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Recent advances in biomolecular vibrational spectroelectrochemistry
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Review Article Recent advances in biomolecular vibrational spectroelectrochemistry Ángela I. López-Lorente 1 and Christine Kranz 2,∗ Spectroelectrochemical studies of biomolecules have gained attraction due to the breadth of information that can be derived from combined in situ bioelectrochemistry–spectroelectrochemistry experiments. This current opinion article provides a comprehensive overview focusing on surface-enhanced IR and Raman spectroscopic techniques applied in bioelectrochemical studies published within the past 3 years. Next to fundamental studies also sensing applications will be discussed taking advantage of advanced spectroelectrochemical techniques. Addresses 1 Departamento de Química Analítica, Instituto Universitario de Investigación en Química Fina y Nanoquímica IUIQFN, Universidad de Córdoba, Campus de Rabanales, Edificio Marie Curie Anexo, E-14071 Córdoba, Spain 2 Institute of Analytical and Bioanalytical Chemistry, Ulm University, Ulm, Germany ∗
Corresponding author: Kranz, Christine ([email protected])
biomolecules [1• ] including redox enzymes [2], proteins [3], and membrane components [4]. In particular, midinfrared spectroscopy (MIR; 2.5–20 μm; 4000–500 cm−1 ) and Raman spectroscopy provide a wealth of information on changes in the vibrational signature, i.e., the molecular fingerprint regime. Complementary in nature, both techniques have been applied to investigate electron transfer processes in cytochrome c or metalloenzymes, for in situ studies of microbial biofilms, peptides, cells, and proteins, and for elucidating protein reactions and protein folding/unfolding. Misfolding and aggregation of specific proteins have been extensively studied via IR spectroscopy, as neurogenerative diseases (e.g., Alzheimer, Parkinson, Creutzfeldt–Jakob) are frequently associated with such changes in protein structure [5• ]. In particular, group vibrations of the protein backbone providing information on the secondary structure including the amide I, amide II, and amide III band have been analyzed using IR spectroscopy, as recently summarized [6].
Current Opinion in Electrochemistry 2017, 5:106–113 This review comes from a themed issue on Bioelectrochemistry Edited by Nicolas Plumeré For a complete overview see the Issue and the Editorial Available online 4 August 2017 http://dx.doi.org/10.1016/j.coelec.2017.07.011 2451-9103/© 2017 Elsevier B.V. All rights reserved.
Introduction Spectroelectrochemical experiments are performed across almost the entire range of the electromagnetic spectrum, i.e., from electrochemical NMR experiments to in situ X-ray absorption spectroelectrochemical studies. The major benefit derives from the combination of chemical and electronic information, which is of particular relevance in bioelectrochemistry. Besides sensing applications (e.g., DNA), such studies are focused on characterizing biological systems and complex processes including but not limited to electron–proton transport in cells, membrane potentials of cells, and electrode reactions of proteins and redox enzymes without the need of labeling the biological component. Several reviews have been published on spectroelectrochemical studies of Current Opinion in Electrochemistry 2017, 5:106–113
A disadvantage of these vibrational spectroscopic techniques is the inherently limited absorption in the IR, and the rather small Raman scattering cross section, if the biological component is covalently attached or adsorbed at an electrode surface. For a densely packed protein monolayer assuming an average protein diameter of approx. 5 nm, this yields a maximum surface coverage on the order of 8 pmol cm−2 [1• ]. Consequently, using surface-enhanced infrared absorbance spectroscopy (SEIRAS), surface-enhanced Raman spectroscopy (SERS), and surface-enhanced resonance Raman spectroscopy (SERRS) techniques is almost mandatory for overcoming this limitation. Utilizing rough metal surfaces, deliberately controlled nanostructures [7,8], or nanoantennas [9,10] not only gives rise to enhanced analytical signals via antenna and/or plasmonic effects, but simultaneously provides an interface potentially serving as working electrode during spectroelectrochemical experiments. Hence, thermodynamic and kinetic information obtained via electrochemistry may be directly correlated with vibrational information revealing structural and conformational changes of the biological constituent. In the following, recent progress in bio/spectroelectrochemistry using IR and Raman techniques will be reviewed focusing on the use of surface-enhanced signatures for studying redox processes at proteins, enzymes, and biofilms, and on their application in www.sciencedirect.com
Recent advances in biomolecular vibrational spectroelectrochemistry López-Lorente and Kranz
sensing scenarios. Also, ATR-SEIRA experiments utilizing IR-transparent boron-doped diamond (BDD) waveguides providing a broad spectroscopic and electrochemical window will be highlighted. Surface- and tipenhanced Raman techniques, in combination with electrochemistry, have also advanced for biomolecular studies. In particular, tip-enhanced Raman spectroscopy (TERS) combined with microelectrochemistry may show great promise for nanoscale spectroelectrochemistry in the future. This article will highlight the current state of the art and future potential of bio/spectroelectrochemistry.
IR-transparent materials for spectroelectrochemistry Besides the limited inherent sensitivity, infrared absorption methods are affected by pronounced water absorptions during in situ studies. In particular, strong bending vibrations around 1640 cm−1 convolute the protein amide I band, while the O–H stretching mode around 3000 cm−1 masks N–H stretching vibrations of amines. IR-ATR spectroscopy on biomolecules attached or adsorbed at an ATR crystal surface ensures a limited penetration depth of the evanescent field into the adjacent medium, thereby minimizing the water interference. Also water may be replaced by deuterium oxide (D2 O) shifting the solvent bands to lower wavenumbers, which avoids masking bands in the amide I region. Typically, IR-transparent materials (e.g., Ge, ZnSe, ZnS, Si, diamond) are insulators or semiconductors, and hence, are not suitable as electrodes. Thin films of BDD with typically doping levels of 1019 –1021 atoms cm−3 are transparent in the MIR with the exception of two-phonon (around 2670 cm−1 and 1744 cm−1 ), have low resistivity, and large potential window. Surface enhancement effects via plasmonic resonances have been achieved using stabilizer-free gold nanoparticles (AuNPs) modifying such IR-transparent BDD-coated diamond ATR crystals [11]. Using these waveguides, combined IR-ATR, electrochemical, and atomic force microscopy (AFM) studies on bovine serum protein films have been demonstrated as shown in Figure 1A [12• ].
SEIRA spectroelectrochemistry Non-conductive ATR crystals have been modified with thin metal layers leading to plasmonic resonances based on strong light–matter interactions at sub-wavelength localization, a.k.a., ATR-SEIRAS [15]. This strategy has been used for in situ spectroscopic [9] and spectroelectrochemical studies on biomolecules [16]. However, it should be noted that the signal enhancement, which depends on the roughness of the SEIRA substrate rapidly decays at a length scale on the order of the average size of protein molecules. Consequently, not all structures of a protein may equally contribute to the obtained SEIRA spectra. www.sciencedirect.com
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Nevertheless, SEIRAS has been applied for biosensing [17–19], in protein assays [20], and for studying conformational changes of proteins [12,21,22] such as redoxrelated processes at electrochemical interfaces [16]. Spectroelectrochemical studies using ATR-SEIRAS at thin gold films have been applied to investigate electron transfer processes at redox active molecules, e.g., conformational changes of cytochrome c (cyt c) oxidase induced by direct electron transfer [23,24] as well as dynamics of charge–transfer and protein orientation of immobilized cyt c on a gold electrode [25]. Recently, the kinetics of cyt c oxidase derived from Rhodobacter sphaeroides were investigated via time-resolved SEIRAS using step-scan spectroscopy applying periodic potential pulses between −800 mV and open circuit potential [26]. Redox changes during the electron transfer of the active centers embedded within the protein secondary structure were investigated via changes in the amide I band at anaerobic and aerobic conditions. Protein film infrared electrochemistry has been demonstrated with enzymes adsorbed on carbon particle electrodes, which were deposited onto the surface of an Si ATR crystal to study the catalytic turnover of H2 oxidation by E. coli hydrogenase 1 (Hyd-1) [13•• ] (see Figure 1B). Relevant in apoptosis, interactions of cyt c with cardiolipin-containing lipid bilayers have been studied on supported lipid bilayers at 1-dodecanethiol functionalized gold electrode using cyclic voltammetry (CV) and SEIRAS measurements [27]. The effect of SiO2 NPs on the electron transfer behavior of cyt c adsorbed onto 11-mercaptoundecanoic acid was also investigated [28]. The presence of nanoparticles modulates the heme microenvironment, and its orientation during adsorption, thereby leading to enhanced electron transfer in dependence on the applied potential. A real-time infrared plasmonic biosensor was demonstrated for chemicalspecific detection of biomimetic lipid membranes using surface-modified Au nanoantennas as shown in Figure 1C [14• ]. The formation kinetics of planar biomimetic membranes in aqueous environments has been monitored in real time using the vibrational fingerprints of the lipid molecules. The effect of the membrane electric field on the KcsA potassium channel has been investigated using voltammetric SEIRA studies [29], which also enabled the characterization of membrane-bound hydrogenase co-immobilized with a phospholipid bilayer on a gold electrode [30]. Moreover, SEIRAS combined with electrical impedance spectroscopy allowed studying the transmembrane proton gradient in bilayer lipid membranes produced by cyt bo3 [31]. Graphene has revealed a significant potential for electrochemical SEIRAS experiments, and also for the label-free quantitative detection of protein monolayers (Figure 2) [32•• ]. A bias voltage was varied to control the Fermi level of graphene, while an IR-beam excited a plasmon resoCurrent Opinion in Electrochemistry 2017, 5:106–113
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Figure 1
A: Schematic setup for combined AFM-IR-EC measurements. a) IR absorption signals and average integral values (N = 4), corresponding to the amide I and II bands from 1.0 (gray), 2.5 (red) and 5.0 (blue) mg mL−1 BSA solution. Dashed lines in a) and dashed filling in b) reflect the data obtained at the non-modified BDD crystal. Adapted with permission from [12• ]. B: Diagram of the working-electrode configuration and solution flow (blue arrows) over the multi-bounce Si IRE. a) Current–time traces of Hyd-1 using the ATR-IR cell in Ar-saturated (gray) and H2 -saturated (black) buffer; b), c) spectra showing the nCO region at each potential under Ar (b) and H2 (c). Adapted with permission from [13•• ]. C Schematic illustration showing the fluidic chamber and experimental configuration for detection of SLB using Au nanoantennas. a) Reflectance within the lipid fingerprint region as a function of time during lipid vesicle adsorption and rupture on a nanoantenna array with L = 1.00 μm, P = 1.37 μm, and 15 nm thick SiO2 layer and the corresponding absorbance spectra. The vertical dashed lines mark the spectra position of the symmetric (blue) and asymmetric (red) CH2 stretching modes, schematically represented in the inset figure. Integrated absorbance over CH2 bands as a function of time for nanoantenna modified with b) 15 nm thick SiO2 ; c) modified with hydrophilic 16-mercapto hexadecanoic acid (MHDA), and d) 15 nm thick Al2 O3 layer. Adapted with permission from [14• ].
nance across graphene nanoribbons. At the ribbon edge, a significantly enhanced light interaction with the adsorbed protein molecules was observed.
SERS/SERRS spectroelectrochemistry Surface-enhanced Raman techniques including SERS, SERRS, and TERS are associated with local field enhancements at metal nanostructures as well as chemical enhancement effects. Enhancement effects are dependent on the shape and size of the nanostructure, Current Opinion in Electrochemistry 2017, 5:106–113
but also on the plasmon coupling of nanoparticle aggregates. A wide variety of electrochemical SERS studies have demonstrated the potential of this technique using, e.g., Nile Blue as a model analyte [33–35]. SERS has also been applied for spectroelectrochemical studies of cyt c, and electrochemical SERRS experiments have been used to study the nature of the biofilm–electrode interaction of cyt OmcB from Desulfuromonas acetoxidans microbial biofilms, which causes the wiring of the cell metabolism to the electrode [36]. Also, the effect of oxywww.sciencedirect.com
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Figure 2
Tunable graphene-based SEIRAS protein sensor. A: Schematic view of the IR biosensor device. An IR-beam excites a plasmon resonance across the graphene nanoribbons, enhancing light interaction with the protein molecules. The plasmonic resonance is electrostatically tuned to sweep continuously over the protein vibrational bands. B: Extinction spectra of the graphene nanoribbon array (width W = 30 nm, period P = 80 nm) for bias voltages Vg from –20 V to –130 V before (dashed curves) and after (solid curves) protein bilayer formation. Extinction is calculated as the relative difference in transmission between regions with and without graphene nanoribbons. Gray vertical strips indicate amide I and II bands of the protein. Adapted with permission from [32•• ].
gen on the electroactivity of microbial biofilms has been investigated [37]. Recently, time-resolved investigations on the structural evolution of surface species equaling the temporal resolution of transient electrochemical methods have been shown via transient electrochemical SERS (TEC-SERS) [38• ]. SERRS and SEIRAS combined with electrochemistry have allowed the study of the calciuminduced reorientation of cellobiose dehydrogenase immobilized on electrodes and the effect on the oxidation of carbohydrates [39]. SERS and SERRS have been also used in sensing applications, e.g., for detecting DNA [40,41]. An ECSERS-based DNA aptasensor has been described for the detection of Mycobacterium tuberculosis DNA using a AgNP-modified screen-printed electrodes following SERS bands of adenine under potential control [41]. Electrochemically driven denaturation assays monitored by SERS were able to discriminate DNA amplicons genwww.sciencedirect.com
erated from pathogenic Yersinia pestis bacteria [42]. Also, EC-SERS biosensors have been designed for analyzing DNA interactions with anticancer drugs such as doxorubicin [43], and mitoxantrone [44]. Yuan et al. [45• ] reported an electrochemical SERS study of a metalloporphyrin hemin system using a microfluidic chamber (Figure 3) via a nanostructured Au surface. Shifts in ν(C–C) and ν(C–N) stretching vibrations of the porphyrin ring in the range of 1300–1700 cm−1 are typically associated with the oxidation state of iron. In the frequency range of 1368–1377 cm−1 , the ν 4 mode is observed for ferric (Fe3+ ) hemin, and in the range of 1344– 1364 cm−1 for ferrous (Fe2+ ) hemin. At a potential of −0.2 V vs. Ag/AgCl, two bands were resolved at 1345 and 1366 cm−1 , which were associated with the partial oxidation of the iron cores. In contrast, at −0.5 V only one ν 4 band at approx. 1349 cm−1 is observed, which is indicative for the reduction of the iron cores. Current Opinion in Electrochemistry 2017, 5:106–113
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Figure 3
In situ SERS SEC set-up. A: Schematic representation of the in situ SERS spectroelectrochemical (SEC) setup with microfluidic chamber. B: Schematic of the MPy/hemin modified Au surface. C: SERS spectra from MPy/hemin modified nanostructured Au WE in the marker band region (background corrected). (a) Electrode potential E = −0.2 V. (b) Electrode potential E = −0.5 V. Raw data: blue traces; modeled data: red traces. Adapted with permission from [45• ].
EC-SERS using AuNP-modified gold electrodes enabled the investigation of the adsorption of 1-thio-β-d-glucose onto the AuNPs, and the extent of oxidation of the monolayer as a function of the applied electrode potential [46]. Likewise, the detection of uric acid as biomarker of preeclampsia using an Au/Ag substrate has been shown [47]. Electrochemical shell-isolated nanoparticle-enhanced Raman spectroscopy (EC-SHINERS) has been applied for electrochemical interface studies on catalytic processes, molecule adsorption, etc. [48]. This technique has also been applied to study the electrochemical behavior of adenine nucleobases [49], as well as for investigating the adsorption of four DNA bases at Au electrodes [50].
TERS spectroelectrochemistry More recently, TERS has been combined with electrochemical studies [51• ,52], yet to date has not been applied for investigating biomolecules. Current Opinion in Electrochemistry 2017, 5:106–113
TERS combines superior sensitivity and molecular selectivity of enhanced Raman signals with the nanoscale spatial resolution of scanning probe microscopy (SPM) techniques such as AFM or scanning tunneling microscopy (STM). TERS at a sharp metal tip (e.g., Au, Ag) may achieve sub-nanometer spatial resolution [53], and has been applied in biochemical studies and in surface science. To date, TERS has predominantly been used for biomolecule detection schemes including targets such as DNA [54], proteins [55], and bacteria/viruses [56] taking advantage of the pronounced sensitivity. (Nano)electrochemical TERS studies are still rare. Analogous to EC-SERS, Nile Blue has served as model analyte to explore the potential of electrochemical TERS (ECTERS) [51• ]. It has been also used to monitor arrangements of 4 -(pyridin-4-yl)biphenyl-4-yl) methanethiol (4PBT) at Au surfaces [52]. While bioelectrochemical TERS experiments have not yet been reported, it may be expected that EC-TERS applications in the field of biomolecular detection will be emerging within few years. www.sciencedirect.com
Recent advances in biomolecular vibrational spectroelectrochemistry López-Lorente and Kranz
Concluding remarks Spectroelectrochemical investigations of biomolecules using surface-enhanced IR and Raman spectroscopic techniques have significantly contributed advancing the understanding on molecular orientation at electrified interfaces, and on electron transfer processes involving complex biomolecules. Signal-enhanced techniques readily support the development of sensitive label-free biosensors, which has been demonstrated, e.g., for DNA sensing. Future directions in bio/spectroelectrochemical studies are clearly targeted toward improving the spatial and temporal resolution (a.k.a., high-resolution imaging), which may lead to obtaining dynamic interfacial in formation at the single molecule level. Although in its infancy, EC-TERS studies on biomolecules may enable understanding structure-related electrochemical activity and surface chemistry in molecular detail. The IR-equivalent to TERS, termed nano-IR or scanning nearfield infrared microscopy (SNIM) has been successfully applied to characterize biomolecules. However, tipenhanced IR-techniques suffer from limited applicability in aqueous solutions, which are usually demanded in bio/spectroelectrochemistry. Recently, an IR-compatible liquid cell architecture has been demonstrated using large-area graphene sheets, which act as an impermeable monolayer barrier facilitating the investigation of tobacco mosaic virus in water trapped underneath graphene [57•• ].
Acknowledgment A.I. López-Lorente thanks the Alexander von Humboldt Foundation for the award of a Postdoctoral Fellowship.
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: •
Paper of special interest. Paper of outstanding interest.
••
1.
Ash PA, Vincent KA: Vibrational spectroscopic techniques for probing bioelectrochemical systems. Adv Biochem Eng/Biotechnol 2017, 158:75–110. This recent review gives a comprehensive overview on spectroelectrochemistry of biomolecules using enhanced IR and Raman techniques such as SEIRAS, SERS, SERRS. •
2.
Ash PA, Vincent KA: Spectroscopic analysis of immobilised redox enzymes under direct electrochemical control. Chem Commun 2012, 48:1400–1409.
3.
Murgida DH, Hildebrandt P: Disentangling interfacial redox processes of proteins by SERR spectroscopy. Chem Soc Rev 2008, 37:937–945.
4.
Melin F, Hellwig P: Recent advances in the electrochemistry and spectroelectrochemistry of membrane proteins. Biol Chem 2013, 394:593–609.
5.
Nabers A, Ollesch J, Schartner J, Kötting C, Genius J, Hafermann H, Klafki H, Gerwert K, Wiltfang J: Amyloid-β-secondary structure distribution in cerebrospinal fluid and blood measured by an immuno-infrared-sensor: a biomarker candidate for Alzheimer’s disease. Anal Chem 2016, 88:2755–2762.
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This paper demonstrates the potential of IR spectroscopy for a label-free diagnostic tool detecting Dementia Alzheimer type (DAT) using the downshift of the amide I band frequency of Aβ-peptides in cerebrospinal fluid and blood plasma. 6.
López-Lorente AI, Mizaikoff B: Mid-infrared spectroscopy for protein analysis: potential and challenges. Anal Bioanal Chem 2016, 408:2875–2889.
7.
Bedford EE, Boujday S, Pradier C-M, Gu FX: Nanostructured and spiky gold in biomolecule detection: improving binding efficiencies and enhancing optical signals. RSC Adv 2015, 5:16461–16475.
8.
Bibikova O, Haas J, López-Lorente AI, Popov A, Kinnunen M, Meglinski I, Mizaikoff B: Towards enhanced optical sensor performance: SEIRA and SERS with plasmonic nanostars. Analyst 2017, 142:951–958.
9.
Adato R, Altug H: In-situ ultra-sensitive infrared absorption spectroscopy of biomolecule interactions in real time with plasmonic nanoantennas. Nat Commun 2013, 4:1–10.
10. Neubrech F, Huck C, Weber K, Pucci A, Giessen H: Surface-enhanced infrared spectroscopy using resonant nanoantennas. Chem Rev 2017, 117:5110–5145. 11. Izquierdo J, Mizaikoff B, Kranz C: Surface-enhanced infrared spectroscopy on boron-doped diamond modified with gold nanoparticles for spectroelectrochemical analysis. Phys Status Solidi A 2016, 213:2056–2062. 12. López-Lorente AI, Izquierdo J, Kranz C, Mizaikoff B: Boron-doped • diamond modified with gold nanoparticles for the characterization of bovine serum albumin protein. Vib Spectrosc 2017, 91:147–156. The paper demonstrates the first combined SEIRA-ATR-EC-AFM multi-analytical platform for the characterization of BSA protein films using spectroscopic, electrochemical methods and AFM imaging. The BDD surface modified with AuNPs via a stainless steel assisted method served as conductive SEIRAS substrate. Globular and fibril aggregates of BSA could be discriminated using curve fitting of the amide I band showing major contributions of crossed β-sheet structures within the fibril aggregates. 13. Hidalgo R, Ash PA, Healy AJ, Vincent KA: Infrared spectroscopy •• during electrocatalytic turnover reveals the Ni–L active site state during H2 oxidation by a NiFe hydrogenase. Angew Chem Int Ed 2015, 54:7110–7113. Evidence is given in these spectroelectrochemical ATR-IR experiments that the fast catalytic turnover of hydrogen oxidation by E. coli hydrogenase 1 (Hyd-1) involves sequential proton and electron transfer via an NiIFeII active site state termed Ni–L. Experiments were performed using a high surface-area carbon electrode deposited onto a Si internal reflection element under Ar and H2 atmosphere. The spectra reveal that the active site state Ni–L, observed in other NiFe hydrogenases only under illumination or at cryogenic temperatures, can be generated reversibly in the dark at ambient temperature under both turnover and non-turnover conditions. As Ni–L is present at all potentials during turnover under H2 , it is concluded that the final steps in the catalytic cycle of H2 oxidation involve sequential proton and electron transfer via Ni–L. 14. Limaj O, Etezadi D, Wittenberg NJ, Rodrigo D, Yoo D, Oh S, • Altug H: Infrared plasmonic biosensor for real-time and label-free monitoring of lipid membranes. ACS Nano 2016, 16:1502–1508. This paper describes the near-field enhancement using engineered and surface-modified Au nanoantennas for real-time and label-free detection of biomimetic lipid bilayers in aqueous solution. Using the vibrational fingerprints of lipid molecules, the real time formation kinetics of planar biomimetic membranes could be monitored. 15. Nedelkovski V, Schwaighofer A, Wraight CA, Nowak C, Naumann RLC: Surface-enhanced infrared absorption spectroscopy (SEIRAS) of light-activated photosynthetic reaction centers from Rhodobacter sphaeroides reconstituted in a biomimetic membrane system. J Phys Chem C 2013, 117:16357–16363. 16. Murgida DH, Hildebrandt P: Redox and redox-coupled processes of heme proteins and enzymes at electrochemical interfaces. Phys Chem Chem Phys 2005, 7:3773–3784.
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17. Boujday S, de la Chapelle ML, Srajer J, Knoll W: Enhanced vibrational spectroscopies as tools for small molecule biosensing. Sensors 2015, 15:21239–21264. 18. Hornemann A, Eichert D, Flemig S, Ulm G, Beckhoff B: Qualifying label components for effective biosensing using advanced high-throughput SEIRA methodology. Phys Chem Chem Phys 2015, 17:9471–9479. 19. Leidner L, Stäb J, Adam JT, Gauglitz G: Surface-enhanced infrared absorption studies towards a new optical biosensor. Beilstein J Nanotechnol 2016, 7:1736–1742. 20. López-Lorente AI, Wang P, Mizaikoff B: Towards label-free mid-infrared protein assays: in-situ formation of bare gold nanoparticles for surface enhanced infrared absorption spectroscopy of bovine serum albumin. Microchim Acta 2017, 184:453–462. 21. Fallah MA, Stanglmair C, Pacholski C, Hauser K: Devising self-assembled-monolayers for surface-enhanced infrared spectroscopy of pH-driven poly-l-lysine conformational changes. Langmuir 2016, 32:7356–7364. 22. Zeng L, Wu L, Liu L, Jiang X: Analyzing structural properties of heterogeneous cardiolipin-bound cytochrome c and their regulation by surface-enhanced infrared absorption spectroscopy. Anal Chem 2016, 88:11727–11733. 23. Ataka K, Heberle J: Use of surface enhanced infrared absorption spectroscopy (SEIRA) to probe the functionality of a protein monolayer. Biopolymers 2006, 82:415–419. 24. Nowak C, Laredo T, Gebert J, Lipkowski J, Gennis RB, Ferguson-Miller S, Knoll W, Naumann RLC: 2D-SEIRA spectroscopy to highlight conformational changes of the cytochrome c oxidase induced by direct electron transfer. Metallomics 2011, 3:619–627.
light interaction with the adsorbed protein molecules could be observed. 33. Wilson AJ, Molina NY, Willets KA: Modification of the electrochemical properties of Nile Blue through covalent attachment to gold as revealed by electrochemistry and SERS. J Phys Chem C 2016, 120:21091–21098. 34. Weber ML, Wilson AJ, Willets KA: Characterizing the spatial dependence of redox chemistry on plasmonic nanoparticle electrodes using correlated super-resolution surface-enhanced Raman scattering imaging and electron microscopy. J Phys Chem C 2015, 119:18591–18601. 35. Wilson AJ, Willets KA: Unforeseen distance-dependent SERS spectroelectrochemistry from surface-tethered Nile Blue: the role of molecular orientation. Analyst 2016, 141:5144–5151. 36. Alves A, Ly HK, Hildebrandt P, Louro RO, Millo D: Nature of the surface-exposed cytochrome−electrode interactions in electroactive biofilms of Desulfuromonas acetoxidans. J Phys Chem B 2015, 119:7968–7974. 37. Millo D, Ly HK: Towards the understanding of the effect of oxygen on the electrocatalytic activity of microbial biofilms: a spectroelectrochemical study. RSC Adv. 2015, 5:92042–92044. 38. Zong C, Chen CJ, Zhang M, Wu DY, Ren B: Transient • electrochemical surface-enhanced Raman spectroscopy: a millisecond time-resolved study of an electrochemical redox process. J Am Chem Soc 2015, 137:11768–11774. This paper demonstrates transient electrochemical surface-enhanced Raman spectroscopy (TEC-SERS) to study the structural evolution of surface species at a time resolution matching the electrochemical cyclic voltammetric and chronoamperometric experiments. With this approach the Raman signal providing the molecular signature information could be precisely correlated with the electrochemical current signal.
25. Jin B, Wang G, Millo D, Hildebrandt P, Xia X: Electric-field control of the pH-dependent redox process of cytochrome c immobilized on a gold electrode. J Phys Chem C 2012, 116:13038–13044.
39. Kielb P, Sezer M, Katz S, Lopez F, Schulz C, Gorton L, et al.: Spectroscopic observation of calcium-induced reorientation of cellobiose dehydrogenase immobilized on electrodes and its effect on electrocatalytic activity. ChemPhysChem 2015, 16:1960–1968.
26. Steininger C, Reiner-Rozman C, Schwaighofer A, Knoll W, Naumann RLC: Kinetics of cytochrome c oxidase from R. sphaeroides initiated by direct electron transfer followed by tr-SEIRAS. Bioelectrochemistry 2016, 112:1–8.
40. Johnson RP, Richardson JA, Brown T, Bartlett PN: A label-free, electrochemical SERS-based assay for detection of DNA hybridization and discrimination of mutations. J Am Chem Soc 2012, 134:14099–14107.
27. Liu L, Zeng L, Wu L, Jiang X: Label-free surface-enhanced infrared spectroelectrochemistry studies the interaction of cytochrome c with cardiolipin-containing membranes. J Phys Chem C 2015, 119:3990–3999.
41. Karaballi RA, Nel A, Krishnan S, Blackburn J, Brosseau CL: Development of an electrochemical surface-enhanced Raman spectroscopy (EC-SERS) aptasensor for direct detection of DNA hybridization. Phys Chem Chem Phys 2015, 17:21356–21363.
28. Zhang X, Zeng L, Liu L, Ma Z, Jiang X: Surface-enhanced infrared absorption spectroscopy and electrochemistry reveal the impact of nanoparticles on the function of protein immobilized on mimic biointerface. Electrochim Acta 2016, 211:148–155. 29. Yamakata A, Shimizu H, Oiki S: Surface-enhanced IR absorption spectroscopy of the KcsA potassium channel upon application of an electric field. Phys Chem Chem Phys 2015, 17:21104–21111. 30. Gutiérrez-Sanz O, Marques M, Pereira IAC, De Lacey AL, Lubitz W, Rüdiger O: Orientation and function of a membrane-bound enzyme monitored by electrochemical surface-enhanced infrared absorption spectroscopy. J Phys Chem Lett 2013, 4:2794–2798. 31. Wiebalck S, Kozuch J, Forbrig E, Tzschucke CC, Jeuken LJC, Hildebrandt P: Monitoring the transmembrane proton gradient generated by cytochrome bo 3 in tethered bilayer lipid membranes using SEIRA spectroscopy. J Phys Chem B 2016, 120:2249–2256. 32. Rodrigo D, Limaj O, Janner D, Etezadi D, Garcia de Abajo FJ, •• Pruneri V, Altug H: Mid-infrared plasmonic biosensing with graphene. Science 2015, 349:165–168. The unique potential of graphene for electrochemical SEIRAS experiments is shown for label-free quantitative detection of protein monolayers. The Fermi level of graphene is tuned by applying a bias voltage while an IR-beam excites a plasmon resonance across the graphene nanoribbons. At the ribbon edge, a significantly enhanced
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42. Papadopoulou E, Goodchild SA, Cleary DW, Weller SA, Gale N, Stubberfield MR, Brown T, Bartlett PN: Using surface-enhanced Raman spectroscopy and electrochemically driven melting to discriminate Yersinia pestis from Y. pseudotuberculosis based on single nucleotide polymorphisms within unpurified polymerase chain reaction amplicons. Anal Chem 2015, 87:1605–1612. 43. Ilkhani H, Hughes T, Li J, Zhong CJ, Hepel M: Nanostructured SERS-electrochemical biosensors for testing of anticancer drug interactions with DNA. Biosens Bioelectron 2016, 80:257–264. 44. Meneghello M, Papadopoulou E, Ugo P, Bartlett PN: Using electrochemical SERS to measure the redox potential of drug molecules bound to dsDNA—a study of mitoxantrone. Electrochim Acta 2016, 187:684–692. 45. Yuan T, Le Thi Ngoc L, Van Nieuwkasteele J, Odijk M, Van Den • Berg A, Permentier H, Bischoff R, Carlen ET: In situ surface-enhanced Raman spectroelectrochemical analysis system with a hemin modified nanostructured gold surface. Anal Chem 2015, 87:2588–2592. The combination of a small volume microfluidic sample chamber with SERS spectroelectrochemical analysis is demonstrated based on a on-chip nanostructured Au WE and micro-fabricated Pt CE, directly bonded to the microfluidic sample chamber with a volume below 100 μL. Shifts in ν(C–C) and ν(C–N) stretching vibrations of the porphyrin ring of hemin were investigated in dependence of the applied potential.
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46. Smith SR, Seenath R, Kulak MR, Lipkowski J: Characterization of a self-assembled monolayer of 1-thio-β-d-glucose with electrochemical surface enhanced Raman spectroscopy using a nanoparticle modified gold electrode. Langmuir 2015, 31:10076–10086. 47. Zhao L, Blackburn J, Brosseau CL: Quantitative detection of uric acid by electrochemical-surface enhanced Raman spectroscopy using a multilayered Au/Ag substrate. Anal Chem 2015, 87:441–447. 48. Ji-Yang, Dong J-C, Kumar VV, Li J-F, Tian Z-Q: Probing electrochemical interfaces using shell-isolated nanoparticles-enhanced Raman spectroscopy. Curr Opin Electrochem 2017, 1:16–21. 49. Li CY, Chen SY, Zheng YL, Chen SP, Panneerselvam R, Chen S, Xu Q-C, Chen Y-X, Yang Z-L, Wu D-Y, Li J-F, Tian Z-Q: In-situ electrochemical shell-isolated Ag nanoparticles-enhanced Raman spectroscopy study of adenine adsorption on smooth Ag electrodes. Electrochim Acta 2016, 199:388–393. 50. Wen B-Y, Jin X, Li Y, Wang Y-H, Li C-Y, Liang M-M, Panneerselvam R, Xu Q-C, Wu D-Y, Yang Z-L, Li J-F, Tian Z-Q: Shell-isolated nanoparticle-enhanced Raman spectroscopy study of the adsorption behaviour of DNA bases on Au(111) electrode surfaces. Analyst 2016, 141:3731–3736. 51. Kurouski D, Mattei M, Van Duyne RP: Probing redox reactions at • the nanoscale with electrochemical tip-enhanced Raman spectroscopy. Nano Lett 2015, 15:7956–7962. This paper shows the first electrochemical TERS study investigating the two electron, 1 proton reduction of Nile Blue (NB) while monitoring the structural changes of NB. The influence of the tip on the local electrochemistry was also investigated. The TERS tip locally perturbs the structure of the electrical double-layer but has no detectable impact on the electron transfer kinetics.
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52. Zeng Z-C, Huang S-C, Wu D-Y, Meng L-Y, Li M-H, Huang T-X, Zhong J-H, Wang X, Yang Z-L, Ren B: Electrochemical tip-enhanced Raman spectroscopy. J Am Chem Soc 2015, 137:11928–11931. 53. Meng L, Yang Z, Chen J, Sun M: Effect of electric field gradient on sub-nanometer spatial resolution of tip-enhanced Raman spectroscopy. Sci Rep 2015, 5:9240. 54. Lo H-C, Hsiung H-I, Chattopadhyay S, Han H-C, Chen C-F, Leu JP, Chen K-H, Chen L-C: Label free sub-picomole level DNA detection with Ag nanoparticle decorated Au nanotip arrays as surface enhanced Raman spectroscopy platform. Biosens Bioelectron 2011, 26:2413–2418. 55. Cowcher DP, Deckert-Gaudig T, Brewster VL, Ashton L, Deckert V, Goodacre R: Detection of protein glycosylation using tip-enhanced Raman scattering. Anal Chem 2016, 88:2105–2112. 56. Sharma G, Deckert-Gaudig T, Deckert V: Tip-enhanced Raman scattering—targeting structure-specific surface characterization for biomedical samples. Adv Drug Deliv Rev 2015, 89:42–56. 57. Khatib O, Wood JD, Mcleod AS, Goldflam MD, Wagner M, •• Damhorst GL, Koepke JC, Doidge GP, et al.: Graphene-based platform for infrared near-field nanospectroscopy of water and biological materials in an aqueous environment. ACS Nano 2015, 9:7968–7975. Infrared scattering-based near-field techniques are highly interesting for studying biomolecules, however cannot be achieved in aqueous environment. Here a special arrangement based on graphene is demonstrated, which allowed imaging of a biomolecule in aqueous environment. Tobacco mosaic virus was trapped together with water underneath a large-area monolayer graphene, which served basically as a cover enabling the investigation of single virion inside the graphene liquid cell.
Current Opinion in Electrochemistry 2017, 5:106–113
Review Article Recent advances in biomolecular vibrational spectroelectrochemistry Ángela I. López-Lorente 1 and Christine Kranz 2,∗ Spectroelectrochemical studies of biomolecules have gained attraction due to the breadth of information that can be derived from combined in situ bioelectrochemistry–spectroelectrochemistry experiments. This current opinion article provides a comprehensive overview focusing on surface-enhanced IR and Raman spectroscopic techniques applied in bioelectrochemical studies published within the past 3 years. Next to fundamental studies also sensing applications will be discussed taking advantage of advanced spectroelectrochemical techniques. Addresses 1 Departamento de Química Analítica, Instituto Universitario de Investigación en Química Fina y Nanoquímica IUIQFN, Universidad de Córdoba, Campus de Rabanales, Edificio Marie Curie Anexo, E-14071 Córdoba, Spain 2 Institute of Analytical and Bioanalytical Chemistry, Ulm University, Ulm, Germany ∗
Corresponding author: Kranz, Christine ([email protected])
biomolecules [1• ] including redox enzymes [2], proteins [3], and membrane components [4]. In particular, midinfrared spectroscopy (MIR; 2.5–20 μm; 4000–500 cm−1 ) and Raman spectroscopy provide a wealth of information on changes in the vibrational signature, i.e., the molecular fingerprint regime. Complementary in nature, both techniques have been applied to investigate electron transfer processes in cytochrome c or metalloenzymes, for in situ studies of microbial biofilms, peptides, cells, and proteins, and for elucidating protein reactions and protein folding/unfolding. Misfolding and aggregation of specific proteins have been extensively studied via IR spectroscopy, as neurogenerative diseases (e.g., Alzheimer, Parkinson, Creutzfeldt–Jakob) are frequently associated with such changes in protein structure [5• ]. In particular, group vibrations of the protein backbone providing information on the secondary structure including the amide I, amide II, and amide III band have been analyzed using IR spectroscopy, as recently summarized [6].
Current Opinion in Electrochemistry 2017, 5:106–113 This review comes from a themed issue on Bioelectrochemistry Edited by Nicolas Plumeré For a complete overview see the Issue and the Editorial Available online 4 August 2017 http://dx.doi.org/10.1016/j.coelec.2017.07.011 2451-9103/© 2017 Elsevier B.V. All rights reserved.
Introduction Spectroelectrochemical experiments are performed across almost the entire range of the electromagnetic spectrum, i.e., from electrochemical NMR experiments to in situ X-ray absorption spectroelectrochemical studies. The major benefit derives from the combination of chemical and electronic information, which is of particular relevance in bioelectrochemistry. Besides sensing applications (e.g., DNA), such studies are focused on characterizing biological systems and complex processes including but not limited to electron–proton transport in cells, membrane potentials of cells, and electrode reactions of proteins and redox enzymes without the need of labeling the biological component. Several reviews have been published on spectroelectrochemical studies of Current Opinion in Electrochemistry 2017, 5:106–113
A disadvantage of these vibrational spectroscopic techniques is the inherently limited absorption in the IR, and the rather small Raman scattering cross section, if the biological component is covalently attached or adsorbed at an electrode surface. For a densely packed protein monolayer assuming an average protein diameter of approx. 5 nm, this yields a maximum surface coverage on the order of 8 pmol cm−2 [1• ]. Consequently, using surface-enhanced infrared absorbance spectroscopy (SEIRAS), surface-enhanced Raman spectroscopy (SERS), and surface-enhanced resonance Raman spectroscopy (SERRS) techniques is almost mandatory for overcoming this limitation. Utilizing rough metal surfaces, deliberately controlled nanostructures [7,8], or nanoantennas [9,10] not only gives rise to enhanced analytical signals via antenna and/or plasmonic effects, but simultaneously provides an interface potentially serving as working electrode during spectroelectrochemical experiments. Hence, thermodynamic and kinetic information obtained via electrochemistry may be directly correlated with vibrational information revealing structural and conformational changes of the biological constituent. In the following, recent progress in bio/spectroelectrochemistry using IR and Raman techniques will be reviewed focusing on the use of surface-enhanced signatures for studying redox processes at proteins, enzymes, and biofilms, and on their application in www.sciencedirect.com
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sensing scenarios. Also, ATR-SEIRA experiments utilizing IR-transparent boron-doped diamond (BDD) waveguides providing a broad spectroscopic and electrochemical window will be highlighted. Surface- and tipenhanced Raman techniques, in combination with electrochemistry, have also advanced for biomolecular studies. In particular, tip-enhanced Raman spectroscopy (TERS) combined with microelectrochemistry may show great promise for nanoscale spectroelectrochemistry in the future. This article will highlight the current state of the art and future potential of bio/spectroelectrochemistry.
IR-transparent materials for spectroelectrochemistry Besides the limited inherent sensitivity, infrared absorption methods are affected by pronounced water absorptions during in situ studies. In particular, strong bending vibrations around 1640 cm−1 convolute the protein amide I band, while the O–H stretching mode around 3000 cm−1 masks N–H stretching vibrations of amines. IR-ATR spectroscopy on biomolecules attached or adsorbed at an ATR crystal surface ensures a limited penetration depth of the evanescent field into the adjacent medium, thereby minimizing the water interference. Also water may be replaced by deuterium oxide (D2 O) shifting the solvent bands to lower wavenumbers, which avoids masking bands in the amide I region. Typically, IR-transparent materials (e.g., Ge, ZnSe, ZnS, Si, diamond) are insulators or semiconductors, and hence, are not suitable as electrodes. Thin films of BDD with typically doping levels of 1019 –1021 atoms cm−3 are transparent in the MIR with the exception of two-phonon (around 2670 cm−1 and 1744 cm−1 ), have low resistivity, and large potential window. Surface enhancement effects via plasmonic resonances have been achieved using stabilizer-free gold nanoparticles (AuNPs) modifying such IR-transparent BDD-coated diamond ATR crystals [11]. Using these waveguides, combined IR-ATR, electrochemical, and atomic force microscopy (AFM) studies on bovine serum protein films have been demonstrated as shown in Figure 1A [12• ].
SEIRA spectroelectrochemistry Non-conductive ATR crystals have been modified with thin metal layers leading to plasmonic resonances based on strong light–matter interactions at sub-wavelength localization, a.k.a., ATR-SEIRAS [15]. This strategy has been used for in situ spectroscopic [9] and spectroelectrochemical studies on biomolecules [16]. However, it should be noted that the signal enhancement, which depends on the roughness of the SEIRA substrate rapidly decays at a length scale on the order of the average size of protein molecules. Consequently, not all structures of a protein may equally contribute to the obtained SEIRA spectra. www.sciencedirect.com
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Nevertheless, SEIRAS has been applied for biosensing [17–19], in protein assays [20], and for studying conformational changes of proteins [12,21,22] such as redoxrelated processes at electrochemical interfaces [16]. Spectroelectrochemical studies using ATR-SEIRAS at thin gold films have been applied to investigate electron transfer processes at redox active molecules, e.g., conformational changes of cytochrome c (cyt c) oxidase induced by direct electron transfer [23,24] as well as dynamics of charge–transfer and protein orientation of immobilized cyt c on a gold electrode [25]. Recently, the kinetics of cyt c oxidase derived from Rhodobacter sphaeroides were investigated via time-resolved SEIRAS using step-scan spectroscopy applying periodic potential pulses between −800 mV and open circuit potential [26]. Redox changes during the electron transfer of the active centers embedded within the protein secondary structure were investigated via changes in the amide I band at anaerobic and aerobic conditions. Protein film infrared electrochemistry has been demonstrated with enzymes adsorbed on carbon particle electrodes, which were deposited onto the surface of an Si ATR crystal to study the catalytic turnover of H2 oxidation by E. coli hydrogenase 1 (Hyd-1) [13•• ] (see Figure 1B). Relevant in apoptosis, interactions of cyt c with cardiolipin-containing lipid bilayers have been studied on supported lipid bilayers at 1-dodecanethiol functionalized gold electrode using cyclic voltammetry (CV) and SEIRAS measurements [27]. The effect of SiO2 NPs on the electron transfer behavior of cyt c adsorbed onto 11-mercaptoundecanoic acid was also investigated [28]. The presence of nanoparticles modulates the heme microenvironment, and its orientation during adsorption, thereby leading to enhanced electron transfer in dependence on the applied potential. A real-time infrared plasmonic biosensor was demonstrated for chemicalspecific detection of biomimetic lipid membranes using surface-modified Au nanoantennas as shown in Figure 1C [14• ]. The formation kinetics of planar biomimetic membranes in aqueous environments has been monitored in real time using the vibrational fingerprints of the lipid molecules. The effect of the membrane electric field on the KcsA potassium channel has been investigated using voltammetric SEIRA studies [29], which also enabled the characterization of membrane-bound hydrogenase co-immobilized with a phospholipid bilayer on a gold electrode [30]. Moreover, SEIRAS combined with electrical impedance spectroscopy allowed studying the transmembrane proton gradient in bilayer lipid membranes produced by cyt bo3 [31]. Graphene has revealed a significant potential for electrochemical SEIRAS experiments, and also for the label-free quantitative detection of protein monolayers (Figure 2) [32•• ]. A bias voltage was varied to control the Fermi level of graphene, while an IR-beam excited a plasmon resoCurrent Opinion in Electrochemistry 2017, 5:106–113
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Figure 1
A: Schematic setup for combined AFM-IR-EC measurements. a) IR absorption signals and average integral values (N = 4), corresponding to the amide I and II bands from 1.0 (gray), 2.5 (red) and 5.0 (blue) mg mL−1 BSA solution. Dashed lines in a) and dashed filling in b) reflect the data obtained at the non-modified BDD crystal. Adapted with permission from [12• ]. B: Diagram of the working-electrode configuration and solution flow (blue arrows) over the multi-bounce Si IRE. a) Current–time traces of Hyd-1 using the ATR-IR cell in Ar-saturated (gray) and H2 -saturated (black) buffer; b), c) spectra showing the nCO region at each potential under Ar (b) and H2 (c). Adapted with permission from [13•• ]. C Schematic illustration showing the fluidic chamber and experimental configuration for detection of SLB using Au nanoantennas. a) Reflectance within the lipid fingerprint region as a function of time during lipid vesicle adsorption and rupture on a nanoantenna array with L = 1.00 μm, P = 1.37 μm, and 15 nm thick SiO2 layer and the corresponding absorbance spectra. The vertical dashed lines mark the spectra position of the symmetric (blue) and asymmetric (red) CH2 stretching modes, schematically represented in the inset figure. Integrated absorbance over CH2 bands as a function of time for nanoantenna modified with b) 15 nm thick SiO2 ; c) modified with hydrophilic 16-mercapto hexadecanoic acid (MHDA), and d) 15 nm thick Al2 O3 layer. Adapted with permission from [14• ].
nance across graphene nanoribbons. At the ribbon edge, a significantly enhanced light interaction with the adsorbed protein molecules was observed.
SERS/SERRS spectroelectrochemistry Surface-enhanced Raman techniques including SERS, SERRS, and TERS are associated with local field enhancements at metal nanostructures as well as chemical enhancement effects. Enhancement effects are dependent on the shape and size of the nanostructure, Current Opinion in Electrochemistry 2017, 5:106–113
but also on the plasmon coupling of nanoparticle aggregates. A wide variety of electrochemical SERS studies have demonstrated the potential of this technique using, e.g., Nile Blue as a model analyte [33–35]. SERS has also been applied for spectroelectrochemical studies of cyt c, and electrochemical SERRS experiments have been used to study the nature of the biofilm–electrode interaction of cyt OmcB from Desulfuromonas acetoxidans microbial biofilms, which causes the wiring of the cell metabolism to the electrode [36]. Also, the effect of oxywww.sciencedirect.com
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Figure 2
Tunable graphene-based SEIRAS protein sensor. A: Schematic view of the IR biosensor device. An IR-beam excites a plasmon resonance across the graphene nanoribbons, enhancing light interaction with the protein molecules. The plasmonic resonance is electrostatically tuned to sweep continuously over the protein vibrational bands. B: Extinction spectra of the graphene nanoribbon array (width W = 30 nm, period P = 80 nm) for bias voltages Vg from –20 V to –130 V before (dashed curves) and after (solid curves) protein bilayer formation. Extinction is calculated as the relative difference in transmission between regions with and without graphene nanoribbons. Gray vertical strips indicate amide I and II bands of the protein. Adapted with permission from [32•• ].
gen on the electroactivity of microbial biofilms has been investigated [37]. Recently, time-resolved investigations on the structural evolution of surface species equaling the temporal resolution of transient electrochemical methods have been shown via transient electrochemical SERS (TEC-SERS) [38• ]. SERRS and SEIRAS combined with electrochemistry have allowed the study of the calciuminduced reorientation of cellobiose dehydrogenase immobilized on electrodes and the effect on the oxidation of carbohydrates [39]. SERS and SERRS have been also used in sensing applications, e.g., for detecting DNA [40,41]. An ECSERS-based DNA aptasensor has been described for the detection of Mycobacterium tuberculosis DNA using a AgNP-modified screen-printed electrodes following SERS bands of adenine under potential control [41]. Electrochemically driven denaturation assays monitored by SERS were able to discriminate DNA amplicons genwww.sciencedirect.com
erated from pathogenic Yersinia pestis bacteria [42]. Also, EC-SERS biosensors have been designed for analyzing DNA interactions with anticancer drugs such as doxorubicin [43], and mitoxantrone [44]. Yuan et al. [45• ] reported an electrochemical SERS study of a metalloporphyrin hemin system using a microfluidic chamber (Figure 3) via a nanostructured Au surface. Shifts in ν(C–C) and ν(C–N) stretching vibrations of the porphyrin ring in the range of 1300–1700 cm−1 are typically associated with the oxidation state of iron. In the frequency range of 1368–1377 cm−1 , the ν 4 mode is observed for ferric (Fe3+ ) hemin, and in the range of 1344– 1364 cm−1 for ferrous (Fe2+ ) hemin. At a potential of −0.2 V vs. Ag/AgCl, two bands were resolved at 1345 and 1366 cm−1 , which were associated with the partial oxidation of the iron cores. In contrast, at −0.5 V only one ν 4 band at approx. 1349 cm−1 is observed, which is indicative for the reduction of the iron cores. Current Opinion in Electrochemistry 2017, 5:106–113
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Figure 3
In situ SERS SEC set-up. A: Schematic representation of the in situ SERS spectroelectrochemical (SEC) setup with microfluidic chamber. B: Schematic of the MPy/hemin modified Au surface. C: SERS spectra from MPy/hemin modified nanostructured Au WE in the marker band region (background corrected). (a) Electrode potential E = −0.2 V. (b) Electrode potential E = −0.5 V. Raw data: blue traces; modeled data: red traces. Adapted with permission from [45• ].
EC-SERS using AuNP-modified gold electrodes enabled the investigation of the adsorption of 1-thio-β-d-glucose onto the AuNPs, and the extent of oxidation of the monolayer as a function of the applied electrode potential [46]. Likewise, the detection of uric acid as biomarker of preeclampsia using an Au/Ag substrate has been shown [47]. Electrochemical shell-isolated nanoparticle-enhanced Raman spectroscopy (EC-SHINERS) has been applied for electrochemical interface studies on catalytic processes, molecule adsorption, etc. [48]. This technique has also been applied to study the electrochemical behavior of adenine nucleobases [49], as well as for investigating the adsorption of four DNA bases at Au electrodes [50].
TERS spectroelectrochemistry More recently, TERS has been combined with electrochemical studies [51• ,52], yet to date has not been applied for investigating biomolecules. Current Opinion in Electrochemistry 2017, 5:106–113
TERS combines superior sensitivity and molecular selectivity of enhanced Raman signals with the nanoscale spatial resolution of scanning probe microscopy (SPM) techniques such as AFM or scanning tunneling microscopy (STM). TERS at a sharp metal tip (e.g., Au, Ag) may achieve sub-nanometer spatial resolution [53], and has been applied in biochemical studies and in surface science. To date, TERS has predominantly been used for biomolecule detection schemes including targets such as DNA [54], proteins [55], and bacteria/viruses [56] taking advantage of the pronounced sensitivity. (Nano)electrochemical TERS studies are still rare. Analogous to EC-SERS, Nile Blue has served as model analyte to explore the potential of electrochemical TERS (ECTERS) [51• ]. It has been also used to monitor arrangements of 4 -(pyridin-4-yl)biphenyl-4-yl) methanethiol (4PBT) at Au surfaces [52]. While bioelectrochemical TERS experiments have not yet been reported, it may be expected that EC-TERS applications in the field of biomolecular detection will be emerging within few years. www.sciencedirect.com
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Concluding remarks Spectroelectrochemical investigations of biomolecules using surface-enhanced IR and Raman spectroscopic techniques have significantly contributed advancing the understanding on molecular orientation at electrified interfaces, and on electron transfer processes involving complex biomolecules. Signal-enhanced techniques readily support the development of sensitive label-free biosensors, which has been demonstrated, e.g., for DNA sensing. Future directions in bio/spectroelectrochemical studies are clearly targeted toward improving the spatial and temporal resolution (a.k.a., high-resolution imaging), which may lead to obtaining dynamic interfacial in formation at the single molecule level. Although in its infancy, EC-TERS studies on biomolecules may enable understanding structure-related electrochemical activity and surface chemistry in molecular detail. The IR-equivalent to TERS, termed nano-IR or scanning nearfield infrared microscopy (SNIM) has been successfully applied to characterize biomolecules. However, tipenhanced IR-techniques suffer from limited applicability in aqueous solutions, which are usually demanded in bio/spectroelectrochemistry. Recently, an IR-compatible liquid cell architecture has been demonstrated using large-area graphene sheets, which act as an impermeable monolayer barrier facilitating the investigation of tobacco mosaic virus in water trapped underneath graphene [57•• ].
Acknowledgment A.I. López-Lorente thanks the Alexander von Humboldt Foundation for the award of a Postdoctoral Fellowship.
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: •
Paper of special interest. Paper of outstanding interest.
••
1.
Ash PA, Vincent KA: Vibrational spectroscopic techniques for probing bioelectrochemical systems. Adv Biochem Eng/Biotechnol 2017, 158:75–110. This recent review gives a comprehensive overview on spectroelectrochemistry of biomolecules using enhanced IR and Raman techniques such as SEIRAS, SERS, SERRS. •
2.
Ash PA, Vincent KA: Spectroscopic analysis of immobilised redox enzymes under direct electrochemical control. Chem Commun 2012, 48:1400–1409.
3.
Murgida DH, Hildebrandt P: Disentangling interfacial redox processes of proteins by SERR spectroscopy. Chem Soc Rev 2008, 37:937–945.
4.
Melin F, Hellwig P: Recent advances in the electrochemistry and spectroelectrochemistry of membrane proteins. Biol Chem 2013, 394:593–609.
5.
Nabers A, Ollesch J, Schartner J, Kötting C, Genius J, Hafermann H, Klafki H, Gerwert K, Wiltfang J: Amyloid-β-secondary structure distribution in cerebrospinal fluid and blood measured by an immuno-infrared-sensor: a biomarker candidate for Alzheimer’s disease. Anal Chem 2016, 88:2755–2762.
•
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This paper demonstrates the potential of IR spectroscopy for a label-free diagnostic tool detecting Dementia Alzheimer type (DAT) using the downshift of the amide I band frequency of Aβ-peptides in cerebrospinal fluid and blood plasma. 6.
López-Lorente AI, Mizaikoff B: Mid-infrared spectroscopy for protein analysis: potential and challenges. Anal Bioanal Chem 2016, 408:2875–2889.
7.
Bedford EE, Boujday S, Pradier C-M, Gu FX: Nanostructured and spiky gold in biomolecule detection: improving binding efficiencies and enhancing optical signals. RSC Adv 2015, 5:16461–16475.
8.
Bibikova O, Haas J, López-Lorente AI, Popov A, Kinnunen M, Meglinski I, Mizaikoff B: Towards enhanced optical sensor performance: SEIRA and SERS with plasmonic nanostars. Analyst 2017, 142:951–958.
9.
Adato R, Altug H: In-situ ultra-sensitive infrared absorption spectroscopy of biomolecule interactions in real time with plasmonic nanoantennas. Nat Commun 2013, 4:1–10.
10. Neubrech F, Huck C, Weber K, Pucci A, Giessen H: Surface-enhanced infrared spectroscopy using resonant nanoantennas. Chem Rev 2017, 117:5110–5145. 11. Izquierdo J, Mizaikoff B, Kranz C: Surface-enhanced infrared spectroscopy on boron-doped diamond modified with gold nanoparticles for spectroelectrochemical analysis. Phys Status Solidi A 2016, 213:2056–2062. 12. López-Lorente AI, Izquierdo J, Kranz C, Mizaikoff B: Boron-doped • diamond modified with gold nanoparticles for the characterization of bovine serum albumin protein. Vib Spectrosc 2017, 91:147–156. The paper demonstrates the first combined SEIRA-ATR-EC-AFM multi-analytical platform for the characterization of BSA protein films using spectroscopic, electrochemical methods and AFM imaging. The BDD surface modified with AuNPs via a stainless steel assisted method served as conductive SEIRAS substrate. Globular and fibril aggregates of BSA could be discriminated using curve fitting of the amide I band showing major contributions of crossed β-sheet structures within the fibril aggregates. 13. Hidalgo R, Ash PA, Healy AJ, Vincent KA: Infrared spectroscopy •• during electrocatalytic turnover reveals the Ni–L active site state during H2 oxidation by a NiFe hydrogenase. Angew Chem Int Ed 2015, 54:7110–7113. Evidence is given in these spectroelectrochemical ATR-IR experiments that the fast catalytic turnover of hydrogen oxidation by E. coli hydrogenase 1 (Hyd-1) involves sequential proton and electron transfer via an NiIFeII active site state termed Ni–L. Experiments were performed using a high surface-area carbon electrode deposited onto a Si internal reflection element under Ar and H2 atmosphere. The spectra reveal that the active site state Ni–L, observed in other NiFe hydrogenases only under illumination or at cryogenic temperatures, can be generated reversibly in the dark at ambient temperature under both turnover and non-turnover conditions. As Ni–L is present at all potentials during turnover under H2 , it is concluded that the final steps in the catalytic cycle of H2 oxidation involve sequential proton and electron transfer via Ni–L. 14. Limaj O, Etezadi D, Wittenberg NJ, Rodrigo D, Yoo D, Oh S, • Altug H: Infrared plasmonic biosensor for real-time and label-free monitoring of lipid membranes. ACS Nano 2016, 16:1502–1508. This paper describes the near-field enhancement using engineered and surface-modified Au nanoantennas for real-time and label-free detection of biomimetic lipid bilayers in aqueous solution. Using the vibrational fingerprints of lipid molecules, the real time formation kinetics of planar biomimetic membranes could be monitored. 15. Nedelkovski V, Schwaighofer A, Wraight CA, Nowak C, Naumann RLC: Surface-enhanced infrared absorption spectroscopy (SEIRAS) of light-activated photosynthetic reaction centers from Rhodobacter sphaeroides reconstituted in a biomimetic membrane system. J Phys Chem C 2013, 117:16357–16363. 16. Murgida DH, Hildebrandt P: Redox and redox-coupled processes of heme proteins and enzymes at electrochemical interfaces. Phys Chem Chem Phys 2005, 7:3773–3784.
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17. Boujday S, de la Chapelle ML, Srajer J, Knoll W: Enhanced vibrational spectroscopies as tools for small molecule biosensing. Sensors 2015, 15:21239–21264. 18. Hornemann A, Eichert D, Flemig S, Ulm G, Beckhoff B: Qualifying label components for effective biosensing using advanced high-throughput SEIRA methodology. Phys Chem Chem Phys 2015, 17:9471–9479. 19. Leidner L, Stäb J, Adam JT, Gauglitz G: Surface-enhanced infrared absorption studies towards a new optical biosensor. Beilstein J Nanotechnol 2016, 7:1736–1742. 20. López-Lorente AI, Wang P, Mizaikoff B: Towards label-free mid-infrared protein assays: in-situ formation of bare gold nanoparticles for surface enhanced infrared absorption spectroscopy of bovine serum albumin. Microchim Acta 2017, 184:453–462. 21. Fallah MA, Stanglmair C, Pacholski C, Hauser K: Devising self-assembled-monolayers for surface-enhanced infrared spectroscopy of pH-driven poly-l-lysine conformational changes. Langmuir 2016, 32:7356–7364. 22. Zeng L, Wu L, Liu L, Jiang X: Analyzing structural properties of heterogeneous cardiolipin-bound cytochrome c and their regulation by surface-enhanced infrared absorption spectroscopy. Anal Chem 2016, 88:11727–11733. 23. Ataka K, Heberle J: Use of surface enhanced infrared absorption spectroscopy (SEIRA) to probe the functionality of a protein monolayer. Biopolymers 2006, 82:415–419. 24. Nowak C, Laredo T, Gebert J, Lipkowski J, Gennis RB, Ferguson-Miller S, Knoll W, Naumann RLC: 2D-SEIRA spectroscopy to highlight conformational changes of the cytochrome c oxidase induced by direct electron transfer. Metallomics 2011, 3:619–627.
light interaction with the adsorbed protein molecules could be observed. 33. Wilson AJ, Molina NY, Willets KA: Modification of the electrochemical properties of Nile Blue through covalent attachment to gold as revealed by electrochemistry and SERS. J Phys Chem C 2016, 120:21091–21098. 34. Weber ML, Wilson AJ, Willets KA: Characterizing the spatial dependence of redox chemistry on plasmonic nanoparticle electrodes using correlated super-resolution surface-enhanced Raman scattering imaging and electron microscopy. J Phys Chem C 2015, 119:18591–18601. 35. Wilson AJ, Willets KA: Unforeseen distance-dependent SERS spectroelectrochemistry from surface-tethered Nile Blue: the role of molecular orientation. Analyst 2016, 141:5144–5151. 36. Alves A, Ly HK, Hildebrandt P, Louro RO, Millo D: Nature of the surface-exposed cytochrome−electrode interactions in electroactive biofilms of Desulfuromonas acetoxidans. J Phys Chem B 2015, 119:7968–7974. 37. Millo D, Ly HK: Towards the understanding of the effect of oxygen on the electrocatalytic activity of microbial biofilms: a spectroelectrochemical study. RSC Adv. 2015, 5:92042–92044. 38. Zong C, Chen CJ, Zhang M, Wu DY, Ren B: Transient • electrochemical surface-enhanced Raman spectroscopy: a millisecond time-resolved study of an electrochemical redox process. J Am Chem Soc 2015, 137:11768–11774. This paper demonstrates transient electrochemical surface-enhanced Raman spectroscopy (TEC-SERS) to study the structural evolution of surface species at a time resolution matching the electrochemical cyclic voltammetric and chronoamperometric experiments. With this approach the Raman signal providing the molecular signature information could be precisely correlated with the electrochemical current signal.
25. Jin B, Wang G, Millo D, Hildebrandt P, Xia X: Electric-field control of the pH-dependent redox process of cytochrome c immobilized on a gold electrode. J Phys Chem C 2012, 116:13038–13044.
39. Kielb P, Sezer M, Katz S, Lopez F, Schulz C, Gorton L, et al.: Spectroscopic observation of calcium-induced reorientation of cellobiose dehydrogenase immobilized on electrodes and its effect on electrocatalytic activity. ChemPhysChem 2015, 16:1960–1968.
26. Steininger C, Reiner-Rozman C, Schwaighofer A, Knoll W, Naumann RLC: Kinetics of cytochrome c oxidase from R. sphaeroides initiated by direct electron transfer followed by tr-SEIRAS. Bioelectrochemistry 2016, 112:1–8.
40. Johnson RP, Richardson JA, Brown T, Bartlett PN: A label-free, electrochemical SERS-based assay for detection of DNA hybridization and discrimination of mutations. J Am Chem Soc 2012, 134:14099–14107.
27. Liu L, Zeng L, Wu L, Jiang X: Label-free surface-enhanced infrared spectroelectrochemistry studies the interaction of cytochrome c with cardiolipin-containing membranes. J Phys Chem C 2015, 119:3990–3999.
41. Karaballi RA, Nel A, Krishnan S, Blackburn J, Brosseau CL: Development of an electrochemical surface-enhanced Raman spectroscopy (EC-SERS) aptasensor for direct detection of DNA hybridization. Phys Chem Chem Phys 2015, 17:21356–21363.
28. Zhang X, Zeng L, Liu L, Ma Z, Jiang X: Surface-enhanced infrared absorption spectroscopy and electrochemistry reveal the impact of nanoparticles on the function of protein immobilized on mimic biointerface. Electrochim Acta 2016, 211:148–155. 29. Yamakata A, Shimizu H, Oiki S: Surface-enhanced IR absorption spectroscopy of the KcsA potassium channel upon application of an electric field. Phys Chem Chem Phys 2015, 17:21104–21111. 30. Gutiérrez-Sanz O, Marques M, Pereira IAC, De Lacey AL, Lubitz W, Rüdiger O: Orientation and function of a membrane-bound enzyme monitored by electrochemical surface-enhanced infrared absorption spectroscopy. J Phys Chem Lett 2013, 4:2794–2798. 31. Wiebalck S, Kozuch J, Forbrig E, Tzschucke CC, Jeuken LJC, Hildebrandt P: Monitoring the transmembrane proton gradient generated by cytochrome bo 3 in tethered bilayer lipid membranes using SEIRA spectroscopy. J Phys Chem B 2016, 120:2249–2256. 32. Rodrigo D, Limaj O, Janner D, Etezadi D, Garcia de Abajo FJ, •• Pruneri V, Altug H: Mid-infrared plasmonic biosensing with graphene. Science 2015, 349:165–168. The unique potential of graphene for electrochemical SEIRAS experiments is shown for label-free quantitative detection of protein monolayers. The Fermi level of graphene is tuned by applying a bias voltage while an IR-beam excites a plasmon resonance across the graphene nanoribbons. At the ribbon edge, a significantly enhanced
Current Opinion in Electrochemistry 2017, 5:106–113
42. Papadopoulou E, Goodchild SA, Cleary DW, Weller SA, Gale N, Stubberfield MR, Brown T, Bartlett PN: Using surface-enhanced Raman spectroscopy and electrochemically driven melting to discriminate Yersinia pestis from Y. pseudotuberculosis based on single nucleotide polymorphisms within unpurified polymerase chain reaction amplicons. Anal Chem 2015, 87:1605–1612. 43. Ilkhani H, Hughes T, Li J, Zhong CJ, Hepel M: Nanostructured SERS-electrochemical biosensors for testing of anticancer drug interactions with DNA. Biosens Bioelectron 2016, 80:257–264. 44. Meneghello M, Papadopoulou E, Ugo P, Bartlett PN: Using electrochemical SERS to measure the redox potential of drug molecules bound to dsDNA—a study of mitoxantrone. Electrochim Acta 2016, 187:684–692. 45. Yuan T, Le Thi Ngoc L, Van Nieuwkasteele J, Odijk M, Van Den • Berg A, Permentier H, Bischoff R, Carlen ET: In situ surface-enhanced Raman spectroelectrochemical analysis system with a hemin modified nanostructured gold surface. Anal Chem 2015, 87:2588–2592. The combination of a small volume microfluidic sample chamber with SERS spectroelectrochemical analysis is demonstrated based on a on-chip nanostructured Au WE and micro-fabricated Pt CE, directly bonded to the microfluidic sample chamber with a volume below 100 μL. Shifts in ν(C–C) and ν(C–N) stretching vibrations of the porphyrin ring of hemin were investigated in dependence of the applied potential.
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Recent advances in biomolecular vibrational spectroelectrochemistry López-Lorente and Kranz
46. Smith SR, Seenath R, Kulak MR, Lipkowski J: Characterization of a self-assembled monolayer of 1-thio-β-d-glucose with electrochemical surface enhanced Raman spectroscopy using a nanoparticle modified gold electrode. Langmuir 2015, 31:10076–10086. 47. Zhao L, Blackburn J, Brosseau CL: Quantitative detection of uric acid by electrochemical-surface enhanced Raman spectroscopy using a multilayered Au/Ag substrate. Anal Chem 2015, 87:441–447. 48. Ji-Yang, Dong J-C, Kumar VV, Li J-F, Tian Z-Q: Probing electrochemical interfaces using shell-isolated nanoparticles-enhanced Raman spectroscopy. Curr Opin Electrochem 2017, 1:16–21. 49. Li CY, Chen SY, Zheng YL, Chen SP, Panneerselvam R, Chen S, Xu Q-C, Chen Y-X, Yang Z-L, Wu D-Y, Li J-F, Tian Z-Q: In-situ electrochemical shell-isolated Ag nanoparticles-enhanced Raman spectroscopy study of adenine adsorption on smooth Ag electrodes. Electrochim Acta 2016, 199:388–393. 50. Wen B-Y, Jin X, Li Y, Wang Y-H, Li C-Y, Liang M-M, Panneerselvam R, Xu Q-C, Wu D-Y, Yang Z-L, Li J-F, Tian Z-Q: Shell-isolated nanoparticle-enhanced Raman spectroscopy study of the adsorption behaviour of DNA bases on Au(111) electrode surfaces. Analyst 2016, 141:3731–3736. 51. Kurouski D, Mattei M, Van Duyne RP: Probing redox reactions at • the nanoscale with electrochemical tip-enhanced Raman spectroscopy. Nano Lett 2015, 15:7956–7962. This paper shows the first electrochemical TERS study investigating the two electron, 1 proton reduction of Nile Blue (NB) while monitoring the structural changes of NB. The influence of the tip on the local electrochemistry was also investigated. The TERS tip locally perturbs the structure of the electrical double-layer but has no detectable impact on the electron transfer kinetics.
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52. Zeng Z-C, Huang S-C, Wu D-Y, Meng L-Y, Li M-H, Huang T-X, Zhong J-H, Wang X, Yang Z-L, Ren B: Electrochemical tip-enhanced Raman spectroscopy. J Am Chem Soc 2015, 137:11928–11931. 53. Meng L, Yang Z, Chen J, Sun M: Effect of electric field gradient on sub-nanometer spatial resolution of tip-enhanced Raman spectroscopy. Sci Rep 2015, 5:9240. 54. Lo H-C, Hsiung H-I, Chattopadhyay S, Han H-C, Chen C-F, Leu JP, Chen K-H, Chen L-C: Label free sub-picomole level DNA detection with Ag nanoparticle decorated Au nanotip arrays as surface enhanced Raman spectroscopy platform. Biosens Bioelectron 2011, 26:2413–2418. 55. Cowcher DP, Deckert-Gaudig T, Brewster VL, Ashton L, Deckert V, Goodacre R: Detection of protein glycosylation using tip-enhanced Raman scattering. Anal Chem 2016, 88:2105–2112. 56. Sharma G, Deckert-Gaudig T, Deckert V: Tip-enhanced Raman scattering—targeting structure-specific surface characterization for biomedical samples. Adv Drug Deliv Rev 2015, 89:42–56. 57. Khatib O, Wood JD, Mcleod AS, Goldflam MD, Wagner M, •• Damhorst GL, Koepke JC, Doidge GP, et al.: Graphene-based platform for infrared near-field nanospectroscopy of water and biological materials in an aqueous environment. ACS Nano 2015, 9:7968–7975. Infrared scattering-based near-field techniques are highly interesting for studying biomolecules, however cannot be achieved in aqueous environment. Here a special arrangement based on graphene is demonstrated, which allowed imaging of a biomolecule in aqueous environment. Tobacco mosaic virus was trapped together with water underneath a large-area monolayer graphene, which served basically as a cover enabling the investigation of single virion inside the graphene liquid cell.
Current Opinion in Electrochemistry 2017, 5:106–113