Excited-State Lifetime Assay for Protein Detection on Gold Colloids

Jan 26, 2009 - ... Piazza della Scienza 3, 20126, Milano, Italy and Laboratorio Nazionale MDM, INFM-CNR, via C. Olivetti, 2 I-20041 Agrate Brianza (Mi...
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J. Phys. Chem. C 2009, 113, 2722–2730

Excited-State Lifetime Assay for Protein Detection on Gold Colloids-Fluorophore Complexes S. Freddi,† L. D’Alfonso,† M. Collini,† M. Caccia,† L. Sironi,† G. Tallarida,‡ S. Caprioli,‡ and G. Chirico*,† Dipartimento di Fisica, UniVersita` di Milano Bicocca, Piazza della Scienza 3, 20126, Milano, Italy and Laboratorio Nazionale MDM, INFM-CNR, Via C. OliVetti, 2 I-20041 Agrate Brianza (Mi) Italy ReceiVed: October 28, 2008; ReVised Manuscript ReceiVed: December 18, 2008

The interaction of the surface plasmons of gold nanoparticles (NP) a few nanometers in size with fluorophores can be used to engineer their fluorescence properties. This possibility can be exploited in principle to obtain nanodevices for protein-protein recognition. We studied different types of constructs based on gold NPs on which derivatives of fluorescein were bound. The interaction of this fluorophore with the gold surface plasmon resonances, mainly occurring through quenching, affects its excited-state lifetime that is measured by fluorescence burst analysis in standard solutions. The binding of proteins to the gold NPs through antigen-antibody recognition further modifies the dye excited-state lifetime. This change can therefore be used to measure the protein concentration. The data reported here indicate that one can measure the concentration of bovine serum albumine in solution with an apparent limit of detection of 5 ( 2 pM. I. Introduction Biotechnology is pushing the minimum amount of detectable material toward lower and lower values.1 The limiting values of detection depend on the experimental method, and they are of the order of 1-10 pM that, for a 60 000 Da molecular weight protein, corresponding to about 6-60 ng/L.2,3 One of the most promising properties that can be exploited in this field is related to the plasmonic resonances of noble-metal nanoparticles (NPs). These resonances, present also in bulk metals but shifted toward higher energy gaps with respect to the nanoparticle case, lie in the visible range of the spectrum and are due to excitation of surface waves of electrons on the nanoparticles or in a thin ( 7 nm, the fluorescence is actually less quenched.57 Quenching is dominating for distances up to a few nanometers,17,54,55 and it is largely determined by the shape of the metal structure, by the dipole orientation with respect to the surface, by the size of the metal particle,51 and by the difference between the dielectric permittivities of the metal and the surface layer.18,58 Since in our case the distance between the metal and FITC is only ca. 3-4 nm, we believe that the main result reported here, namely, the change in the FITC lifetime upon BSA-NP binding, is mostly due to a change of quenching efficiency rather than to an effective emission enhancement. To partially support this hypothesis, we can bring the observation of the initial decrease in the lifetime of FITC upon binding to the gold surface (Table 2) and of the additional larger decrease induced by the BSA binding (Table 4). The tiny increase of the emitted photons per burst, 〈NBP〉, at rising BSA concentrations (Figure 6A), on the other hand, can be taken as an indication of the presence of high (and heterogeneous) local electric fields on the surface of the nanoclusters that produces a concomitant increase in the molecular brightness. The gold-induced quenching of FITC is directly related to the Fresnel coefficients at the metal-surface boundary.18 Their change upon binding of proteins to the surface is determined then by the change in the surface layer dielectric permittivity related to the protein relative concentration on the surface layer. For this reason, we tried to keep the relative concentration of BSA on the surface high while keeping the FITC signal per gold NP cluster at measurable levels. This reasoning has also driven our choice of the ratio [Ab]:[FITC] ) 3:1 reported above. As anticipated in the Materials and Methods section, the choice of the ratio [Ab]:[FITC] ) 3:1 has provided us with the better sensitivity. FITC bound to constructs prepared at the lower value of [Ab]:[FITC] ) 1:1 displays a reduced average excited-state lifetime already at [BSA]:[Ab] ) 0 with respect to the case [Ab]:[FITC] ) 3:1. For example, we found 〈τ〉 ) 2.1 ( 0.3 ns for the 10 nm constructs prepared at [Ab]:[FITC] ) 1:1, more than 30% lower than the [Ab]:[FITC] ) 3:1 case. Moreover, upon BSA addition the FITC average lifetime increases in the [Ab]:[FITC] ) 1:1 constructs up to 30% but with very large uncertainty ((22%, see Figure 6A2, inset). This behavior, which makes the construct prepared at [Ab]:[FITC] ) 1:1 less adequate than the case [Ab]:[FITC] ) 3:1 for protein assay applications, is due to the interplay of the different mechanisms that have been discussed above. In particular, we find that the increase in the FITC lifetime measured in the [Ab]:[FITC] ) 1:1 case is due to a marked increase of the long lifetime component which is also affected by a large variability (data not shown). This behavior is probably related to the increase in the local surface field53 and its inhomogeneity on the surface of the larger gold NP nanoclusters59 present in the 10 nm gold-dye constructs. These issues may be also at the basis of the observed reduced sensitivity of the 10 nm gold NPs constructs with respect to the 5 nm constructs. In fact, small colloids are expected to quench fluorescence more efficiently than larger ones since the absorption component of the extinction coefficient is dominant

J. Phys. Chem. C, Vol. 113, No. 7, 2009 2729 over the scattering one, which is responsible for the plasmoninduced fluorescence enhancement,51,60 and this would result in a reduced lifetime. On the other hand, the local field enhancement is expected to be larger and more inhomogeneous on larger, possibly aggregated, structures, such as those expected for the 10 nm NPs constructs, and this would result in an increase of the FITC lifetime53 for these larger constructs. The fine balancing of these two opposite behaviors would then determine the reduced sensitivity of the 10 nm particles constructs compared to those based on the 5 nm gold NPs described here. IV. Conclusions We reported a detailed analysis of the effect of binding the FITC dye to gold NPs of size in the 5-10 nm range and explored the possibility of using the fluorescence emission of FITC bound to their surface in order to monitor traces of proteins in standard solutions at pH = 7. The data presented and discussed here indicate that the FITC excited-state lifetime is a very sensitive parameter in order to detect tiny amounts of protein in solution with an estimated limit of detection of ca. 5 pM, mostly determined by the statistical accuracy of the lifetime measurement. This value, compatible with the sensitivity of most nanotechnology-based assays, encourages application of this method to other protein systems. It must be considered that for a direct application of the effect discussed in this report to a protein detection assay in cellular extracts we should also evaluate its specificity. We expect that the degree of specificity is largely determined by the aspecific protein binding to the gold NP surface with respect to the antibody-protein recognition. Moreover, an understanding of the fine balance of metal-induced quenching and enhancement that occurs on constructs of different size and aggregation would be invaluable for the possibility to design specific protein or DNA assays. These issues will be the subject of our future efforts. Acknowledgment. We gratefully acknowledge Dr. P. Pallavicini (Univ. Pavia) for the useful discussions and suggestions. This research has been funded by a MIUR PRIN grant to G.C. for the years 2006-2008 and by the grant 2005-1079 to G.C. from Fondazione Cariplo (Milano, I). References and Notes (1) Anker, J. N.; Hall, W. P.; Lyandres, O.; Shah, N. C.; Zhao, J.; Van Duyne, R. P. Nat. Mater. 2008, 7, 442–453. (2) Pita, M.; Cui, L.; Gaikwad, R. M.; Katz, E.; Sokolov, I. Nanotechnology 2008, 19, 375502. (3) Bizzarri, A. R.; Cannistraro, S. Nanomed. Nanotechnol. Biol. Med. 2007, 3, 306–310. (4) Haes, A. J.; Van Duyne, P. R. Anal. Bioanal. Chem. 2004, 379, 920–930. (5) Malmqvist, M. Nature 1993, 361, 186–187. (6) He, L.; Musick, M. D.; Nicewarner, S. R.; Salinas, F. G.; Benkovic, S. J.; Natan, M. J.; Keating, C. D. J. Am. Chem. Soc. 2000, 122, 9071– 9077. (7) Aslan, K.; Lakowicz, J. R.; Geddes, C. D. Curr. Opin. Chem. Biol. 2005, 9, 538–544. (8) Aslan, K.; Lakowicz, J. R.; Geddes, C. D. Anal. Bioanal. Chem. 2005, 382, 926–933. (9) Hutter, E.; Fendler, J. H. AdV. Mater. 2004, 16, 1685–1706. (10) Lakowicz, J. R. Anal. Biochem. 2005, 337, 171–194. (11) Stuart, D. A.; Haes, A. J.; Yonzon, C. R.; Hicks, E. M.; Van Duyne, R. P. IEE Proc. Nanobiotechnol. 2005, 152, 13–32. (12) Dulkeith, E.; Morteani, A. C.; Niedereichholz, T.; Klar, T. A.; Feldmann, J.; Levi, S. A.; van Veggel, F. C. J. M.; Reinhoudt, D. N.; Moeller, M.; Gittins, D. I. Phys. ReV. Lett. 2002, 89, 203002. (13) Barnes, W. L. J. Mod. Opt. 1998, 45, 661–699. (14) Aslan, K.; Lakowicz, J. R.; Szmacinski, H.; Geddes, C. D. J. Fluoresc. 2004, 14, 677–679.

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