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  • www.rsc.org/pccp Volume 10 | Number 28 | 28 July 2008 | Pages 40694200

    ISSN 1463-9076

    Physical Chemistry Chemical Physics

    PERSPECTIVEKremsCold controlled chemistry

    COVER ARTICLEBlackie et al.Bi-analyte SERS with isotopically edited dyes

  • Bi-analyte SERS with isotopically edited dyes

    E. Blackie,*ab E. C. Le Ru,ab M. Meyer,ab M. Timmer,bc B. Burkett,b

    P. Northcoteb and P. G. Etchegoinab

    Received 4th March 2008, Accepted 21st April 2008

    First published as an Advance Article on the web 3rd June 2008

    DOI: 10.1039/b803738h

    Isotopically substituted rhodamine dyes provide ideal probes for the study of single-molecule

    surface enhanced Raman scattering (SM-SERS) events through multiple-analyte techniques.

    Isotopic editing should, in principle, provide probes that have identical chemical properties (and

    surface chemistries); while exhibiting at the same time distinct Raman features which enable us to

    identify single-molecule SERS events. We present here a specific example of two-analyte

    SM-SERS based on the isotopic substitution of a methyl ester rhodamine dye. The dyes are

    carefully characterized (in both standard and SERS conditions) to confirm experimentally their

    similar chemical properties. We then demonstrate their utility for bi-analyte SERS (BiASERS)

    experiments and, as an example, highlight the transition from a single, to a few, to many

    molecules in the statistics of SM-SERS signals.

    I. Introduction

    The detection of single-molecule surface enhanced Raman

    scattering (SM-SERS) has recently entered a new phase in

    its development with the introduction of multiple-analyte

    techniques15 to identify, categorize, and classify single mole-

    cule events in SERS. The technique (together with the math-

    ematical groundwork based on a modification of principal

    component analysis6 (PCA) for a fast and unbiased analysis of

    the data) has recently been explained in full length in ref. 7,

    which will serve, accordingly, as a basis for the discussion of

    the results presented here. Multiple analyte techniques, like the

    bi-analyte SERS (BiASERS) method proposed in ref. 1, work

    basically as a contrast method to observe the statistics of single

    molecule events related to one dye in the background of the

    signals produced by the other (or others). Hence, the method

    provides an additional degree of freedom with respect to plain

    intensity fluctuations8 to decide on the single molecule char-

    acter of the signal. It has, for example, been used for an

    accurate estimation of single-molecule SERS enhancement

    factors.4 Underlying the comprehension of SM-SERS spectra

    is the extreme nature of the statistics of single molecules events

    in-and-around the so-called electromagnetic (EM) hot-spots,

    which typically display a long-tail distribution of enhance-

    ments; a topic studied in full detail in ref. 9. In a recent

    development, Dieringer et al.5 moved the BiASERS technique

    to a new level of sophistication with the introduction of

    isotopic editing. We believe this to be an important develop-

    ment in the field and our work here builds on this previous

    work. In what follows, we provide a brief overview of the

    present status, an outlook into the application of isotopic

    editing for SM-SERS, and justification for its importance.

    From a purely experimental point of view, there are many

    variables that can be optimized in multiple analytes techniques

    for SM-SERS to pin down single molecule events more effi-

    ciently and simplify the interpretation. The nature of the

    probes stands out as one of the most important first steps to

    a successful implementation of the concept. As a case in point:

    in the two analyte version of the technique (BiASERS) studied

    in ref. 13 and 7 one would ideally like to study (for example)

    two molecules that have different SERS spectra but identical

    chemical properties. In particular, one would like the surface

    chemistry of the probes (in connection with their interaction

    with the metal substrate producing the SERS enhancement;

    typically silver or gold) to be as similar as possible; if not

    identical. One would also like to compare single molecule

    fluctuations of SERS peaks that have very similar SERS cross

    sections (and should therefore have a similar resonance condi-

    tion with the excitation wavelength). Otherwise, the statistics of

    single molecule events in SERS could conceivably be biased

    towards one dye (or the other), depending on the different

    sticking properties to the metal surface, which in turn affects

    the assumed nominal concentrations for the statistical analysis

    of the results. This results in relatively strong constraints for

    the selection of the probes, and one usually has to recourse to a

    compromise. In the previous reports of BiASERS experiments,

    a compromise was achieved among the desired properties by

    means of specific dyes. In ref. 1 the partner probes were a

    benzotriazole dye10 and Rhodamine 6G (RH6G). Ref. 3, on

    the other hand, used n-pentyl-5-salicylimidoperylene and octa-

    decylrhodamine B together with the technique of Lang-

    muirBlodgett films, while ref. 2 made use of the

    combination of the two related probes 4,40-bipyridine and

    2,20-bipyridine. More recently4,7, Nile Blue (NB) and RH6G

    have also been used as successful BiASERS partners for SM-

    SERS. While all of these combinations proved the multiple

    analyte concept was a successful indicator of SM-SERS, one

    a The MacDiarmid Institute for Advanced Materials andNanotechnology, Victoria University of Wellington, PO Box 600,Wellington, New Zealand

    b School of Chemical and Physical Sciences, Victoria University ofWellington, PO Box 600, Wellington, New Zealand

    c The Malaghan Institute of Medical Research, PO Box 7060,Wellington, New Zealand

    This journal is c the Owner Societies 2008 Phys. Chem. Chem. Phys., 2008, 10, 41474153 | 4147

    PAPER www.rsc.org/pccp | Physical Chemistry Chemical Physics

  • could argue that the combination of 4,40-bipyridine and 2,20-

    bipyridine in ref. 2 is perhaps the best among this list, in the

    sense that it uses two probes which are the closest in both

    resonance conditions and surface chemistry.

    Isotopic editingwith a long and well established tradition

    in spectroscopyappears as one of the best solutions to this

    problem. If isotopic editing of a useful SERS probe (with high

    SERS cross sections) results in a spectrum with distinguishable

    features compared to the standard (unedited) version of the

    same molecule, we have then two probes thatfor all practical

    purposesshould have exactly the same chemical properties

    but can still be distinguished by their SERS signals. This is

    particularly important from the point of view of the surface

    chemistry of the probes.

    It is interesting to note that isotopic editing is actually not a

    new concept in SERS either. Zhang and coworkers11 have already

    demonstrated recently the usefulness of isotopic editing for

    analytical studies in SERS, but have not extended the study to

    the SM-SERS regime (despite the fact that all the ingredients were

    already present in their study). As mentioned earlier, Dieringer

    and coworkers5,12 have also recently suggested and applied the

    concept of isotope editing to bi-analyte SERS experiments.

    Arguably, the results in ref. 5 will establish new standards on

    the study of single molecule SERS conditions in years to come.

    In order to produce quantitative studies of single molecules in

    the future, an important initial step is the full characterization of

    the molecules themselves. In particular, it is not always possible

    to simply assume the Raman cross sections of isotopically shifted

    modes to be exactly the same as the unedited counterparts;

    because oscillator strength redistributions, changes in linewidths,

    and even splitting of modes (or appearance of new modes) can

    occur. It is also possible to test experimentally (rather than

    assume) that the isotopic substitution effectively does not change

    the surface chemistry or binding to the surface. This can be done

    through the comparison of enhancement factors (measured

    independently under identical experimental conditions) for

    modes that are not affected by the isotopic substitution in the

    edited and unedited versions of the molecule.

    Along these lines, we show in this paper an explicit im-

    plementation of isotopic editing for BiASERS in solution,

    together with a full experimental verification of these assump-

    tions. The isotopic editing is achieved in a methyl ester version

    of Rhodamine 6G (instead of the ethyl ester version used in

    ref. 5), which we shall call RH6M hereafter (shown in Fig. 1).

    The structure of rhodamine is broadly characterized by a

    three-ring chromophore (xanthen moiety) and a phenyl side-

    chain, which is in a different plane from the main backbone.

    The isotopic editing is performed by replacing the four hydro-

    gens of the phenyl moiety with deuterium, thus resulting in d4-

    RH6M. We shall show the usefulness of these probes as

    BiASERS partners for SM-SERS and provide a full charac-

    terization of them (including fluorescence, absorption, NMR,

    and bare Raman cross sections) for future use and reference as

    standard isotopic probes.

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