Plug and play anisotropy-based nanothermometers

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Plug and play anisotropy-based nanothermometers Sebastian A. Thompson, Ignacio A. Martinez, Patricia Haro- González, Alejandro Adam, Daniel Jaque, Jana B. Nieder, and Roberto de la Rica ACS Photonics, Just Accepted Manuscript • DOI: 10.1021/acsphotonics.8b00292 • Publication Date (Web): 18 Jun 2018 Downloaded from http://pubs.acs.org on June 19, 2018

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Plug and play anisotropy-based nanothermometers. Sebastian A. Thompson‡†*, Ignacio A. Martínez+, Patricia Haro- González%, Alejandro Adam||, Daniel Jaque %, Jana B. Nieder§* and Roberto de la Rica†,#*. †

Department of Pure and Applied Chemistry, WestCHEM, University of Strathclyde,

Technology and Innovation Centre, 99 George Street, Glasgow, G1 1RD, Scotland, UK. #

Department of Chemistry, University of the Balearic Islands, Carretera de Valldemossa km 7.5 Palma, 07122, Spain. ‡

Department of Chemistry and Biochemistry, Hunter College - City University of New York New York 10065, USA §

Ultrafast Bio- and Nanophotonics Group, INL - International Iberian Nanotechnology Laboratory, Av. Mestre José Veiga s/n, 4715-330 Braga, Portugal

%

Fluorescence Imaging Group, Departamento de Física de Materiales, C-IV, Universidad Autónoma de Madrid, C/Francisco Tomás y Valiente 7, Madrid 28049, Spain +

Departamento de Estructura de la Materia, Física Térmica y Electrónica and Grupo

Interdisciplinar de Sistemas Complejos, Universidad Complutense de Madrid, Madrid 28040, Spain

||

Department of Molecular and Cellular Physiology and Department of Ophthalmology, Albany

Medical Center, Albany NY 12208, USA

ACS Paragon Plus Environment

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ABSTRACT

Temperature is a crucial parameter in biology, nanoelectronics, nanophotonics and microfluidics. Optical methods excel for measuring temperature because they are non-invasive, spatially accurate and can measure real time local changes in temperature. Among these, fluorescence anisotropy-based methods are particularly advantageous because they are less affected by changes in the probe concentration and irradiation conditions. However, at physiologically relevant temperature ranges in aqueous solution, fluorescence anisotropy contrast can only be achieved with rather large fluorescent proteins such as the green fluorescent protein (GFP), which can limit the range of applications through this method. Here, we propose a method to add thermosensitivity to any protein thereby transforming them into fluorescence anisotropy-based thermoprobes. It consists of covalently attaching a dye to the protein, which increases the rotational time of the dye-protein system compared to the free dye, and confers thermosensitivity to the resulting bioconjugates. With this method we transformed bovine serum albumin, glucose oxidase and catalase into nanothermothers. This also allowed us to analyze the anisotropy signal changes occurring during the catalytic cycle of catalase, as well as their correlation with the reaction exothermicity. The potential of this method ensures applicability in extending temperature measurements to any protein-based experiments.


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KEYWORDS

Nanothermometers, fluorescence anisotropy, temperature, enzyme.

Temperature influences all biological mechanisms and processes, such as gene and metabolic regulation, protein expression and intracellular uptake1–4. Temperature-altering therapies are used to induce the death of cancer cells in anti-tumoral treatments, including photothermal and magnetothermal therapies5,6. Consequently, the possibility of accurately measuring temperature variations inside biological tissues has drawn increasing attention7. Among the different approaches for measuring intracellular temperature, photonic methods excel because the measurements can be performed in non-contact mode and in real time. Several different types of optical probes have been developed as temperature indicators, including fluorescent polymers8, nanodiamonds9, fluorescent DNA-based thermometers10, infrared rare earth doped nanocrystals11 and fluorescent proteins12,13. Among these, the green fluorescent protein (GFP) is particularly advantageous as a thermoprobe due to its inherent biocompatibility and temperature-dependent fluorescence polarization anisotropy (FPA)12,13. Together with ratiometric luminescent probes14,15, FPA-based probes present important advantages over luminescence intensity-based probes for detecting temperature. For example, they are not affected by changes in the absolute intensity of the fluorescence, which can be a consequence of photobleaching, variations in illumination intensity or fluorochrome migration. Fluorescence intensity variations make other fluorescence-based nanothermometry methods prone to artifacts. So far, FPA-based temperature measurements in aqueous solution have only been reported using genetically expressed fluorescent proteins as thermoprobes12,13. The genetically expressed


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fluorescent proteins are manly used in intracellular environments and its applications are limited to genetically modifiable systems. So, it is highly desirable to have a method that shows the outstanding sensitivity and robustness of FPA detection with GFP but can utilize the common fluorescent dyes currently available. These dye-based nanothermometers will have large potential in various other fields that also require measuring local changes in temperature as well as determining the temperature of the extracellular environment and in clinical applications. However, a serious limitation must be overcome when designing FPA-based thermoprobes16. FPA-based temperature measurements depend on how much the dye rotates (rotational correlation time, θr) during its fluorescent lifetime τf . Experimentally, FPA is measured as follows: =

||

||

,

[1]

where and || correspond to the intensities of the fluorescence polarized orthogonal and parallel to the incident light respectively. As the anisotropy changes following Perrin’s equation: = 1 +

,

[2]

where is the delimiting anisotropy, a constant value of 0.4. It can be easily inferred that the best temperature sensitivity is achieved when θr has the same order of magnitude than the fluorescent lifetime of the molecule16, as the equation [2] derivative maximizes in the limit / → 1 . For GFP, θr = 4.1 ns and the fluorescent lifetime τf = 2.5 ns at physiological temperature and pH13, thus making this protein an excellent probe for FPA-based measurements. For common fluorescent dyes the rotational correlation time is significantly shorter compared to the fluorescence lifetime, which makes them unsuitable for FPA measurements. For example, fluorescein has a rotational correlation time θr = 0.17 ns and a fluorescence lifetime τf = 4.1 ns.


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Due to the mismatch between θr and τf the sensitivity of fluorescein as an anisotropy-based thermoprobe is extremely poor16. This is the crucial factor that limits the utilization of common fluorescent dyes as thermoprobes. To overcome this limitation, the rotational correlation time θr of the reporter dye needs to be increased to the same order of magnitude as the fluorescence lifetime τf. Here we employ a method that meets this demand and allows transforming common fluorescent probes, such as fluorescein and other dyes, into FPA-based thermoprobes. It is based on increasing the θr of the reporter dye via attachment to a larger protein molecule. A schematic presentation of this concept is shown in Figure 1.

Figure 1. Schematic representation of the method used here for transforming a simple dye (e.g. fluorescein) into a fluorescence polarization anisotropy (FPA) - based nanothermometer. Dye


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molecules are conjugated to a protein (e.g. glucose oxidase). After the conjugation, the hydrodynamic radius (Rhyd) and therefore the rotational correlation time θr of the hybrid increases three orders of magnitude, from picoseconds to nanoseconds, which is the key factor to increase the sensitivity of the hybrid dye-protein thermoprobes. The θr is directly related to the hydrodynamic volume of the molecule, as described by the Debye-Stoke-Einstein equation: =

!"($) &$

,

[3]

with V as hydrodynamics volume of the system, '(() is the dynamic viscosity, T the absolute temperature and k the Boltzmann constant. We propose that the hydrodynamic volume is increased, by covalently binding the dye to a non-fluorescent protein, thereby increasing θr of the bioconjugate. As the rotational time of a globular protein depends essentially on its molecular weight, the rotational time of a fluorescent protein conjugate can be designed to match any requirement. This modification is routinely used to study biomolecular interactions, for example protein-protein and protein-DNA interactions17,18. Here, we report the first use of protein-dye hybrids as FPA-based thermoprobes. In addition to this, the proteins in the hybrids retain their native activity, which allows preparing multifunctional thermoprobes that incorporate biomolecular functions such as biorecognition and biocatalysis. In this manuscript, we also demonstrate this concept by preparing dye-enzyme hybrids that can monitor changes in anisotropy derived from the biocatalytic activity of the enzyme. The proposed method is easily generalizable to other dyes-protein conjugates, and therefore it is a universal approach for the development of FPA-based thermoprobes. Multiple fluorescent dyes are commercially available


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with a wide range of fluorescence lifetimes. Further, altering the size of the protein allows for exquisite control of the rotational speed, leading to optimization Proteins were modified with fluorescent dyes following standard procedures, consisting in the reaction of N-hydroxysuccinimide (NHS) derivatives of fluorescein with primary amine groups in proteins, followed by simple column filtering to remove unbound dye from the protein-dye conjugate. A vast array of NHS-derivatives of fluorescent dyes are commercially available, and all globular proteins have primary amine groups available for bioconjugation, which makes this method a universal approach for obtaining protein-dye hybrids. Here we demonstrate this idea by covalently attaching fluorescein to three different proteins: glucose oxidase (GOx), catalase (Cat) and bovine serum albumin (BSA) and measuring the corresponding changes in fluorescence polarization anisotropy. As it was expected, the FPA values increase when the fluorescein is bound to the GOx, Cat and BSA, (SI Fig. S1). The temperature-dependent FPA properties of the hybrid and the repeatability of thermosensing during temperature cycles were first tested by changing the temperature of a sample containing fluorescein bound to glucose oxidase (GOx-F) (Figure 2 a, b). As it can be observed in Figure 2a, decreasing the sample temperature from 18 to 35 oC, increases the anisotropy of the hybrid from 0.070 to 0.089. Likewise, increasing the sample temperature from 20 to 35 oC in Figure 2b results in a continuous increase in anisotropy from 0.085 to 0.070. These results demonstrate that by binding fluorescein to GOx, the hybrid system shows measurable temperature-dependent FPA, allowing for sensitive determinations in a physiologically relevant temperature range.


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Figure 2. Temperature-dependent fluorescence polarization anisotropy (FPA) of bioconjugates. Real-time FPA of the glucose oxidase-fluorescein hybrid during temperature decrease from 35 o

C to 18 oC (a), and while heating the cuvette holder back to 35 oC (b). In both cases, the black

dashed line corresponds to the stationary value of the FPA at low temperature while the red dotted line represents the high temperature case. The temperature is changed using a tunable thermal bath connected to the sample, and temperature measured at the bottom of the cuvette during cooling (a) and heating (b). Notice that the delay is due to the distance between the probed sample volume and the independent thermometer (around 2 centimeters). Next, we determined the temperature sensitivity for free fluorescein and for three fluoresceinprotein hybrids GOx-F, Cat-F and BSA-F (Figure 3a-d). The sensitivity s is determined based on the FPA characterization at various set temperatures in the physiological range. The Peltier cell is set at a certain value, allowing the system to thermalize. Then, the FPA at this given temperature is registered, before setting the temperature to the next value. The results are compared with the sensitivity value as a function of the hydrodynamic volume obtained with a theoretical model (Figure 3e). The experiments are also performed with GFP in order to compare with the gold standard in FPA-based nanothermometry (SI Fig. S2). All hybrids clearly show


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measurable changes in anisotropy as a function of temperature. The sensitivity is calculated as the slope of the resulting calibration curves. All the hybrids perform much better than the unconjugated fluorescein, whose FPA change is so low that it is almost undetectable in PBS16. Moreover, GOx-F and BSA-F hybrids displayed sensitivity comparable with GFP (Table 1), while Cat-F shows a somewhat lower sensitivity (s = 0.534 10-3 oC-1) than the other hybrids, although still higher that fluorescein alone. Table 1: Thermal sensitivities of the studied FPA probes FPA-based Thermoprobe

Sensitivity

GOx-F

0.93.10-3 C-1

BSA-F

0.95. 10-3 C-1

Cat-F

0.534 10-3 C-1

GFP

1.7x10-3 C-1

Fluorescein

0.36 10-3 C-1

For a deeper understanding of the relationship between protein size and sensitivity we linearized the Perrin equation around a given temperature ( , as follows: (() ≈ (( ) + *+,+( - (( − ( ) = 0( + 1 . $.

[4]

We justify this expansion as the range of interest is small enough to develop eq. [1] as a Taylor series over the desired range (the associated error of the approximation is below 5 % in the


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studied interval). Here, we define 0 ≡ *+,+( -

$.

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as the sensitivity of the probe and c as the

residual anisotropy, which has no physical interpretation in our method. As seen in equation [3], the temperature appears twice in . On the one hand, the absolute temperature appears in the denominator, if the temperature rises from 10.0ºC to 40ºC, this term will change around a 10%. On the other hand, the dynamic viscosity appears in the numerator. If we focus in the 10ºC-40ºC range, '(() will decrease from 1.4 mPa s up to 0.65 mPa s, much more than the direct

contribution of the temperature. In the Taylor expansion, the evolution of the viscosity '(() with temperature can be assumed to vary with the Arrhenius expression ' = 4 5/$ where A and B

are given constants and T is the absolute temperature19. We choose this approximation to ease the calculus, the same reasoning can be done using a more accurate expression for the dynamic viscosity. Then, expanding the function around ( , the sensitivity will be defined as: 0 = −

78 < 78 = 9:(;. ) :(;. );. 9 E

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@&$. D A ? !"($. ) C >

, [5]

B

where '(( ) is the dynamic viscosity evaluated in ( . Assuming that fluorescent lifetime corresponding to fluorescein is 4 ns, and additionally that the viscosity response to temperature and the thermal energy for all the samples is the same, the sensitivity of a FPA based probe is a direct function of both the hydrodynamic volume V of the rotating emitter and the temperature.

Notice that, at fixed of 4 ns, a maximal sensitivity of 0FGH ≈ −2.5 · 10M N is obtained for hydrodynamic volumes around 20 nm3 and decreases for smaller and larger hydrodynamic volumes. This behavior is shown in Fig 3e, where the predicted behavior corresponds to the solid


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blue line, while the experimental values described before are plotted with their experimental errors in form of squares. The theoretical calculations are in an agreement with the experimental result for fluorescein and all three protein-dye hybrids tested. Note that the behavior of the sensitivity is represented in semilogarithmic scale in volumes. The reported hydrodynamic Stokes radius of BSA is about 3,5 nm resulting in a volume of about 180 nm3. Assuming a spherical shape for one of the asymmetric GOx dimers with dimensions of about radius

O G , P , Q R = O6.0, 5.2, 7.7R nm respectively, then the volume is calculated as U = 4,3 G P Q resulting in a volume of about 125 nm3. To extend the capabilities of this method to other dyes, the sensitivity value (as a function of the hydrodynamic volume obtained with a theoretical model) was also performed for rhodamine-6 τf of 1.68 ns and lucifer yellow τf of 5.7 ns (SI Fig S3).


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Figure 3. Temperature-dependent response of the fluorescence polarization anisotropy (FPA) curves determined for (a) fluorescein alone, (b) catalase-fluorescein (Cat-F), (c) glucose oxidase- fluorescein (GOx-F), and (d) bovine serum albumin-fluorescein hybrid (BSA-F). (e) Theoretical prediction for the thermosensitivity in dependence of the FPA probe volume (blue


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line) and experimentally determined sensitivities for fluorescein (red solid square), and for the three studied protein – fluorescein hybrids: GOx-F (green solid square), BSA-F (grey solid square) and Cat-F (violet solid square). The dimension of the square equals the estimated error in sensitivity and volume determination. These results shown above demonstrate that the proposed method of bioconjugation of dyes with proteins can be used to assemble thermoprobes from commercially available dyes and different proteins. Therefore, the temperature can be inferred from the changes in the anisotropy using a

linear relation within certain range: ( = / 0. The thermoprobe design is highly flexible and can be applied to proteins with unique biomolecular functions, for example biorecognition or

biocatalytic functions, which makes it promising for measuring changes in temperature derived from biomolecular reactions. Since the method proposed here can be used to obtain thermoprobes using any protein as a basic building block, it should be possible to apply it in situ to the study of exothermicity or endothermicity of biological reactions. Here we support this idea by measuring variations in FPA originating in the biocatalytic activity of catalase. Catalase transforms hydrogen peroxide into oxygen and water through a strongly exothermic reaction20. Cat-F thermoprobes were prepared with the method described above. Figure 4 shows the timedependent anisotropy curve measured after the addition of the hydrogen peroxide H2O2 substrate to the fluorescein-labeled enzyme. The FPA does not decrease in control experiments in the absence of the enzyme substrate, which corroborates that the biocatalytic transformation of H2O2 is responsible for the observed change in anisotropy. H2O2 was added to a final concentration of ca. 0.1 M, which is higher than the Michaelis constant (KM) of catalase from bos taurus about 30 mM. This means that the enzyme is saturated and is working at the highest rate possible under the chosen experimental conditions. The activity of the enzyme provided by the manufacturer is


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≥ 104 units/mg. The FPA measurements were interleaved by approximately 100 seconds in order to have time in between measurements for the removal of oxygen bubbles from the solution. As it can be observed in Figure 4, the FPA reached its minimum once the system enters in its stationary state, being all the enzymes working at saturation (considering that the enzyme is saturated, 0.1 M H2O2 are consumed in ca. 60 s.). After this, the FPA signal returns to the previous value following an exponential relaxation while the substrate is totally consumed. These changes in FPA can be correlated to alterations in different parameters of the system under study. On one hand, the apparent local temperature (SI fig S4) reached by the enzyme is in contradiction with the estimations based on Fourier law. This estimated calculation suggests that the temperature should increase only a few hundreds of miliKelvins21. Thus, it is necessary to correlate the observed changes in FPA with processes or changes that occur during the catalytic activity. On one hand, the changes in FPA signal can be correlated with the chemoacoustic process as suggested by Riedel et al21. In this process the heat released during the catalytic activity enhances the enzyme diffusion (ergo its rotation that will decrease rotational correlation time of the hybrid). On the other hand, this variation in the FPA can be also interpreted as a change in the enzyme conformation induced by the catalytic activity. In this scenario, the mean rotational time varies due to geometrical changes, i.e. varying the hydrodynamic volume of the studied sample but not the surrounding temperature. The FPA measure in such described experiment will be an ensemble average of the different conformations of the operating enzyme, as recently pointed by Illien et al22. Such conformational changes could be studied with alternative fluorescence techniques such as fluorescence correlation spectroscopy. This would allow isolating the contribution of temperature changes originated solely by the


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exothermicity of the enzymatic reaction, therefore providing a unique approach for sensing temperature changes occurring at the biomolecular level.

Figure 4. Time-lapse fluorescence polarization anisotropy changes (∆FPA) of Cat-F after addition of hydrogen peroxide substrate to the enzymatic thermoprobe solution. Blue empty circles represent the experimental data while the dashed black line is a guide to eye based on a smoothing spline (p=5.5 10-5). In conclusion we have successfully demonstrated a new design for fluorescence polarization anisotropy (FPA)-based nanothermoprobes consisting of common fluorescent dyes covalently bound to proteins. This design principle can be applied to a wide range of available fluorescent dyes proteins, allowing for excellent temperature sensitivity optimization for various applications. The bioconjugation used does not affect the biomolecular function of the protein. Thus, this method can be used to enrich the protein functionality and produce proteins with simultaneous thermal nanosensing capability. The resulting protein thermoprobes therefore can


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be used to follow temperature changes in the nanoscale environment of the constituent protein, e.g. happening as a result of the biomolecular function. This could be used to study changes in temperature induced by biological reactions in situ. METHODS Protein-dye hybrids synthesis and purification: The hybrids were obtained by adding NHSfluorescein (0.2 mg mL-1, 6-[fluorescein-5(6)-carboxamido]hexanoic acid N-hydroxysuccinimide ester, Sigma) to the target protein (1 mg mL-1 in PBS) for 30 min followed by purification with a PD-10 desalting column. Anisotropy measurements: Optical measurements were performed using a fluorescence spectrometer in orthogonal excitation – detection geometry (ChronosBH spectrometer, ISS Inc), equipped with a pulsed picosecond 476 nm diode laser (Hamamatsu), broad band polarizers in excitation and detection path and additionally a 473 LP filter (Semrock) in the detection path with PMT detector (H7422P-40, Hamamatsu). Fluorescence lifetime measurements are based on time correlated photon counting (TCSPC) measurements using fast electronics (SPC130, Becker&Hickl) and were performed with the analyzer at magic angle position (54.7°). The device control and data analysis were performed with the ISS Vinci software. The temperature of the sample solutions was either controlled or temperature measured at the bottom of the cuvette with Peltier-based temperature controller (Turret 4™, Quantum Northwest, Inc.). All the experiments were performed in the same aqueous buffered solution (phosphate buffer saline, PBS) in order to avoid changes in fluorescence polarization anisotropy originating from variations in the viscosity of the solution. AUTHOR INFORMATION


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Corresponding Author * Corresponding authors: [email protected]; [email protected], [email protected] SUPPORT INFORMATION Figure SI1. Effect of bioconjugation on anisotropy and fluorescence lifetime. Figure SI2. Temperature-dependent fluorescence polarization anisotropy (FPA) curve determined for Green Fluorescent Protein (GFP) Figure SI3. Theoretical predictions for the thermosensitivity of Rhodamine 6 and Lucifer Yellow. Figure SI4. Apparent local temperature changes (DT) reached by the fluoresceine functionalized enzyme Cat-F.

Figure SI5. Time stable anisotropy of the fluorescein-catalase (Cat-F) hybrid

AUTHOR INFORMATION Author Contributions S.T. and J.B.N., R.R. conceived and planned the experiments. R.R. synthesis and purification of the thermoprobes; S.T., J.B.N. spectroscopic experiments, I.A.M developed the theory and performed the computations, R.R., S.T., I.A.M., J.B.N. analysis and interpretation of the data; S.T., I.A.M., J. B. N., R.R. drafting the manuscript; D.J., A.A., P. G., critical revision. ACKNOWLEDGMENT


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S. T. acknowledges a PEER/PECRE travel grant from WestCHEM. IAM acknowledges financial support from Spanish Government TerMic (FIS2014-52486-R) and CONTRACT (FIS201783709-R) and Juan de la Cierva formación program. R. R. acknowledges a Ramón y Cajal contract from Ministerio de Economía, Industria y Competitividad, Agencia estatal de investigación, Universitat de les Illes Balears, Conselleria d’Innovació, Recerca i Turisme and the European Social Fund. J.B.N. acknowledges funding by the CCDR-N via the project “Nanotechnology based functional solutions”, grant no.: NORTE-01-0145-FEDER-000019. A.P.A acknowledges funding by the American Heart Association, grant 13SDG17100110. We are thankful to Dr. J. S. Donner for critical discussions. ABBREVIATIONS BSA, Bovine Serum Albumin; GOx, Glucose Oxidase; Cat Catalase. θr, rotational correlation time; τf, fluorescence lifetime; NHS, N-hydroxysuccinimide; FPA, Fluorescence Polarization Anisotropy. REFERENCES

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ACS Photonics

Dye

Protein

-Emission -Emission -Fast -FastRotation Rotation

Hybrid: Nanothermometer -Emission -Slow Rotation

-No Emission -Slow Rotation


Plug and play anisotropy-based nanothermometers

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