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Cite this: DOI: 10.1039/c4nr02069c
Received 16th April 2014 Accepted 24th June 2014
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Exploring luminescence-based temperature sensing using protein-passivated gold nanoclusters† Xi Chen, Jeremy B. Essner and Gary A. Baker*
DOI: 10.1039/c4nr02069c www.rsc.org/nanoscale
We explore the analytical performance and limitations of optically monitoring aqueous-phase temperature using protein-protected gold nanoclusters (AuNCs). Although not reported elsewhere, we find that these bio-passivated AuNCs show pronounced hysteresis upon thermal cycling. This unwanted behaviour can be eliminated by several strategies, including sol–gel coating and thermal denaturation of the biomolecular template, introducing protein-templated AuNC probes as viable nanothermometers.
As atomically-precise thiolate-protected clusters of a few to tens of metal atoms, metal nanoclusters (MNCs) represent an intriguing link between organometallic complexes and crystalline solids.1,2 With a size ranging from well below 1 nm to a few nanometers, MNCs possess intriguing optical and catalytic features.3–6 Recently, luminescent MNCs have shown broad promise for the detection of hydrogen peroxide,7 transition metal ions,8,9 dopamine,10 glucose,11 ascorbic acid,12 cholesterol,13 and protease activity.14 A popular approach to the formation of MNCs, rst shown by Ying and co-workers in 2009, involves using a protein scaffold (typically, bovine serum albumin) as a mineralizing agent under alkaline conditions.15 Temperature is probably the most fundamental parameter measured in all of science, representing an 80% share of the world's sensor market.16 Optical sensors hold a number of particular advantages, including their wireless operation, making contactless and large-scale surface imaging possible even in harsh, corrosive environments or in strong magnetic elds. Frequently, luminescence-based thermometry is used by exploiting some temperature-dependent parameter (e.g., intensity, spectral shape, width or position, polarization/anisotropy, excited-state lifetime, cyclization kinetics) of a molecular probe.17 Nanoscale thermometry is also gaining momentum for mapping temperature uctuations with high spatial resolution Department of Chemistry, University of Missouri-Columbia, Columbia, MO 65211, USA. E-mail: [email protected] † Electronic supplementary information (ESI) available: Supplemental gures and experimental details. See DOI: 10.1039/c4nr02069c
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on the sub-micron scale, with emerging applications in micro/ nano-electronics, microuidics, integrated photonics, and biomedicine (e.g., monitoring laser-induced hyperthermia treatments).16,18,19 A number of ground-breaking examples have been reported, including nanoscale thermometry based on lanthanide-included functional organic–inorganic hybrids,20 dual-emitting alloyed Zn1xyCdxMnySe semiconductor nanocrystals,21 photon upconverting NaYF4:Yb, Er nanoparticles,22 diphenylamine-modied water-dispersible silicon nanoparticles,23 and organosilane-functionalized carbon dots.24 A growing number of researches have been carried out on the catalytic activity, kinetics, ligand effects, and optical features (e.g., polarization, intensity decays) of thiol- or amine-protected MNCs.25–30 Among these, a number of examples exist exploring the temperature dependence of MNC optical features. In an early contribution, Pradeep and co-workers looked at magic numbered Au25SG18 (SG ¼ glutathione thiolate) quantum clusters in the range from 80 to 300 K.26 Although this represents the earliest report on the distinct temperature-modulated solid-state emission of such clusters, the increased intensity with temperature proved difficult to rationalize and hysteresis was clearly evident. Recently, Tang and co-workers investigated the uorescence behavior of histidine-protected Au10NCs as a function of temperature in solution, suggesting thermally-activated nonradiative trapping.31 The Calvaresi group also reported on an interesting molecular thermometer comprising ve copper atoms bound to three highly conjugated ligands.32 Of particular note, the Nienhaus group developed lipoic acid-protected AuNCs as probes for lifetime-based intracellular thermometry.33 Protein-passivated MNCs are enticing nanoscale candidates for mapping temperature distributions within cells. However, to date, the performance of AuNCs@BSA in nanothermometry remains an open question. This forms the topic of the current work. AuNCs@BSA were prepared following methods reported elsewhere.34 As is already established, under UV excitation, AuNCs@BSA display dual emission: a blue peak ascribed to protein surface oxidation species and a prominent red emission
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arising from the core Au25 NC unit. Interestingly, the blue and red emissions give differential responses to temperature, as summarized in Fig. 1A. The red emission from AuNCs@BSA displays greater sensitivity to temperature, exhibiting a roughly 40% drop in intensity upon raising the temperature from 10 to 45 C compared to a 25% decrement for the blue emission. Given the proven benets of ratiometric optical sensors in terms of simplicity and analytical robustness,35 we initially considered whether these disparate responses might form the basis for a self-referenced nanothermometer. It quickly became clear, however, that the temperature-dependence of the blueemitting species was unpredictable and could not be made reliable (refer to Fig. S1†). For this reason, the remainder of the work focuses on the intense red emission arising from the gold cluster itself within AuNCs@BSA rather than on the luminescence originating from presumed but ill-dened BSA oxidation by-product(s). As shown in Fig. S2,† the temperature-dependent
Temperature dependence of the emission from AuNCs@BSA in water. (A) Fluorescence emission spectra measured in the range 10– 45 C (top to bottom) under excitation of 250 nm. The peak marked * arises from second-order Rayleigh scattering. Normalized fluorescence intensities (F/F0) versus temperature and the corresponding linear regression results are provided in the inset. (B) Thermal cycling curves constructed from the fluorescence emission intensity integrated from 640 to 650 nm for (a) virgin AuNCs@BSA (green curves) alongside results for (b) thermally “annealed” AuNCs@BSA (red curves). Heating and cooling segments are indicated by filled and open symbols, respectively, and F/F0 denotes the fraction of fluorescence remaining relative to that measured at 10 C. Data for the annealed AuNCs@BSA sample is offset by 0.2 for clarity. lexc ¼ 400 nm.
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response for the red band is not strongly excitation wavelength dependent. Although the red emission from AuNCs@BSA exhibits a monotonous decrease upon increasing the temperature from 10 to 45 C (a percent variation of the intensity per degree of 1.1% per C), upon re-cooling to 10 C, pronounced hysteresis is evident, resulting in a signal 12% higher than the initial value (prole a in Fig. 1B; see also Fig. S3†). Interestingly, the normalized emission spectra reveal a temperature-dependent bandshape, with a shoulder becoming more prominent at 690 nm as the temperature increases (Fig. S4†). This behaviour might prove attractive for self-referenced nanothermometry but clearly such hysteresis is a serious impediment to the development of protein-templated MNCs as probes of the cellular environment, for instance. In order to combat this problem, several different strategies were evaluated, as outlined in Fig. 2. This hysteresis is thought to reect slight changes in the local environment (protein shell) proximal to the AuNC aer refolding of the BSA scaffold. One approach that we initially proposed to address this was iterative, gentle cycling designed to thermally “anneal” the protein. This annealing sequence consisted of three controlled ramp-hold-cool cycles between 10 C and 60 C (refer to Scheme S1 of the ESI† for details). An upper temperature of 60 C was selected on the basis of serum albumins exhibiting reversible unfolding in this range.36 As can be seen, this approach resulted in marked improvement, although the nal signal aer one thermal cycle is still 3% higher than the original signal value (prole b in Fig. 1B). Similar dris in recovered uorescent signal were observed in previous studies of lipoic acid-protected AuNCs but were not discussed.33 With the goal of achieving a fully reversible response to temperature in mind, we developed additional strategies toward eliminating this hysteresis. In all cases, a nal annealing step was also performed before making measurements. One such strategy involved employing a sol–gel coating, an approach well known to enhance the thermal stability and modulate the dynamics of sequestered biomolecules.37–40 Two different
Fig. 1
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Fig. 2 Strategies evaluated for generating reproducible AuNCs@BSA luminescent nanothermometers: (i) protein reduction prior to AuNC synthesis, and post-synthetic (ii) sol–gel coating, (iii) halide treatment, and (iv) iterative thermal cycling (“annealing”).
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silicate (tetramethylorthosilicate, TMOS and diglycerylsilane, DGS) and one aluminum-based (aluminum isopropoxide, Al(O-i-Pr)3) sol–gel precursor were tested in this work (refer to the ESI† for details). TEM images of the resulting sol–gel modied AuNCs@BSA are provided in Fig. S5† and reveal clusters ca. 1 nm in size, similar to the unmodied AuNCs@BSA. The results of temperature-dependent luminescence experiments carried out on sol–gel coated AuNCs@BSA, summarized in Fig. 3A, reveal extents of hysteresis of 1.7%, 1.4%, and 5.2% aer one 10–45–10 C cycle for DGS-, TMOS-, and Al2O3-based treatments, respectively. Although the silicate coatings proved to be quite effective in enhancing the reproducibility of AuNCs@BSA as nanoscale thermometers, there remains scope for improvement, especially anticipating demanding applications like cellular and device (e.g., lab-on-a-chip) thermal mapping. The next strategy we followed was inspired by the nding that halide ions increased the thermal stability of HSA according to the lyotropic series F < Cl < Br < I.41 Consistent with this earlier observation, AuNCs@BSA were incubated in 0.33 M NaCl, KI, and KBr for 48 h followed by the annealing sequence used earlier. Remarkably, whereas the presence of NaCl resulted in a signal highly dependent upon the sample history (i.e., 16% hysteresis aer one thermal cycle up to 45 C), both KBr and KI led to dramatic improvements in this behaviour (2.4% and 1.6% total variation, respectively; Fig. 3B). We attribute this favourable outcome to a tightening in the overall BSA structure and an accompanying increase in the melting point and enthalpy of unfolding. Unexpectedly, KI treatment of
AuNCs@BSA resulted in very small negative hysteresis for reasons not immediately clear. Overall, however, this approach appears to largely alleviate memory effects associated with thermal history in vitro to a similar extent as silica sol–gel functionalization. It should of course be pointed out that the latter appears much more relevant to bioimaging efforts where it will not be possible to control the neutral salt concentration within the cellular milieu. A recent report argues that a number of cysteine (Cys) residues in BSA remain linked via disulde bridges during the formation of [email protected] It is thought that the AuNCs are primarily stabilized by Cys residues via Au–S bonds. Given this, it might be reasoned that BSA containing a multiplicity of free Cys residues would provide an opportunity for efficient Au–S bonding, to achieve AuNCs@BSA with improved stability. Accordingly, we employed NaBH4 as a reducing agent to produce AuNCs@BSA templated by BSA wherein disulde linkages have been cleaved; we term these clusters AuNCs@rBSA to denote the fact that the protein has been reduced in advance of synthesis. Unfortunately, AuNCs@rBSA offered little improvement over conventionally-made (untreated) AuNCs@BSA, showing a 7.7% hysteresis (Fig. S6†). A nal approach we considered is thermal protein denaturation prior to production of AuNCs@BSA. It is known that the ahelical content of serum albumins decreases upon thermal denaturation beyond 75 C and does not fully recover upon subsequent cooling.36,43 Consequently, thermal denaturation of BSA was achieved by incubation at 80 C for 30 min before proceeding with the nanocluster synthesis. The resulting protein-passivated clusters are referred to as AuNCs@hBSA. As synthesized AuNCs@hBSA display similar emission proles as conventional AuNCs@BSA. In stark contrast, however, cooling of an annealed AuNCs@hBSA sample resulted in recovery of the intensity initially obtained. Indeed, the response curves for AuNCs@hBSA are completely overlapping in the heating and cooling segments (Fig. 4), making rigorous temperature tracking possible. Based on the uncertainty in F/F0, the average
Fig. 3 Temperature dependence of the integrated fluorescence emission (640–650 nm) for (A) sol–gel coated AuNCs@BSA and (B) halide salt treated AuNCs@BSA, normalized to the initial intensity. The closed and open symbols denote the heating and cooling segments, respectively. Profiles are shifted vertically by 0.1 for clarity. lexc ¼ 400 nm.
Fig. 4 Thermometric response curve constructed from the integrated fluorescence intensity from AuNCs@hBSA measured during heating (red) and cooling (blue) processes. The solid fit shows a slope of 1.02 102 C1. Inset: integrated fluorescence of AuNCs@hBSA upon cycling the temperature three times between 10 C and 45 C. lexc ¼ 400 nm.
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error in the determination of temperature in the range shown is just 0.2 C. This performance (which notably spans physiological temperature) coupled with insensitivity to oxygen concentration (Fig. S7†) suggests a promising path forward for AuNCs@hBSA in tracking intracellular thermal uctuations, as well as other applications such as identifying heat bottlenecks within integrated circuits. In order to better quantify the sensitivity of these stabilized AuNCs@BSA to temperature and to place these results in context with examples from the open literature, we tted the data from Fig. 4 to an Arrhenius-type expression of the form 1 1 Ea ¼ þ A exp kB T F ðTÞ F0 where F(T) is the temperature-dependent uorescence signal, F0 is the signal at 0 K, the preexponential factor A is a dimensionless constant, Ea is the activation energy for thermal quenching, and kB is Boltzmann's constant. The tting results, presented in Fig. 5 reveal a mean Ea value of 206 17 meV. By comparison, Ea values of 74 meV and 130 meV were determined from data for histidine-protected Au10NCs31 and lipoic acidprotected AuNCs,33 respectively. In other words, the luminescence from AuNCs@BSA is inherently more sensitive to temperature than that from these small thiolate ligand-capped AuNCs. Our results also compare favourably with non-MNCs such as organosilane-functionalized carbon dots (Ea ¼ 59.5 meV) which were recently proposed as optical probes for cell imaging.24 With the well-behaved AuNCs@BSA now in hand, we returned our attention to the prospect of employing bandshape analysis as an alternate means of determining the local temperature. Namely, the superb reproducibility in the optical properties of AuNCs@hBSA during iterative heating and cooling cycles allows us to leverage the variation in emission spectral bandshape for self-referenced nanothermometry. Employing the intensity ratio measured at 700 and 610 nm (F700/F610), we
Fig. 5 Arrhenius plots of AuNCs@hBSA during heating and cooling portions of a single thermal cycle. As benchmarks, curves representing the sensitivity to temperature of known MNCs taken from the literature are also shown. The Ea values presented correspond to activation energies for thermal quenching. For an expanded view of this plot, showing all data from ref. 31, refer to Fig. S8.†
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Fig. 6 (A) The difference spectrum constructed by subtracting the normalized emission spectrum for AuNCs@hBSA measured at 10 C from that measured at 45 C. Vertical marks denote the wavelengths 610 nm and 700 nm used to track temperature. (B) Normalized steadystate fluorescence emission spectra taken of AuNCs@hBSA during controlled heating (shown as solid curves) and cooling (symbols) segments of one thermal cycle. lexc ¼ 400 nm. The corresponding results for untreated AuNCs@BSA are given in Fig. S4 of the ESI.†
can reliably estimate the temperature to within one degree (Fig. 6). We are not aware of any previous report showing a consistent variation in the spectral properties of MNCs with temperature. Given the benets of utilizing ratiometric changes to monitor temperature,17,44 however, our current results suggest that this approach merits consideration in future efforts seeking to optically track temperature using various protein- or ligand-stabilized uorescent MNCs. As a nal point, we note that, for consistency with our earlier work,13,34 we have opted to employ UV and blue excitation wavelengths when seeking dual (i.e., blue and red) and solely red emission from AuNCs, respectively. However, as we demonstrate in Fig. S9,† it is entirely possible to employ visible wavelengths to excite red emission from AuNCs@BSA, including the use of common argon-ion laser lines (488 and 514 nm), suggesting a clear avenue for effective cellular thermometry. Two-photon excited uorescence (TPEF) is also an enticing possibility for high-resolution bioimaging or deep tissue studies with minimal excitation of endogenous cellular uorescence (e.g., avins, bilirubin).45 Of note, Oh, Medintz and co-workers recently applied biocompatible near-infrared-emitting PEGylated AuNCs for two-photon cellular imaging,46 suggesting feasibility for TPEF thermometry using AuNCs@BSA probes within cells as well. To summarize, we report the rst study seeking to employ protein-passivated AuNCs as optical probes for accurate temperature monitoring at physiological temperatures. Although not previously known, we nd that the original luminescence intensity arising from AuNCs@BSA is not recovered following a single heating and cooling cycle. We have solved this issue by evaluating several disparate strategies to
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alleviate the hysteresis, erasing the thermal history of AuNCs@BSA to yield a highly promising nanoscale thermometer. The most effective strategies involved halide doping, sol– gel incarceration and, most particularly, thermal denaturation of the starting protein to yield an expedient route to rigorously precise optical probes with a temperature resolution of 0.2 C.
References 1 G. Schmid, Chem. Rev., 1992, 92, 1709–1727. 2 L. Shang, S. Dong and G. U. Nienhaus, Nano Today, 2011, 6, 401–418. 3 M. D. Hughes, Y.-J. Xu, P. Jenkins, P. McMorn, P. Landon, D. I. Enache, A. F. Carley, G. A. Attard, G. J. Hutchings and F. King, Nature, 2005, 437, 1132–1135. 4 A. A. Herzing, C. J. Kiely, A. F. Carley, P. Landon and G. J. Hutchings, Science, 2008, 321, 1331–1335. 5 H. Qian, M. Zhu, Z. Wu and R. Jin, Acc. Chem. Res., 2012, 45, 1470–1479. 6 G. Li and R. Jin, Acc. Chem. Res., 2013, 46, 1749–1758. 7 F. Wen, Y. Dong, L. Feng, S. Wang, S. Zhang and X. Zhang, Anal. Chem., 2011, 83, 1193–1196. 8 S. Liu, F. Lu and J.-J. Zhu, Chem. Commun., 2011, 47, 2661– 2663. 9 H. Liu, X. Zhang, X. Wu, L. Jiang, C. Burda and J.-J. Zhu, Chem. Commun., 2011, 47, 4237–4239. 10 P. Biji and A. Patnaik, Analyst, 2012, 137, 4795–4801. 11 L. Jin, L. Shang, S. Guo, Y. Fang, D. Wen, L. Wang, J. Yin and S. Dong, Biosens. Bioelectron., 2011, 26, 1965–1969. 12 X. Wang, P. Wu, X. Hou and Y. Lv, Analyst, 2013, 138, 229– 233. 13 X. Chen and G. A. Baker, Analyst, 2013, 138, 7299–7302. 14 J. Zheng, P. R. Nicovich and R. M. Dickson, Annu. Rev. Phys. Chem., 2007, 58, 409–431. 15 J. Xie, Y. Zheng and J. Y. Ying, J. Am. Chem. Soc., 2009, 131, 888–889. 16 X.-d. Wang, O. S. Woleis and R. J. Meier, Chem. Soc. Rev., 2013, 42, 7834–7869. 17 G. A. Baker, S. N. Baker and T. M. McCleskey, Chem. Commun., 2003, 2932–2933. 18 D. Jaque and F. Vetrone, Nanoscale, 2012, 4, 4301–4326. 19 C. D. Brites, P. P. Lima, N. J. Silva, A. Mill´ an, V. S. Amaral, F. Palacio and L. D. Carlos, Nanoscale, 2012, 4, 4799–4829. 20 C. D. Brites, P. P. Lima, N. J. Silva, A. Millan, V. S. Amaral, F. Palacio and L. D. Carlos, New J. Chem., 2011, 35, 1177– 1183. 21 E. J. McLaurin, M. S. Fataah and D. R. Gamelin, Chem. Commun., 2013, 49, 39–41. 22 A. Sedlmeier, D. E. Achatz, L. H. Fischer, H. H. Gorris and O. S. Woleis, Nanoscale, 2012, 4, 7090–7096. 23 Q. Li, Y. He, J. Chang, L. Wang, H. Chen, Y.-W. Tan, H. Wang and Z. Shao, J. Am. Chem. Soc., 2013, 135, 14924–14927.
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24 P.-C. Chen, Y.-N. Chen, P.-C. Hsu, C.-C. Shih and H.-T. Chang, Chem. Commun., 2013, 49, 1639–1641. 25 M. S. Devadas, S. Bairu, H. Qian, E. Sinn, R. Jin and G. Ramakrishna, J. Phys. Chem. Lett., 2011, 2, 2752–2758. 26 E. Shibu, M. H. Muhammed, T. Tsukuda and T. Pradeep, J. Phys. Chem. C, 2008, 112, 12168–12176. 27 J. T. v. Wijngaarden, O. Toikkanen, P. Liljeroth, B. M. Quinn and A. Meijerink, J. Phys. Chem. C, 2010, 114, 16025–16028. 28 S. Raut, R. Chib, R. Rich, D. Shumilov, Z. Gryczynski and I. Gryczynski, Nanoscale, 2013, 5, 3441–3446. 29 S. L. Raut, R. Fudala, R. Rich, R. A. Kokate, R. Chib, Z. Gryczynski and I. Gryczynski, Nanoscale, 2014, 6, 2594– 2597. 30 G. Wang, R. Guo, G. Kalyuzhny, J.-P. Choi and R. W. Murray, J. Phys. Chem. B, 2006, 110, 20282–20289. 31 P. Yu, X. Wen, Y.-R. Toh and J. Tang, J. Phys. Chem. C, 2012, 116, 6567–6571. 32 D. Cauzzi, R. Pattacini, M. Delferro, F. Dini, C. D. Natale, R. Paolesse, S. Bonacchi, M. Montalti, N. Zaccheroni and M. Calvaresi, Angew. Chem., Int. Ed., 2012, 51, 9662–9665. 33 L. Shang, F. Stockmar, N. Azadfar and G. U. Nienhaus, Angew. Chem., Int. Ed., 2013, 52, 11154–11157. 34 C. M. Hofmann, J. B. Essner, G. A. Baker and S. N. Baker, Nanoscale, 2014, 6, 5425–5431. 35 S. Pandey, S. N. Baker, S. Pandey and G. A. Baker, Chem. Commun., 2012, 48, 7043–7045. 36 K. Flora, J. D. Brennan, G. A. Baker, M. A. Doody and F. V. Bright, Biophys. J., 1998, 75, 1084–1096. 37 A. Rutenberg, V. V. Vinogradov and D. Avnir, Chem. Commun., 2013, 49, 5636–5638. 38 Y. Yang, K. Xiang, Y.-X. Yang, Y.-W. Wang, X. Zhang, Y. Cui, H. Wang, Q.-Q. Zhu, L. Fan and Y. Liu, Nanoscale, 2013, 5, 10345–10352. 39 B. Satishkumar, S. K. Doorn, G. A. Baker and A. M. Dattelbaum, ACS Nano, 2008, 2, 2283–2290. 40 M. A. Doody, G. A. Baker, S. Pandey and F. V. Bright, Chem. Mater., 2000, 12, 1142–1147. 41 G. A. Pic´ o, Biochem. Mol. Biol. Int., 1996, 38, 1–6. 42 H. Li, Y. Guo, I. Xiao and B. Chen, Analyst, 2013, 139, 285– 289. 43 R. Wetzel, M. Becker, J. Behlke, H. Billwitz, S. B¨ ohm, B. Ebert, H. Hamann, J. Krumbiegel and G. Lassmann, Eur. J. Biochem., 1980, 104, 469–478. 44 N. Chandrasekharan and L. A. Kelly, J. Am. Chem. Soc., 2001, 123, 9898–9899. 45 G. A. Baker, S. Pandey and F. V. Bright, Anal. Chem., 2000, 72, 5748–5752. 46 E. Oh, F. K. Fatemi, M. Currie, J. B. Delehanty, T. Pons, A. Fragola, S. L´ evˆ eque-Fort, R. Goswami, K. Susumu, A. L. Huston and I. L. Medintz, Part. Part. Syst. Charact., 2013, 30, 453–466.
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Cite this: DOI: 10.1039/c4nr02069c
Received 16th April 2014 Accepted 24th June 2014
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Exploring luminescence-based temperature sensing using protein-passivated gold nanoclusters† Xi Chen, Jeremy B. Essner and Gary A. Baker*
DOI: 10.1039/c4nr02069c www.rsc.org/nanoscale
We explore the analytical performance and limitations of optically monitoring aqueous-phase temperature using protein-protected gold nanoclusters (AuNCs). Although not reported elsewhere, we find that these bio-passivated AuNCs show pronounced hysteresis upon thermal cycling. This unwanted behaviour can be eliminated by several strategies, including sol–gel coating and thermal denaturation of the biomolecular template, introducing protein-templated AuNC probes as viable nanothermometers.
As atomically-precise thiolate-protected clusters of a few to tens of metal atoms, metal nanoclusters (MNCs) represent an intriguing link between organometallic complexes and crystalline solids.1,2 With a size ranging from well below 1 nm to a few nanometers, MNCs possess intriguing optical and catalytic features.3–6 Recently, luminescent MNCs have shown broad promise for the detection of hydrogen peroxide,7 transition metal ions,8,9 dopamine,10 glucose,11 ascorbic acid,12 cholesterol,13 and protease activity.14 A popular approach to the formation of MNCs, rst shown by Ying and co-workers in 2009, involves using a protein scaffold (typically, bovine serum albumin) as a mineralizing agent under alkaline conditions.15 Temperature is probably the most fundamental parameter measured in all of science, representing an 80% share of the world's sensor market.16 Optical sensors hold a number of particular advantages, including their wireless operation, making contactless and large-scale surface imaging possible even in harsh, corrosive environments or in strong magnetic elds. Frequently, luminescence-based thermometry is used by exploiting some temperature-dependent parameter (e.g., intensity, spectral shape, width or position, polarization/anisotropy, excited-state lifetime, cyclization kinetics) of a molecular probe.17 Nanoscale thermometry is also gaining momentum for mapping temperature uctuations with high spatial resolution Department of Chemistry, University of Missouri-Columbia, Columbia, MO 65211, USA. E-mail: [email protected] † Electronic supplementary information (ESI) available: Supplemental gures and experimental details. See DOI: 10.1039/c4nr02069c
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on the sub-micron scale, with emerging applications in micro/ nano-electronics, microuidics, integrated photonics, and biomedicine (e.g., monitoring laser-induced hyperthermia treatments).16,18,19 A number of ground-breaking examples have been reported, including nanoscale thermometry based on lanthanide-included functional organic–inorganic hybrids,20 dual-emitting alloyed Zn1xyCdxMnySe semiconductor nanocrystals,21 photon upconverting NaYF4:Yb, Er nanoparticles,22 diphenylamine-modied water-dispersible silicon nanoparticles,23 and organosilane-functionalized carbon dots.24 A growing number of researches have been carried out on the catalytic activity, kinetics, ligand effects, and optical features (e.g., polarization, intensity decays) of thiol- or amine-protected MNCs.25–30 Among these, a number of examples exist exploring the temperature dependence of MNC optical features. In an early contribution, Pradeep and co-workers looked at magic numbered Au25SG18 (SG ¼ glutathione thiolate) quantum clusters in the range from 80 to 300 K.26 Although this represents the earliest report on the distinct temperature-modulated solid-state emission of such clusters, the increased intensity with temperature proved difficult to rationalize and hysteresis was clearly evident. Recently, Tang and co-workers investigated the uorescence behavior of histidine-protected Au10NCs as a function of temperature in solution, suggesting thermally-activated nonradiative trapping.31 The Calvaresi group also reported on an interesting molecular thermometer comprising ve copper atoms bound to three highly conjugated ligands.32 Of particular note, the Nienhaus group developed lipoic acid-protected AuNCs as probes for lifetime-based intracellular thermometry.33 Protein-passivated MNCs are enticing nanoscale candidates for mapping temperature distributions within cells. However, to date, the performance of AuNCs@BSA in nanothermometry remains an open question. This forms the topic of the current work. AuNCs@BSA were prepared following methods reported elsewhere.34 As is already established, under UV excitation, AuNCs@BSA display dual emission: a blue peak ascribed to protein surface oxidation species and a prominent red emission
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arising from the core Au25 NC unit. Interestingly, the blue and red emissions give differential responses to temperature, as summarized in Fig. 1A. The red emission from AuNCs@BSA displays greater sensitivity to temperature, exhibiting a roughly 40% drop in intensity upon raising the temperature from 10 to 45 C compared to a 25% decrement for the blue emission. Given the proven benets of ratiometric optical sensors in terms of simplicity and analytical robustness,35 we initially considered whether these disparate responses might form the basis for a self-referenced nanothermometer. It quickly became clear, however, that the temperature-dependence of the blueemitting species was unpredictable and could not be made reliable (refer to Fig. S1†). For this reason, the remainder of the work focuses on the intense red emission arising from the gold cluster itself within AuNCs@BSA rather than on the luminescence originating from presumed but ill-dened BSA oxidation by-product(s). As shown in Fig. S2,† the temperature-dependent
Temperature dependence of the emission from AuNCs@BSA in water. (A) Fluorescence emission spectra measured in the range 10– 45 C (top to bottom) under excitation of 250 nm. The peak marked * arises from second-order Rayleigh scattering. Normalized fluorescence intensities (F/F0) versus temperature and the corresponding linear regression results are provided in the inset. (B) Thermal cycling curves constructed from the fluorescence emission intensity integrated from 640 to 650 nm for (a) virgin AuNCs@BSA (green curves) alongside results for (b) thermally “annealed” AuNCs@BSA (red curves). Heating and cooling segments are indicated by filled and open symbols, respectively, and F/F0 denotes the fraction of fluorescence remaining relative to that measured at 10 C. Data for the annealed AuNCs@BSA sample is offset by 0.2 for clarity. lexc ¼ 400 nm.
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response for the red band is not strongly excitation wavelength dependent. Although the red emission from AuNCs@BSA exhibits a monotonous decrease upon increasing the temperature from 10 to 45 C (a percent variation of the intensity per degree of 1.1% per C), upon re-cooling to 10 C, pronounced hysteresis is evident, resulting in a signal 12% higher than the initial value (prole a in Fig. 1B; see also Fig. S3†). Interestingly, the normalized emission spectra reveal a temperature-dependent bandshape, with a shoulder becoming more prominent at 690 nm as the temperature increases (Fig. S4†). This behaviour might prove attractive for self-referenced nanothermometry but clearly such hysteresis is a serious impediment to the development of protein-templated MNCs as probes of the cellular environment, for instance. In order to combat this problem, several different strategies were evaluated, as outlined in Fig. 2. This hysteresis is thought to reect slight changes in the local environment (protein shell) proximal to the AuNC aer refolding of the BSA scaffold. One approach that we initially proposed to address this was iterative, gentle cycling designed to thermally “anneal” the protein. This annealing sequence consisted of three controlled ramp-hold-cool cycles between 10 C and 60 C (refer to Scheme S1 of the ESI† for details). An upper temperature of 60 C was selected on the basis of serum albumins exhibiting reversible unfolding in this range.36 As can be seen, this approach resulted in marked improvement, although the nal signal aer one thermal cycle is still 3% higher than the original signal value (prole b in Fig. 1B). Similar dris in recovered uorescent signal were observed in previous studies of lipoic acid-protected AuNCs but were not discussed.33 With the goal of achieving a fully reversible response to temperature in mind, we developed additional strategies toward eliminating this hysteresis. In all cases, a nal annealing step was also performed before making measurements. One such strategy involved employing a sol–gel coating, an approach well known to enhance the thermal stability and modulate the dynamics of sequestered biomolecules.37–40 Two different
Fig. 1
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Fig. 2 Strategies evaluated for generating reproducible AuNCs@BSA luminescent nanothermometers: (i) protein reduction prior to AuNC synthesis, and post-synthetic (ii) sol–gel coating, (iii) halide treatment, and (iv) iterative thermal cycling (“annealing”).
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silicate (tetramethylorthosilicate, TMOS and diglycerylsilane, DGS) and one aluminum-based (aluminum isopropoxide, Al(O-i-Pr)3) sol–gel precursor were tested in this work (refer to the ESI† for details). TEM images of the resulting sol–gel modied AuNCs@BSA are provided in Fig. S5† and reveal clusters ca. 1 nm in size, similar to the unmodied AuNCs@BSA. The results of temperature-dependent luminescence experiments carried out on sol–gel coated AuNCs@BSA, summarized in Fig. 3A, reveal extents of hysteresis of 1.7%, 1.4%, and 5.2% aer one 10–45–10 C cycle for DGS-, TMOS-, and Al2O3-based treatments, respectively. Although the silicate coatings proved to be quite effective in enhancing the reproducibility of AuNCs@BSA as nanoscale thermometers, there remains scope for improvement, especially anticipating demanding applications like cellular and device (e.g., lab-on-a-chip) thermal mapping. The next strategy we followed was inspired by the nding that halide ions increased the thermal stability of HSA according to the lyotropic series F < Cl < Br < I.41 Consistent with this earlier observation, AuNCs@BSA were incubated in 0.33 M NaCl, KI, and KBr for 48 h followed by the annealing sequence used earlier. Remarkably, whereas the presence of NaCl resulted in a signal highly dependent upon the sample history (i.e., 16% hysteresis aer one thermal cycle up to 45 C), both KBr and KI led to dramatic improvements in this behaviour (2.4% and 1.6% total variation, respectively; Fig. 3B). We attribute this favourable outcome to a tightening in the overall BSA structure and an accompanying increase in the melting point and enthalpy of unfolding. Unexpectedly, KI treatment of
AuNCs@BSA resulted in very small negative hysteresis for reasons not immediately clear. Overall, however, this approach appears to largely alleviate memory effects associated with thermal history in vitro to a similar extent as silica sol–gel functionalization. It should of course be pointed out that the latter appears much more relevant to bioimaging efforts where it will not be possible to control the neutral salt concentration within the cellular milieu. A recent report argues that a number of cysteine (Cys) residues in BSA remain linked via disulde bridges during the formation of [email protected] It is thought that the AuNCs are primarily stabilized by Cys residues via Au–S bonds. Given this, it might be reasoned that BSA containing a multiplicity of free Cys residues would provide an opportunity for efficient Au–S bonding, to achieve AuNCs@BSA with improved stability. Accordingly, we employed NaBH4 as a reducing agent to produce AuNCs@BSA templated by BSA wherein disulde linkages have been cleaved; we term these clusters AuNCs@rBSA to denote the fact that the protein has been reduced in advance of synthesis. Unfortunately, AuNCs@rBSA offered little improvement over conventionally-made (untreated) AuNCs@BSA, showing a 7.7% hysteresis (Fig. S6†). A nal approach we considered is thermal protein denaturation prior to production of AuNCs@BSA. It is known that the ahelical content of serum albumins decreases upon thermal denaturation beyond 75 C and does not fully recover upon subsequent cooling.36,43 Consequently, thermal denaturation of BSA was achieved by incubation at 80 C for 30 min before proceeding with the nanocluster synthesis. The resulting protein-passivated clusters are referred to as AuNCs@hBSA. As synthesized AuNCs@hBSA display similar emission proles as conventional AuNCs@BSA. In stark contrast, however, cooling of an annealed AuNCs@hBSA sample resulted in recovery of the intensity initially obtained. Indeed, the response curves for AuNCs@hBSA are completely overlapping in the heating and cooling segments (Fig. 4), making rigorous temperature tracking possible. Based on the uncertainty in F/F0, the average
Fig. 3 Temperature dependence of the integrated fluorescence emission (640–650 nm) for (A) sol–gel coated AuNCs@BSA and (B) halide salt treated AuNCs@BSA, normalized to the initial intensity. The closed and open symbols denote the heating and cooling segments, respectively. Profiles are shifted vertically by 0.1 for clarity. lexc ¼ 400 nm.
Fig. 4 Thermometric response curve constructed from the integrated fluorescence intensity from AuNCs@hBSA measured during heating (red) and cooling (blue) processes. The solid fit shows a slope of 1.02 102 C1. Inset: integrated fluorescence of AuNCs@hBSA upon cycling the temperature three times between 10 C and 45 C. lexc ¼ 400 nm.
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error in the determination of temperature in the range shown is just 0.2 C. This performance (which notably spans physiological temperature) coupled with insensitivity to oxygen concentration (Fig. S7†) suggests a promising path forward for AuNCs@hBSA in tracking intracellular thermal uctuations, as well as other applications such as identifying heat bottlenecks within integrated circuits. In order to better quantify the sensitivity of these stabilized AuNCs@BSA to temperature and to place these results in context with examples from the open literature, we tted the data from Fig. 4 to an Arrhenius-type expression of the form 1 1 Ea ¼ þ A exp kB T F ðTÞ F0 where F(T) is the temperature-dependent uorescence signal, F0 is the signal at 0 K, the preexponential factor A is a dimensionless constant, Ea is the activation energy for thermal quenching, and kB is Boltzmann's constant. The tting results, presented in Fig. 5 reveal a mean Ea value of 206 17 meV. By comparison, Ea values of 74 meV and 130 meV were determined from data for histidine-protected Au10NCs31 and lipoic acidprotected AuNCs,33 respectively. In other words, the luminescence from AuNCs@BSA is inherently more sensitive to temperature than that from these small thiolate ligand-capped AuNCs. Our results also compare favourably with non-MNCs such as organosilane-functionalized carbon dots (Ea ¼ 59.5 meV) which were recently proposed as optical probes for cell imaging.24 With the well-behaved AuNCs@BSA now in hand, we returned our attention to the prospect of employing bandshape analysis as an alternate means of determining the local temperature. Namely, the superb reproducibility in the optical properties of AuNCs@hBSA during iterative heating and cooling cycles allows us to leverage the variation in emission spectral bandshape for self-referenced nanothermometry. Employing the intensity ratio measured at 700 and 610 nm (F700/F610), we
Fig. 5 Arrhenius plots of AuNCs@hBSA during heating and cooling portions of a single thermal cycle. As benchmarks, curves representing the sensitivity to temperature of known MNCs taken from the literature are also shown. The Ea values presented correspond to activation energies for thermal quenching. For an expanded view of this plot, showing all data from ref. 31, refer to Fig. S8.†
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Fig. 6 (A) The difference spectrum constructed by subtracting the normalized emission spectrum for AuNCs@hBSA measured at 10 C from that measured at 45 C. Vertical marks denote the wavelengths 610 nm and 700 nm used to track temperature. (B) Normalized steadystate fluorescence emission spectra taken of AuNCs@hBSA during controlled heating (shown as solid curves) and cooling (symbols) segments of one thermal cycle. lexc ¼ 400 nm. The corresponding results for untreated AuNCs@BSA are given in Fig. S4 of the ESI.†
can reliably estimate the temperature to within one degree (Fig. 6). We are not aware of any previous report showing a consistent variation in the spectral properties of MNCs with temperature. Given the benets of utilizing ratiometric changes to monitor temperature,17,44 however, our current results suggest that this approach merits consideration in future efforts seeking to optically track temperature using various protein- or ligand-stabilized uorescent MNCs. As a nal point, we note that, for consistency with our earlier work,13,34 we have opted to employ UV and blue excitation wavelengths when seeking dual (i.e., blue and red) and solely red emission from AuNCs, respectively. However, as we demonstrate in Fig. S9,† it is entirely possible to employ visible wavelengths to excite red emission from AuNCs@BSA, including the use of common argon-ion laser lines (488 and 514 nm), suggesting a clear avenue for effective cellular thermometry. Two-photon excited uorescence (TPEF) is also an enticing possibility for high-resolution bioimaging or deep tissue studies with minimal excitation of endogenous cellular uorescence (e.g., avins, bilirubin).45 Of note, Oh, Medintz and co-workers recently applied biocompatible near-infrared-emitting PEGylated AuNCs for two-photon cellular imaging,46 suggesting feasibility for TPEF thermometry using AuNCs@BSA probes within cells as well. To summarize, we report the rst study seeking to employ protein-passivated AuNCs as optical probes for accurate temperature monitoring at physiological temperatures. Although not previously known, we nd that the original luminescence intensity arising from AuNCs@BSA is not recovered following a single heating and cooling cycle. We have solved this issue by evaluating several disparate strategies to
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alleviate the hysteresis, erasing the thermal history of AuNCs@BSA to yield a highly promising nanoscale thermometer. The most effective strategies involved halide doping, sol– gel incarceration and, most particularly, thermal denaturation of the starting protein to yield an expedient route to rigorously precise optical probes with a temperature resolution of 0.2 C.
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