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#### Anisotropy (Particle Sizing) Probes for the Life and Materials Sciences

Ursa BioScience™ offers a patent protected range of anisotropy fluorophores, designed to bind to a range of biologicals, proteins, colloidal silica (SiO2, TiO2 etc.) nanoparticles and more, and using either time-resolved or steady-state Fluorescence Anisotropy, readily allow for nanoparticle sizes, folding, small molecule binding, and structural growth mechanisms to be determined. Our anisotropy probes offer customers the ability to bind fluorophores electrostatically to their biomolecules / nanoparticles. Ursa BioScience™ offers 5 different fluorescence anisotropy probes in our electrostatic binding category. Each probe has either slightly different water solubility’s, facilitating affinities for different types of nanoparticles, and/or slightly different fluorescence / photophysical properties, e.g. excitation and emission wavelength etc. In addition, Ursa BioScience also offers convenient particle sizing kits, with Ursa’s range of particle sizing probes already dispensed within plastic 1-1-4 cm cuvettes for easy, quick and no hassle use. Our probes typically have mean lifetimes around 20 ns, which is ideal for determining particle / protein sizes < 10 nm in size.

#### Advantages of Ursa BioScience’s Approach to Nanoparticle Metrology and Structure Elucidation

It is well known that fluorescence techniques are highly suited for determining the size and volume of nanoparticles / proteins etc in both static volume (such as colloidal dispersions) [1-4] and dynamic samples, i.e. samples which are changing volume with time, such as in the sol-gel process. [5,6]

Ursa BioScience’s range of silica probes has been specifically designed to electrostatically or covalently bind silica and other nanoparticles as well as proteins, readily allowing the size of the nanoparticles to simply and rapidly be determined in both static and dynamic samples, allowing material structural formation and hierarchy to be elucidated. In addition, our probes offer the flexibility of measuring nanoparticle sizes as little as 1 nm and at convenient wavelengths, compatible with nearly all fluorometers and time-resolved fluorescence instruments today.

Figure 1. Fluorophore labelling of colloidal nanoparticles is conveniently achieved by using Ursa’s dye preloaded cuvettes. The rotational diffusion of the colloidal particles is monitored using a standard spectrofluorometer with polarizers to rapidly determine the nanoparticle size.

Figure 2. Illustration of the fluorescence anisotropy experiment. The labelled colloidal particles are excited and the polarization properties of the emitted fluorescence light is recorded. The anisotropy spectra and the relation to the particle size is given by the Perrin equation as discussed later in this article.

#### Ursa Blue™

Ursa Blue™ is a cationic, highly water soluble fluorophore which can readily be excited from 300-400 nm, with emission ≈ 440 nm. Ursa Blue™ readily binds electrostatically to colloidal silica nanoparticles allowing customers the choice of determining nanoparticle sizes using either steady-state or time resolved fluorescence analysis. In colloidal silica nanoparticle solutions, Ursa Blue™ shows triple exponential fluorescence decay with mean lifetimes of 16.1, 18.0 and 16.8 ns for DuPont’s SM30, AM30 and AS40 respectively.

Figure 3. Excitation-Emission intensity Maps constructed for data recorded for Ursa Blue™ mixed with silica nanoparticles, Ludox SM 30 diluted to 5 % w/w SiO2. (A) Contour data constructed for sum data and (B) the corresponding anisotropy map. Note that the contour lines represent intensity data from panel A. For this probe we recommend exciting at 350 nm and to record the anisotropy at 410 nm. Although a better fluorescence signal (i.e. intensity) would be obtained by recording at 430 nm the dynamic range available in the anisotropy would diminish. For all our probes we provide the 3D emission spectra and the anisotropy contour map, overlaid with the emission contour map, allowing customers the ability to optimize their excitation, emission and anisotropy wavelengths / dynamic range.

Steady-State Fluorescence Perrin-Plots readily allow Ursa Blue™ to determine the size of nanoparticles by simply determining the anisotropy as a function of temperature. A plot of 1 / r vs. Temp / Viscosity gives a straight line from which the radius of the nanoparticles spheres can accurately be determined from the slope (gradient).

Figure 4. Steady-state anisotropies recorded for Ursa Blue™ at different tempertures, from +5 °C to + 80 °C, when dissolved in Ludox SM 30 diluted to 5 % w/w. The sample was in a standard 1 cm quarz cuvette. Note that a typical constructed sum curve also is shown. (B) Constructted Perrin graph from steady-sate anisotropies presented in panel A. From the slope of the line the particle radius is calculated to 6.35 nm, slightly larger than the manufacturers reported 3.5 nm, and possibly indicating a degree of agglomerationn in the sample.

In a similar manner, time-resolved fluorescence anisotropy measurements, readily allow nanoparticle sizes to be determined using the well-known Stokes-Einstein expression. For Ursa Blue™ in Ludox SM30, the two recovered rotational correlation times conveniently reveal both the bulk solution microviscosity (Φ1 = 0.63 ns) as well as the Ludox nanoparticle size, 3.27 nm (Φ2 = 35.5 ns). While we show examples for uptake of the probes onto silica nanoparticles, similar approaches can be applied to protein uptake, binding and protein sizing / segmental motion studying.

A recent paper describes the use of Ursa Blue™ for silica Nanoparticle metrology.

#### Dipole Blue™

Ursa BioScience™ offers a shorter wavelength absorption/emission anisotropy probe, 320 and ≈ 410 nm respectively. Dipole Blue™ is slightly less water soluble than Ursa Blue™, and better suited to less polar nanoparticle surfaces. In colloidal silica nanoparticle solutions, Dipole Blue™ shows triple exponential fluorescence decay with mean lifetimes of 17.1, 21.9 and 15.9 ns for DuPont’s SM30, AM30 and AS40 respectively. Similar to Ursa Blue™, Dipole blue™ can readily size nanoparticles in both a fluorescence steady-state and time-resolved manner.

Figure 5. Excitation-Emission intensity Maps constructed for data recorded for Dipole Blue™ mixed with silica nanoparticles, Ludox SM 30 diluted to 5 % w/w SiO2. (A) Contour data constructed for sum data and (B) the corresponding anisotropy map. Note that the contour lines represent intensity data from panel A. For this probe we recommend exciting at 350 nm and to record the anisotropy at 400 nm. Although a better fluorescence signal (i.e. intensity) would be obtained by exciting at 325 nm and recording at 420 nm the dynamic range available in the anisotropy would diminish.

For all our probes we provide the 3D emission spectra and the anisotropy contour map, overlaid with the emission contour map, allowing customers the ability to optimize their excitation, emission and anisotropy wavelengths / dynamic range.

Figure 6. (A) Steady-state emission ansiotropies recorded at different teperatures for Diploe Blue™ dispersed in Ludox  SM 30 diluted to 5 % w/w SiO2. The excitation wavelength was 350 nm. The sample was placed in a standard 1 cm quartz cuvette. (B) Consturced Perrin graph for anisotropies presented in panel A.

A recent paper describes the use of Dipole Blue™ for 3D anisotropy contour mapping of nanoparticles in sols, J. Phys. Chem. Lett., 2015, 6 (6), pp 918–922.

#### Rotational Blue 1™

Similar to Ursa Blue™, but less polar and water soluble, Rotational Blue 1™ is ideally suited for a wide variety of fluorescence-anisotropy based particle sizing applications and depending on the polarity of the bulk phase and nanoparticle / protein surface, can partition itself accordingly. In colloidal silica nanoparticle solutions, Rotational Blue 1™ shows triple exponential fluorescence decay with mean lifetimes of 16.1, 18.6 and 17.5 ns for DuPont’s SM30, AM30 and AS40 respectively. Rotational Blue 1™ is a cationic dye, which can readily be excited 300-400 nm, with an emission centered ≈ 445 nm.

Figure 7. Excitation-Emission intensity Maps constructed for data recorded for Rotational Blue 1™ mixed with silica nanoparticles, Ludox SM 30 diluted to 5 % w/w SiO2. (A) Contour data constructed for sum data and (B) the corresponding anisotropy map. Note that the contour lines represent intensity data from panel A. For this probe we recommend exciting at 350 nm and to record the anisotropy at 410 nm. Although a better fluorescence signal (i.e. intensity) would be obtained by recording at 430 nm the dynamic range available in the anisotropy would diminish. For all our probes we provide the 3D emission spectra and the anisotropy contour map, overlaid with the emission contour map, allowing customers the ability to optimize their excitation, emission and anisotropy wavelengths / dynamic range.

#### Rotational Blue 2™

Rotational Blue 2™ is considerably less water soluble than Ursa Blue™ and less soluble than Rotational Blue 1™ and is more ideally suited for nanoparticle metrology measurements in less polar media. Rotational Blue 2™ shows triple exponential fluorescence decay with mean lifetimes of 13.9, 16.2 and 14.8 ns for DuPont’s SM30, AM30 and AS40 respectively. Rotational Blue 2™ is a cationic dye at acidic-neutral pH’s and zwitterionic above pH 7. Rotational Blue 2™, can readily be excited 300-400 nm, with an emission centered ≈ 445 nm.

Figure 8. Excitation-Emission intensity Maps constructed for data recorded for Rotational Blue 2™ mixed with silica nanoparticles, Ludox SM 30 diluted to 5 % w/w SiO2. (A) Contour data constructed for sum data and (B) the corresponding anisotropy map. Note that the contour lines represent intensity data from panel A. For this probe we recommend exciting at 350 nm and to record the anisotropy at 410 nm. Although a better fluorescence signal (i.e. intensity) would be obtained by recording at 430 nm the dynamic range available in the anisotropy would diminish. For all our probes we provide the 3D emission spectra and the anisotropy contour map, overlaid with the emission contour map, allowing customers the ability to optimize their excitation, emission and anisotropy wavelengths / dynamic range.

#### Rotational Blue 3™

Rotational Blue 3™ has comparable water solubility as well as similar excitation and emission wavelengths as Ursa Blue™, and can therefore be used for similar applications. Rotational Blue 3™ readily binds to both Ludox SM30 and AM30 and can readily determine there respective nanoparticle colloidal sizes, 6-7 nm and 7-8 nm respectively, using both steady-state and time-resolved fluorescence anisotropy. Rotational Blue 3™ shows triple exponential fluorescence decay with mean lifetimes of 15.1 and17.1 ns for DuPont’s SM30 and AM30 respectively.

Figure 9. Excitation-Emission intensity Maps constructed for data recorded for Rotational Blue 3™ mixed with silica nanoparticles, Ludox SM 30 diluted to 5 % w/w SiO2. (A) Contour data constructed for sum data and (B) the corresponding anisotropy map. Note that the contour lines represent intensity data from panel A. For this probe we recommend exciting at 350 nm and to record the anisotropy at 410 nm. Although a better fluorescence signal (i.e. intensity) would be obtained by recording at 440 nm the dynamic range available in the anisotropy would diminish. For all our probes we provide the 3D emission spectra and the anisotropy contour map, overlaid with the emission contour map, allowing customers the ability to optimize their excitation, emission and anisotropy wavelengths / dynamic range.

#### Steady-State Anisotropy and the Perrin Expression for Nanoparticle Sizing

The degree of polarization of fluorescent light that remains after polarized excitation carries information about the rotational diffusion of fluorophores. The phenomenon was first studied by Perrin who published a classic paper on the topic in 1926. [7] The technique of combining fluorescence polarization with the equations describing diffusion was further developed by Weber and has since found widespread use, particular in biochemical applications where the size of biomolecules, e.g. proteins and viruses, can be determined in-situ using intrinsic fluorophores such as tryptophan.1 However, in many cases there are no natural occurring fluorophores present, or the fluorescent emitters are not suitable for sizing applications, in which case extrinsic fluorophores must be introduced. Ursa Bioscience has developed a range of particle sizing fluorophores, optimized and designed for colloidal particle solutions, and further, fluorophores with charge properties that allow for electrostatic attachment directly on the surface of macromolecules removing the additional complexity of covalent labeling.

The application of the Perrin technique for particle sizing requires suitable fluorophores to be rigidly attached to the targeted macromolecule, e.g. a protein, a virus particle, or a silica particle. Further, photophysical properties of the fluorophore must also be suitable for particle sizing; of particular importance are the fluorescence lifetime, τ, and the fundamental anisotropy r0. The fluorescence lifetime should be on a similar timescale as the rotational diffusion, typically in the region 5-100 ns. The fundamental anisotropy is a parameter that reflects on the available dynamic range for a particle sizing experiment, it is a parameter that for a perfect fluorophore can take the maximum vale 0.4, assuming one photon excitation, but in practice it is often found that r0 < 0.4 due to an internal angle between transition dipoles within the fluorophore.

The degree of polarization of fluorescence light is conveniently measured in a steady-state anisotropy experiment. The steady-state anisotropy rem) is calculated from the fluorescence intensities Ixyem) measured for different setting of the excitation, x, and emission, y, polarizers at a constant excitation wavelength, i.e.

$\small&space;r(\lambda_e_m)=\frac{I_v_v(\lambda_e_m)-gI_v_h(\lambda_e_m)}{I_v_v(\lambda_e_m)+gI_v_h(\lambda_e_m)}(1)$

Here v and h indicates vertical and horizontal alignment of the polarizers relative a laboratory fixed coordinate system, respectively. Different transmission properties of polarized light through the optical detection system is corrected for by introducing the g-factor, defined as

$\small&space;g(\lambda_e_m)=\frac{I_h_v(\lambda_e_m)}{I_h_h(\lambda_e_m)}(2)$

Perrine showed that there is a relationship between the steady-state anisotropy measured on a sample where the fluorophores are attached to macromolecules of radius R that reads

$\small&space;\frac{1}{r}=\frac{1}{r_0}+\frac{3\tau&space;k_b&space;}{4\pi&space;r_0R^{3}}\frac{T}{\eta&space;}(3)$

where kb, T and η indicates Boltzmann constant, the temperature, and the viscosity, respectively. Thus, by graphically plotting 1/r as function of T/η it is possible to calculate the macromolecule radius R from the gradient. The fundamental anisotropy, r0, can then be found from the intercept on the y-axis. Note that for simplicity the emission wavelength, λem, has not been written out in the Perrin equation.

Figure 10. (A) Photophysical decay recorded for Ursa Blue™ dissolved Ludox SM30. (B) Time-resolved anisotropies recorded for Ursa Blue electrostatically attached to colloidal particles of different sizes.

#### Time-Resolved Fluorescence Anisotropy for Nanoparticle Sizing

In the previous section the Perrin technique for nanoparticle sizing was discussed. The advantage of the Perrin technique is the simplicity of the experiment, i.e. from the temperature dependence of the anisotropy the particle size is easily found by linear regression. A requirement of the Perrin technique is however a high uptake of the dye on the macromolecule, although this criteria often is fulfilled, there are situation where a fraction of the fluorophores will remain free in the bulk solution, thus reporting a low anisotropy and the overall steady-state anisotropy will be biased towards smaller R values. This problem can be overcome by performing time-resolved anisotropy experiments. The experimental equipment is more complex, however, today several manufacturer offer boxed in solutions, computer controlled, and with software for data analysis. The key expression for the time-resolved anisotropy is similar to the steady-state anisotropy, i.e. Equation 3, however, it is assumed the anisotropy is only measured for a fixed emission wavelength, and obviously the time t needs to be introduced,

$\small&space;r(t)=\frac{I_v_v(t)-gI_v_h(t)}{I_v_v(t)+gI_v_h(t)}(4)$

The fluorescent intensity at time t is thus indicated by Ixy(t) where x and y refers to the orientation of the excitation and emission polarizers, respectively. Similar, the g-factor corrects for transmission properties of polarized light through the detection system, including the polarization response of the detector, and is calculated from

$\small&space;g=\frac{\int_{0}^{\infty&space;}I_h_v(t)dt}{\int_{0}^{\infty&space;}I_h_h(t)dt}(5)$

For a perfect detection system, g should equal 1, however, in most cases this is not the case, especially if monochromators are used to the select the emission wavelength.

The fluorescence anisotropy recorded from fluorophores dispersed in a colloidal solution can often be parameterized in terms of a sum of two correlation functions, Φ’s, i.e.

$\small&space;r(t)=b_1\exp&space;\left&space;(&space;-\frac{t}{\phi_f_r_e_e}&space;\right&space;)+b_2\exp&space;\left&space;(&space;-\frac{t}{\phi_b_o_u_n_d}&space;\right&space;)(6)$

where “free” and “bound” referees to the contribution from free and macromolecular bound fluorophores. The sum of the pre-exponential factors equals the fundamental anisotropy, i.e. b1 + b2 = r0. Geddes et al reported in several publications that equation 6 adequate described the anisotropy recorded for dynamic silica sol-gel system, i.e. colloidal system where the particles are growing as function of time. [8-10] However, it was in some cases necessary to introduce a limiting anisotropy in equation 6 that likely reflected a fraction of particles to large to be measured during the lifetime of the fluorophore.

The macromolecular size can be calculated from the Stokes-Deby equation

$\small&space;\phi_b_o_u_n_d=\frac{\eta&space;V_h}{k_bT}(7)$

where Vh indicates the hydrodynamic volume of the particle, i.e. the volume of the particle including the layer of solvent molecules that is dragged along during the diffusion. If it is assumed that the particles are spherical with radius R, then Equation 7 can be rewritten

$\small&space;R=\sqrt[3]{\frac{3k_bT\phi_b_o_u_n_d}{4\pi\eta}}(8)$

The successful application of equation 8 requires the viscosity to be measured. In an elegant approach Geddes et al [8] showed that the signal contribution from the free dye can be used for this purpose provided that the hydrodynamic volume of the particle sizing fluorophore, Vprobe, is known. Equation [8] then takes the form

$\small&space;R=\sqrt[3]{\frac{V_p_r_o_b_e}{4\pi}\frac{\phi_b_o_u_n_d}{\phi_f_r_e_e}}(9)$

Thus, by measuring the time-resolved anisotropy and analyzing the data in terms of the model presented in Equation 6, the calculation of the particle size is straightforward from Equation 9.

#### References

(1) Jameson, D. M.; Ross, J. A. Fluorescence Polarization/Anisotropy in Diagnostics and Imaging. Chem. Rev. 2010, 110, 2685.

(2) Eftink, M. R. Fluorescence techniques for studying protein structure. Methods of biochemical analysis 1991, 35, 127.

(3) Steiner, R. Fluorescence Anisotropy: Theory and Applications. In Topics in Fluorescence Spectroscopy; Lakowicz, J., Ed.; Springer US, 2002; Vol. 2; pp 1.

(4) Tleugabulova, D.; Sui, J.; Ayers, P. W.; Brennan, J. D. Evidence for rigid binding of rhodamine 6G to silica surfaces in aqueous solution based on fluorescence anisotropy decay analysis. J. Phys. Chem. B 2005, 109, 7850.

(5) Geddes, C. D.; Chevers, J. M.; Birch, D. J. S. Probing the sol-gel transition in SiO2 hydrogels - A new application of near-infrared fluorescence. J. Fluoresc. 1999, 9, 73.

(6) Birch, D. J. S.; Geddes, C. D. Cluster dynamics, growth and syneresis during silica hydrogel polymerisation (vol 320, pg 229, 2000). Chem Phys Lett 2000, 322, 300.

(7) Perrin, F. Polarisation de la lumière de fluorescence. Vie moyenne des molécules dans l'etat excité. J. Phys. Radium 1926, 7, 390.

(8) Birch, D. J. S.; Geddes, C. D. Sol-gel particle growth studied using fluorescence anisotropy: An alternative to scattering techniques. Phys. Rev. E 2000, 62, 2977.

(9) Geddes, C. D.; Birch, D. J. S. Nanometre resolution of silica hydrogel formation using time-resolved fluorescence anisotropy. J. Non-Cryst. Solids 2000, 270, 191.