ARTICLE pubs.acs.org/JPCC
Decrease in Luminescence Lifetime Indicating Nonradiative Energy Transfer from Upconverting Phosphors to Fluorescent Acceptors in Aqueous Suspensions Terhi Riuttam€aki,*,†,§ Iko Hypp€anen,*,‡,§ Jouko Kankare,‡ and Tero Soukka† † ‡
Department of Biotechnology, University of Turku, Tykist€okatu 6A, FI-20520 Turku, Finland Department of Chemistry, Laboratory of Materials Chemistry and Chemical Analysis, University of Turku, Vatselankatu 2, FI-20014 Turku, Finland
bS Supporting Information ABSTRACT: Utilization of energy transfer from anti-Stokes photoluminescent upconverting phosphors (UCPs) to acceptor fluorophores has been successfully described in various homogeneous bioanalytical assays with subnanomolar detection limits. However, only limited evidence has been shown concerning the nature of the energy transfer mechanism involved, especially in aqueous environment. Energy may be transferred in a photon-mediated way (e.g., photon reabsorption) or through strongly distance-dependent dipoledipole interactions between resonant energy levels (resonance energy transfer). To study the mechanism, we determined the luminescence lifetimes of the UCP donor in aqueous suspension by using the frequencydomain measurement and examined the lifetime changes in systems involving acceptor molecules in close proximity or within undefined distance. A decreased lifetime component in the presence of close-proximity acceptors was confirmed (6472% reduction), and strong support for the contribution of the nonradiative resonance energy transfer in the process was obtained, although photon reabsorption is always present to some extent.
’ INTRODUCTION Upconverting phosphors (UCPs) providing unique luminescent features were introduced to bioanalytical applications in the 1990s,1 and papers demonstrating homogeneous bioaffinity assays based on energy transfer from UCP donors were published in 2005.2,3 UCPs consist of an inorganic host lattice (e.g., NaYF4) doped with certain lanthanide ions (e.g., Yb3+ combined with Er3+, Tm3+, or Ho3+). They are capable of converting lowerenergy near-infrared (NIR) radiation to visible light via sequential absorption of two or more photons.4 The anti-Stokes photoluminescence enables total elimination of autofluorescence and, thus, highly sensitive detection. Considering the homogeneous assays utilizing energy transfer from a donor to an acceptor fluorophore, the bright and very narrow-banded emission of the UCPs, typical for lanthanides, is an outstanding advantage as the sensitized acceptor emission can be measured without crosstalk from the donor. By employing a NIR excitation source, the direct excitation of acceptors is also avoided, and practically all emission collected at the wavelength characteristic to the acceptor originates from the energy transfer. In addition, even optically challenging sample materials, such as whole blood,5 are compatible with the UCP technology as it is possible to exploit far-red and NIR wavelengths (>650 nm), where even the red-colored blood is practically transparent. r 2011 American Chemical Society
Fluorescence-based homogeneous bioanalytical assay concepts avoid the need for separating bound and free fraction of the labeled probe because the fluorescence is modulated by ligand binding itself or alternatively by enzymatic activity. Since 2005, several papers describing homogeneous assays based on photon upconversion combined with energy transfer have been published.512 Two basic requirements for F€orster-type resonance energy transfer13 are fulfilled: the donor and the acceptor are in close proximity due to the biomolecule recognition, and the emission spectrum of the donor overlaps with the excitation spectrum of the acceptor. Even though the results in the above-mentioned papers indicate that the distance between the UCP donor and the acceptor fluorophore plays an important role in modulation of the fluorescence signal, to the best of our knowledge, no evidence concerning the nonradiative nature of the upconversion energy transfer mechanism has previously been reported in aqueous solution, which is the fundamental environment for bioanalytical assays. Both the nonradiative energy transfer and photon reabsorption result in the decrease of the observed donor luminescence intensity as part of the energy is transferred to the acceptor molecule and, consequently, the sensitized emission of the Received: June 17, 2011 Published: July 19, 2011 17736
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Figure 1. Emission spectrum of the UCP donor and fluorescence spectra of (a) BPE, (b) ATTO565, and (c) DY556 acceptors. The rectangle indicates the half-width of the emission band-pass filter used for the lifetime measurements. The dashed lines represent excitation spectra and the solid lines emission spectra.
acceptor is increased in the case of a fluorescent acceptor. Photon reabsorption is a photon-mediated process where the acceptor laying at a distance from the donor is excited due to the absorption of photons emitted by the donor. As there is no interaction between the molecules, no changes in the luminescence lifetimes take place. Nonradiative resonance energy transfer (RET) relies on the near proximity of the two interacting molecules, and energy is transferred between the resonant electronic excited states resulting in significantly more efficient energy transfer compared to the photon reabsorption. RET decreases the luminescence lifetime of the donor because it offers an additional relaxation pathway. Nonradiative resonance energy transfer from a lanthanide donor has some advantages over the traditional fluorescence resonance energy transfer (FRET). The F€orster radius R0 (the distance at which 50% of the energy is transferred to the acceptor) for conventional organic dyes is typically 1055 Å but evidently longer for lanthanide donors (up to >75 Å), allowing efficient energy transfer over longer distances (possibly 100200 Å).14 In addition, due to the excellent signal-to-background ratios the lanthanide-based reporter technologies utilizing the timeresolved fluorometry (e.g., lanthanide chelates) or anti-Stokes emission (UCPs) enable the measurement of even the lowintensity sensitized emission corresponding to relatively long distances between the donor and the acceptor molecules. Previously, RET-related changes in the luminescence lifetime of UCPs have been studied with time-domain methods (i.e., pulse fluorometry) in solid state and without any surface functionalization of the donor crystals.15,16 The UCP and the acceptor were brought together by simply drying them on a solid surface. Yan et al.15 used a rod-shaped hexagonal NaYF4:Yb3+, Er3+ (tens of nanometers in width and few micrometers in length) and discovered two luminescence lifetimes (around 90 and 230 μs), which were both reduced (84% and 44%, respectively) when UCPs were mixed with a tetramethylrhodamine isothiocyanate (TRITC) acceptor. However, for some reason the lifetime of the TRITC alone was reported to be several microseconds, which disagrees greatly with the generally known lifetime of rhodamine dyes around a few nanoseconds.17 In addition, a blue laser was used as an excitation source for the UCPs instead of NIR radiation and control measurements defining possible direct excitation of the acceptor were excluded. Bednarkiewicz et al.16
studied the lifetime changes of oleic acid capped cubic NaYF4: Yb3+,Er3+ (diameter ∼30 nm) induced by the presence of trioctylphosphine oxide capped CdSe quantum dots. They discovered a reduction of 15% in the UCP luminescence lifetime (only one reported in this paper; 150 μs), and the reasons for the low RET efficiency were extensively discussed. These include the low quantum yield of the UCP nanocrystal itself, inability of the Er3+ ions in the core parts to participate in RET, and the multiphonon relaxation pathways of the near-surface Er3+ ions resulting in luminescence quenching. One important factor inducing quenching is the aqueous environment with a myriad of OH vibrations, which was omitted in these experiments due to the solid-state measurements. Our research group has previously published several homogeneous bioanalytical assays based on the energy transfer from UCPs.3,5,6,9,18,19 In this work, we studied possible changes in luminescence lifetimes of the UCP donor in an aqueous environment to confirm the assumed nonradiative RET. The lifetimes were determined by utilizing the frequency-domain (FD) technique capable of more efficient deconvolution of overlapping decay processes compared to the time-domain measurements. This technique is especially suitable for lanthanide compounds having a long luminescence lifetime.20 Three acceptors were utilized in studies to confirm the results: a relatively large fluorescent protein (B-phycoerythrin) with high molar absorptivity and two organic fluorophores (ATTO565 and DY556) with significantly smaller size.
’ EXPERIMENTAL METHODS UCP Material. The upconverting material NaYF4:Yb3+,Er3+
(xYb: 0.17, xEr: 0.03) was synthesized with a coprecipitation method.21 The synthesis protocol is described more precisely in the Supporting Information file and in the publication by Hypp€anen et al.,22 which also includes the characterization of luminescent properties. Briefly, solutions of rare earth chlorides as well as EDTA solution were mixed with aqueous solution of NaF. After 1 h stirring, the precipitate was collected, washed with water and anhydrous ethanol, dried under vacuum, and annealed at 400 °C for 5 h. The diameter of the UCP particles was about 110 nm according to the transmission electron microscopy (TEM) images (Figure S1 in Supporting Information). 17737
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Figure 2. Schematic representation of the frequency domain luminometer. PM, photomultiplier; GPIB, general purpose interface bus.
UCP Conjugates. Surface functionalization of the UCPs by poly(acrylic acid) adsorption and conjugation to fluorescent acceptor molecules containing amino groups are described in detail in the Supporting Information file. B-phycoerythrin (BPE; Cyanotech Corp., Kailua-Kona, HI) is a large fluorescent protein (molecular weight (MW) 240 000 g mol1) with high molar absorptivity (ε545nm 2 410 000 M1 cm1) due to ∼40 chromophores per protein. This acceptor was chosen for this study based on its intense emission facilitating the measurements. Amino-modified fluorophores ATTO565 (MW 666 g mol1, ε563nm 120 000 M1 cm1; ATTO-TEC GmbH, Siegen, Germany) and DY556 (MW 736 g mol1, ε548nm 100 000 M1 cm1; Dyomics GmbH, Jena, Germany) were conjugated to UCPs similarly with the BPE, but much higher molar amounts were used due to hundreds of times smaller molecular size. Fluorescence spectra of the UCP and the acceptors are illustrated in Figure 1. In addition to the UCPacceptor conjugates, also control samples were prepared. The UCPs were conjugated with a bovine serum albumin (BSA, fraction V; Bioreba, Nyon, Switzerland) that is not fluorescent but acts as a barrier on the UCP surface to eliminate nonspecific binding of any other molecules. Lifetime Measurements. All measurements were carried out in 10 mM borate buffer (pH 8.5) containing 1 g L1 Tween 20, and the UCP concentration was fixed to 0.2 g L1. Different samples were coded with names where “D” denotes the donor and “A” the acceptor. The “DfA” samples consisted of an UCPacceptor conjugate possibly featuring the RET due to the close proximity of the donor and the acceptor. The “D+A” samples were prepared by mixing the BSA-blocked UCPs with the free acceptor molecules (2.6 nM BPE, 28 nM ATTO565, or 48 nM DY556), which dispersed evenly to the buffer solution without any specific mutual interaction. We created as similar an environment as possible for the DfA and D+A samples with respect to the donor and acceptor concentrations. The amount of acceptor in the UCP conjugates was calculated based on the fluorescence measurements. The possible concentration differences do not have any influence on the luminescence lifetimes, but only the luminescence intensity is affected. To confirm the trends in lifetime changes, the “D+(5)A” samples with 5-fold acceptor concentrations were also prepared. The “D only” sample did not contain any acceptor but only the UCPs blocked with BSA. The luminescence lifetimes were measured on a homemade modular FD luminometer (Figure 2). The sine-wave modulation between 10 Hz and 100 kHz was generated with a SR850
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dual-phase lock-in amplifier (Stanford Research Systems, Sunnyvale, CA). A DC bias was added to the sine-wave, and the modulation signal was fed into the external voltage input of a WLD3343 laser driver (Wavelength Electronics, Inc., Bozeman, MT). The laser driver was used to modulate the current of a NIR laser diode L9418-04 (Hamamatsu Photonics, Hamamatsu City, Japan) providing max 1.2 W at 976 nm that was used as the excitation source. The optical part of the luminometer consisted of the tubular excitation and emission chambers (Thorlabs, Inc., Newton, NJ) in the right angle configuration. The sample chamber was a cage with an aluminum sample holder for a short-cut quartz NMR tube (outer diameter 5 mm). In the excitation chamber, a RG850 long-pass filter with a cutoff at 850 nm (Edmund Optics, Barrington, NJ) was used to ensure a pure NIR excitation. The excitation radiation was focused on the sample with a C350TMEB aspheric lens (Thorlabs). In the emission chamber, a short-pass filter Schott KG5 (Elliot Scientific Ltd., Hertfordshire, UK) with a good transmission at visible wavelengths was used to exclude the scattered excitation radiation. An interference filter with a suitable band-pass, 544/10 nm for the UCP donor (center wavelength 544 nm, half-width 10 nm; Thorlabs) and 600/ 40 nm for the acceptors (Chroma Thechnology Corp., Rockingham, VT), was used to select the emission wavelength. The emission light was focused to a photomultiplier with two BK7 optical glass plano convex lenses (Thorlabs). At the end of the emission chamber was the head-on R1464 photomultiplier (Hamamatsu). The signal from the photomultiplier was amplified with a highspeed current amplifier (DHPCA-100; Femto Messtechnik GmbH, Berlin, Germany). The in-phase and out-of-phase components of the amplified signal were recorded with the same dualphase lock-in amplifier, which was used for the sine-wave generation. The lock-in amplifier was connected to a personal computer with a GPIB interface to control the instrument and to collect data using a program written in LabVIEW (National Instruments, Austin, TX). Luminescence was measured at 200 separate frequencies divided equidistantly in the logarithmic scale between 10 Hz and 100 kHz. The frequency range corresponds to the lifetimes from 16 ms to 1.6 μs. The duration of one full-range measurement was 15 min. The modulation degree of diode laser current was 10%. For compensating the instrumental gain and phase shifts, scattered light from a LUDOX sample was measured without any emission band-pass filters. LUDOX HS-40 (SigmaAldrich, St. Louis, MO), colloidal silica, does not induce phase shift into the signal within the used frequency range, hence it can be used to compensate the systematic deviations of instrumental origin. Data Analysis. The analysis of data is based on the principles described in prior publications.20,23 Assuming the discrete distribution of N lifetimes, the in-phase (Sx) and out-of-phase (Sy) signals of the dual-phase lock-in amplifier as the functions of the angular frequency can be written as Sx ðωÞ ¼
Hτ
N
k k ; ∑ 1 þ ω2 τ2k k¼1
Sy ðωÞ ¼
N
H ωτ2
∑ k 2k 2 k ¼ 1 1 þ ω τk
ð1Þ
Here Hk is the amplitude factor, τk the corresponding lifetime, 17738
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and ω the angular modulation frequency of excitation. Only the out-of-phase signal Sy was used for fitting the observed and calculated data. Parameters Hk and τk are obtained by minimizing the sum of squares Fλ ¼
N
2 obs calc ∑ σ2 k ½Sy ðωk Þ Sy ðωk Þ k¼1
ð2Þ
The standard deviations σk were estimated by smoothing the by using the cubic spline approximation observed signal Sobs y with the cross-validation (program DCSSCV of the IMSL Fortran Numerical Library) and calculating the deviation between the smoothed and unsmoothed signal on a moving window with 20 data points. The goodness of fit was estimated by using the reduced chi-squared parameter (χR2), and the residuals were analyzed by using the lag plot and autocorrelation. Fitting of the data was done with a computer program written in Visual Fortran 90 equipped with the DISLIN graphics package (www.dislin.de). Compensation with LUDOX measurement data was used in all fits. In the present case, it was found feasible to minimize the sum of expressions 2 with the observed signals measured at the wavelengths of donor and acceptor emission, respectively Ftot ¼ Fdonor þ Facceptor
ð3Þ
The relative amplitudes are defined as Ak ¼ 100
Hk τ k N
∑ Hi τi
ð4Þ
i¼1
Depending on the sign of Hk, the relative amplitude may attain also negative values, corresponding to the “rise time” of the emission pulse in the pulse excitation. The sum of relative amplitudes is normalized to 100. Another parameter used in this work is F, the relative sum of amplitude factors, defined as N
F ¼ 100
∑ Hk k¼1 N
∑ jHk j
ð5Þ
k¼1
Obviously F may attain values between 100% and +100%. As shown previously,20 the sum of amplitude factors is in most cases zero. A near-zero value of F indicates a consistent set of lifetimes within the measurement range.
’ RESULTS AND DICUSSION Characterization of UCP Conjugates. To find a suitable acceptor dye load on the UCP conjugates, different amounts of fluorophores were used for conjugation. Those UCPacceptor conjugates with the highest amount of fluorescent molecules without evident self-quenching were chosen for the lifetime measurements (Figure S2 in Supporting Information). The amount of acceptor molecules attached to the UCPs was determined based on the intensity of their fluorescence. A better method for deciding on the amount of the dye would be based on the absorbance, taking into account that too densely conjugated fluorophores may be prone to self-quenching and distort the fluorescence intensity-based result. However, the absorbance
was impossible to read reliably due to the strong scattering caused by the UCP particles. The chosen conjugates consisted of approximately 12.8 pmol of BPE, 141 pmol of ATTO565, or 241 pmol of DY556 per 1 mg of UCPs. These conjugates were prepared by combining 2 mg of the surface functionalized UCPs and 0.05 nmol of BPE, 24 nmol of ATTO565, or 134 nmol of DY556 in the conjugation reaction. Energy transfer to the acceptor could be confirmed based on the emission spectra of the conjugates (Figure S3 in Supporting Information), even though the intensive donor emission was dominating. Lifetime Measurements. The emission was collected at 544 nm (specific band-pass filter for the UCP emission) and at 600 nm (specific band-pass filter for the acceptor emission) to determine the luminescence lifetimes in the different samples. No significant changes in luminescence lifetimes were observed at 544 nm. This finding was obvious due to the large size of the UCP particle (∼110 nm), which is many times greater than the F€orster radius R0. Only the near-surface emitting ions of the UCP can contribute to RET, while a major part of the intense antiStokes photoluminescence is emitted by the ions too far away from the acceptors. The dominating luminescence from the core parts complicates the detection of the effects caused by the potential RET. However, measuring the sensitized emission of the acceptor molecule at 600 nm ignores the emitting donor ions not participating in the RET process. Energy transfer from a long-lived lanthanide excited state to an acceptor molecule will take place as long as the donor remains at the excited state. In other words, the appearance of excited acceptors depends on the donor excited state decay, and as a result, the RET-induced acceptor emission will follow the donor decay.24 Furthermore, due to the NIR excitation employed in the case of a UCP donor, there is no disturbing emission from directly excited acceptor molecules. By collecting the sensitized emission of the acceptor, all luminescence not related to the energy transfer (either nonradiative or radiative) was eliminated, and measurements of low signals and consequently large distances were allowed. The method of choice for data analysis was to fit the signals of the donor (D only at 544 nm) and the acceptor (DfA or D+A at 600 nm) samples together. As a result, both samples attain the same set of lifetime values (τ) but different values of relative amplitudes and F. The idea was to show that the amplitudes of the D+A sample fitted together with the D only sample are equal, and the lifetimes unchanged compared to the results of the D only sample fitted alone. On the other hand, the DfA sample does not behave similarly, and a good fit requires an additional lifetime that has a negligible amplitude for the D only sample and a relatively strong amplitude for the DfA sample. Figure S5 and Table S1 in the Supporting Information file demonstrate with examples how the number of lifetimes (3 or 4) affects the parameters describing the goodness of the fit of an observed distribution compared to a theoretical one (F approaching 0, reduced Chi-square χR2 approaching 1, and lag plot showing random distribution of deviation). As a result, clear evidence referring to the nonradiative RET was found: 6472% reduction in the lifetime (Δτ in Table 1). The decreased luminescence lifetime was not the dominating component (see the relative amplitudes at 600 nm measurement in Table 1), but it is reasonable to assume that some of the energy transfer originates from the reabsorption mechanism that inevitably is always present to some extent and enables energy transfer without affecting the lifetime τ. Particularly the emission 17739
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Table 1. Apparent Luminescence Lifetimes of the Different UCPAcceptor Pairsa,b 544 nm,c specific for the UCP donor τ μs
sample
Fe
Akd %
%
17
1.36
600 nm, specific for the acceptors Akd %
Fe %
χR2f
-
17.1
13.6
9.3
5.6
9.5
5.9
9.9
21.1
9.4
6.5
9.0
15.7
9.3
7.0
9.6
no dye D only
τ1
57
(0.33
τ2
310
(1.1
91
-
τ3
3007
(25
25
-
-
BPE D+A
D+(5)A
DfA
τ1
57
(0.32
τ2
310
(1.1
91
89
τ3
3006
(24
25
24
τ1 τ2
57 309
τ3
3002
(23
25
(0.33 (1.1
17
1.36
17 91
1.36
13
15 90 25
τ1
60
(1.3
20
τ2
315
(5.0
89
τ3
3012
(27
25
24
τ4
113
(12
6
39
1.53
30 68
Δτg = 202 μs (64%) ATTO565 D+A
DfA
τ1
57
(0.32
τ2
309
(1.1
91
87
τ3
3005
(25
25
24
τ1
60
(2.0
21
τ2 τ3
312 2998
(3.0 (22
90 25
65 23
88
(7.0
6
59
τ4
17
1.37
1.30
11
47
Δτg = 224 μs (72%) DY556 D+A
DfA
τ1
57
(0.32
τ2
309
(1.1
17 91
88
τ3
3005
(24
25
24
1.37
12
τ1
60
(24
20
τ2
312
(8.0
90
τ3
3009
(30
25
24
τ4
92
(15
5
52
1.41
41 65
Δτg = 220 μs (71%)
Samples featuring potential RET (DfA samples) were fitted to four lifetimes and samples involving mainly photon reabsorption (D+A samples) or no energy transfer at all (D only sample) to three lifetimes. b Abbreviations: BPE, B-phycoerythrin; D only, the UCP donor only; D+A, the donor and the acceptor mixed in solution (undefined distance); D+(5)A, similar to the previous but 5 times higher acceptor concentration compared to other samples; DfA, the donor and the acceptor conjugated together (close proximity). c Data from D only sample. d Relative amplitude, eq 4. e Relative sum of amplitude factors, eq 5. f Reduced Chi-square (Chi-square/number of degrees of freedom). g Δτ = τ4(DfA) τ2(DfA), where τ4 is the additional lifetime component at 600 nm and τ2 is the dominating τ at 544 nm a
from the core parts of the large donor particle contributes to the photon reabsorption. It must be emphasized that to increase the
overall emission intensity levels the concentrations of UCPs and acceptors in this study were about 10-fold and 2-fold, respectively, 17740
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The Journal of Physical Chemistry C compared to the homogeneous assays reported earlier.3,5,6,18,19 As the degree of photon reabsorption is strongly proportional to the concentration of luminescent species in the sample, the reabsorption mechanism is likely to be less evident in the upconversionRET-based assays compared to the results presented in this study. The measured lifetimes τ1 and τ2 of the D only sample at green (57 and 310 μs at 544 nm) and also at red (181 and 489 μs at 650 nm) wavelengths agreed with those reported earlier by Lin et al.25 In that paper, a different method (time-domain instead of frequency-domain) was used, and the longest lifetime τ3 was not reported. The operation of the modular FD luminometer was regarded to be valid and the results reliable. RET implies a component of reduced lifetime superposed on the existing lifetime of the D only sample.26 Actually, in the present case, the system is more complex as the donor is a relatively large particle. In reality, the RET would induce a continuous distribution of UCP lifetimes due to the continuum of distances between the acceptor and the emitting ions of the donor particle. The data show that the additional lifetime τ4 in the DfA samples (113, 88, or 92 μs depending on the acceptor) was reduced compared to the dominating donor lifetime of the D only sample at 544 nm (∼310 μs; the lifetime having the highest absolute value for the relative amplitude). In addition, the amplitude of the reduced lifetime τ4 in the D only sample (at 544 nm) was only 56%, while in measurements specific for the acceptors (at 600 nm) the amplitude was 710-fold and thus represented a significant lifetime component in DfA samples. On the contrary, the amplitudes of the D+A samples followed those of the D only samples with only minor divergences. Accordingly, the behavior of the DfA samples differed significantly from the D+A and D only samples and conformed to the hypothesis of a superposed RET process. Naturally, the emission intensity from the D+A samples at 600 nm under NIR excitation was considerably lower compared to the DfA samples (see Figure S4 in Supporting Information), but it was high enough to be analyzed. The D+(5)A samples with 5-fold acceptor concentration showed even slightly greater equality in amplitudes with the D only sample, which probably results from more reliable data due to higher emission intensity clearly distinguishable from the noise. The better confidence degree of the fit (F values in Table 1 and Table S1, Supporting Information) supports this assumption. Resolution of the decay times becomes increasingly difficult when they are more closely spaced. Elevated standard deviations for the lifetimes of the DfA samples result probably from the several different distances between the individual donoracceptor pairs influencing the efficiency of RET. The lifetime announced to be the τ4 for the DfA samples in Table 1 rather represents the average luminescence lifetime for a complex situation instead of one precise lifetime, and the standard deviation is naturally reflecting the distance continuum. The relatively large values of the reduced Chisquare may be due to the too optimistic values of measurement errors estimated by the spline smoothing or alternatively due to the non-normal distribution of errors. The lag plots show that the residuals are quite randomly distributed in those cases where the proper number of lifetimes was assigned to the system.
’ CONCLUSIONS This study assured the capability of the UCP donors to nonradiative RET in the aqueous environment. The decrease
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in the donor luminescence lifetime (around 200 μs corresponding >60% reduction) indicates that there is an alternative relaxation pathway strongly suggesting the nonradiative energy transfer. The decreased lifetime component was not the dominating one implying that photon reabsorption also plays an important role in systems involving acceptor molecules in close proximity with relatively large UCPs. Part of the RET may also remain undetected as the weak RET related to longer donoracceptor distances causes only tiny changes in the donor luminescence lifetime and is not distinguished from the standard deviation. The emitting ions in core parts of the UCP can never be close enough to acceptors unless the UCP is a real nanocrystal (diameter below 20 nm). The UCP material used in this study had an average diameter of 110 nm. It is well-known that reduction in the UCP particle size decreases steeply the efficacy of the upconversion due to an increase in surface area-tovolume ratio.27,28 The surface effects can quench the long-lived intermediate excited states hindering upconversion mechanisms. However, in the past few years, publications about the synthesis methods for efficient UCP nanocrystals10,2933 as well as about the surface passivation techniques28,3436 have emerged, and utilization of the entire UCP crystal to the RET process might be possible leaving less room for the radiative energy transfer. Also, the thickness of the surface modification of the UCP particle plays a remarkable role. A thick coating will inhibit the RET to some extent, and the monolayer coatings are therefore preferred. Another extra spacer between the donor and the acceptor is present in bioaffinity assays: a biomolecule responsible for the analyte recognition. The physical dimensions of large proteins are several nanometers. Nevertheless, the lanthanide-based donors enable elimination of most of the background luminescence, and even the RET with low efficiency can still be distinguished from the background noise. To conclude, the data suggest that the distance-dependent nonradiative RET is the mechanism exploited in the proximitybased homogeneous bioanalytical assays utilizing UCP donors. However, a proper control reaction to quantify the emission arising from the photon reabsorption should always be included in the studies. Nanosized UCP crystals will probably reduce the proportion of radiative energy transfer and favor the RET.
’ ASSOCIATED CONTENT
bS
Supporting Information. Synthesis of the UCP material and conjugation of the functionalized UCPs with fluorescent molecules and proteins. Examples of the frequency-domain measurement data of different samples and the data analysis to either three or four luminescence lifetimes. This material is available free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION Corresponding Author
*E-mail: tetrant@utu.fi. Tel.: +358 2 333 8065 (T. Riuttam€aki). E-mail: ikohyppa@utu.fi. Tel.: +358 2 333 6714 (I. Hypp€anen). Author Contributions §
The authors wish it to be known that they contributed equally to this manuscript. 17741
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’ ACKNOWLEDGMENT We thank Jorma H€ols€a and Laura Pihlgren for providing the UCP crystals, Sami Blom and Emilia Engstr€om for preparing the UCP conjugates, and Riikka Arppe for the TEM images. This study was funded by the Finnish Funding Agency for Technology and Innovation (Tekes) and the Academy of Finland (grant numbers 119497 and 140758). We also thank the Graduate school of Chemical Sensors and Microanalytical Systems (CHEMSEM; Espoo, Finland) and the Graduate School of Materials Research (GSMR; Turku, Finland) for funding of Hypp€anen and Pihlgren, respectively.
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