and others by measuring the absorption of the formed ferrocinium cation at 620 nm. Owing to the improved stability and water solubility, inclusion complexes of ferrocene and β-cyclodextrin were applied instead of pure ferrocene [133]. The spectrophotometric determination of the ferrocene content may be rapidly performed after release of iron from the ferrocene complex by trichloroacetic acid. Iron(III) in the acidic filtrate is reduced to iron(II) and after complexation with ferrozine [3-(2′-pyridyl)-5,6-bis(4′-phenylsulfonic acid)1,2,4-triazine], spectrophotometric analysis at 564 nm is possible. The method was applied to determine the number of labeled functional groups in proteins. The detection limit was 10 times lower than that of AAS, especially when a 198 protein matrix was present [134]. The goal of this method was to determine the number of labels attached to the protein. Conclusions It is evident that the role of ferrocene derivatization in analytical chemistry does by far exceed the function as mediators in biosensors. By derivatization, an electroactive label is introduced. The reversible redox behavior of the ferrocene/ferrocinium couple at low potentials is a unique property, which finds widespread application. The ECD of ferrocenes is cheap and sensitive, and it is possible to detect minor differences in electrode potentials, which allow the analysis of differentially labeled analytes. Several ferrocene labels can be determined within one measurement. The application of ferrocenes in HPLC/ECD is well established. However, the limitation to isocratic elution and problems with the limited separation efficiency of HPLC and identification of coeluted analytes restrict the more general use. Capillary electrophoresis techniques coupled with ECD are promising owing to their large separation efficiency. This is particularly valid for DNA and RNA detection as well as for immunoassays, which are to date the main applications of ECD combined with separations subsequent to ferrocene-based derivatization. ECD methods on microchips have some significant advantages over fluorescence detection: They offer sensitivity, compatibility with microfabrication, simplicity and inexpensive instrumentation. As for the fluorescence approach, derivatization is mostly needed to enhance the general applicability of the method. Ferrocenes are readily available for this purpose. However, strong research activities are still required to develop new and improved ferrocenebased derivatizing agents for all of these applications. The electrochemical ionization of ferrocene-derivatized analytes is a useful tool to determine nonpolar compounds, but a functional group has to be present in the analyte that undergoes facile reaction in order to introduce a ferrocene label. For more polar functionalities, the improvement in LC separation is important. Furthermore, the possibility of selective enhancement in LC/EC/MS compared with LC/MS is an option. Dedicated fragmentation is an additional opportunity that should be investigated in the future for other functionalities, too. Ferrocene derivatives of nucleic acids have not been investigated with LC/EC/MS yet, although this appears to be promising. The detection of ferrocene derivatives by ICP/MS and ICP/OES seems to be particularly promising as well, especially combined with other metal complexes, thus using differential derivatization. The hyphenation with LC, CE or GC adds selectivity. The reduction of interferences with Ar species and reduction of operating costs are Anal Bioanal Chem (2008) 390:181–200 important challenges, which remain. GC/MS and GC/AED might be promising alternatives for volatile derivatives. Acknowledgements Financial support by the Deutsche Forschungsgemeinschaft (Bonn, Germany) and the Fonds der Chemischen Industrie (Frankfurt, Germany) is gratefully acknowledged. References 1. Arrayas GR, Adrio J, Carretero JC (2006) Angew Chem Int Ed Engl 45:7674–7715 2. 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