Several Förster resonance energy transfer (FRET) lasers have been realized by employing the robust and versatile streptavidin-biotin (SPB) biocomplex as the acceptor–donor linkage. SPB offers a fixed acceptor–donor separation (“ruler”) of <6 nm, which lies within the Förster radius for a broad range of donors and acceptors. A Cy3-SPB-Cy5 conjugate laser (where Cy3 and Cy5 are cyanine dyes) peaking at λ ∼ 708 nm has been observed, and its bandwidth and threshold pump energy (at 532 nm) have been measured to be ∼4.5 nm and 118 µJ (corresponding to a pump energy density of 179 ± 5 µJ/mm2), respectively. Depolarization of the linearly polarized pump optical field by this FRET process is found to be <12%. To tether the acceptor and donor, the SPB complex requires only that either be conjugated, thereby allowing FRET processes to be examined among an extensive set of biomolecules, inorganics, and nanoantenna acceptors, for example. As a result, fluorophore-nanoparticle lasers having characteristics of both FRET lasers and plasmonic emitters have been demonstrated. Laser spectra and the phase shift induced by a 10 or 100 nm gold nanoparticle tethered to the Cy3-SPB complex suggest that both the fluorescent protein and nanoparticle are able to act as an acceptor. The brightness associated with this new class of fluorophore/nanostructure FRET lasers will broaden the scope of accessible biomedical diagnostics, including cellular imaging and the detection of DNA and proteins.

In 1948, Förster reported the molecular energy-exchange process that has proven to be an invaluable tool in chemistry and biology for more than 70 years, known as Förster (or fluorescence) resonance energy transfer (FRET).1,2 A milestone in the development of FRET as a biochemical diagnostic was the demonstration by Stryer and Haugland3 in 1967 of a “spectroscopic ruler,” a synthesized oligomer of poly-L-proline that served to separate donor and acceptor fluorophores by 1.2–4.6 nm. During the intervening years, FRET has been studied extensively and shown to be a precise means for examining and visualizing interactions between DNA, drug/biomolecular complexes, and fluorescent proteins. With regard to the latter, acceptor–donor energy exchange processes involving two fluorescent proteins have quite literally revolutionized the field of cellular imaging.4 

Only a few lasers employing biomolecular tethers and the FRET process as the mechanism for pumping the lasant species have been reported, the first of which was based on DNA scaffolds to provide the linkage between the donor and acceptor fluorescent proteins.5 Subsequent studies adopted dye-labeled DNA tetrahedrons,6 colloidal suspensions of latex nanoparticles co-doped with organic dyes,7 or layers of dried fluorescent proteins8 as media for promoting donor–acceptor excitation transfer and subsequent lasing in the visible or near-infrared spectral regions. Drawbacks of previously reported FRET lasers include the inability to precisely specify, or maintain, the donor–acceptor separation or provide a robust chemical linkage system able to accommodate a wide range of molecular and nanostructure/nanoparticle moieties. The former is a priority for FRET lasers, in particular, because of the R−6 dependence of the FRET energy transfer mechanism efficiency on R, the donor–acceptor separation. DNA scaffold tethers are also susceptible to secondary formations.5 

Here, we report the realization of fluorophore–fluorophore FRET and fluorophore-nanoparticle FRET/plasmonic lasers which exploit the unique properties of the streptavidin-biotin (SPB) complex as an acceptor–donor binding system. Prominent among these is the stability of SPB complexes, which is exemplified by the strongest known non-covalent interaction between a ligand and a protein. Specifically, the affinity constant for the SPB complex has been measured to be 1015 M−1.9 In addition, the geometry of streptavidin (volume of 4.2 × 4.2 × 5.6 nm3) provides up to four binding (“docking”) sites per molecule and sets the donor–acceptor separation at ∼6 nm, a value comparable to (or less than) the Förster radius for the well-known fluorophores Cy3 and Cy5 (cyanine dyes).10 Tethering a moiety such as a fluorescent protein or nanoparticle to streptavidin requires only that the molecular acceptor or donor be conjugated. This condition is easily met because a wide range of biotinylated and streptavidin-conjugated molecules, including nucleotides and DNA, are now available commercially.

Experiments have demonstrated a Cy3-SPB-Cy5 FRET laser, which generates maximum intensity at ∼708 nm and a spectral bandwidth of 4.5 nm when pumped in a Fabry–Pérot cavity at 532 nm. The first FRET lasers based on fluorescent protein-nanoparticle energy transfer are also reported here. Binding of Cy3 and a 100 nm-diameter, biotinylated gold nanoparticle (AuNP) to streptavidin yields peak lasing at ∼617 nm, and the peak intensity in the longitudinal mode spectrum is red-shifted by ∼1.4 cm−1 (∼42 GHz) with respect to the Cy3-streptavidin spectrum. Optical pumping of the Cy3-SPB-AuNP conjugate results in a double-FRET process in which both the fluorescent protein and the plasmonic nanoparticle are able to act as an acceptor. The experimental results reported here demonstrate that the streptavidin-biotin conjugate provides a versatile and robust acceptor–donor linkage for realizing efficient FRET lasers with reproducible characteristics. Furthermore, replacing the acceptor (fluorophore or nanoparticle) with a nanoantenna array designed for a particular wavelength range and a near-isotropic field-of-view is expected to promote optical interactions among Cy3-SPB-nanoantenna complexes in solution. Optical cooperative effects are also expected to be evident, and the experiments described here are the initial step in that direction.

Two laser spectra are presented in Fig. 1 for the Cy3-SPB-Cy5 conjugate pumped at 532 nm. As illustrated in panel (a) of Fig. 1, peak intensity is produced at ∼708 nm, and the spectral bandwidth (FWHM) is ∼4.5 nm when the mirror–mirror separation (L) is set at 1.2 mm. More than 60 longitudinal modes are observed owing to the spectrometer resolution of ∼0.3 cm−1 (10 GHz), and the free-spectral range (FSR) is 97 GHz. When L is reduced to 550 µm [Fig. 1(b)], however, the spectrum is red-shifted, maximum intensity occurs at 730 nm, and the gain bandwidth has fallen to ∼2 nm because the single-pass gain for the oscillator has been reduced. Note, too, that the FSR has increased to 205 GHz and the number of longitudinal modes able to reach threshold declines. The dependence of the output pulse energy (Eo) on the 532 nm pump pulse energy (Ep) is presented in Fig. 2 where the multiple measurements shown for specific values of Ep are intended to illustrate the degree of scatter in the data. Laser threshold occurs for Ep ∼ 118 µJ, which corresponds to a pump energy density of 179 ± 5 µJ/mm2 and is approximately a factor of two larger than the value measured for Cy3-streptavidin alone. The increase in laser threshold is presumably attributed to the FRET energy transfer efficiency from Cy3 to Cy5, which is 50% at the Förster radius. Before leaving this subject, it should be emphasized that the inset illustration of Fig. 2 is intended only to show the capacity of streptavidin for binding up to four biotin molecules. For these experiments, the average biotin occupancy is ∼1/2 of that in the illustration.

FIG. 1.

Laser spectra for the Cy3-SPB-Cy5 complex when pumped at 532 nm and the mirror spacing (L) set at (a) 1.2 mm and (b) 550 µm. For these experiments, the pump pulse energy was fixed at 750 µJ. Note that the number of observed longitudinal modes declines for L = 550 µm and the spectrum is red-shifted with respect to (a) because the single-pass gain has been reduced.

FIG. 1.

Laser spectra for the Cy3-SPB-Cy5 complex when pumped at 532 nm and the mirror spacing (L) set at (a) 1.2 mm and (b) 550 µm. For these experiments, the pump pulse energy was fixed at 750 µJ. Note that the number of observed longitudinal modes declines for L = 550 µm and the spectrum is red-shifted with respect to (a) because the single-pass gain has been reduced.

Close modal
FIG. 2.

Dependence of the Cy3-SPB-Cy5 laser output energy (Eo) on the 532 nm pump pulse energy (Ep) for 0 < Ep < 200 µJ. Threshold is reached for Ep ∼ 118 µJ, which corresponds to an incident energy density of ∼179 µJ/mm2. The red and black circles of the illustration represent Cy3 and Cy5, respectively, and are intended to show only the capacity of streptavidin for binding up to four biotin molecules. For the current experiments, the biotin occupancy is ∼1/2 of that in the illustration. SPB also provides a tether to Cy3.

FIG. 2.

Dependence of the Cy3-SPB-Cy5 laser output energy (Eo) on the 532 nm pump pulse energy (Ep) for 0 < Ep < 200 µJ. Threshold is reached for Ep ∼ 118 µJ, which corresponds to an incident energy density of ∼179 µJ/mm2. The red and black circles of the illustration represent Cy3 and Cy5, respectively, and are intended to show only the capacity of streptavidin for binding up to four biotin molecules. For the current experiments, the biotin occupancy is ∼1/2 of that in the illustration. SPB also provides a tether to Cy3.

Close modal

Depolarization of the FRET laser emission, relative to the pump optical field, is known to be a valuable diagnostic of biomolecular lasers11 and the resonant energy transfer process, in particular.12,13 Consequently, the polarization characteristics of the Cy3-SPB-Cy5 conjugate laser were measured with the optical arrangement illustrated by the inset in Fig. 3. A polarizing beamsplitter (denoted PBS) inserted into the optical path resolves the vertically and horizontally polarized components of the FRET laser output (illustrated in Fig. 3 by the red and black traces, respectively) and matched photodetectors with risetimes of <1.5 ns, recording the waveforms at 712 nm near the peak of the Fig. 1(a) spectrum. Because the peak intensity of the vertically polarized portion of the laser pulse is ∼12% of that for the horizontally polarized component, one concludes that depolarization of the pump by the FRET process is minimal. As discussed in Ref. 11, the weak vertical polarization component of the FRET laser output, shown in Fig. 3, demonstrates the gain medium to be anisotropic and the rotational correlation time for Cy5 to be greater than the molecule’s radiative lifetime.12,13 Specifically, the degree of anisotropy in the gain medium, following the arrival of a polarized pump pulse, is determined by the rotational correlation time as compared to the upper laser level lifetime. Long rotational correlation times prevent rotational relaxation in the liquid gain medium prior to the emission of the laser pulse.11 

FIG. 3.

Laser emission waveforms for the Cy3-SPB-Cy5 complex, recorded separately for the horizontally and vertically polarized components of the FRET laser output with matched photodetectors. These measurements were conducted at 712 nm for L = 1.2 mm with the optical arrangement illustrated by the inset, where the acronyms PBS, DM, and M represent a polarizing beamsplitter, dichroic mirror, and mirror, respectively. The position of the laser resonator is also indicated, and the pump laser (Nd:YAG) beam was horizontally polarized.

FIG. 3.

Laser emission waveforms for the Cy3-SPB-Cy5 complex, recorded separately for the horizontally and vertically polarized components of the FRET laser output with matched photodetectors. These measurements were conducted at 712 nm for L = 1.2 mm with the optical arrangement illustrated by the inset, where the acronyms PBS, DM, and M represent a polarizing beamsplitter, dichroic mirror, and mirror, respectively. The position of the laser resonator is also indicated, and the pump laser (Nd:YAG) beam was horizontally polarized.

Close modal

Subsequent experiments investigated biotinylated gold nanoparticles, with a diameter of 10 or 100 nm, as the acceptor, and Fig. 4 compares the FRET laser spectra for Cy3-streptavidin (black curve) with that recorded for the Cy3-SPB-AuNP complex having a 100 nm nanoparticle. Introducing a gold nanoparticle of a large surface area into the bioconjugate red-shifts the laser spectrum because of the dissipative properties of the surface plasmon resonance of the nanoparticle, which is excited directly by the pump optical field or indirectly by the acceptor. In this regard, the gold nanoparticle behaves classically. The peak emission of the Cy3-SPB-AuNP conjugate lies near 617 nm, whereas the maximum intensity of the Cy3-streptavidin complex is at 610 nm. Close examination of the Cy3-SPB-AuNP (100 nm) spectrum in Fig. 4 reveals the appearance of a secondary longitudinal mode structure, which suggests that higher-order transverse modes are excited by the introduction of the nanoparticle. In addition, the mean red shift of the longitudinal mode peaks, relative to the Cy3-streptavidin spectrum, is 1.4 cm−1 or 42 GHz. Normalized spectra for the Cy3-streptavidin and Cy3-SPB-AuNP lasers are compared in Fig. 5 for both 10 and 100 nm gold particles. The onset of non-radiative quenching by the 10 nm gold nanoparticles is evidenced by the red-shift of the laser spectral envelope in Fig. 5(b) and the relative strength of the longitudinal modes beyond λ = 618 nm. Quenching is much more pronounced for the 100 nm gold particles [Fig. 5(a)] as all modes below λ ∼ 615 nm are strongly suppressed. This observation is somewhat surprising because the concentration of the 10 nm particles is several orders of magnitude higher than that for the 100 nm nanoparticles, a result we attribute to the partial offset provided by the surface area of the larger gold particles. Note, too, that the intensity envelope for the spectrum in Fig. 5(b) is red-shifted with respect to that in Fig. 5(c) by a mean value of 983 pm (31.9 cm−1) [the S2 manifold (left) and S1 manifold (right) are shifted by 1.14 nm (32.8 cm−1) and 826 pm (30.9 cm−1), respectively]. Therefore, the gold nanoparticles can be regarded as a secondary acceptor capable of non-radiatively transferring energy to the dye. Because the extinction spectra for 10 and 100 nm AuNPs peak at ∼520 and ∼570 nm, respectively, both are efficiently coupled to the pump optical field.

FIG. 4.

FRET laser spectra for Cy3-streptavidin and the Cy3-SPB-AuNP bioconjugate (black and blue curves, respectively) where the gold nanoparticle has a diameter of ∼100 nm.

FIG. 4.

FRET laser spectra for Cy3-streptavidin and the Cy3-SPB-AuNP bioconjugate (black and blue curves, respectively) where the gold nanoparticle has a diameter of ∼100 nm.

Close modal
FIG. 5.

Comparison of the normalized FRET laser spectra resulting from photoexcitation of (a) the Cy3-SPB-100 nm AuNP bioconjugate, (b) Cy3-SPB-10 nm AuNP, and (c) Cy3-streptavidin at 532 nm. The dual-maxima envelopes for the spectra of (b) and (c) are presumed to be associated with laser transitions from the S1 and S2 excited state manifolds of the Cy3 molecule.

FIG. 5.

Comparison of the normalized FRET laser spectra resulting from photoexcitation of (a) the Cy3-SPB-100 nm AuNP bioconjugate, (b) Cy3-SPB-10 nm AuNP, and (c) Cy3-streptavidin at 532 nm. The dual-maxima envelopes for the spectra of (b) and (c) are presumed to be associated with laser transitions from the S1 and S2 excited state manifolds of the Cy3 molecule.

Close modal

The variation in the FRET laser output energy with pump pulse energy is illustrated in Fig. 6. For the Cy3-streptavidin complex, the estimated threshold pump energy is 75 µJ. With the introduction of a 100 nm nanoparticle, however, the pump energy threshold rises by 50% to ∼115 µJ, a result consistent with the well-known dissipative properties of gold nanoparticles. In addition, the laser slope efficiency is considerably smaller than that measured in the absence of the nanoparticle, which also attests to non-radiative quenching of Cy3 emission through coupling of the donor to the plasmon surface resonance of the acceptor.

FIG. 6.

Measurements of the FRET laser output pulse energy for Cy3-streptavidin (blue open circles) and Cy3-SPB-100 nm AuNP (black symbols) when the pump pulse energy is varied up to 190 µJ. The red and gold circles in the lower drawing (not to scale) represent Cy3 and the AuNP, respectively, which are linked by SPB as shown in blue. Note that the ordinate scale is logarithmic.

FIG. 6.

Measurements of the FRET laser output pulse energy for Cy3-streptavidin (blue open circles) and Cy3-SPB-100 nm AuNP (black symbols) when the pump pulse energy is varied up to 190 µJ. The red and gold circles in the lower drawing (not to scale) represent Cy3 and the AuNP, respectively, which are linked by SPB as shown in blue. Note that the ordinate scale is logarithmic.

Close modal

Several FRET lasers in which the donor and acceptor are linked by the streptavidin-biotin bioconjugate have been demonstrated and characterized. Owing to its strong non-covalent interaction, SPB provides a robust tether to fluorescent molecules, such as dyes, proteins, and nanoparticles, which is resilient in aggressive chemical environments. Since the average streptavidin/biotin concentration ratio chosen for these studies is 4:9, the experimental FRET laser measurements reported here examine, in effect, energy transfer processes occurring between two biotin molecules and Cy3. The incorporation of a nanoparticle into a FRET laser provides the opportunity to significantly increase the signal-to-noise ratio (SNR) for optical biomedical diagnostics that presently rely on fluorescence quenching.10,13,14 The extensive longitudinal mode spectra for the lasers reported here also offer frequency combs, extending over several nm, that are valuable for precisely detecting small changes in the refractive index of a solution. Finally, the Au nanoparticles employed in these experiments are far from optimal with respect to optical interactions with the optical excitation source or visible and infrared emission from other bioconjugates (or other species) in solution. Linking an efficient fluorescent protein with a single nanoantenna (or an array) with SPB provides a unique optical platform for transmitting or receiving optical signals in a biochemical environment. Careful selection of two or more optical excitation wavelengths and interspersing SPB FRET bioconjugates having different nanostructure acceptors will allow for laser emission to be generated or quenched on command.

Cy3-SPB-Cy5 conjugates were synthesized from 40 µM Cy3-streptavidin and 90 µM Cy5-biotin stock concentrations obtained from Thermo Fisher Scientific and Click Chemistry, respectively. The optimal streptavidin/biotin ratio was chosen empirically by starting with an equal parts mixture of 40 µM Cy3-streptavidin and 40 µM Cy5-biotin. Upon varying the biotin concentration in the mixture in steps of 10 µM, lasing was observed for 90 µM Cy5-biotin (given a static Cy3-streptavidin concentration), and the 40 µM Cy3-streptavidin/90 µM Cy5-biotin mixture was incorporated into all Cy3/Cy5 FRET experiments. The observed sensitivity of this FRET laser to the biotin concentration is presumed to be the result of nonlinear quenching dynamics occurring between the donor and acceptor. At lower biotin concentrations, the acceptor number density in solution is insufficient for the laser to reach threshold, whereas high concentrations of the acceptor presumably lead to unacceptably large non-radiative quenching rates. To date, no effort has yet been made to optimize streptavidin/biotin ratios.

Ten microliters of the Cy3-streptavidin and Cy5-biotin solutions were mixed and incubated for 30 min at room temperature. Experiments with Cy3-SPB-AuNP bioconjugates required replacing Cy5-biotin with biotinylated Au nanoparticles having diameters of 10 or 100 nm. Solutions of biotinylated Au nanoparticles were purchased from Cytodiagnostics with a stock concentration of 1.92 × 1011/ml (for 100 nm particles) and diluted with DI water by two orders of magnitude, resulting in nanoparticle concentrations of 1.92 × 109/ml and 2.99 × 1012/ml for the 100 and 10 nm particles, respectively. Ten microliter solutions of the gold nanoparticles were then incubated with 10 µl solutions of stock Cy3-streptavidin for 20 min.

These gain medium solutions were dispensed onto the surface of a horizontally oriented, planar dielectric mirror having a reflectivity R > 99.9% in the 600–1100 nm interval and transmission T > 90% at 532 nm. A second, identical mirror was positioned over the lower mirror with one of several precision spacers, thereby forming a Fabry–Pérot cavity, which was aligned with a He–Ne laser operating in a single transverse mode. Linearly polarized pump radiation was provided by a Q-switched, frequency-doubled Nd:YAG laser, which generates 8 ns, 532 nm pulses that are delivered to the resonator by a dichroic beamsplitter and a 4X Olympus objective. Laser radiation exiting the optical cavity was focused onto the entrance slit of a 0.75 m spectrograph having a Czerny-Turner configuration and a gated, intensified CCD array at the exit plane. Temporal and polarization properties of the laser pulses were characterized with fast photodiodes (<1.5 ns risetime) and a polarizing beamsplitter. Absolute pulse energies of the FRET and pump lasers were measured with calibrated silicon and pyroelectric detectors, respectively.

The support of this work by the U.S. Air Force Office of Scientific Research (AFOSR) under Grant Nos. FA9550-14-1-0002 and FA9550-18-1-0380 (H. Schlossberg, J. Luginsland, and G. Pomrenke) is gratefully acknowledged.

The authors have no conflicts to disclose.

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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