Front.Phys.,2014,9(1):31–46DOI 10.1007/s11467-013-0347-3
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Fundamental studies on enhancement and blinking mechanism of surface-enhanced Raman scattering (SERS)and basic
applications of SERS biological nsing
Yuko S.Yamamoto 1,Mitsuru Ishikawa 2,Yukihiro Ozaki 3,Tamitake Itoh 1,†
1Nano-Bioanalysis
Rearch Group,Health Rearch Institute,National Institute of Advanced Industrial Science and
Technology (AIST),Takamatsu,Kagawa 761-0395,Japan
2Department
of Chemistry,Josai University,Itado,Saitama 350-0295,Japan
3Department
of Chemistry,School of Science and Technology,Kwani Gakuin University,Sanda,Hyogo 669-1337,Japan
Corresponding author.E-mail:†jp
Received April 1,2013;accepted May 20,2013
We review recent our results in the fundamental study of surface-enhanced Raman scattering (SERS)with emphasis on experiments that attempted to identify the enhancement and blinking mechanism using single Ag nanoparticle dimers attached to dye molecules.The results are quantitatively discusd in the framework of electromagnetic mechanism.We also review recent our results in basic SERS applications for biological nsing regarding detections of cell surface molecules and distinction of dia marker molecules under single cell and single molecule level.
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Keywords plasmonics,surface-enhanced Raman scattering (SERS),surface-enhanced fluorescence,Ag nanoparticle
PACS numbers 78.30.-j,78.67.Bf,78.67.Sc,33.20.Fb
Contents
1Introduction
312Enhancement and blinking of SERS
quantitatively evaluated by EM mechanism 322.1Introduction
322.2Two-fold EM enhancement in SERS
evaluated by EM mechanism
322.3SERS blinking quantitatively treated by
EM mechanism 332.4Summary
363Applications of SERS to biological nsing 363.1Introduction
363.2Advantages of SERS for bionsing 363.3Applications of SERS to cell nsing
373.4Applications of SERS to biomarker nsing 383.5Summary 424Conclusion
42Acknowledgements 42References
42
1Introduction
Nanostructures and nanoparticle (NP)aggregates of plasmonic metals (e.g.Ag and Au)generate strong en-hancement of Raman scattering intensity from molecules adsorbed on their surfaces.This phenomenon is widely known as surface-enhanced Raman scattering (SERS)[1–4].In particular,huge enhancement factors of SERS (1010to 1014)from molecules located in the NP gaps of aggregates allow us to nsitively measure spectra of analytes at single molecule (SM)level [5–9].The high-nsitivity and lectivity of SERS motivate rearchers to extend its application towards bionsing [10–13].However,despite the significant impact of SERS in basic rearch,fundamental issues such as lack of conclusive experimental evidence for validating the mechanism un-derlying the enhancement and blinking have prevented us from clear understanding of SERS,and furthermore difficulty in finding out potential SERS applications lim-its its public recognition.
It is propod by many rearchers that there are two
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enhancement mechanisms of SERS[3,14].The two mechanisms are called the electromagnetic mechanism (EM)and the chemical mechanism,respectively.The EM mechanism is characterized by twofold plasmonic EMfield enhancement of Raman scattering signals from molecules adsorbed on pl
asmonic metal NPs or NP ag-gregates[3,7,14–20].Chemical mechanism is character-ized by shifting of Raman scattering in non-resonance to that in resonance through the formation of charge trans-fer complexes between adsorbed molecules and metal surfaces[2,21–25].Both mechanisms have been exper-imentally investigated in detail and have been found to be correct[16,22,23,26–28].Accordingly quantitative evaluation of SERS bad on exclusive one mechanism is important in an effort tofind out which mechanism is dominant.EM mechanism has universality for every molecular specy.Thus,we have investigated the origin of enhancement and blinking in the framework of EM mech-anism.Bad on the mechanism we have studied SERS bionsing regarding detections of cell surface molecules and distinction of dia marker molecules to explore the real application of SERS[29–34].
In the prent mini-review,we focus our attention on two topics below in SERS studies.First,in Section2, we summarize results of our recent experimental inves-tigations to validate EM mechanism by quantitatively evaluate enhancement factors of SERS[16,35].Micro-spectroscopy using single Ag NP dimers enables us to quantitatively evaluate SERS spectra by EM mecha-nism excluding inhomogeneity induced by NP dimer-by-dimer variations in SERS spectra[16,35,36].Ag NP dimers,who nano-gaps are the minimum unit generat-ing SM SERS,directly illustrate relationship
among the enhancement factors,SERS spectra and plasmon reso-nance spectra by comparing with shapes of Ag NP dimers [37].The relationship provided us not only quantitative verification of EM mechanism in SERS but also mecha-nism of SERS blinking as an extension of EM mechanism [38].Second,in Section3,we explored basic applications of SERS using four types of biological targets;yeast, helicobacter li,and hemoglobin A1c,as po-tentially being incorporated to future progress in SERS bionsing[29–34].The equipment ud in the ction is common to that for studies on EM mechanism as a platform so that we succeeded to identify the molecu-lar species of proteins generating SERS signals on living single yeast cell wall at single molecular level.In this c-tion we also briefly summarize the advantages of SERS overfluorescence spectroscopy for label-free detection of biomolecules by taking examples from our own investi-gations.Finally,in conclusion,we correlate our recent findings with potential applications of SERS.2Enhancement and blinking of SERS quantitatively evaluated by EM mechanism 2.1Introduction
In this ction,we describe EM mechanism of SERS and quantitatively demonstrate its absolute validity.Ag NP dimer-by-dimer variations are well-explained by the lective enhancement of SERS bands who maxima are clo to the plasmon resonance maxima through cond EM enhancement,that is,coupling of plasmon resonance and Raman light[16].Experimental obrva-tion
s of plasmon resonance,SERS,and shapes of the Ag NP dimers were compared with FDTD(Finite-difference time-domain)calculations,which enable us to reproduce spatial and spectral distribution of EMfields around Ag NP dimers[16].The experimental enhancement factors were quite consistent with the calculations,indicating that EM mechanism has dominant role in SERS.Thus, we applied EM mechanism to evaluate SERS blinking [38].
2.2Two-fold EM enhancement in SERS evaluated by EM mechanism
To quantitatively evaluate the EM mechanism,we have examined two types of experimental obrvations:(i)Ag NP dimer-by-NP dimer variations in SERS spectra[35]; (ii)Quantitative evaluation of SERS spectra bad on EM mechanism[16].
We explain here an theoretical outline of the EM mech-anism.A Raman process is compod of an excitation and an emission process.In the process,a molecule and afield exchange light energy.The rates of the ex-change are enlarged by increasing in local mode density of thefield in the vicinity of plasmonic metal NPs be-cau of their large conduction electron density and their long oscillation time.Thanks to the increasing in local mode density,both Raman excitation and emission pro-cess become efficient.In other words,Raman excitation obtains the enhancement due to couplin
g of incident light with plasmon,which is calledfirst EM enhancement[15, 17],and Raman emission obtains that due to coupling of Raman light with plasmon,which is called cond EM enhancement[15,17].Note that a broad plasmon reso-nance line width(∼200meV)enables EM enhancement to work on both Raman excitation and the Raman emis-sion transition rates of a molecule.The highest enhance-ment factors of SERS are theoretically and experimen-tally obrved for molecules located at junction of metal NP dimers.The factors are up to1010−14,allowing us to
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measure SERS spectra at SM level.
The twofold EM enhancement provides us with a sim-ple expression for total SERS enhancement becau it is a product of EM enhancement of the incident and scattered light coupled with plasmon.Thus,the total enhancement factor M EM of SERS is given by [15,17]|M EM (λL ,λL ±λR )|2
= E Loc (λL )E I (λL ) 2×
E Loc (λL ±λR )E I (λL ±λR ) 2
=|M 1(λL )|2×|M 2(λL ±λR )|2e的用法
(1)
where E Loc and E I is localized and incident EM field
amplitude,respectively.M 1and M 2are first and cond EM enhancement,respectively.λL and λL ±λR are ex-citation light and Raman light wavelength,respectively.More details in the Eq.(1)have been provided elwhere [36].It is not difficult to understand |M 1|2becau it is increment in Raman excitation EM field intensity by plasmon resonance,but not so for |M 2|2.Thus,demon-stration of |M 2
|2is a key to understand “twofold”EM enhancement.The important point is that the spectrum of |M 2|2is expected to be similar to that of plasmon resonance becau the cond enhancement is produced by scattering of Raman light through plasmon resonance [15,17].Thus,to experimentally evaluate two-fold EM enhancement,we identified the cond EM enhancement factor |M 2|2as dependence of SERS spectra on plasmon resonance maxima.
We summarize here how we experimentally evaluated |M 2|2.It is difficult to identify |M 2|2as dependence of SERS spectra on plasmon resonance maxima,becau EM fields on larger Ag NP aggregates are complex due to overlapping between dipolar and multipolar plasmonic EM fields and the overlapping may break optical reci-procity.This difficulty can be resolved by lecting Ag NP aggregates which mainly show dipolar plasmon reso-nance exclusively coupled with SERS.Such Ag NP aggre-gates satisfy the following two criteria:(i)Polarization dependence of a plasmon resonance maximum follows a cosine-squared law,(ii)SERS maxima and plasmon res-onance maxima have the same polarization dependence to each other [16].We experimentally confirmed that Ag NP aggregates satisfying such criteria are always dimers when plasmon resonance maxima located around 600–680nm [16].We detected Ag NP dimer-by-dimer varia-tions of SERS spectra of rhodamin 6G (R6G)molecules (∼10−8M)bad on the criteria and the variations well explained in ter
ms of the dependence of plasmon res-onance spectra [35].The explanations mean that EM mechanism certainly exists in SERS.The result moti-vates us to quantitatively reproduce SERS spectra also
by EM mechanism.SERS spectra from single Ag NP dimers were evaluated to determine one-to-one relation-ship among plasmon resonance,SERS,and the shapes of the dimers measured by scanning electron microscopy (SEM).The experimental obrvations were compared with FDTD calculations of the EM field induced by plas-mon resonance using individual shapes of the dimers.The experimental enhancement factors of SERS ∼109were well consistent with that of the calculations within a factor of ∼2.The consistency fortifies the indispensible importance of EM mechanism in SERS [35,37].2.3SERS blinking quantitatively treated by EM mechanism
We discuss here the origin of SERS blinking bad on EM mechanism.Blinking is well known as intrin-sic fluctuation and intermittent of emission light from single quantum systems, e.g.quantum dots,single-molecular detections including SERS.SERS blinking has extensively been studied for more than a decade by various approaches [5–7,39,40].Here reprenta-tive “blinking mechanisms”for SERS are briefly sum-marized:molecules activated to metastable nonabsorb-ing and nonemissive states [41,42],the molecules in thermal diffusion in-and-out of hot spots [43],molec
ules in thermal diffusion on the nanoparticle surface coupled with photo-induced electron transfer,the structural re-laxation of surface active sites [44],thermally-stimulated molecular reorientation and chemical process [45],pho-toionization via charge-transfer states [46],and the mor-phology rearrangement of the metallic substrate [47].However,analys of SERS blinking in the previous works are limited to qualitative approaches.Complex-ity in the blinking mechanisms including unclear SERS mechanism prevent us from examining direct correlation between blinking and its origins.Thus,we quantitatively analyzed SERS blinking in terms of intensity and spec-tral instability bad on EM mechanism under the com-mon experimental conditions.In short,we lected Ag NP dimers adsorbed to R6G as a target molecule to ex-clusively analyze blinking by EM mechanism [38].
thefreeFor quantitatively analysis in SERS blinking,we -lected Ag NP dimers bad on the criteria [16]and mea-sured SERS,surface-enhanced fluorescence (SEF),and plasmon resonance spectra.Ag NPs were prepared by the Lee and Meil method [48]and added NaCl (10mM)and R6G (∼10−8M)to form SERS-active Ag NP aggre-gates.The R6G-adsorbed Ag NP aggregates including dimers were randomly immobilized onto a glass plate by spin-coating.A green lar beam from a cw Nd 3+:YAG lar (532nm)was introduced as a light source of SERS
R EVIEW ARTICLE and SEF.Plasmon resonance spectra coming from each
Ag NP dimer were measured under dark-field condenr
using white-light from a50-W halogen lamp as described
tramplein Ref.[49].The morphology of each Ag NP dimer was
obtained using SEM.To addfluctuations which inducenoneofyourbusiness
SERS blinking on Ag NP dimers,we ud1064-nm lar
puls from a cw Nd3+:YAG lar.Further detailed ex-
perimental condition has been described in the report
[38].
Here we outline EM enhancement factor of SEF M SEF.
M SEF includes M EM,which is shown in Eq.(1),and ad-
ditionally includes an enhancement factor of the decay
rate M d for a molecule in the excited state due to res-
onance energy transfer from a molecule to a Ag dimer.
Thus,
|M SEF(λL,λL±λR)|2
=|M1(λL)|2×|M2(λL±λR)|2
|M d(d eff)|2
=|M EM(λL,λL±λR)|2
|M d(d eff)|2(2)
where d effis effective distance between a molecule and a
metal surface.More details in the Eq.(2)have been pro-
vided elwhere[38].To quantitatively confirm the ori-
gin of SERS blinking,we lected SERS and SEF active
Ag NP dimers.Figures1(a1)–(a3)shows Ag NP aggre-
gates showing dipolar plasmon resonance who maxima
located around600–680nm.As indicated in the previ-
ous ction such Ag NP aggregates generating detectable
SERS and SEF spectra like Figs.1(b1)–(b3)were always
dimers as Figs.1(c1)–(c3)[16].We evaluated plasmon
resonance and shapes of12Ag NP dimers that show
parallelism
SERS and SEF activity then calculated plasmon res-
masqueradeonance and SERS spectra by FDTD calculation bad
on EM mechanism.The calculated spectra are quanti-
tatively consistent with experimental ones as described
[16].
SERS blinking means both drastic spectral changes
and total intensity changes,meaning temporal spectral
intensity instability in SERS.To quantitatively analyze
the temporal spectral instability,we examined spec-numeric
tral changes in SERS and SEF for the same Ag NP
dimer and theoretically restructured the spectra
using
Fig.1Fundamental obrvation from Ag NP dimer adsorbed by R6G:(a1)–(a3)spectra of plasmon resonance,(b1)–(b3)tho of SERS and SEF,and(c1)–(c3)SEM images obtained from three Ag dimers.Model for a Ag dimer generating SERS:(d1)electricfield distribution around a Ag dimer calculated by FDTD,(d2)enlarged image of a crevas in the Ag dimer,and(d3)assumed location of a R6G molecule at the crevas.Reproduced from Ref.[38].
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pupil怎么读音Fig.2Origin of SERS blinking:(a)Temporal profile of SERS intensity blinking including SEF blinking from a repren-tative SERS generated from an Ag NP dimer.Intensity is integrated from 540to 700nm,(b1)–(b3)spectral changes in SERS and SEF from the common Ag dimer in (a),time lap for each measurement is indicated in each panel.Exposure time for spectral detection is 5s,(c)definition of SERS and SEF intensities along wavelength for analysis of S (normalized standard deviation scores of blinking)dependence of |M d |2in SERS fluctuated region,(d)S dependence of |M d |2in fluc-tuated region for eight dimers,(e)|M EM |dependence of |M d |2for the eight dimers,(f )schematic of the origin of SERS and SEF blinking.Reproduced from Ref.[38].
EM mechanism.Figure 2shows the summary of the analysis [38].Figure 2(a)shows temporal total inten-sity instability in both SERS and SEF from a Ag dimer.Figures 2(b1)–(b3)show temporal spectral instability in SERS and SEF.Figure 2(c)indicates definition of SERS and SEF intensities.Using the definition,we can exper-imentally derive |M EM |and |M d |2[38].We quantified the intensity instability in SERS by normalized stan-dard deviation scores (S )dependence of |M d |2.Regard-ing that |M d |2is nsitive to effective distance between a molecule and a metal surface d eff[38],S dependence of |M d |2shown as Fig.2(d)indicates that the instabil-ity is induced by the increasing in d eff.|M EM | |M d |2shown as Fig.2(e)is reasonable,becau |M EM |induced by radiati
ve plasmons and |M d |2induced by both radia-tive and nonradiative plasmons.Regarding that |M d |2is more nsitive to d effthan |M EM |,the instability may be
a main fluctuation in SEF.Figure 2(f)shows a schematic of origin of SERS instability as a molecular fluctuation within veral angstroms [38].
In short,experimental evaluation of changes in EM en-hancement factor spectra has been demonstrated along the changes in plasmon resonance,explaining spectral instability in SERS and SEF spectra.The quantitative analysis reveals two new physical insights into blinking as follows.(i)The intensity instability is inverly pro-portional to the enhancement factors of decay rate of molecules.The estimation using the proportionality sug-gests that intensity instability is induced by fluctuations in paration of the molecules from Ag NP surfaces by veral angstroms.(ii)The spectral instability is induced by blue-shifts in EM enhancement factors,which have spectral shapes similar to the plasmon resonance (data shown in Ref.[38]).This analysis provides us a quantita-
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tive picture for intensity and spectral instability in SERS and SEF within the framework of EM mechanism.The work reveals that SERS blinking is quantitatively clar-ified by EM mechanism.Note t
hat in the ca of non-resonant molecules,the effect of EM mechanism for the SERS blinking activity is unclear.However,we comment that following three points may be the keys for determin-ing the blinking activity of non-resonant molecules:(i) more confined hotspots are required to obtain higher EM enhancement from non-resonant molecules so that non-resonant molecules may be more nsitive to molecular fluctuation than resonant molecules,(ii)SERS blinking is expected to be smaller for non-resonant molecules be-cau of weaker contribution of SEFfluctuation,and(iii) forming CT complexes will give weaker SERS blinking. We note that the evaluation does not mean that the instability in SERS and SEF can be totally explained by EM mechanism excluding other ones compod of other Ag NPs and pyridine molecules show-ing strong chemical effect[28,50].We also underline that the current work does not identify the exclusive ori-gin of blinking.The point is that blinking is correlated with the photo-excitation/emission mediated with the plasmon resonance[38].The fact therefore suggests that the underlying reason for blinking is a photo-induced ef-fect,such as thermal heating,photo-bleaching,or photo-induced diffusion,rather than purely chemical effects in the ca of the system compod of Ag NPs and R6G molecules.We need further evaluation in SERS blinking to clear the exclusive origin of blinking in each system and we also believe our recent studies give help for fur-ther investigation.
2.4Summary四六级口语
We have quantitatively investigated the EM mechanism of SERS and demonstrated its absolute validity[16].The intensity and spectral instability in SERS and SEF were also quantitatively analyzed.Experimental evaluation of SERS blinking revealed that changes in molecular loca-tions and plasmon resonance cau intensity and spectral instability in SERS spectra,respectively.The prent work reveals thatfluctuation in SERS including blink-ing is quantitatively clarified by EM mechanism[38]. However,the prent evaluation does not mean that the SERS enhancement mechanism can be totally explained by EM mechanism excluding other ones becau we -lected Ag NPs/R6G system to quantitatively evaluate the phenomena of SERS by only one mechanism.In par-ticular SERS blinking,we believe the prent achieve-ment could help us to evaluate the degree of other mech-anisms.3Applications of SERS to biological nsing
3.1Introduction
A large number of reviews and articles on various ap-plications of SERS to biological nsing have been pub-lished[51–98]including biomedical applications[51–62], cellular probing[63–68],in vivo cell probing[69–79],in vitro cell analysis[80,81],imaging of individual cells[82, 83],differentiating cancer cells[84],imaging of proteins [85–88],bacteria detection[89–93],virus detection[89–93]and etc.The rearches have mainly been carried out using confocal Raman microscopic systems under non-res
onant Raman excitation conditions.The stand-points of our SERS applications compared with others are u of simple non-confocal Raman microscopic sys-tems,resonant Raman excitation conditions,and plas-monic imaging with dark-field microscopy.The stand-points enable us to easily measure resonanct SERS im-ages at single molecule conditions checking adsorption of plasmonic metal nanoparticles on samples;for example, we succeeded to identify proteins generating SERS sig-nals from single Ag NP dimers on living single yeast cell wall at single molecular level.
In this ction,we briefly summarize the advantageous point of SERS for label-free detection of biomolecules overfluorescence spectroscopy.Our rent progress in SERS applications for biological nsing using four types of biological targets;yeast,helicobacter li, and Hemoglobin A1c(HbA1c)are also introduced here.
3.2Advantages of SERS for bionsing
For the analysis of single biomolecules,fluorescent la-bels,such as dye molecules or quantum dots,have been well developed[100].However,photobleaching or pH-dependence of such labels still prevents us from stable and long-time obrvation.Raman spectroscopy has cer-tain advantages becau the vibrational bands especially in thefingerprint region are sharper thanfluorescence ones.
The u offluorescent tags is suffered from con-fud overlappingfluorescence spectra,which are broader than Raman spectra.Non-uniform photobleaching rates of eachfluorescent tags also lead us to veral potential complications[10].Thus,Raman spectroscopy is uful for well-defined discrimination of molecular species in-cluding biomolecules.However,cross-ctions of Raman scattering(∼10−30cm2)is quite low compared with ab-sorption cross-ctions offluorescence(∼10−16cm2)so that there is a problem of nsitivity in Raman spec-troscopy for bionsing[44].SERS resolves the problem
