Raman and surface‐enhanced Raman studies of α‐aminophosphinic inhibitors of metalloenzymes

Here, we report the nature of new di‐α‐amino (L¹–L³) and α‐amino‐α‐hydroxyphosphinic (L⁴–L⁶) acids, which are considered potential inhibitors of the aminopeptidase N, adsorbed on a colloidal silver surface by means of surface‐enhanced Raman scattering (SERS) spectroscopy. In order to reveal the adsorption mechanism of these species from their SERS spectra, Fourier‐transform Raman (FT‐RS) spectra of these nonadsorbed molecules were measured. By examining the enhancement, shift in wavenumbers, and changes in breadth of the SERS bands due to the adsorption process, we revealed that the tilted compounds interact with the colloidal silver substrate mainly through the benzene ring, amino group, and phosphinic moiety in the following way. The benzene ring of L² and L³ is ‘standing up’ on the colloidal silver surface, and the C N bond is almost vertical to it, while the tilt angle between the O PO bond and this surface is greater than 45°. On the other hand, for L¹, L⁴, and L⁵, the aromatic ring and C N bond are arranged more or less tilted, and the tilt angle between the O PO bond and the silver substrate is smaller than 45°. The elongation of the bond to the benzene ring, the L⁶ case, produces an almost horizontal orientation of the benzene ring and the O PO bond on the silver nanoparticles. For these ligands, the complement inhibition IC₅₀ tested in vitro using porcine kidney leucine aminopeptidase was correlated mainly with the behavior of the O PO and C CH N fragments on the silver surface. Copyright © 2009 John Wiley & Sons, Ltd.     

Adsorption of 4,4′‐thiobisbenzenethiol on silver surfaces: surface‐enhanced Raman scattering study

Adsorption of 4,4′‐thiobisbenzenethiol (4,4′‐TBBT) on a colloidal silver surface and a roughened silver electrode surface was investigated by means of surface‐enhanced Raman scattering (SERS) for the first time, which indicates that 4,4′‐TBBT is chemisorbed on the colloidal silver surface as dithiolates by losing two H‐atoms of the S H bond, while as monothiolates on the roughened silver electrode. The different orientations of the molecules on both silver surfaces indicate the different adsorption behaviors of 4,4′‐TBBT in the two systems. It is inferred from the SERS signal that the two aromatic rings in 4,4′‐TBBT molecule are parallel to the colloidal silver surface as seen from the disappearance of ν_{C H} band (3054 cm⁻¹), which is a vibrational mode to be used to determine the orientation of a molecule on metals according to the surface selection rule, while on the roughened silver electrode surface they are tilted to the surface as seen from the enhanced signal of ν_{C H}. The orientation of the C‐S bond is tilted with respect to the silver surface in both cases as inferred from the strong enhancement of the ν_{C S}. SERS spectra of 4,4′‐TBBT on the roughened silver electrode with different applied potentials reveal that the enhancement of 4,4′‐TBBT on the roughened silver electrode surface may be related to the chemical mechanism (CM). More importantly, the adsorption of 4,4′‐TBBT on the silver electrode is expected to be useful to covalently adsorb metal nanoparticles through the free S H bond to form two‐ or three‐ dimensional nanostructures. Copyright © 2007 John Wiley & Sons, Ltd.     

Electrochemical and in situ SERS spectroelectrochemical investigations of 4‐methyl‐4H‐1, 2, 4‐triazole‐3‐thiol monolayers at a silver electrode

Electrochemically anticorrosive behavior of 4‐methyl‐4H‐1, 2, 4‐triazole‐3‐thiol (MTTL) self‐assembled monolayers (SAMs) on the silver electrode was studied by means of electrochemical impedance spectroscopy (EIS) and polarization measurements. The promising inhibition effect of the MTTL for silver had been affirmed. Results of surface‐enhanced Raman scattering (SERS) experiments indicated that the MTTL molecule in a tilted orientation was self‐assembled on the silver surface through S⁶ and N² atoms to form monolayers. An in situ electrochemical SERS experiment implied the changes of adsorption fashion of MTTL momolayers on the silver surface with the potential shifted to more negative direction. Copyright © 2009 John Wiley & Sons, Ltd.     

Experimental and theoretical Raman and surface‐enhanced Raman scattering study of cysteine

Raman spectra in solid and 1 M solution of L ‐cysteine and surface‐enhanced Raman scattering (SERS) spectra of this molecule in the zwitterionic form, by using colloidal silver nanoparticles, have been recorded. Density functional theory with the B3LYP functional was used for the optimizations of the ground state geometries and simulation of the vibrational spectrum of this amino acid. The SERS spectrum with a large silver cluster as a model metallic surface was simulated for the first time. Taking into account the experimental and calculated Raman and SERS vibrations and the corresponding assignments, as well as a comparison of force constants and geometrical parameters between the free zwitterion cysteine and the one in the presence of the colloidal silver nanoparticles, we can confirm the presence of gauche (P_H) and trans (P_N) rotamers in the solid state, the formation of a S S bond in the solution state, the dissociation of the peptide bond and mixing of rotamers because of the SERS effect, and the relative importance of the interaction of sulphyldryl, NH₃⁺, and carboxylate groups with the metallic surface. Copyright © 2009 John Wiley & Sons, Ltd.     

Vibrational spectroscopic studies and computational study of methyl(2‐methyl‐4,6–dinitrophenylsulfanyl)ethanoate

FT‐IR and FT‐Raman spectra of methyl(2‐methyl‐4,6–dinitrophenylsulfanyl)ethanoate (MDIE) were recorded and analyzed. Surface‐enhanced Raman scattering (SERS) spectra were recorded in silver colloid and silver electrode. The vibrational wavenumbers were computed using HF/6‐31G* and B3LYP/6‐31G* basis. The data obtained from vibrational wavenumber calculations are used to assign vibrational bands obtained in infrared and Raman spectroscopies as well as in SERS of the studied molecule. The first hyperpolarizability and infrared intensities are reported. The geometrical parameters of the title compound are in agreement with the reported similar derivatives. The presence of new bands at 1045 and 948 cm⁻¹ in the SERS spectrum in silver electrode is related to the change in orientation of the molecule with respect to the metal surface. In silver colloid SERS spectrum, the methyl group attached to the methoxy carbonyl group is close to the metal surface, whereas on silver electrode the methyl group attached to the phenyl ring is close to the metal surface. Copyright © 2009 John Wiley & Sons, Ltd.     

Adsorbed states of substituted α‐aminophosphinic acids on a silver electrode surface: comparison with a colloidal silver substrate

We investigated the interfacial structures of various aromatic (each compound contains one or two phenyls) di‐α‐amino ( L¹ – L³ ) and α‐amino‐α‐hydroxyphosphinic ( L⁴ – L⁶ ) acids immobilized onto an electrochemically roughened silver electrode surface in an aqueous solution using surface‐enhanced Raman scattering (SERS). These structures were compared to those on a colloidal silver surface to determine the relationship between adsorption strength and geometry. The presence of an enhanced ν_19a ring band in the SERS spectra of L⁶ , L² , and L³ on the electrode indicated that the benzene rings of those molecules interact with the electrode surface through localized CC bond(s). We observed significant band broadening of the benzene ring modes for all α‐hydroxyphosphinic acids on both substrates, except for L¹ deposited onto the electrode surface. This suggests the possibility of direct interaction between the ring and Ag, although the benzene ring–surface π overlap is weaker for the colloidal silver than for the Ag electrode. The downward shift in wavenumber and alternations in the enhancement of a ν₁₂ ring band indicate a general increase of tilt angle on both silver substrates in the order L³ < L⁴ < L⁵ < L⁶ . The altered enhancement of the bands due to the vibrations of the  NH₂ and O PO fragments, a finding observed on both silver substrates, strongly suggests that the groups interact with different strength and geometry with these substrates. Copyright © 2009 John Wiley & Sons, Ltd.     

FT‐IR,FT‐Raman and SERS spectra of anilinium sulfate

The FT‐IR and FT‐Raman spectra of anilinium sulfate were recorded and analyzed. The surface‐enhanced Raman scattering (SERS) was recorded from a silver electrode. The vibrational wavenumbers of the compound have been computed using the Hartree‐Fock/6‐31G* basis and compared with the experimental values. The molecule is adsorbed on the silver surface with the benzene ring in a tilted orientation. The presence of amino and sulfate group vibrations in the SERS spectrum reveal the interaction between amino and sulfate groups with the silver surface. The direction of the charge transfer contribution to SERS has been discussed from the frontier orbital theory. Copyright © 2009 John Wiley & Sons, Ltd.     

Vibrational characterization of L‐valine phosphonate dipeptides: FT‐IR,FT‐RS,and SERS spectroscopy studies and DFT calculations

Four L ‐valine (L ‐Val) phosphonate dipeptides that are potent inhibitors of zinc metalloproteases, namely, L ‐Val‐C(Me)₂‐PO₃H₂ (V1), L ‐Val‐CH(iP)‐PO₃H₂ (V2), L ‐Val‐CH(iB)‐PO₃H₂ (V3), and L ‐Val‐C(Me)(iP)‐PO₃H₂ (V4), are studied by Fourier‐transform infrared (FT‐IR) spectroscopy, Fourier‐transform Raman spectroscopy (FT‐RS), and surface‐enhanced Raman scattering (SERS). The band assignment (wavenumbers and intensities) is made based on (B3LYP/6‐311 + + G**) calculations. Comparison of theoretical FT‐IR and FT‐RS spectra with those of SERS allows to obtain information on the orientation of these dipeptides as well as specific‐competitive interactions of their functionalities with the silver substrate. More specifically, V1 and V4 appear to interact with the silver substrate mainly via a  C_sgCH₃ moiety localized at the  N_amideC_sg(CH₃)P molecular fragment. In addition, the  POH and isopropyl units of V4 assist in the adsorption process of this molecule. In contrast, the  C_αNH₂ and  PO₃H⁻ groups of V2 and V3 interact with the silver nanoparticles, whereas their isopropyl and isobutyl fragments seem to be repelled by the silver substrate (except for the  CH₂  of V3), similar to the  C_β(CH₃)₂ fragment of L ‐Val for all L ‐Val phosphonate dipeptides investigated in this work. The adsorption mechanism of these molecules onto the colloidal silver surface is also affected by amide bond behavior. Copyright © 2010 John Wiley & Sons, Ltd.     

pH‐dependent surface‐enhanced Raman scattering studies of N‐acetylalanine monolayers self‐assembled on a silver surface

Monolayers of N‐acetylalanine on a metallic surface can serve as a biocompatible functional interface to construct biosensors. In the present paper, the surface‐enhanced Raman scattering (SERS) spectra of N‐acetylalanine monolayers self‐assembled on a silver surface under different pH were recorded. Assignments of the obtained spectra were carried out by density functional theory (DFT) calculations (BLYP/6‐311G). On the basis of the SERS effect, the nature of adsorption of N‐acetylalanine on a silver surface was deduced. It can be concluded that the fully protonated N‐acetylalanine is adsorbed on the silver surface via the imine group together with the carboxylate group, while it anchored onto the surface not only through both the imine and the carboxylate groups but also through the amide group after being completely deprotonated in the basic solution. Copyright © 2007 John Wiley & Sons, Ltd.     

Surface‐enhanced Raman scattering study of L‐tryptophan

Surface‐enhanced Raman scattering (SERS) spectra of tryptophan (Trp) were obtained. A unique SERS spectrum of Trp, corresponding to the most stable conformation and orientation on the metal surface, is observed after a stabilization period. The Trp molecules interact with the surface through both the carboxylate and amino groups; the aliphatic moiety is close to the surface. The pyrrole ring of the indole moiety is farther from the surface than the benzene fragment. The observed spectra vary depending on both the preparation of the silver colloid and the aggregation time. The interpretation of the experimental results is supported by theoretical treatment of the molecule on the silver surface. Copyright © 2008 John Wiley & Sons, Ltd.     

Effect of Pb2+ on L‐glutathione monolayers on a silver surface investigated by surface‐enhanced Raman scattering spectroscopy

Surface‐enhanced Raman scattering (SERS) spectroscopy was applied to observe reduced L ‐glutathione [L‐Glut(R)] molecules self‐assembled on a silver surface and the effect of Pb²⁺ on them. The adsorption structure suggests that the mercapto group of the L‐Glut(R) molecule is covalently bonded to the silver surface along with the imine group, amino group and entire carboxyl group in a perpendicular orientation after self‐reorganization. Results of SERS experiment show that Pb²⁺ influences the structure of L‐glutathione monolayers as a result of the binding reaction possibly occurring between Pb²⁺ and the carboxyl and the amino groups. Copyright © 2009 John Wiley & Sons, Ltd.     

Effect of an aliphatic spacer group on the adsorption mechanism on the colloidal silver surface of L‐proline phosphonodipeptides

A comparative study of molecular structures of five L ‐proline (L ‐Pro) phosphonodipeptides: L ‐Pro‐NH‐C(Me,Me)‐PO₃H₂ (P1), L ‐Pro‐NH‐C(Me,iPr)‐PO₃H₂ (P2), L ‐Pro‐L ‐NH‐CH(iBu)‐PO₃H₂ (P3), L ‐Pro‐L ‐NH‐CH(PA)‐PO₃H₂ (P4) and L ‐Pro‐L ‐NH‐CH(BA)‐PO₃H₂ (P5) has been carried out using Raman and absorption infrared techniques of molecular spectroscopy. The interpretation of the obtained spectra has been supported by density functional theory calculations (DFT) at the B3LYP; 6–31 + + G** level using Gaussian 2003 software. The surface‐enhanced Raman scattering (SERS) on Ag‐sol in aqueous solutions of these phosphonopeptides has also been investigated. The surface geometry of these molecules on a silver colloidal surface has been determined by observing the position and relative intensity changes of the Pro ring, amide, phosphonate and so‐called spacer (−R) groups vibrations of the enhanced bands in their SERS spectra. Results show that P4 and P5 adsorb onto the silver as anionic molecules mainly via the amide bond (∼1630, ∼1533, ∼1248, ∼800 and ∼565 cm⁻¹), Pro ring (∼956, ∼907 and ∼876 cm⁻¹) and carboxylate group (∼1395 and ∼909 cm⁻¹). Coadsorption of the imine nitrogen atom and PO group with the silver surface, possibly by formation of a weaker interaction with the metal, is also suggested by the enhancement of the bands at 1158 and 1248 cm⁻¹. P1, P2 and P3 show two orientations of their main chain on the silver surface resulting from different interactions of the  C CH₃,  NH and  CONH fragments with this surface. Bonding to the Ag surface occurs mainly through the imino atom (1166 cm⁻¹) for P2, while for P1 and P3 it occurs via the methyl group(s) (1194–1208 cm⁻¹). The amide group functionality (CONH) is practically not involved in the adsorption process for P1 and P2, whereas the C_s P bonds do assist in the adsorption. Copyright © 2008 John Wiley & Sons, Ltd.     

2-Amino-5-(4-pyridinyl)-1,3,4-thiadiazole film at the silver surface: Observation by Raman spectroscopy and electrochemical methods

Surface enhanced Raman scattering (SERS) spectrum of the 2-amino-5-(4-pyridinyl)-1,3,4-thiadiazole (4-APTD) on the silver surface was recorded and assigned with the help of B3LYP/6-311G** method. SERS result explored that 4-APTD molecule with a tilted orientation anchored at the silver surface via N¹², S¹ and C² atoms. In situ SERS spectroelectrochemical experiment indicated 4-APTD molecule experienced an intermediate adsorption process of its thiadiazole ring moiety with the vertical orientation at the surface before the 4-APTD molecule detached completely from the surface as the potential applied at −1.3 V vs. SCE. Electrochemical impedance spectroscopy (EIS) and polarization experiments exhibited the sound anticorrosive effect of the 4-APTD film on silver surface with an efficiency of 89.5%.     

Single‐molecule surface‐enhanced Raman scattering of fullerene C60

We achieved single‐molecule surface‐enhanced Raman scattering (SM‐SERS) spectra from ultralow concentrations (10⁻¹⁵ M) of fullerene C₆₀ on uniformly assembled Au nanoparticles. It was found that resonant excitation at 785 nm is a powerful tool to probe SM‐SERS in this system. The appearance of additional bands and splitting of some vibrational modes were observed because of the symmetry reduction of the adsorbed molecule and a relaxation in the surface selection rules. Time‐evolved spectral fluctuation and ‘hot spot’ dependence in the SM‐SERS spectra were demonstrated to result from the single‐molecule Raman behavior of the spherical C₆₀ on Au nanoparticles. Copyright © 2010 John Wiley & Sons, Ltd.     

Surface‐enhanced Raman scattering of benzenesulfonamide and sulfanilamide adsorbed on silver nanoparticles

The Surface‐enhanced Raman scattering of benzenesulfonamide and sulfanilamide adsorbed on silver sols was studied. On the basis of the noticeable shifts observed for wavenumbers of the ν_s(OSO), ν(CS), and ν(SN) vibrations with respect to the Raman spectra of the solids and the ionic solutions, we conclude that these molecules are adsorbed on silver nanoclusters at pH ≥ 7 with the aminosulfonyl groups partially deprotonated. The benzenesulfonamide links to the metal through the nitrogen atom of the corresponding azanion, while the sulfanilamide interacts in turn through the nitrogen atoms of the –NH₂ and –SO₂NH^– groups in the para‐position. Additionally, it was found that the most enhanced surface‐enhanced Raman scattering bands, especially the 8a;ν_ring mode, are related to the presence of the charge transfer mechanism. Copyright © 2011 John Wiley & Sons, Ltd.     

Food additives characterization by infrared,Raman, and surface‐enhanced Raman spectroscopies

Fourier‐transform infrared (FT‐IR), Raman (RS), and surface‐enhanced Raman scattering (SERS) spectra of β‐hydroxy‐β‐methylobutanoic acid (HMB), L ‐carnitine, and N‐methylglycocyamine (creatine) have been measured. The SERS spectra have been taken from species adsorbed on a colloidal silver surface. The respective FT‐IR and RS band assignments (solid‐state samples) based on the literature data have been proposed. The strongest absorptions in the FT‐IR spectrum of creatine are observed at 1398, 1615, and 1699 cm⁻¹, which are due to ν_s(COOH) + ν(CN) + δ(CN), ρ_s(NH₂), and ν(C O) modes, respectively, whereas those of L ‐carnitine (at 1396/1586 cm⁻¹ and 1480 cm⁻¹) and HMB (at 1405/1555/1585 cm⁻¹ and 1437–1473 cm⁻¹) are associated with carboxyl and methyl/methylene group vibrations, respectively. On the other hand, the strongest bands in the RS spectrum of HMB observed at 748/1442/1462 cm⁻¹ and 1408 cm⁻¹ are due to methyl/methylene deformations and carboxyl group vibrations, respectively. The strongest Raman band of creatine at 831 cm⁻¹ (ρ_w(R NH₂)) is accompanied by two weaker bands at 1054 and 1397 cm⁻¹ due to ν(CN) + ν(R NH₂) and ν_s(COOH) + ν(CN) + δ(CN) modes, respectively. In the case of L ‐carnitine, its RS spectrum is dominated by bands at 772 and 1461 cm⁻¹ assigned to ρ_r(CH₂) and δ(CH₃), respectively. The analysis of the SERS spectra shows that HMB interacts with the silver surface mainly through the  COO⁻, hydroxyl, and  CH₂ groups, whereas L ‐carnitine binds to the surface via  COO⁻ and  N⁺(CH₃)₃ which is rarely enhanced at pH = 8.3. On the other hand, it seems that creatine binds weakly to the silver surface mainly by  NH₂, and C O from the  COO⁻ group. Copyright © 2006 John Wiley & Sons, Ltd.     

Exploring excited‐state proton transfer mechanism for 9,10‐dihydroxybenzo[h]quinolone

In this work, we mainly focus on the excited‐state intramolecular proton transfer mechanism of a new molecule 9,10‐dihydroxybenzo[h]quinoline (9‐10‐HBQ). Within the framework of density functional theory and time‐dependent density functional theory methods, we have theoretically investigated its excited‐state dynamical process and our theoretical results successfully reappeared previous experimental electronic spectra. The ultrafast excited‐state intramolecular proton transfer process occurs in the first excited state (S₁ state) forming 9‐10‐HBQ‐PT1 structure without potential energy barrier along with hydrogen bond (O₃–H₄···N₅). Then the second proton may transfer via another intramolecular hydrogen bonded wire (O₁–H₂···N₃) with a moderate potential energy barrier (about 7.69 kcal/mol) in the S₁ state forming 9‐10‐HBQ‐PT2 configuration. After completing excited‐state dynamical process, the molecule on the first excited electronic state would come back to the ground state. We not only clarify the excited‐state dynamical process for 9‐10‐HBQ but also put forward new predictions and successfully explain previous experimental results.     

Theoretical elucidation of the origin of surface‐enhanced Raman spectra of PCB52 adsorbed on silver substrates

To better understand experimentally observed surface‐enhanced Raman Scattering (SERS) of polychlorinated biphenyls (PCBs) adsorbed on nanoscaled silver substrates, a systematic theoretical study was performed by carrying out density functional theory and time‐dependent density functional theory calculations. 2,2′,5,5′‐tetrachlorobiphenyl (PCB52) was chosen as a model molecule of PCBs, and Ag_n (n = 2, 4, 6, and 10) clusters were used to mimic active sites of substrates. Calculated normal Raman spectra of PCB52–Ag_n (n = 2, 4, 6, and 10) complexes are analogical in profile to that of isolated PCB52 with only slightly enhanced intensity. In contrast, the corresponding SERS spectra calculated at adopted incident light are strongly enhanced, and the calculated enhancement factors are 10⁴ ~ 10⁵. Thus, the experimentally observed SERS phenomenon of PCBs supported on Ag substrates should correspond to the SERS spectra rather than the normal Raman spectra. The dominant enhancement in Raman intensities origins from the charge transfer resonance enhancement between the molecule and clusters. Copyright © 2014 John Wiley & Sons, Ltd.     

Surface‐enhanced Raman evidence for Rhodamine 6 G and its derivative with different adsorption geometry to colloidal silver nanoparticle

Some high‐affinity functional groups or resonant molecules were often used as probe molecules adsorbed on silver nanoparticles for Surface‐enhanced Raman scattering (SERS). However, it is still unclear how the attached molecules interact with the silver nanoparticles' surface, and how the anchoring groups affect the optical and electronic properties of molecules. Here, we report that surface‐enhanced Raman studies of two organic compounds; rhodamine 6G (R6G) and its aminated derivative (R‐NH₂) have very different functional groups for surface binding but nearly identical SERS spectroscopic properties at pH = 7 and UV–vis at pH = 3, respectively. A surprise was found that under the same experimental conditions, the SERS signal intensity for R6G is nearly 50‐fold higher than that of R‐NH₂. Furthermore, the pH‐dependent study reveals that the structure of R6G is irreversibly stabilized or ‘locked’ in its form and no longer responsive to pH changes. In contrast, R‐NH₂ is still sensitive to pH, and can be switched between its open‐ring and closed‐ring structures. Copyright © 2013 John Wiley & Sons, Ltd.     

Near‐infrared surface‐enhanced Raman spectroscopy (NIR‐SERS) studies on oxyheamoglobin (OxyHb) of liver cancer based on PVA‐Ag nanofilm

A near‐infrared surface‐enhanced Raman spectroscopy (NIR‐SERS) method was employed for oxyheamoglobin (OxyHb) detection to develop a simple blood test for liver cancer detection. Polyvinyl alcohol protected silver nanofilm (PVA‐Ag nanofilm) used as the NIR‐SERS active substrate to enhance the Raman scattering signals of OxyHb. High quality NIR‐SERS spectrum from OxyHb adsorbed on PVA‐Ag nanofilm can be obtained within 16 s using a portable Raman spectrometer. NIR‐SERS measurements were performed on OxyHb samples of healthy volunteers (control subjects, n = 30), patients (n = 40) with confirmed liver cancer (stage I, II and III) and the liver cancer patients after surgery (n = 30). Meanwhile, the tentative assignments of the Raman bands in the measured NIR‐SERS spectra were performed, and the results suggested cancer specific changes on molecule level, including a decrease in the relative concentrations and the percentage of aromatic amino acids of OxyHb, changes of the vibration modes of the C_aH_m group and pyrrole ring of OxyHb of liver cancer patients. In this paper, principal component analysis (PCA) combined with independent sample T test analysis of the measured NIR‐SERS spectra separated the spectral features of the two groups into two distinct clusters with the sensitivity of 95.0% and the specificity of 85.7%. Meanwhile, the recovery situations of the liver cancer patients after surgery were also assessed using the method of discriminant analysis‐predicting group membership based on PCA. The results show that 26.7% surgeried liver cancer patients were distinguished as the normal subjects and 63.3% were distinguished into the cancer. Our study demonstrated great potentials for developing NIR‐SERS OxyHb analysis into a novel clinical tool for non‐invasive detection of liver cancers. Copyright © 2013 John Wiley & Sons, Ltd.