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Professor Stefano Corni and assistant professor Nicolò Maccaferri deeepen their collaboration through UCMR Seminar Series support
my impression is that the research performed at Umeå University is of world-class scientific level
“I am very grateful to Umeå Centre of Microbial Research
for funding my short visit to Umeå and allowing me to connect with a new reality
since this is my first time in Sweden and I am happy with my experience,” says Stefano Corni
professor at the Department of Chemical Sciences at the University of Padova in Italy
Nicolò Maccaferri and his research group had the pleasure to host Stefano Corni in the context of UCMR Seminar Series
Corni’s research is focusing on using the basic principles of chemistry and physics to model the properties of complicated systems involving molecules
nanostructures and light and he is already an important collaborator of Nicolò Maccaferri in the Pathfinder project ‘iSenseDNA’ funded by the European Innovation Council
“Stefano Corni’s team is helping our group to model the experimental results that we obtain here
We use a homemade multidimentional spectroscopy setup to measure the dynamics of molecules at very short timescales and from this we extract structural information useful to understand their elementary functions,” says Nicolò Maccaferri
assistant professor and group leader of the Ultrafast Nanoscience Unit at the Department of Physics at Umeå University
As a mandatory part of the UCMR Seminar Series programme
His talk covered multiscale modelling of molecules interacting with nanostructures
a topic at the intersection between chemistry and physics
partially connected to some other on-going projects in the Nicolò Maccaferri lab
a lot of time was devoted to informal discussions about on-going experiments and projects and future new ideas involving researchers in different units
Stefano Corni has started a collaboration with Magnus Andersson lab at the Department of Physics
where the idea is to validate his theoretical models using Raman spectroscopy
Corni’s team can help us in modeling experiments we plan to do together with André Mateus lab at the Department of Chemistry,” says Nicolò Maccaferri
PhD student Hinduja Bhuvanendran and ‘Excellence by Choice’ postdoctoral fellow Ben Johns are showing professor Stefano the experimental setup used in both the Pathfinder project ‘iSenseDNA’ and the UCMR postdoctoral project funded by Kempestiftelserna
Corni’s expertise is also complementary to that of Ludvig Lizana
who is co-supervisor to postdoctoral fellow Ben Johns together with André Mateus (co-supervisor) and Nicolò Maccaferri (main supervisor)
This additional collaboration will strengthen the Umeå University team in performing a challenging interdisciplinary research project within the ‘Excellence by Choice’ postdoctoral fellow programme in life science
Stefano Corni thinks that the highlight of his stay was meeting researchers that were so enthusiastic about their work and visiting their labs
“I was particularly impressed by the strong research in materials science and photonics carried out at the Department of Physics
funded by prestigious grants such as the European Research Council and Knut och Alice Wallenbergs Stiftelse (KAW)
Even though I was here only for a short time
I have a strong feeling that Umeå University is a pleasant place to study at and to do research in an open and collaborative manner.”
PhD student Mouna Rafei is explaining to professor Stefano Corni how they make nanomaterials using chemical synthesis techniques
UCMR is a Linnaeus Centre of Excellence promoting cutting-edge microbial research at Umeå University
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Electronic Energy Transfer (EET) between chromophores is fundamental in many natural light-harvesting complexes
serving as a critical step for solar energy funneling in photosynthetic plants and bacteria
The complicated role of the environment in mediating this process in natural architectures has been addressed by recent scanning tunneling microscope experiments involving EET between two molecules supported on a solid substrate
These measurements demonstrated that EET in such conditions has peculiar features
such as a steep dependence on the donor-acceptor distance
reminiscent of a short-range mechanism more than of a Förster-like process
By using state of the art hybrid ab initio/electromagnetic modeling
here we provide a comprehensive theoretical analysis of tip-enhanced EET
we show that this process can be understood as a complex interplay of electromagnetic-based molecular plasmonic processes
whose result may effectively mimic short range effects
the established identification of an exponential decay with Dexter-like effects does not hold for tip-enhanced EET
and accurate electromagnetic modeling is needed to identify the EET mechanism
our calculations show how the proper accounting of all the relevant molecule-metal and molecule-molecule electromagnetic interaction pathways results in a trend that deceptively mimics an exponential decay
we disclose a situation where the popular criterion to distinguish between Förster-like and Dexter-like energy transfer mechanisms is no longer appropriate due to the relevance of plasmonic nanoscale effects occurring in tip-molecule-substrate STM junctions
In the experimental work of Cao et al.30
is based on emission intensities of the acceptor (IA) and donor molecules (ID) upon excitation of the donor
we highlight that the above quantity RETeff does not depend only on the theoretical efficiency of the EET step unless special restrictive conditions are met
such as the donor and acceptor molecules being identical
More generally, such empirical energy transfer efficiency depends on multiple radiative and nonradiative decay processes within the system, and the presence of the metallic tip may influence and modify these processes, as schematically illustrated in Fig. 1.
The STM tip excites the donor molecule through a tunneling current
and the donor may decay to its ground state radiatively
it subsequently may decay to its ground state radiatively or nonradiatively
The excitation energy of the donor’s first excited state is larger than that of the acceptor’s
which typically prevents energy flow back to the donor
All possible decay processes in both donor and acceptor are affected by the presence of the metallic tip
the tip may not only modify the radiative emission of each emitter
but also provides an additional source of nonradiative decay for the molecular excited states
Given the fluorescence quantum yield of the donor ΦD
where Γex is the excitation rate (in this case promoted by tunneling electrons)
Γrad,D is the radiative decay rate of the donor in presence of the metal tip
Γnr,met,D is the nonradiative decay rate induced by the metal tip
and ΓEET is the EET rate from the donor to the acceptor
which is also modified by the presence of the metal tip
assuming that the donor undergoes EET to the acceptor upon excitation
Γnr,0,A and Γnr,met,A are the radiative decay rate
and metal-induced nonradiative decay rate of the acceptor
We note that the quantity ηEET is the general theoretical definition of the efficiency of EET from the donor to the acceptor
Equations (2) and (3) assume only one donor state and one acceptor state
which is a good approximation in the absence of degenerate excited states
for the donor molecule (palladium-phthalocyanine
PdPc) the first two excited states are degenerate
so they both may be excited by the tip and participate in the EET process
the acceptor molecule (free-base phthalocyanine
H2Pc) has two excited states that are close in energy and may both be excited by EET from the donor
we consider the emission intensity of the donor from state i
where the index i indicates the i-th excited state of the donor
and the index j indicates the j-th excited state of the acceptor
the total acceptor emission intensity after tip-induced excitation of donor state i (\({I}_{{{{{{{{\rm{A}}}}}}}}}^{{{{{{{{\rm{i}}}}}}}}}\)) is the sum over emission intensities from possible acceptor states j
after EET between donor state i and acceptor state j
where \({\Phi }_{{{{{{{{\rm{A}}}}}}}}}^{{{{{{{{\rm{j}}}}}}}}}\) is the jth state emission quantum yield of the acceptor, as defined by the third term in Eq. (3). Thus, the net RET efficiency, as defined in Eq. (1)
as excitation rates \({\Gamma }_{{{{{{{{\rm{ex}}}}}}}}}^{{{{{{{{\rm{i}}}}}}}}}\) cancel out
it is possible to formulate the RET efficiency directly in terms of decay rates
Equation (7) reveals that the RET efficiency upon exciting the i-th donor state is independent of all nonradiative decay properties of the donor
where f is a complicated function that depends on the EET rate from donor to acceptor
radiative decay rate of the donor and all decay rates of the acceptor
where gxc is the exchange-correlation kernel and the third term in the right-hand side is the overlap between molecular transition densities weighted by the transition energy
Transition densities for donor emission and acceptor absorption are used instead of dipolar or multipolar approximations to take into account the charge distribution within each molecule during electronic excitations
Response charges are multiplied by the acceptor transition potential evaluated at the same spatial coordinates
with εmet(ω) being the nanostructure’s frequency-dependent dielectric function that enters Eq. (17)
thus not affecting results whether or not it is included
PdPc (donor) and H2Pc (acceptor) molecules shown at center-center distances of 1.68 nm (a) and 3.20 nm (b)
Aromatic lobes of both molecules are oriented 30 degrees from the x and y axes
The relative orientation between donor and acceptor is kept rigid throughout the samples separation range
a Transition dipoles of the first two excited states of the donor molecule PdPc
The black dots correspond to two different locations of the STM tip apex above the molecule
b Computed HOMO density of both donor and acceptor molecules
it is important to assess the impact of the frequency-dependent response of the metal tip on the EET process
We find that in all theoretical calculations
RETeff decreases monotonically with the distance between molecules
similar to observed trends in the experimental data
in these experiments it was noted that the observed fast decay of RET with D-A distance could not be understood in terms of simple dipole-dipole interactions (Förster mechanism)
and it was speculated that both multipolar RET and Dexter-like energy transfer may be possible explanations for the observed behavior
The finding that a combination of purely electromagnetic effects may mimic an exponential decay with distance is the main result of this present work
The shape of the decay curve is universally used to classify Förster versus Dexter EET mechanisms
and we show here that in case of tip-mediated EET
such a simple indicator cannot be reliably used
This suggests the possibility to further tune the STM tip geometry and absorption spectrum to either maximize or minimize EET efficiency between donor and acceptor molecules and to control how steeply EET drops off as the molecules are brought farther apart
the main result of this work is that for tip-mediated EET
an almost exponential decay is obtained as a consequence of the complex interplay of purely electromagnetic effects
the established identification of an exponential decay with a Dexter-like mechanism does not hold for tip-mediated EET
we also observed that the frequency-dependent response of the plasmonic nanostructure
which is strongly dictated by the nanostructure’s geometric shape and metallic composition
can drastically impact the efficiency of energy transfer in such systems
thus paving the way for engineering these systems to control energy flow at the nanoscale
We believe the theoretical model proposed here may be a valuable starting point for exploring the role of plasmonic nanostructures in tailoring energy flow across molecules in more sophisticated multichromophoric architectures
such as artificial and natural light-harvesting complexes
molecular electronic structure is computed using ab initio methods
The resulting molecular charge densities perturb the metal nanostructure
and the nanostructure’s response to the molecule-induced external perturbation is treated classically with the Polarizable Continuum Model (PCM)
making use of the frequency-dependent dielectric function of the nanostructure
The PCM electromagnetic problem is numerically solved using the Boundary Element Method (BEM)
where the surface of the metallic nanostructure is discretized into elementary areas
Each tessera is associated with a polarization charge qi(ω) located in its geometrical center \({\overrightarrow{{{{{{{{\bf{s}}}}}}}}}}_{{{{{{{{\rm{i}}}}}}}}}\) that describes the interaction between the nanostructure and the potential of a nearby molecule
Polarization charges are computed on the nanostructure surface as
where Q(ω) is the PCM response matrix in the frequency domain
Here, A is a diagonal matrix whose elements are the tesserae areas, while the matrices S and D are representative of Calderons’ projectors50
where the vector \({\overrightarrow{{{{{{{{\bf{s}}}}}}}}}}_{{{{{{{{\rm{j}}}}}}}}}\) is representative of the j-th tessera’s position on the nanoparticle surface
and \({\overrightarrow{{{{{{{{\bf{n}}}}}}}}}}_{{{{{{{{\rm{j}}}}}}}}}\) is the unit vector normal to the j-th tessera
Geometry optimization of molecular structures was performed for gas-phase PdPc and H2Pc with DFT calculations using the software Gaussian1669
ground state optimization DFT calculations were performed using the B3LYP functional with the LanL2DZ basis set for palladium (Pd) and the 6-31G(d)**++ basis set for non-metal atoms (C
so both were considered in the simulations
the results presented here would effectively take into account such an issue
since upon tautomerization the S2 state accounted for here would convert to S1 of the tautomer
The TDPlas code used to model the plasmonic systems and couple them with molecules is freely available at https://github.com/stefano-corni/WaveT_TDPlas
The post-processing python code used to evaluate RETeff and to make the corresponding figures is available from the authors upon reasonable request
Multiscale modelling of photoinduced processes in composite systems
Light absorption and energy transfer in the antenna complexes of photosynthetic organisms
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Exciton dynamics of c60-based single-photon emitters explored by hanbury brown–twiss scanning tunnelling microscopy
Visualizing coherent intermolecular dipole–dipole coupling in real space
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Single-molecule investigation of energy dynamics in a coupled plasmon-exciton system
Single-molecule tautomerization tracking through space-and time-resolved fluorescence spectroscopy
Sub-nanometre resolution in single-molecule photoluminescence imaging
Energy funnelling within multichromophore architectures monitored with subnanometre resolution
Wavelike electronic energy transfer in donor–acceptor molecular systems through quantum coherence
Distance and orientation dependence of excitation transfer rates in conjugated systems: beyond the förster theory
Beyond forster resonance energy transfer in biological and nanoscale systems
Conformational disorder in energy transfer: beyond förster theory
Effect of the orientation of donor and acceptor on the probability of energy transfer involving electronic transitions of mixed polarization
Orientation dependence of energy transfer in an anthracene–porphyrin donor–acceptor system
Distance and orientation dependence of excitation energy transfer: from molecular systems to metal nanoparticles
Strong enhancement of the radiative decay rate of emitters by single plasmonic nanoantennas
Controlling spontaneous emission with plasmonic optical patch antennas
and radiative decays in nanogap emitting devices
Plasmon-controlled forster resonance energy transfer
Plasmon-induced photonic and energy-transfer enhancement of solar water splitting by a hematite nanorod array
Plasmon-induced resonance energy transfer for solar energy conversion
Matching nanoantenna field confinement to fret distances enhances forster energy transfer rates
Can we control the electronic energy transfer in molecular dyads through metal nanoparticles
Energy transfer across a metal film mediated by surface plasmon polaritons
Exciton energy transfer in pairs of single-walled carbon nanotubes
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Quantum mechanical continuum solvation models
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optimal setup revealed by the polarizable continuum model
Lifetimes of electronic excited states of a molecule close to a metal surface
Radiative and nonradiative decay rates of a molecule close to a metal particle of complex shape
Semiempirical (zindo-pcm) approach to predict the radiative and nonradiative decay rates of a molecule close to metal particles
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Electronic dynamics of a molecular system coupled to a plasmonic nanoparticle combining the polarizable continuum model and many-body perturbation theory
Coupling of molecular emitters and plasmonic cavities beyond the point-dipole approximation
Sub-nanometre control of the coherent interaction between a single molecule and a plasmonic nanocavity
and Purcell effects at a chromophore-picocavity junction with hyper-resolved fluorescence microscopy
Strong plasmon-molecule coupling at the nanoscale revealed by first-principles modeling
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https://github.com/stefano-corni/WaveT_TDPlas
Current-induced hydrogen tautomerization and conductance switching of naphthalocyanine molecules
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The authors acknowledge financial support from the European Union’s Horizon 2020 research and innovation program through the project PROID (Grant Agreement No
Computational work has been carried out on the C3P (Computational Chemistry Community in Padua) HPC facility of the Department of Chemical Sciences of the University of Padua
acknowledge MIUR “Dipartimenti di Eccellenza” under the project Nanochemistry for energy and Health (NExuS) for funding the Ph.D
acknowledge the USA Department of Energy Office of Science
acknowledge the Defense Advanced Research Projects Agency (DARPA)
performed all the calculations and prepared the images
equally contributed to the first draft of the manuscript
The authors declare no competing interests
Communications Chemistry thanks the anonymous reviewers for their contribution to the peer review of this work
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations
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Volume 3 - 2024 | https://doi.org/10.3389/fchbi.2024.1377357
This article is part of the Research TopicExploring Novel Metal-Based Compounds for Enhanced Therapeutic Efficacy and SafetyView all 4 articles
The Anderson-Evans polyoxometalates (POM) display a promising anticancer activity
The conjugation with the GRP-receptor antagonist peptide Demobesin (fQWAVGHL-NHEt) was exploited to impart cell targeting capabilities and improve the selectivity of such polyanions
the POM interacts with the grafted peptides
inducing chains folding and self-assembly of the resulting hybrids
a hydrophilic one (1,13-diamino-4,7,10-trioxatridecan-succinamic acid
Ttds) and a tetra-anionic one (Glu-Glu-Glu-Glu-βAla
EEEE-βA) was previously utilized to mitigate such interaction
hybrid POMs containing only Ttds or EEEE-βA were prepared and the contribution of the two spacers was separately studied by using 2D NMR
Transmission electron microscopy (TEM) was also used to observe the impact of the different spacers on self-assembly
Owing to the relevant effects observed for EEEE-βA
MD calculations were finally performed to elucidate its behavior when incorporated in the hybrid POM
despite the stronger impact of EEEE-βA spacer
only when both spacer are present together it is possible to observe a significant effect on the retention of peptide's secondary structure and recognition capability
they can be rationally synthesized on a multi-gram scale at a low cost
The resulting POM (4-POM) showed better performance in terms of tumor cell recognition and is thus encouraging a further development of this approach
and structure of hybrids 2-POM and 3-POM (only one pendant is shown; tetrabutyl ammonium cations are also omitted)
In this work, the Mn-Anderson-Evans was functionalized with two 1 analogs containing one of the two portions of the previously described spacer. Peptide 2 contains only the Ttds group, while peptide 3 only the EEEE-βA anionic domain (Figure 1). The corresponding POM constructs (2-POM and 3-POM) were prepared (Figure 1 and Supplementary Figure S1) and compared with spacer-free
and Ttds-EEEE-βA containing POMs (1-POM and 4-POM
to investigate the contribution of different spacers
POM hybrids were characterized by various spectroscopic techniques
3-POM was also investigated by MD calculations to explain the observed behavior
N-Fmoc-N-succinyl-4,7,10-trioxa-1,13-tridecanediamine (Fmoc-Ttds-OH)
2-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HATU)
2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU)
N,N′-dicyclohexylcarbodiimide (DCC)
were purchased from Sigma-Aldrich and used without any further treatment
Nuclear Magnetic Resonance (NMR) Spectroscopy was performed on a Bruker DMX-400 instrument (Bruker Corporation
USA) operating at 399.92 MHz for 1H
are referred to the residual 1H-DMSO solvent signal in DMSO-d6 (δ = 2.49 ppm)
About 4 mg of each sample were dissolved in 600 μL DMSO-d6 for analysis
All NMR experiments have been carried out at temperature of 298 K
One-dimensional (1D) 1H-NMR spectra were acquired using typically 32 scans with 32K data size
For the two-dimensional (2D) 1H NMR spectra
and ROESY (Rotating Frame Overhauser Effect Spectroscopy)
homonuclear pulse programs of the standard Bruker library were used (cosygpmfqf for COSY experiments
512 experiments of 50 scans each were performed: relaxation delay T2 equal to 1; size 4K; 5,597 Hz spectral width in F2
Spectral processing was performed using the software TopSpin
Infrared Spectra were collected by preparing KBr pellets
on a Nicolet 5700 FI-IR instrument (Thermo Fisher Scientific
Electrospray Ionization Mass Spectroscopy (ESI-MS) was performed on a LC/MSD Trap SL Agilent instrument (Agilent Technologies
The samples were dissolved in CH3CN and analyzed in negative mode
High-performance liquid chromatography (HPLC) analyses were run on a Shimadzu system equipped with a binary pump (LC-10AD)
The semi-preparative HPLC was performed on a Shimadzu system equipped with a binary pump (LC-8A)
SPD-6A detector and ERMA (ERC-3562) degasser (Shimadzu Corporation
enabling signal deconvolution by means of the CONTINLL algorithm (encompassing α-helix
Emission fluorescence experiments were collected on a Perkin Elmer LS50B spectrofluorimeter
equipped with FL-WinLab software (PerkinElmer
Samples were measured in 10 mm pathlength quartz cuvettes at 25°C
Emission spectra were recorded in the range 300–550 nm
with excitation at λ=295 nm
3.5 nm excitation and emission slits and 400 nm/min scan speed
Dynamic Light Scattering (DLS) was monitored by a Zetasizer Nano ZS instrument (Malvern Panalytical Ltd
collecting 3 series of measurements from 0.2 mM solutions in phosphate buffer (10 mM
Transmission Electron Microscopy (TEM) was carried out using a FEI Tecnai G2 instrument (Thermo Fisher Scientific
drop casting aqueous solutions (with 3% DMSO) onto copper grids
Cytotoxicity/viability tests were performed as 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide (MTT) assays. For the experiments, HeLa cervical cancer (LGC Standards S.r.l., Sesto San Giovanni, Italy), cultured according to the manufacturer’s instructions, were used. On day 1, 10,000 HeLa cervical cancer cells/well in a volume of 200 µL were plated in 96-wells plates, as previously reported (Tagliavini et al., 2021)
and 48 h to different concentrations of POM-TRIS
Stock solutions with 100 mM POM in water
were prepared by prior solubilization of POMs in DMSO
The results were expressed as the mean ± SD percentage of living cells normalized to the control
The Lennard-Jones parameters for the POM were taken from the universal force field (UFF)
Peptides 1-4 were prepared by solid phase synthesis adopting the Fmoc/HBTU strategy, according to the previously described procedure (Rubini et al., 2010; Ventura et al., 2018; Tagliavini et al., 2021). The N-ethylamide group was introduced ab initio using an ethyl-indole AM Resin. The peptides were purified by HPLC and characterized by ESI-MS, FT-IR and 2D-NMR (see Supplementary Figures S2–S11
showing the characterization of peptides 2 and 3)
The syntheses of the 1-4-POM hybrids were performed in air, at room temperature for 24 h, under continuous stirring (Supplementary Figure S1) (Ventura et al., 2018; Tagliavini et al., 2021)
The obtained solutions were placed in a closed vessel under diethyl ether vapors
to allow the precipitation of the products
which were finally washed with diethyl ether and dried under air
to achieve pale orange solids (yields >85%)
All POM-peptides conjugates were characterized by ESI-MS, FT-IR, 2D-NMR, CD, UV-vis (see Supplementary Figures S12–S27
showing the characterization of 2-POM and 3-POM)
The resonance assignment of amino acid residues in peptides 2 and 3 and their corresponding POM derivatives were obtained by collecting COSY, TOCSY and ROESY spectra for all samples (Supplementary Tables S1–S4)
Due to the limited solubility of POM hybrids in water
The latter is also useful for the study of peptides that interact with membrane receptors
having a dielectric constant (ε 47.5) very close to that of the membrane interface (ε 40)
with a tendency to form the helical structure also at the N-terminus
displays its most negative Δδ value at the His residue
Secondary Hα chemical shift (Δδ) values of DB-1 derivatives (A) or of the corresponding POM-peptide hybrids (B)
The dotted line indicates the threshold value for the presence of a helical secondary structure
To evaluate the spacers’ contribution on the interactions between POM and different peptides, the NH and Hα secondary chemical shift values, determined by subtracting the NH and Hα resonance of each peptide to the resonance of corresponding POM construct were also compared (Figure 3)
The presence of POM cluster had an evident impact in the resonance of the closer N-terminal peptide region in 1-POM and 2-POM
indicating that the introduction of the negatively charged Glu4-βAla sequence (in 3-POM and 4-POM) seems useful to attenuate the interaction between the peptide and the inorganic cluster
Influence of spacer on NH (A) and Hα (B) resonances of DB-1 in POM-peptide constructs
Secondary chemical shift values were determined by subtracting the NH or Hα chemical shift values of DB-1 analogues (peptide 1-4) to the resonance of corresponding POM constructs (POM 1-4)
Values greater than |0.15| ppm (dotted lines) indicate a significant effect on resonances
without the need of screening other solvents
at 10 and 80% (v/v) TFE percentages (indicated)
The spectra were recorded by a Jasco J-1500 spectropolarimeter using a 0.1 cm pathlength Suprasil quartz cuvette
the CD spectra of peptides 2 and 3 show a negative band at 197–200 nm (n → π* electronic transition) and a positive band at λ<190 nm (π → π*)
the CD spectra of peptides 2 and 3 are characterized by a positive band at 192 nm and a negative band around 206 nm
generated by the presence of more ordered structures
involving an increased contribution of the α-helical structure
the differences in the intensity of the bands level off when the compounds are situated in TFE ≥80% v/v
For a sake of comparison, 1-POM and 4-POM showed the highest and the lowest impact on DB-1 folding, favoring, respectively, α-helix and β-strand conformations (Ventura et al., 2018; Tagliavini et al., 2021)
have a relatively similar effect in terms of secondary structure evolution in H2O/TFE
and their behavior is intermediate with respect to the spacer-free and double-spacer POMs
Interestingly, when POM and peptide are not conjugated (see Supplementary Figure S37
reporting the example for peptide 3 in the presence of 0.5 equiv
of POM-succ) no significant difference was observed in the CD spectra of peptide with or without POM at both the examined TFE percentages (10 and 40% v/v)
This suggests that non-covalent interactions between POM and peptides are of intramolecular type and that the increase of dichroic signal observed for the hybrids can be due to an induction of chirality to the POM core in a less polar solvent
where intramolecular hydrogen bonds are promoted
peptides 1-4 showed the expected quenching of the signal
with efficiency in the order 1>2>3=4
in agreement with the repulsion between negative charges of I− and Glu residues in 3 and 4
Fluorescence emission of POM-peptide hybrids (indicated) at increasing amounts of cesium ions (CsCl)
Fluorescence is referred to as the ratio between the fluorescence at a given quencher concentration (F) and the fluorescence in its absence (F0)
These fluorescence experiments highlight a greater impact of the spacers in terms of peptide-POM interactions rather than POM-induced secondary structure evolution
demonstrating the need to attenuate them to increase peptide availability
Complementary interactions (amino groups/POM surface and amino groups/carboxylic groups) are thus also useful to establish directional interactions
10−4 M solutions of a (A) 1-POM
scale bar =100 nm (B) 2-POM in 3% DMSO/water
scale bar =200 nm; (C) 3-POM in H2O
scale bar 100 nm; (D) 4-POM in 3% DMSO/water
Noteworthy, in DMF as solvent, 2-POM showed a gelator effect, likely mediated by the polar Ttds chains (Supplementary Figure S43), as previously observed also for 4-POM (Tagliavini et al., 2021)
These results underly how hybrid POMs may represent versatile building blocks
capable of exploiting both the polyanionic surface and the organic pendants to control the interactions
it is evident that the spacer based on the tetra glutamic acid has a bigger impact than Ttds on improving the accessibility of the peptide chains
• retention of POM-free peptide’s secondary structure in DMSO-d6
• higher accessibility of the peptide chain with respect to quenching by ions
where the time evolution of the fraction of β-structure
helical structure and unstructured peptide is shown for both compounds
It must be noted that a relevant amount of unstructured peptide is present in the MD simulations (≈50% on average)
while in the experimental CD data a lower percentage of unstructured peptides was observed (32–35%)
This can be ascribed to the relatively short simulation time (100 ns for each MD) that does not allow the peptides to assume a stable folded conformation
relevant structural differences between the two compounds can be already observed: in both compounds the peptides assume a β-structure
but in compound 1-POM a non-negligible fraction of helical structure is also present
The formation of helical structure is observed in two of the three simulations of 1-POM: in the third simulation
a particularly high unstructured fraction is present
3-POM shows a bigger amount of β-structures
in the first simulation of compound 3-POM a more relevant percentage of helical structure is present with respect to the other two simulations of the same compound
The statistical sampling of different secondary structures is not surprising in MD simulations starting from an extended conformation
the difference in the average secondary structure content between the two compounds appears to be meaningful
Time evolution of the relative abundance of β-structure (red)
helical structure (blue) and unstructured peptide (gray) along the three independent MD simulations of compound 1-POM (upper panels) and compound 3-POM (lower panels)
the first 10 ns of each MD simulation have been neglected
This fact is important to explain the presence of interactions despite the presence of electrostatic repulsions and deserves attention
in terms or length of the peptide and functionality at the terminal end
Time evolution of the distance between the terminal residue of the two peptides and the center of mass of the POM moiety in one of the MD simulations of compound 3-POM
The black and red lines refer to the two pendants
Some representative snapshots of the POM-peptides conformation are also shown
Fraction of interchain contacts in compounds 1-POM (left) and 3-POM (right)
The color code represents the fraction of MD frames at which an Ri Rj contact is present
Cytotoxicity performances of 3-POM on HeLa cells at increasing concentration
Results are presented after 24 and 48 h
The amount of such transient interactions is likely related to the different conformations observed; in particular
they can make the chains less available for longer range interactions required for β-structures as well as for targeting purposes
These outcomes are consistent with the data herein collected
the significant impact of tetra glutamic acid domain
the need of combining the two spacers to achieve a stronger biological activity
The incorporation of spacers could mitigate the influence of the POM cluster
the first proved to be more useful in minimizing molecular interactions
as 3-POM and 4-POM showed lower CHα shifts compared to the free peptide
compounds 1-POM and 2-POM displayed more differences in the secondary structure compared to bare Demobesin-1
favouring the formation of α-helix in DMSO-d6
the impact of the two spacers on secondary structure seemed not relevant in TFA/H2O solutions: CD spectroscopy did not highlight a meaningful difference between 2-POM and 3-POM
which both displayed intermediate behavior with respect to 1-POM and 4-POM
underscored the positive effect conferred by the charged spacer
with compounds 1-POM and 2-POM exhibiting no dynamic quenching upon the addition of both KI and CsCl
while 3-POM and 4-POM showed better responsivity of the side chain Trp
MD calculation showed that 3-POM secondary structure is mostly composed by β-sheet and random coil conformations
Despite the high accessibility of the chains
they still tend to fold on the POM structure
exploiting the terminal residues for the interactions
the biological activity of 3-POM towards HeLa cells appears still low
and justifies the need for a longer spacer
This observation may be crucial to overcome the general problem of undesired POM-triggered peptide folding
and will be useful to drive the design of a next generation of POM-based drugs
with possibility to control targeting and delivery
while enabling mechanistic studies for the still unclear mechanism of action of anticancer POMs
Further studied will evaluate the nature of POM scaffold
The original contributions presented in the study are included in the article/Supplementary Material
further inquiries can be directed to the corresponding authors
The author(s) declare financial support was received for the research
This work was supported by the Department of Chemical Sciences of the University of Padova (grant number P-DiSC#11NExuS_BIRD2019-UNIPD) and by MUR (project PRIN2022CAS9ZT)
HY thanks the China Scholarship Council for funding her PhD in Molecular Science at Padova University
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest
All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations
Any product that may be evaluated in this article
or claim that may be made by its manufacturer
is not guaranteed or endorsed by the publisher
The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fchbi.2024.1377357/full#supplementary-material
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Received: 27 January 2024; Accepted: 12 March 2024;Published: 25 March 2024
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‘Quality evaluation based on color grading’ is one of the features used in Chinese medicine discrimination
In order to assess the feasibility of electronic eye (E-eye) in implementing ‘quality evaluation based on color grading’
the present study applied an IRIS VA400 E-eye to test 58 batches of Corni Fructus samples
Their optical data were acquired and combined with their corresponding classes
A total of four quality discrimination models were produced according to discrimination analysis (DA)
least squares support vector machine (LS-SVM)
partial least squares-discrimination analysis (PLS-DA)
and principal component analysis-discrimination analysis (PCA-DA)
The accuracy rate of the aforementioned 4 cross evaluation models were 86.21%
the PCA-DA method was used to build the final discrimination model for classifying Corni Fructus or discriminating its quality
it is rarely reported in Chinese medicine quality evaluation
A survey demonstrated that Corni Fructus could change its color to purplish-black from its original bright red color if stored improperly
This in turn reduced the purchasing value and medicinal quality of the herb
thereby influencing its production and clinical usage
the specification and grade of Corni Fructus is primarily determined by medicinal farmers and practitioners with considerable experience
who can make decisions based on the medicinal material origin
This strategy hardly meets the current market requirements and it is not sufficient to guide the rapid development of the Corni Fructus decoction market
inadequate experience of the medicinal practitioners may be one of the contributing factors that can hinder the accurate recognition of the Corni Fructus
the aim of the present study was to produce a discrimination model for the Corni Fructus specification and grade
and to build a more scientific and objective quality evaluation system by the concept of ‘quality evaluation based on color grading’ in order to rapidly and precisely identify the specification and grade of medicinal materials
The standards of the 4 classes were customed for the Corni Fructus samples tested in the present study (Table 1). According to these standards, 58 samples were graded (Table 2)
The landscape of Corni Fructus samples depicted with 26 color numbers
The accuracy rate of the building models constructed for the overall samples was 98.28%
and the RBF kernel function parameters gamma and sig2 were 0.355 and 17.245
(a) Chart of “latent variables-error rate”; (b) chart of “latent variables-percentage of not assigned samples”.
Variance contribution percentage of latent variables on independent variables (a) and dependent variables (b)
Chart of scores on latent variables in PLS-DA model
The relation of principal components-error rate.
(a) Variance contribution percentage of individual principle components on variables; (b) cumulative variance contribution percentage of principle components on variables
Chart of scores on principle components in the PCA-DA model
Chart of scores on canonical variables of the PCA-DA model
Variation information determined by 26 variables
Loading diagram of latent variables in the PCA-DA full-sample model
only the PLS-DA model exhibited 9 unassigned samples
This result could be attributed to the discrimination mechanism of the PLS-DA model
These 9 samples failed to meet the standards required for the 4 classes
This indicated that the PLS-DA method was inappropriate for building discrimination models of the 58 Corni Fructus samples tested in the present study
The unassigned samples further indicated that PLS-DA exhibited more stringent requirements over the other 3 modeling methods
The adequate size of the training set ensured that unassigned samples exhibited no significant influence on the 4 discrimination models
the limited size of the training set resulted in unassigned samples that could affect the model margin when recruited into other discrimination models
These discrepancies may be attributed to offset values that could be corrected by enlarging the training set and inspecting the sample processing
the new PLS-DA discrimination model was built
of which the accurate discrimination rate was increased to 97.92% by cross validation in the absence of unassigned samples
The data indicated that the PLS-DA model could probably act as a way to screen the sample library
Chart of scores on principle components in the PCA-DA model in the presence of only 3 classes
the mechanism of the e-eye sensor referred to sample discrimination by acquiring sample color information
The traditional “eye view” used for discrimination contained appearance information that was defined by “color” and “shape” (such as “quality evaluation based on shape grading”)
The E-eye sensor can provide valuable biological information and is an important tool that can be used for further development
The present study adopted 4 different mathematical statistics to construct models for analysis of Corni Fructus information collected by e-eye
The accurate discrimination rate of the constructed cross validation discrimination models was as follows: PCA-DA > LS-SVM > DA > PLS-DA
The models were all within the range of 81–92%
and optimal classification power was achieved
the accuracy rate was 91.38% and 98.28% for the selected PCA-DA cross validation and the full-sample models
indicating optimal predictive results in the classification of the quality of the Corni Fructus
of which the quality was closely related to color
the use of the e-eye sensor in this model provided a new conception to evaluate the quality or to implement grading that was different from the previous methods used by sensory organs or chemical constituents
The current study validated the feasibility of the e-eye in the ‘quality evaluation based on color grading’ for this type of medicinal materials
E-eye experiments were conducted with an IRIS VA400 e-eye (Alpha M.O.S
The instrument was equipped with 16 million color industrial camera and 6700 K color temperature D65 international standard light source
A total of 58 samples of Corni Fructus were from the primary places of origin and medicinal material markets distributed in Henan
Suiqing CHEN at Henan University of CM as the pulp of the ripe fruit of Cornus officinalis Sieb
in accordance with the “Chinese Pharmacopoeia I” (edition 2015)
the components Monoside (C17H26O11) and Loganin (C17H26O10) were identified in the water-soluble extracts of all individual samples examined
Sample grading was conducted by 6 specialists from Henan University with expertise in Chinese medicinal quality standards or authenticity analysis
They were familiar with all processes of Corni Fructus planting
the grading standard for Corni Fructus was established according to the “Chinese Pharmacopoeia” (edition 2015) and Corni Fructus commodity specification requirements stated in “commodity specification and grade standards for 76 Chinese medicinal materials”
and in accordance to relevant literature and the market circulation guidelines
A total of 6 specialists were trained according to the standard guidelines prior to the grading of 58 samples
The class of individual samples was determined by the principle of no less than 2/3 proportion
The samples with less than 2/3 specialists supporting a certain grade were excluded from this study
The E-eye was switched on and checked for light stability (green light) prior to the initiation of the detection
The calibration was performed with a 24-color correction board
and the background was eliminated by simultaneous turning on the upper and lower backlights with a 5 mm aperture
the samples of the unified thickness were placed evenly on the watch glass
followed by image acquisition for each sample
Triplicate images were acquired for each sample at exchanged positions
Raw images were processed with the software equipped with a RGB/L*a*b*/HSV multicolor processing system
The images were split into 4096 colors for analysis and similar colors were subjected to normalizing
thus generating the result of color distribution
Discrimination models were constructed respectively by four distinct methods including discrimination analysis (DA)
least squares-support vector machine (LS-SVM)
Linear discrimination analysis aimed to identify a projection direction in the high dimension space
which could separate samples of different classes
while cluster samples of the same class were remained as closely as possible at the projection point
It aimed to identify which samples enabled the minimum objective function:
referred to between-cluster variance and variance sum within clusters
new samples were projected with w and the location of the projection point determined the class of the new samples
SVM classification was essentially defined as the maximum margin linear classifier in the feature space
aiming to identify a hyperplane in the p-dimensional space and to separate two classes of samples as correctly as possible
LS-SVM was a simplified and improved version of SVM that preserved the capability of processing small sample sizes
The improvement was represented by two factors: (1) inequality constraints in SVM were revised to equality constraints and (2) loss function was revised to squares-error loss function
The objective function defined in LS-SVM was:
LS-SVM was used to map vector X with regard to the primitive space
the weight coefficient w could not be solved directly
the solution of the objective equation of LS-SVM could be achieved by constructing Lagrange function of the objective equation and converting to its dual problem
New samples were classified by the equation below:
PLS-DA was essentially a special form of PLS regression
It defined dummy variables of classes as Y according to a certain rule
thereby enabling classification by regression
PLS-DA merely solved binary classification problems
although the PLS algorithm could process multivariate dependent variable Y
it could be employed for the classification of multiple classes
with the exception of the PC number optimization
the other elements of the model were the same with those of DA
multi-class classification was implemented by expanding class variables into matrices
Since LS-SVM could merely solve binary classification problems
the realization of multi-class classification also relied on decomposing the raw multi-class classification problems into multiple binary classifications
of which the results were further processed
thus achieving the classification for new samples
Brief introduction of research methods of commodity specification and grade of Chinese medicinal materials
Authenticity analyses of Rhizoma Paridis using barcoding coupled with high resolution melting (Bar-HRM) analysis to control its quality for medicinal plant product
BOKP: A DNA Barcode Reference Library for Monitoring Herbal Drugs in the Korean Pharmacopeia
Identification of Dioscorea nipponica and its sects based on psbA-trnH barcode
Research progress on identification and quality evaluation of glues medicines
Exploring lipid markers of the quality of coix seeds with different geographical origins using supercritical fluid chromatography mass spectrometry and chemometrics
Research on quality standard of processed Cinnamomi Cortex decoction pieces containing volatile oil
Research on standard decoction of processed Curcumae Longae Rhizoma containing volatile oil
Analysis of visual quality attributes of white shrimp by machine vision
Analysis of Skin Color Development in Live Goldfish Using a Color Machine Vision System
Projective Mapping and Product Positioning of Deep Fat Fried Snack
Automated image analysis for high-content screening and analysis
High content screening of a kinase-focused library reveals compounds broadly-active against dengue viruses
near real-time acquisition of particle motion in rotating pan coating equipment using imaging techniques
Modeling the motion and orientation of various pharmaceutical tablet shapes in a film coating pan using DEM
Corni Fructus: a review of chemical constituents and pharmacological activities
et al A green ionic liquid-based vortex-forced MSPD method for the simultaneous determination of 5-HMF and iridoid glycosides from Fructus Corni by ultra-high performance liquid chromatography
et al Quality Evaluation of Polar and Active Components in Crude and Processed Fructus Corni by Quantitative Analysis of Multicomponents with Single Marker
et al Simultaneous Determination of 11 High-Polarity Components from Fructus Corni: A Quantitative LC-MS/MS Method for Improved Quality Control
UPLC-Q-TOF/MS-Based Metabolic Profiling Comparison of Two Major Bioactive Components and Their Metabolites in Normal and CKD Rat Plasma
Urine and Feces Following Oral Administration of Fructus Corni Extract
Todeschini R. Milano Chemometrics and QSAR Research Group. Available online, http://michem.disat.unimib.it/chm/download/classificationinfo.htm (accessed on 18 October 2018)
Brabanter, K. D. et al. LS-SVM Lab. Available online, http://www.esat.kuleuven.be/sista/lssvmlab/
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The present study was supported by the following funding: 2017 Chinese medicine public healthcare subsidy project “National Chinese Medicine Resource Survey Project” (Finance Society [2017] No
66); General Program of National Natural Science Foundation of China (No
81773892 & 81774452); Youth Program of National Natural Science Foundation of China (No
81603396); Special Research Project on Traditional Chinese in Henan Province (No
2018ZY2131); Henan Administration of Traditional Chinese Medicine National Clinical Research base of Traditional Chinese Medicine Research Project (No
2018JDZX087); Henan Province Natural Science Foundation Projects (No
162300410187); Henan Province Science and Technology Tackling Plan Project (No
Present address: Beijing Hospital of Traditional Chinese Medicine
The First Affiliated Hospital of Henan University of CM
Beijing Institute of Chinese Materia Medica
analyzed data and wrote the manuscript; Suiqing CHEN and Ruixin LIU designed experiments
analyzed data,wrote and revised the manuscript and coordinated the resources; Zhaozhou LIN and Pengju CHEN analyzed data; Yanli WANG and Lili WANG contributed reagents/materials and performed the E-eye test
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DOI: https://doi.org/10.1038/s41598-019-53210-5
Current Food Science and Technology Reports (2024)
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Introduction: Corni Fructus (CF) is a Chinese herbal medicine used for medicinal and dietary purposes. It is available commercially in two main forms: raw CF (unprocessed CF) and wine-processed CF. Clinical observations have indicated that wine-processed CF exhibits superior hypoglycemic activity compared to its raw counterpart. However, the mechanisms responsible for this improvement are not well understood.
Methods: To address this gap in knowledge, we conducted metabolomics analysis using ultra-performance liquid chromatography-quadrupole/time-of-flight mass spectrometry (UPLC-QTOF-MS) to compare the chemical composition of raw CF and wine-processed CF. Subsequently, network analysis, along with immunofluorescence assays, was employed to elucidate the potential targets and mechanisms underlying the hypoglycemic effects of metabolites in CF.
Discussion: These findings support the notion that CF exerted hypoglycemic activity via multiple components and targets, shedding light on the impact of processing methods on the chemical composition and hypoglycemic activity of Chinese herbal medicine.
Volume 14 - 2023 | https://doi.org/10.3389/fphar.2023.1173747
Introduction: Corni Fructus (CF) is a Chinese herbal medicine used for medicinal and dietary purposes
It is available commercially in two main forms: raw CF (unprocessed CF) and wine-processed CF
Clinical observations have indicated that wine-processed CF exhibits superior hypoglycemic activity compared to its raw counterpart
the mechanisms responsible for this improvement are not well understood
we conducted metabolomics analysis using ultra-performance liquid chromatography-quadrupole/time-of-flight mass spectrometry (UPLC-QTOF-MS) to compare the chemical composition of raw CF and wine-processed CF
was employed to elucidate the potential targets and mechanisms underlying the hypoglycemic effects of metabolites in CF
Results: Our results revealed significant compositional differences between raw CF and wine-processed CF
identifying 34 potential markers for distinguishing between the two forms of CF
wine processing led to a marked decrease in iridoid glycosides and flavonoid glycosides
Network analysis predictions provided clues that eight compounds might serve as hypoglycemic metabolites of CF
and glucokinase (GCK) and adenylate cyclase (ADCYs) were speculated as possible key targets responsible for the hypoglycemic effects of CF
Immunofluorescence assays confirmed that oleanolic acid and ursolic acid
significantly upregulated the expression of GCK and ADCYs in the HepG2 cell model
Discussion: These findings support the notion that CF exerted hypoglycemic activity via multiple components and targets
shedding light on the impact of processing methods on the chemical composition and hypoglycemic activity of Chinese herbal medicine
the bioactive chemical changes occurring during the wine processing of CF remain unclear
The strategy to discover potential hypoglycemic metabolites based on metabolomics and network analysis
and caffeic acid (Lot: 110885-201703) were all bought from China National Institutes for Food and Drug Control (Beijing
5-hydroxymethylfurfural (Lot: 191015-037) and cornuside (Lot: 190917-066) were purchased from Beijing Ya Xi’er Technology Co.
Astragalin (Lot: 19092602) was purchased from Chengdu Herbpurify (Chengdu
LC-MS-grade acetonitrile and methanol were supplied by Merck (Darmstadt
and LC-MS-grade formic acid was purchased from Acslabchem (ACS
Ultrapure water was supplied by Shenzhen Watsons Distilled Water Co.
CF samples were purchased from five herbal pieces factories in China and identified by Professor Zhiguo Zhang from The First Hospital of Hunan University of Chinese Medicine. The specific information is shown in Supplementary Table S1
According to the wine-processing methods of CF recorded in the Chinese Pharmacopoeia (National Commission of Chinese Pharmacopoeia
and the temperature set up at 115°C
then steamed with high-pressure wine for 1 h
Each batch of CF sample was weighed 100 g
extracted with 8 volumes of water and refluxed twice for 1 h each at 100°C
and subsequently lyophilized to prepare a CF extract powder
The 3 g powder was weighed precisely
and then was dissolved in a 30 mL 50% methanol
The solution was sonicated for 30 min
filtered and obtained the CF sample solution which was used for LC/MS analysis
added in a 25 mL volumetric bottle
and dissolved in methanol yielding a standard solution at 0.4 mg/mL
Molecule Feature Extractor of Masshunter Qualitative Analysis (Agilent
United States) was applied to analyze the primary and secondary mass spectrometry data
The identification of metabolites in CF were conducted through comparison with standards and MS/MS fragmentation and GNPS platform
UPLC-DAD was further performed to quantitatively analyze metabolites
“Homo sapiens” and p < 0.05 was selected as the standard of the KEGG pathway
HepG2 cells (Procell Life Science & Technology Co.
CL-0103) were cultured at 37°C in 5% CO2
the medium contained 10% fetal bovine serum (FBS) supplemented with 100 U/mL penicillin and 100 g/mL streptomycin (HyClone
United States) in high glucose Dulbecco’s Modified Eagle Medium (DMEM) (Gibco
HepG2 cells in logarithmic phase were seeded into 96-well plates for 24 h
Cell viability was examined using the CCK-8 kit (Bioss
HepG2 cells in the logarithmic phase were cultured into 96-well plates for 24 h
the medium was replaced with DMEM culture medium without FBS containing metabolites groups at different molar concentrations (10 μM
Ten tested groups were divided into control group
morroniside and active metabolites of CF (six standard solutions were mixed in equal proportions)
10 µL of CCK-8 was added to each well and cultured at 37°C for 1 h
The optical density was measured at 450 nm using a microplate reader (PerkinElmer
DMEM culture medium without FBS was replaced and cultured for 12 h
and the culture medium of drug groups was added for 24 h
the content of glucose in the supernatant of the culture medium was tested with a glucose detection kit (Robio
glucose consumption = glucose content of blank group-glucose content of drug group
Treated HepG2 cells were incubated for 24 h
and treated with 0.25% triton-100 for 15 min
blocked with 5% bovine serum albumin for 20 min
incubated with anti-GCK (1:100) and anti-ADCYs (ADCY2
ADCY8 and ADCY9) (1:100) in the dark overnight in a humidified container at 4°C
Following incubation with sheep anti-rabbit IgG (H + L) secondary antibody
The protein expression level and images were observed in laser scanning confocal microscopy
Statistical analysis was performed by SPSS 25.0
and mean ± standard deviation was used to express the data
The comparison between groups was conducted by one-way analysis of variance (ANOVA)
p < 0.05 indicated that the difference was statistically significant
UPLC fingerprints of different batches of CF extracts
(A) raw CF in positive ion mode; (B) wine-processed CF in positive ion mode; (C) raw CF in negative ion mode; (D) wine-processed CF in negative ion mode
The results demonstrated that all experimental samples were effectively separated into two main clusters in negative ion mode
S5 and P10 exhibited complete clustering with no distinguishable differences
Both HCA and PCA model suggested that processing altered chemical composition of CF
Metabolic phenotype differentiation between raw and wine-processed CF
(A) Positive ion mode of PCA score plot (R2X = 0.745
(B) Negative ion mode of PCA score plot (R2X = 0.689
(C) HCA model of raw and wine processed CF in positive ion mode
(D) HCA model of raw and wine-processed CF in negative ion mode
Identification of metabolites in raw CF and wine-processed CF samples by UPLC-QTOF-MS/MS
This phenomenon may be attributed to the compound’s poor stability
as they underwent hydrolysis and aglycon bond breakage under high temperatures
morroniside can transform into sarracenin by losing one sugar moiety
the structure of morroniside would be disrupted
the levels of gallic acid increased in wine-processed CF samples
suggesting a potential transformation of cornuside into gallic acid
Heat map of 34 metabolites of raw CF and wine-processed CF
The relative content changes of 9 metabolic markers
The content of 9 markers in different bathes of Corni Fructus samples (mg/g)
A comprehensive metabolites-targets-pathway interaction network (C)
GO enrichment analysis of potential targets (D)
we conducted the network topology analysis
were implied as key bioactive constituents
and GCK were possible to be the core targets which played important roles in CF against diabetes
these results hinted that CF might exert its hypoglycemic effect through the modulation of endocrine metabolism
To investigate the hypoglycemic activity of the active ingredients on HepG2 cells, we evaluated the cytotoxicity of these compounds (kaempferol, quercetin, oleanolic acid, ursolic acid, loganin, and morroniside) on HepG2 cell growth. The results showed that these tested compounds exhibited significant cytotoxicity towards HepG2 cells at concentrations ranging from 0.01 μM to 10 μM (Supplementary Figure S6)
glucose consumption experiments were conducted within this concentration range
These findings suggest that all these tested compounds have the potential to enhance glucose consumption in HepG2 cells
and metformin treatments led to significant upregulation the proteins of ADCYs
and quercetin specifically upregulated the protein expression of ADCY2
These results showed multi-component of CF may exert hypoglycemic activity via upregulation of GCK and ADCYs
ADCY9) in ten tested groups by immunoluorescence staining and relative luorescence intensity
Representative confocal images (A) and relative luorescence intensity of GCK (B)
Representative confocal images (A) and relative luorescence intensity of ADCY2 (C)
Representative confocal images (B) and relative luorescence intensity of ADCY3 (D)
Representative confocal images (E) and relative luorescence intensity of ADCY8 (G)
Representative confocal images (F) and relative luorescence intensity of ADCY9 (H)
our study employed metabolomics combined with network analysis to discover bioactive metabolites in raw CF and wine-processed CF
and to explore the underlying hypoglycemic mechanisms
significant changes in the composition of flavonoids were observed after wine processing of CF
kaempferol-3-O-α-L-rhamnoside was firstly discovered to generate during wine processing
while the presence of kaempferol-3-O-α-L-rhamnose-(1→6)-O-β-D-glucopyranoside was nearly undetectable based on our findings
through the analysis of downstream metabolites
we compared the levels of the kaempferol glycoside in raw CF and wine-processed CF
and observed a significant increase in kaempferol content following wine production
we cautiously propose that flavonoid glycosides might underwent deglycoside during the wine-process
Further investigation is needed to determine the potential correlation between these changes in chemical composition and the variation in hypoglycemic effects
these results showed CF metabolites significantly upregulated GCK and ADCYS expression
indicating that the interaction of the metabolites in a Chinese herbal concoction during the processing impelled the transformation of chemical metabolites
the efficacy of Chinese herbal medicine does not depend on one component entirely
It is the result of the combined action of a variety of indicator metabolites
our findings indicate that wine processing alterated chemical composition and hypoglycemic activity
the hypoglycemic of wine processed CF were correlated with the alterations of iridoids
Through the integration of network analysis
we have established the correlation between targets and small molecules
while we acknowledging the limitations of network analysis
including uncertainty and unreliability in predicted outcomes
experimental validation is crucial to ensure result reliability
we aim to assess these targets using in vivo studies and gain deeper insights into the underlying molecular mechanisms of action
a strategy by integrating network analysis and plant metabolomics was developed
to uncover the alteration of active metabolites of CF during wine processing and the potential hypoglycemic effects
34 markers in CF were tentatively identified using UPLC-Q-TOF-MS
the OPLS-DA model revealed that iridoids were the primary differential metabolites during wine processing
were speculated to be potential hypoglycemic metabolites of CF
GCK and ADCYs were inferred as hypoglycemic targets
Immunofluorescence assays verified that oleanolic acid and ursolic acid could upregulate the expression of GCK and ADCYs in the HepG2 cells model
suggesting the synergistic hypoglycemic mechanism of CF via multicomponent and multitarget interactions
This research contributes to a novel understanding of the impact of processing on the metabolites and hypoglycemic activity of Chinese herbal medicine
further inquiries can be directed to the corresponding author
JaL conducted cell experiments and wrote the manuscript
LT and JnL contributed to sample preparation
the final manuscript was confirmed by all the authors
All authors contributed to the article and approved the submitted version
This research was funded by the National Natural Science Foundation of China (No
and Hunan Administration of Traditional Chinese Medicine Project (No
Hunan Provincial Department of Education Project (No
Discipline construction of Hunan University of Chinese Medicine (No
The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fphar.2023.1173747/full#supplementary-material
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Ding C and Long H (2023) Changes in metabolites in raw and wine processed Corni Fructus combination metabolomics with network analysis focusing on potential hypoglycemic effects
Received: 07 March 2023; Accepted: 25 July 2023;Published: 07 August 2023
Copyright © 2023 Zhou, Liu, Tan, Wang, Li, Wang, Ding and Long. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use
*Correspondence: Hongping Long, bG9uZ2hvbmdwaW5nODRAMTYzLmNvbQ==
†These authors share first authorship
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Palazzo Madama-Turin’s Museo Civico d’Arte Antica (Museum of Ancient Art ) inaugurates
curated by Palazzo Madama in collaboration with the Francesco Corni Foundation
A visual narrative that welcomes visitors as they enter the museum
to tell the two thousand years of history of Palazzo Madama and Turin
where the architectural traces of this millennial evolution remain
the exhibition develops from some 30 plates created by the Modenese draftsman Francesco Corni: drawings that detail the main moments of the city’s growth and follow the building events of Palazzo Madama
They start from the foundation of Julia Augusta Taurinorum in the Augustan period
when the quadrangular layout of the city was set up based on the two main roads: the decumanus maxim us and the cardus maximus
It was during that period that the four monumental city gates were built
including the decumanus gate on which the Achaia castle was to be built
later to be transformed into Palazzo Madama in the 18th century.The route follows the evolution of the medieval city and the Phibellona gate that gradually replaced the decuman gate
the castle is transformed: in the 14th century Filippo d’Acaia attaches two square towers to the Roman towers and makes the new terracotta facing of the inner courtyard; in the following century Ludovico d’Acaia enlarges the building plan by adding two more towers symmetrical to the Roman ones
The castle became a princely residence with bedrooms
Emanuel Philibert of Savoy moved the capital of the Savoy duchy from Chambéry to Turin (1563) and began the fortification of the city with the creation of the citadel
The three successive enlargements completed the development of the capital city
almost tripling its size: with Charles Emmanuel I toward Porta Nuova (from 1619)
with Charles Emmanuel II toward the Po (from 1673)
Tables of the extensions alternate with drawings of Palazzo Madama at the time of the two Madame Royales of Savoy
Christina of France and Marie Jeanne Baptiste of Savoy Nemours
Christina’s intervention led to the covering of the Medieval Court and the creation of a great hall on the second floor
was responsible for renovating the first-floor apartment and commissioning Filippo Juvarra to build the monumental staircase and facade
a masterpiece of Baroque architecture (1718 - 1721)
This was followed by territorial expansion from the 1840s in the Porta Nuova area
the San Salvario district and the Vanchiglia area
with the construction of the landmark building of the Mole Antonelliana
The halls of Palazzo Madama see the succession of public offices
restoration work by architect Alfredo D’Andrade has led the monument back to its medieval appearance
The itinerary closes with the twentieth century and the arrival in 1934 of the Museo Civico d’Arte Antica in its new home in Palazzo Madama
Francesco Corni’s black ink drawings are based on a solid knowledge of historical
documentary and visual sources relating to the subjects depicted
Drawings that give visitors the opportunity to appreciate the most minute details and get a very concrete idea of how the city and palace looked in each historical period
The accompanying texts in the exhibition provide information about the history
The aim of the project is to restore to visitors’ sensibilities the essential role of Palazzo Madama in its being the beating heart of Turin
whose growth and development is reflected in the evolution of the city itself
A Palazzo Madama pivot of a short city system that sees
the development over the centuries of what has now become one of Europe’s great museum routes: from the Royal Church of San Lorenzo to Palazzo Chiablese
to the Royal Palace with the Chapel of the Holy Shroud and its archaeological areas
But also the State Secretariats with the prefecture
the Cavallerizza Reale and the Cinema Museum
the Accademia Albertina and the National Library
Palazzo Carignano and the Academy of Sciences with the Egyptian Museum
For info: www.palazzomadamatorino.it
Image: Francesco Corni, Palazzo Madama at the time of Marie Jeanne Baptiste of Savoy Nemours (Indian ink on tracing paper; 35 x 30 cm; Strambino, 1997).
Volume 8 - 2020 | https://doi.org/10.3389/fchem.2020.00340
This article is part of the Research TopicRecent Trends in Optical and Mechanical Characterization of NanomaterialsView all 5 articles
quantum tunneling) and recently developed by some of the present authors in Nanoscale
6004-6015 is applied to the calculation of the optical properties of complex Na
each atom of the nanostructures is endowed with an electric charge that can vary according to the external electric field
The electric conductivity between nearest atoms is modeled by adopting the Drude model
which is reformulated in terms of electric charges
Quantum tunneling effects are considered by letting the dielectric response of the system arise from atom-atom conductivity
ωFQ is challenged to reproduce the optical response of metal nanoparticles of different sizes and shapes
and its performance is compared with continuum Boundary Element Method (BEM) calculations
The wide applicability of the BEM approach is strictly related to its intrinsic low computational cost
which is due to the fact that the NP surface is discretized in terms of point complex charges that interact with the external electric field
the atomistic nature of the system needs to be explicitly taken into account
usually based on Density Functional Theory (DFT)
they cannot afford NPs of sizes larger than a few nanometers (hundreds of atoms) due to their high computational cost
An alternative fully atomistic classical model has recently been proposed by some of us (Giovannini et al., 2019e). Such an approach, named ωFQ (frequency-dependent Fluctuating Charges), is based on text-book concepts, i.e., the Drude model for conduction in metals, classical electrostatics, and quantum tunneling (Giovannini et al., 2019e)
Each atom of the NP is endowed with an electric charge that is not fixed but can vary as a response to the externally applied oscillating electric field
ωFQ indeed adopts the atomistic description introduced by Jensen and coworkers
but it sticks to the simplest possible assumptions
Drude-like conductance even between two (bonded) atoms instead of parameterizing the model based on accurate calculations or empirical inputs (nanoparticle size
ωFQ has been successfully applied to the optical response of subnanometer junctions
where quantum tunneling plays a crucial role
we apply the model to reproduce the optical properties of single NPs with different shapes and sizes
the main physical features of both ωFQ and BEM are briefly recalled and compared
the computational methodology is presented
ωFQ is tested against the reproduction of the absorption cross-sections of differently shaped Na
and its performance is compared with BEM results so as to highlight the similarities and differences of the two models
A section focusing on conclusions and future perspectives for the approach ends the presentation
the main features of the atomistic ωFQ and continuum BEM approaches are briefly recalled
the working equations of the two models are presented
and the conceptual differences between the two are discussed in detail
ωFQ is an atomistic approach aimed at describing the optical properties of a metal NP in the quasi-static limit. ωFQ has its theoretical foundation on the Fluctuating Charges (FQ) force field, which, combined with a QM description of a molecular solute, is usually exploited in the modeling of the spectroscopic properties of solvation phenomena (Cappelli, 2016; Giovannini et al., 2018, 2019b)
each classical atom is described in terms of a charge
which value can vary as a response of the external sources
each NP atom is modeled as a charge that varies as a response to an external oscillating electric field [E(ω)]
ωFQ charges are complex because the metal response includes a dissipative part
their imaginary part is directly related to the absorption cross-section (vide infra)
The equation of motion of ωFQs is written in terms of the Drude model of conductance (Bade, 1957), adequately reformulated in case of charges (Giovannini et al., 2019e):
where Aij is the effective area dividing atom i by atom j and Aij is a model parameter optimized to reproduce reference ab-initio data. n0 is the atomic electron density, < p > is the momentum of an electron averaged over all trajectories connecting i and j, and l^ji=-l^ij is the unit vector of the line connecting j to i. By replacing p with its expression in terms of the external electric field E(ω) within the Drude model (Giovannini et al., 2019e)
Equation (1) can be rewritten in the frequency domain as:
where τ is a friction-like constant due to scattering events
By then assuming <E(ω)>·l^ji≈(μjel-μiel)/lij
where μiel is the electrochemical potential of atom i and lij the distance between atoms i and j
with σ0 being the static conductance of the considered metals
a matrix named Kdru with elements Kijdru=2n01/τ-iωAijlij has been introduced
Equation (3) describes the electron transfer between all atoms constituting the metal NP
in order to make the model physically consistent
to avoid electron transfer between atoms that are too far apart
the pairs of atoms in Equation (3) are limited to nearest neighbors only
a Fermi-like f(lij) damping function is introduced to weight the Drude conductive mechanism:
Equation (4) can be rewritten by defining the electrochemical potential μ in terms of the external potential Vext:
where Dij is the electrostatic coupling kernel written in terms of Gaussian charges (Mayer, 2007; Giovannini et al., 2019c,e) and δij is the Kronecker delta
Equation (6) finally gives the complex ωFQs charges from which the complex polarizability α¯ and thus the absorption cross-section σabs can be recovered:
where μ is the complex dipole moment
and l runs over the x,y,z directions of the external electric field
and Im(α¯) is the imaginary part of the complex polarizability α¯
ωFQ has recently been developed and applied to describe the optical response of sodium (Na) nanoparticles (Giovannini et al., 2019e)
it describes the conductive electrons only by neglecting any possible contribution arising from d-electrons
the model can be used in its present form to model the optical properties of any metal at frequencies that are far from interband transitions
valid whenever the size of the NP is much smaller than the wavelength of the incident light
The BEM equation for solving the ASCs per tessera reads:
where En(ω) is the normal component of the applied electric field calculated at each charge position
whereas A is a diagonal matrix containing the tessera areas
D is a matrix that is defined in terms of the tesserae positions si and the outgoing normal unit vector per tessera ni as:
ACSs are complex quantities because dielectric functions are complex themselves
the absorption cross-section σabs is recovered from the definition of the complex polarizability α¯ij
which in turn is defined in terms of the complex dipole moment μ¯:
whereas i and j indicate Cartesian components
Atomistic ωFQ and continuum BEM approaches were challenged to reproduce the optical response of Na, Ag, and Au nanoparticles of different shapes, varying from cylindrical and pentagonal nanorods and spherical nanodomes (see Figures 1, 2 for NP structures)
Atomistic (left) and continuum (right) sodium NP structures
(A) Cylindrical nanorod with length l and radius r
(C) Spherical nanodome with internal radius r
The dome has been cut in half so as to best represent the structure
Atomistic (left) and continuum (right) silver and gold NP structures
(B) Pentagonal nanorod with length l and radius r
(D) Spherical nanodome with internal radius r
retaining the same packing and distances mentioned above in the case of nanorods
Different heights of the obtained dome were considered by retaining all atoms that are placed at a certain distance h from the top of the dome
All atomistic structures exploited for the following ωFQ calculations were obtained by using the Atomic Simulation Environment (ASE) Python module v. 3.17 (Larsen et al., 2017). The numbers of atoms in Na, Ag, and Au structures are reported in Tables S1–S8 (Supplementary Material)
In the case of spherical nanodome portions
the full tessellation contains 600 tesserae
BEM cross-sections were computed by using the MNPBEM Matlab toolbox (v. 17) (Hohenester and Trügler, 2012). Similarly to ωFQ simulations, Equation (8) was solved for steps of 0.01 eV. The dielectric functions defined in Equation (8) were recovered from experimental data (Rakić et al., 1998 for Ag and Au and Althoff and Hertz, 1967; Smith, 1969 for Na)
Both ωFQ and MNPBEM simulations were limited to the quasi-electrostatic (non-retarded) approximation because the size of the largest studied nanoparticle (nanorods with r = 50 Å and l = 150 Å) is much smaller than the computed absorption wavelength
In this section, ωFQ and BEM absorption cross-sections (σabs) of sodium, silver, and gold NPs of different shapes (see Figures 1, 2) are presented and compared so as to highlight the differences arising by exploiting atomistic/continuum approaches
The presentation of the computed results is divided into three sections
the first presenting a comparison between both ωFQ and BEM and ab-initio σabs for silver/gold pentagonal nanorods
The second and third sections are instead focused on Na NPs and Ag and Au NPs
This way of presenting the results is justified by the following: (i) the comparison with ab-initio results permits a quantitative analysis of the performance of the ωFQ and BEM approaches; (ii) Ag and Au are characterized by FCC packing
which makes the number of atoms of the structures rapidly incomparable; (iii) the Ag and Au structures are identical because they are characterized by the same lattice constant; (iv) both Ag and Au present inter-band transitions
whereas Na has a simpler dielectric response (interband transitions are
the d− electron screening is enhanced by the pre-resonance condition
The nanorods considered here have their resonance far from that region
and the shifts are dominated by atomistic effects that are grasped well by ωFQ
Figure 3. BEM(blue), ωFQ (green), and TD-DFT (red) absorption cross-sections σabs of silver (A) and gold (B) pentagonal nanorods with different numbers of atoms (see labels). TD-DFT data are reproduced from Sinha-Roy et al. (2017)
The aspect ratio (AR) of the selected nanorods is also given
Calculated BEM (A) and ωFQ (B) absorption cross-sections σabs of a sodium nanorod with r = 10 Å as a functions of l (see labels)
(C) ωFQ imaginary charges for a Na nanorod with r = 10 Å and l = 150 Å calculated at the PRFs of the two peaks highlighted in (B)
All σabs are normalized with respect to the volume of the considered structure
could in principle be coupled with our approach
We remark that some mixing with the dipolar plasmon is needed to see σabs≠ 0
To further investigate the performance of ωFQ and BEM, we selected a challenging system, i.e., a portion of a spherical Na (see Figure 1C), which has not been widely investigated before from both the theoretical and experimental points of view (Ye et al., 2009; Raja et al., 2016). In Figure 5A
the absorption cross-section of such structure as a function of the height h of the dome is reported
Notice that the internal r and external R radii are kept fixed to 40 and 50 Å
common to both ωFQ and BEM absorption spectra
• All spectra are characterized by an intense and sharp peak at about 1.5 eV (ωFQ) and 1.8 eV (BEM) that redshifts by increasing the height of the dome (i.e., by moving from h=25 to h=45 Å). Such a trend is in agreement with what has been shown above in the case of nanorods (see Figure 4)
increasing the height of the dome results in an increase in the number of atoms
results in a redshift of the absorption spectrum
BEM and ωFQ PRFs differ by almost 0.3 eV; this is probably due to a different description of the nanodome edges
• All spectra present a second low-in-intensity peak in the region between 2 and 3 eV
Both the intensities and PRFs of such bands show the same trends already commented on for the first intense peak
two additional bands arise for all structures in the region between 3 and 4 eV (BEM) and 4-5 eV (ωFQ)
It is worth noticing that for such a composite band
the trend reproduced by ωFQ PRFs is opposite with respect to the low-in-energy peaks
PRFs blueshift as the structures are enlarged
BEM PRFs are instead almost constant with an increase in the system's size
(A) BEM (top) and ωFQ (bottom) isotropic absorption cross-sections σabs of different sodium spherical nanodomes with r = 40 Å and R = 50 Å as a function of the height (h) of the dome (25 < h < 45 Å)
(B,C) Graphical depiction of the model exploited to explain the plasmonic character of the absorption peaks with co-axial and orthogonal polarizations
(D,E) ωFQ imaginary charges for a Na nanodome with r = 40 Å
and h = 45 Å calculated with co-axial and orthogonal polarizations at the PRFs of the two peaks (bonding and anti-bonding) highlighted in (A)
Top and bottom views of the dome are depicted
the basis of the shell is the only portion that is positively charged
and the external surface is globally negatively charged
such a mode is instead characterized by the internal surface negatively charged and the external positively charged
and the basis of the shell is almost zero charged
all the present features can entirely be explained by the suggested model
To conclude the discussion on Na nanodomes, it is worth remarking that Figure 5A reports isotropic cross-sections. Therefore, the bonding and antibonding modes result in a pair of peaks because co-axial and orthogonal polarizations are not degenerate. This can be further appreciated by looking at Figure S2 in Supplementary Material
Figure 6 presents BEM (top) and ωFQ (bottom) σabs of silver (left) and gold (right) pentagonal (Figures 4A,B) and cylindrical (Figures 4C,D) nanorods with radius r = 2.8 Å as a function of the length l (see Figures 2A,B for their structures)
BEM and ωFQ intensities were normalized with respect to nanorod volumes and the number of NP atoms
BEM (top) and ωFQ (bottom) absorption cross-sections σabs of pentagonal (A,B) and cylindrical (C,D) silver (left) and gold (right) nanorods with r = 2.8 Å as a function of l (see labels)
we remark that the atomistic structures exploited in ωFQ are unambiguously determined by the lattice constant of the studied material
different approximations need to be done to represent a given atomistic structure (smooth/sharp edges
and this constitutes a limitation of the BEM approach itself
ωFQ PRFs for cylindrical/pentagonal nanorods are instead almost identical for the largest nanorods (r ≥ 11.2 Å)
thus confirming that edge effects become more and more negligible as the radius of the nanorod increases
BEM (top) and ωFQ (bottom) absorption cross-sections σabs of pentagonal (A,B) and cylindrical (C,D) silver (left) and gold (right) nanorods with l = 150 Å as a function of r (see labels)
co-axial and orthogonal) give rise to two non-degenerate absorption peaks due to the symmetry breaking that has been introduced by cutting the pristine sphere in half (see also the Computational Details Section)
ωFQ predicts a pair of two high-energy peaks
which can instead be related to antibonding plasmonic modes
Notice that the latter bands undergo a blueshift as the NP number of atoms increases
thus resulting in an opposite behavior with respect to that underlined in the case of the bonding modes
it is worth noticing that the discussed antibonding excitations are not fully reported for BEM
This is due to the fact that the experimental permittivity function (ε(ω)) adopted to model the plasmonic response of the two metals (see Equation 8) is not defined in the considered energy range
BEM (top) and ωFQ (bottom) isotropic absorption cross-sections σabs of silver (A) and gold (B) spherical nanodomes with r = 25 Å and R = 30 Å as a function of h (15 < h < 25 Å)
the potentialities of the atomistic ωFQ approach to describe the optical properties of sodium
and gold NPs characterized by different geometrical arrangements have been investigated
and theoretical analogies and differences between ωFQ and BEM have been discussed and analyzed in terms of the physical quantities underlying both approaches
The main difference between ωFQ and BEM lies in the fact that in the former
the atomistic nature of the NP is retained
the NP is modeled through its surface only
both models describe the polarization in terms of complex charges
which are placed on each NP atom in the case of ωFQ
they are located at points defined through the discretization procedure of the NP surface
Similar results have been obtained for Ag and Au structures
In the case of cylindrical and pentagonal nanorods
the discrepancies between ωFQ and BEM PRFs tend to disappear with an increase in the size of the studied structures
The results obtained with the two methods are similar in the case of metal nanodomes
for which bonding and anti-bonding plasmonic modes are correctly described by ωFQ
Antibonding excitations have not been studied by BEM
because the experimental permittivity function (ε(ω)) of the two metals is not available in the considered energy range
Although this drawback can be easily overcome by modeling the permittivity function with a Drude model
the quality of BEM results would inevitably lose accuracy due to the inaccurate description of interband transitions
Such an extension is currently under development and will be a topic of future communications
The datasets generated for this study are available on request to the corresponding author
TG wrote the stand-alone Fortran95 code for ωFQ calculations
and GG analyzed data and wrote the manuscript
and CC discussed and supervised the whole project
This work has received funding from the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation program (grant agreement No
GG and SC acknowledge funding from ERC under the grant ERC-CoG-681285 TAME-Plasmons
TG acknowledges funding from the Research Council of Norway through the grant TheoLight (grant no
We are thankful for the computer resources provided by the high-performance computer facilities of the SMART Laboratory (http://smart.sns.it/)
The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fchem.2020.00340/full#supplementary-material
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Received: 25 November 2019; Accepted: 01 April 2020; Published: 07 May 2020
Copyright © 2020 Bonatti, Gil, Giovannini, Corni and Cappelli. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY)
*Correspondence: Tommaso Giovannini, dG9tbWFzby5naW92YW5uaW5pQG50bnUubm8=; Chiara Cappelli, Y2hpYXJhLmNhcHBlbGxpQHNucy5pdA==