Cyclodextrin-promoted energy transfer for broadly applicable small-molecule detection

Abstract

Reported herein is the development of non-covalent, proximity-induced energy transfer from small-molecule toxicants to organic fluorophores bound in the cavity of γ-cyclodextrin. This energy transfer occurs with exceptional efficiency for a broad range of toxicants in complex biological media, and is largely independent of the spectral overlap between the donor and acceptor. This generally applicable phenomenon has significant potential in the development of new turn-on detection schemes.

1. Introduction

The accurate detection of small-molecule organic toxicants in complex environments has significant implications for public health. Such toxicants are potentially significant contributors to human disease (1–3), and are found in food supplies (4–6), water supplies (7) and in commercial products (8). Current methods for the detection of these chemical toxicants generally require multiple steps: (a) extraction of the toxicants from the environment (9); (b) purification of the toxicants via high-performance liquid chromatography (10) or gas chromatography (11) and (c) detection of the toxicants by mass spectrometry (12) or fluorescence spectroscopy (13). Such detection methods are limited in their ability to distinguish toxicants with identical molecular weights or similar fluorescence spectra.

Small-molecule toxicants can also be detected through fluorescence energy transfer-based methods. Such fluorescence energy transfer, which has been used extensively for biomolecule detection (14–16), often requires significant spectral overlap between the emission spectrum of the donor and the absorption spectrum of the acceptor to achieve efficient energy transfer (i.e. a Förster-type mechanism) (17). This overlap ultimately compromises the sensitivity of the system, as even in the absence of the target analyte there is residual donor emission (18). Efficient energy transfer that is independent of the spectral overlap (i.e. a Dexter-type mechanism) has the potential to lead to improved sensitivities in fluorescent detection schemes (19, 20).

Reported herein is a highly efficient, practical approach for small-molecule detection: using the small molecules directly as energy donors in a non-covalent, macrocycle-promoted energy transfer scheme (21). In such a scheme, both the toxicant and the fluorophore are bound in the interior of γ-cyclodextrin (Figure 1). The enforced proximity of the two molecules allows for non-covalent energy transfer to occur, with excitation of the toxicant (energy donor) resulting in energy transfer to and emission from the fluorophore (energy acceptor). The energy transfer is independent of the spectral overlap between the donor and the acceptor, and has the potential to lead to improved sensitivities in turn-on detection schemes.

Figure 1.

Schematic illustration of cyclodextrin-promoted energy transfer from organic toxicants to fluorophore acceptors.

We recently reported that cyclodextrin-promoted energy transfer occurred from polycyclic aromatic hydrocarbons (PAHs) (compounds 1 –5, Figure 2) and polychlorinated biphenyls (compounds 14–19, Figure 2) to three fluorophores (two of which are shown in Figure 3) (22–24). Proximity-induced energy transfer between the analytes and the fluorophores occurred in the cavity of γ-cyclodextrin, resulting in up to 35% energy transfer efficiencies.

Figure 2.

Known and suspected toxicants investigated as energy donors.

Figure 3.

Fluorophores investigated as energy acceptors.

Reported herein is a substantial expansion of this preliminary report to include (a) a wide range of small-molecule toxicants as energy donors (Figure 2) (25); (b) energy transfer efficiencies as high as 100% and (c) examples of successful energy transfer in complex media: coconut water, plasma (26), breast milk (27) and seawater. The general and highly efficient energy transfer reported herein highlights the robust nature of this phenomenon and the strength of the intermolecular interactions that allow for such energy transfer to occur.

2. Results and discussion

The full chart of examined energy donors is shown in Figure 2. This chart contains several compounds that have been classified as known carcinogens (Group 1) according to the International Agency for Research on Cancer (compounds 3 and 6–10) (28), as well as a variety of other toxicants (29–32). These structures also contain a wide variety of functional groups, steric bulk and photophysical properties, which allows us to probe the donor features necessary for efficient energy transfer.

Energy transfer experiments were conducted by mixing the analyte and fluorophore in a 10 mM γ-cyclodextrin solution in phosphate-buffered saline (PBS), coconut water, seawater, human plasma, or human breast milk. The resulting solution was excited near the analyte’s absorption maximum (defined as ‘analyte excitation’) and near the fluorophore’s absorption maximum (defined as ‘fluorophore excitation’). The energy transfer efficiencies were calculated according to Equation (1):

$$\%\text{energy}\text{transfer}=\frac{I_{\text{DA}}}{I_{\mathrm{A}}}\times 100,$$ (1)

where I_DA is defined as the integrated fluorophore emission from indirect excitation and I_A is the integrated fluorophore emission from direct excitation. A graphical depiction of I_DA and I_A is shown in Figure 4.

Figure 4.

Graphical illustration of I_DA/I_A for a generic donor–acceptor.

Control experiments were also conducted to determine whether the observed fluorophore peaks from analyte excitation were due to legitimate energy transfer rather than a result of the fluorophore having non-zero absorption at the excitation wavelength of the analyte. In these experiments, the fluorophore was mixed with cyclodextrin and excited at the excitation wavelength of the analyte (but in the absence of any analyte). That fluorophore emission was compared with the emission of the fluorophore via analyte excitation in the presence of the analyte. The ratio of these two emissions, defined as the ‘fluorophore ratio’, was calculated according to Equation (2):

$$\text{fluorophore}\text{ratio}=\frac{I_{\text{fluorophore}-\text{control}}}{I_{\text{fluorophore}-\text{analyte}}},$$ (2)

where I_{fluorophore – analyte} is the integration of the fluorophore emission in the presence of the analyte and I_{fluorophore – control} is the integration of the fluorophore emission in the absence of the analyte. Fluorophore ratios substantially less than 1 indicate that the fluorophore emission increases with analyte addition as a result of energy transfer.

The final concentrations of the toxicants were some-what higher than literature-reported concentrations of contaminated biological samples (33–35), although such literature reports vary widely depending on the toxicant identity, biological fluid and sample population. Full results for all donor–acceptor combinations in all media are reported in the Electronic Supporting Information. Particularly exciting results were found using energy donors 7, 8, 11 and 12 with acceptor 20.

2.1 In phosphate-buffered saline

The energy transfer from analytes 7, 8, 11 and 12 to BODIPY 20 in 10 mM γ-cyclodextrin in PBS was exceptionally efficient, with greater than 100% efficiencies observed in all cases (Figure 5). Control experiments with 0 mM γ-cyclodextrin in PBS showed substantially less energy transfer than the 10 mM γ-cyclodextrin solution (Table 1), highlighting the beneficial role of γ-cyclodex-trin in promoting energy transfer.

Figure 5.

Energy transfer in PBS from (a) compound 7, (b) compound 8, (c) compound 11 and (d) compound 12 to fluorophore 20. The black line represents analyte excitation and the grey line represents direct fluorophore excitation.

Table 1.

Selected energy transfer efficiencies in PBS.

Donor	Acceptor	In 10 mM cyclodextrin (%)	In 0 mM cyclodextrin (%)
7	20	121	25
8	20	107	24
11	20	168	32
12	20	119	27

2.2 In coconut water

The composition of coconut water is remarkably similar to that of human plasma, and it has been used as a plasma surrogate during emergencies (36, 37). Analytes 7, 8, 11 and 12 demonstrated efficient energy transfer in 10 mM γ-cyclodextrin dissolved in coconut water (Table 2), albeit with diminished efficiencies compared to energy transfer in pure PBS.

Table 2.

Selected energy transfer efficiencies in complex media.^a

Donor	In coconut water		In plasma		In breast milk
	10 mM CD (%)	0 mM CD (%)	10 mM CD (%)	0 mM CD (%)	10 mM CD (%)	0 mM CD (%)
7	29	29	27	30	24	26
8	26	26	26	27	25	24
11	39	31	17	22	28	30
12	26	18	21	16	19	30

Note: CD, γ-cyclodextrin.

^aFluorophore 20 used as the energy acceptor in all cases.

2.3 In biological media

The ability to achieve cyclodextrin-promoted energy transfer in biological media can provide significant benefit for the detection of toxicants. Efficient energy transfer from compounds 7, 8, 11 and 12 to fluorophore 20 occurred in both human plasma samples and human breast milk samples that were doped with 10 mM γ-cyclodextrin (Table 2).

2.4 Energy transfer in seawater

The detection of toxic oil components in seawater has significant applications in the aftermath of environmental disasters such as the Deepwater Horizon oil spill of 2010 (38) and the Colorado floods of 2013 (http://www.nytimes.com/2013/09/27/us/after-the-floods-a-deluge-of-worry-about-oil.html). Such components include PAHs 1–5, which we have previously shown can participate in energy transfer in purified PBS solution (23). Cyclodextrin-promoted energy transfer using these donors occurred in seawater taken from Narragansett Bay (Rhode Island), with fluorophore 20 as an energy acceptor. All PAHs (1–5) exhibited some degree of energy transfer to fluorophore 20 (Figure 6) under these conditions.

Figure 6.

Energy transfer in seawater to fluorophore 20 from (a) analyte 1, (b) analyte 2, (c) analyte 3, (d) analyte 4 and (e) analyte 5. The black line represents analyte excitation and the grey line represents direct fluorophore excitation.

For all complex fluids, the energy transfer efficiencies were somewhat lower than the efficiencies in pure PBS. These results are not surprising, considering the complex nature of coconut water (39), human plasma (40–43) and breast milk (44, 45), and the high salt content and complex nature of seawater (46, 47). That γ-cyclodextrin-promoted energy transfer from carcinogens to the fluorophores occurred successfully in such complex environments highlights the robust nature of this detection method and the underlying enabling supramolecular interactions.

In contrast to the results obtained in PBS solution, where cyclodextrin clearly promotes efficient energy transfer, many of the analyte–fluorophore pairs in complex media demonstrate equivalent or even greater energy transfer efficiencies in the absence of γ-cyclodextrin than the efficiencies in the presence of cyclodextrin. These results are likely due to two possible phenomena:

1. For cases in which the energy transfer efficiencies are roughly equivalent in the presence and absence of cyclodextrin, it is likely that the donor and acceptor associate without cyclodextrin due to the hydrophobic effect (48). This association leads to energy transfer efficiencies that are essentially identical regardless of the cyclodextrin concentration. Previous research in our laboratory has shown some degree of cyclodextrin-free association as well (23).

2. For cases in which the energy transfer efficiencies are lower in the presence of cyclodextrin, the cyclodextrin might bind one of the two small molecules selectively, thus removing it from the proximity of the second molecule. This removal of one of the energy transfer partners lowers the observed energy transfer efficiencies.

2.5 Comparison with published methods

The ability to detect toxicants via non-covalent energy transfer has a number of advantages compared with previously reported methods, including the ability to tune the emission signal of a single analyte throughout the spectral region through choosing a variety of fluorophores. To achieve this ‘tuning’ ability, preliminary experiments were conducted using a third fluorophore: commercially available coumarin 6 (compound 22) as a fluorescent energy acceptor with selected analytes (10 mM γ-cyclodextrin, PBS solution) as energy donors. Good energy transfer efficiencies were observed for many cases (Table 3), and in most cases the energy transfer efficiencies were substantially higher in the presence of γ-cyclodextrin than in its absence.

Table 3.

Selected energy transfer efficiencies with fluorophore 22.

Donor	10 mM CD (%)	0 mM CD (%)
7	24	8
8	30	38
11	28	26
12	56	39

Moreover, the use of multiple fluorophores allows for the tuning of the fluorescence signal from a single analyte. For this experiment, analyte 12 was mixed with fluorophores 20, 21 and 22 in three vials (in 10 mM γ-cyclodextrin in PBS). Excitation of each solution at 320 nm (the excitation wavelength of the analyte) resulted in three distinct fluorophore signals at 515, 530 and 555 nm for fluorophores 20, 22 and 21, respectively (Figure 7). This tuning of the toxicant signal via judicious choice of fluorophore provides maximum flexibility in developing toxicant detection schemes.

Figure 7.

A comparison of the fluorophore emission peak from toxicant 12 to fluorophores 20–22 in 10 mM γ-cyclodextrin in PBS.

One key challenge of this method compared with published methods for toxicant detection is the difficulty in obtaining quantitative data through non-covalent energy transfer. Preliminary experiments have demonstrated that the fluorescence signal obtained via energy transfer is not proportional to the concentration of the analyte; this is in line with literature reports that demonstrate a complicated relationship between fluorescence energy transfer signals and the concentration of the donor and acceptor (49, 50). This relationship is affected by a multitude of other intermolecular interactions, including donor–donor interactions (51), fluorophore dimerisation and aggregation (52) and undesired fluorophore self-quenching (53).

2.6 General discussion

There are a number of factors that determine whether a particular analyte participates efficiently in cyclodextrin-promoted energy transfer, and the results reported herein provide crucial information towards deconvoluting some of these factors. High energy transfer efficiencies occur in cases in which the analyte–fluorophore pairs (a) form ternary complexes in the cyclodextrin cavity with high affinities and (b) participate in proximity-induced energy transfer. The binding affinities in cyclodextrin are determined by the molecules’ steric and electronic characters (54), and the participation in energy transfer schemes is determined by steric and electronic complementarity between the donor and acceptor (55), molecular orientations of the two guests (56) and the degree of spectral overlap with the fluorophore acceptor (57).

The analytes that demonstrated highly efficient energy transfer in the various media included compounds 7, 8, 11 and 12 (discussed herein) as well as compounds 1–3 (reported in previous publications). The fact that compounds 11 and 12 were efficient energy donors compared with compound 5 is likely due to the presence of the nitrogen substituents, which either enhance the electron-donating ability of the analyte and/or provide favourable electrostatic interactions with the highly polarised fluorophore acceptors. Directly comparing the absorbance spectra, fluorescence spectra and quantum yields of compounds 5, 11 and 12 indicates similar photophysical properties for the three compounds (58, 59), which rules out spectral overlap as a substantial contributing factor.

The success of compound 7 compared with structurally similar compound 6 may be a result of additional amino group enabling compound 7 to form more electrostatic interactions or to bind in cyclodextrin with higher affinities. The similarities in the spectral properties of compounds 6 and 7 again rule out spectral overlap as a significant factor (60, 61). The fact that the photophysical properties of the toxicant energy donors play only a limited role in determining energy transfer efficiencies strongly supports our hypothesis that proximity-induced energy transfer in the cyclodextrin cavity occurs via a Dexter-type, direct orbital overlap mechanism.

One of the most surprising results was the successful use of compound 8 as an energy donor in combination with fluorophore acceptors. Compound 8 has been used as a fluorescence quencher of other small molecules (62, 63), and is only weakly fluorescent. Nonetheless, the weak photophysical activity (455 nm emission maximum from 340 nm excitation) was sufficient for it to participate in proximity-induced energy transfer. The free hydroxyl groups of the molecule likely allow for the formation of hydrogen bonds to the highly polarised fluorophore acceptors. Comparing the results obtained with compound 8 with those of compound 10 (which was relatively inefficient as an energy donor) highlights possible steric constraints (compound 10 is substantially larger than compound 8) and functional group requirements (compound 10 lacks the free hydroxyl moieties) that are necessary for cyclodextrin-promoted energy transfer.

3. Conclusion

In conclusion, highly efficient energy transfer from a variety of organic toxicants occurred to multiple fluorophore acceptors when bound in the cavity of γ-cyclodextrin. The fact that this approach is successful in many environments with a variety of analytes is very beneficial. The robust nature of this approach leaves a wide range of opportunities to expand the scope of the analytes that can be detected, as well as the environments that they can be detected in. Indeed, the only requirement is that the analyte be (at least) weakly fluorescent. Furthermore, sample preparation is simple compared with current methods, as most media simply require dilution with PBS.

The fact that γ-cyclodextrin can bind analytes within its cavity in complex environments means that it can simultaneously isolate the analytes and promote energy transfer so that the analytes can be reliably identified. This method is a significant contribution to the facile and reliable detection of toxic analytes. The ability to tune the emission signal for a particular analyte by varying the choice of fluorophore provides substantial flexibility, and can be used in the development of array-based detection schemes. The development of such an array is currently under investigation, and results of these and other experiments will be reported in due course.

4. Experimental

All chemicals were obtained from Sigma-Aldrich chemical company (St. Louis, Missouri, USA) or Fisher Scientific (Hampton, New Hampshire, USA) and used as received. BODIPY fluorophore 20 was synthesised following literature-reported procedures (64). Human plasma was obtained from Innovative Research, Novi, MI, USA. Human breast milk was obtained from an anonymous donor. Seawater was obtained from the Narragansett Beach in Rhode Island. Coconut water (VitaCoco 100% pure coconut water) was obtained from CVS Pharmacy (Kingston, RI 02881, USA).

The human plasma, seawater and coconut water were used as received. The breast milk was prepared by separating all solids via filtration and centrifugation, followed by dilution with PBS. UV–visible spectra were recorded on an Agilent 8453 spectrometer (Agilent Technologies, Santa Clara, CA, USA). Fluorescence measurements were recorded on a Shimadzu RF 5301 spectrophotometer (Shimadzu Corporation, Columbia, MD, USA) with slit widths of 1.5 nm excitation and 1.5 nm emission slit widths. All fluorescence spectra were integrated versus wavenumber on the x-axis, using OriginPro version 8.6.

The energy transfer experiments were conducted as follows: 2.5 ml of a 10 mM solution of γ-cyclodextrin dissolved in the fluid of interest (PBS, coconut water, Narragansett Bay seawater, human plasma or human breast milk) was measured into a cuvette. Then 20 μl of the analyte (1 mg/ml) and 100 μl of the fluorophore (0.1 mg/ml) were added. After thorough mixing, the solution was excited at two wavelengths: near the analyte’s absorption maximum (defined as ‘analyte excitation’) and near the fluorophore’s absorption maximum (defined as ‘fluorophore excitation’). The energy transfer efficiencies were calculated according to Equation (1):

$$\%\text{energy}\text{transfer}=\frac{I_{\text{DA}}}{I_{\mathrm{A}}}\times 100,$$

where I_DA is defined as the integrated fluorophore emission from indirect excitation and I_A is the integrated fluorophore emission from direct excitation. A graphical depiction of I_DA and I_A is shown in Figure 4. Experiments were also conducted where 0 mM of γ cyclodextrin was used for each fluid, analyte and fluorophore combination, in place of the 10 mM cyclodextrin solution.

Control experiments were conducted as follows: (a) the fluorophore was mixed with γ-cyclodextrin and excited at the excitation wavelength of the analyte (but in the absence of any analyte) and (b) the fluorophore and analyte were both mixed in γ-cyclodextrin and excited at analyte excitation wavelength. The fluorophore emission that resulted from excitation at the analyte wavelength in the absence of the analyte was compared with the fluorophore emission from excitation at the analyte wavelength in the presence of the analyte. The ratio of these two emissions, shown as the ‘fluorophore ratio’, was calculated according to Equation (2):

$$\text{fluorophore}\text{ratio}=\frac{I_{\text{fluorophore}-\text{control}}}{I_{\text{fluorophore}-\text{analyte}}},$$

where I_{fluorophore – analyte} is the integration of the fluorophore emission in the presence of the analyte and I_{fluorophore – control} is the integration of the fluorophore emission in the absence of the analyte. Full tables of energy transfer efficiencies for all analyte–fluorophore combinations and summary figures of all analyte–fluorophore combinations are shown in the Supplementary Material.