Luminescent terbium complexes: Superior Förster resonance energy transfer donors for flexible and sensitive multiplexed biosensing

Coordination Chemistry Reviews 273–274 (2014) 125–138

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Coordination Chemistry Reviews journal homepage: www.elsevier.com/locate/ccr

Review

Luminescent terbium complexes: Superior Förster resonance energy transfer donors for flexible and sensitive multiplexed biosensing Niko Hildebrandt a,∗ , K. David Wegner a , W. Russ Algar b a b

NanoBioPhotonics, Institut d’Electronique Fondamentale, Université Paris-Sud, 91405 Orsay Cedex, France Department of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver, BC V6T 1Z1, Canada

Contents 1. 2.

3. 4. 5. 6.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . LTCs and FRET . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Unpolarized emission . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Long-lived excited states . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Antenna ligand absorption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. Emission spectra . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Multiplexed FRET from LTCs to dyes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Multiplexed FRET from LTCs to QDs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Multiplexed FRET from LTCs to QDs to dyes (multistep FRET) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Summary and perspective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

a r t i c l e

i n f o

Article history: Received 17 October 2013 Received in revised form 14 January 2014 Accepted 18 January 2014 Available online 28 January 2014 Keywords: Terbium Quantum dots Lanthanides Diagnostics FRET Biosensor Multiplexing Time-resolved Spectroscopy Immunoassay Fluorescence

126 126 126 127 127 127 129 131 134 136 136 136

a b s t r a c t Optical quantification of several biomarkers at very low concentrations and nanometric distances has become an important requirement for many biosensing applications. Förster resonance energy transfer (FRET) and, in particular, luminescent terbium complex (LTC)-based FRET, is a valuable tool for sensitive and versatile multiplexed FRET. Here, we review recent progress in the development of novel LTC-FRET photonic sensors for ultra-sensitive and multiplexed diagnostics of various biomarkers and distances (molecular ruler) in different biological systems. The basic concept of FRET, the exceptional photophysical properties of LTCs, and possibilities and opportunities for multiplexed optical sensing are outlined. Sophisticated FRET systems such as multiplexed LTC-to-dye FRET, LTC-to-quantum dot (QD) FRET, and LTC-to-QD-to-dye FRET relays have been assembled with biological recognition molecules such as antibodies, peptides, and oligonucleotides to permit biosensing applications in the form of homogeneous immunoassays, DNA hybridization and enzyme assays, and molecular logic devices. A perspective on the emerging field of multiplexed LTC-based FRET biosensing is given at the end of this review to highlight the promising future of these nanometric biosensors. © 2014 Elsevier B.V. All rights reserved.

Abbreviations: A, acceptor; AF, Life Technologies AlexaFluor dyes; AFP, alpha-fetoprotein; APC, allophycocyanin; BHQ, Black-Hole quencher; CA15.3, Carbohydrate Antigen 15.3; CARM1, coactivator-associated arginine methyltransferase 1; CEA, carcinoembryonic antigen; Cy, GE Healthcare cyanine dyes; Cyfra21-1, Cytokeratin-19 Fragment 21-1; D, donor; DLS, dynamic light scattering; DNA, deoxyribonucleic acid; Dy, dysprosium; EGFR, epidermal growth factor receptor; ELISA, enzyme-linked immunosorbent assay; ER, estrogen receptor; Eu-TBP, europium trisbipyridine cryptate; Eu, europium; F(ab )2 , antigen-binding fragment consisting of two F(ab) fragments; F(ab), antigenbinding fragment of an IgG antibody; FP, fluorescent protein; FRET, Förster resonance energy transfer; GFP, green fluorescent protein; HTS, high-throughput screening; IgG, Immunoglobulin G antibody; LOD, limit of detection; LTC, luminescent terbium complex; MLD, molecular logic device; NSE, neuron specific enolase; OG, Life Technologies OregonGreen dye; PEG, polyethylene glycol; PL, photoluminescence; PRB, probe ssDNA; PSA, prostate specific antigen; QD, quantum dot; RNA, ribonucleic acid; SCC, squamous cell carcinoma antigen; Sm, samarium; SNAPFL, semi-naphthalene fluorescein; ssDNA, single-stranded DNA; Tb, terbium; TEM, transmission electron microscopy; TGT, target ssDNA; TR-FIA, time-resolved fluoroimmunoassay; VH H, single-domain antibodies (or nanobodies); Zn, zinc. ∗ Corresponding author. Tel.: +33 169155581. E-mail address: [email protected] (N. Hildebrandt). 0010-8545/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ccr.2014.01.020

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1. Introduction Technological advances in the bio- and nanosciences have fostered new methods for detecting a myriad of different chemical and biological processes and interactions at extremely low concentrations and extremely short distances [1–3]. Among these advances, luminescent lanthanide coordination compounds containing the trivalent ions of Eu, Tb, and, less frequently, Sm and Dy, have played an important role in developing very sensitive, time-resolved photoluminescence (PL) biosensing because of their exceptional photophysical properties [4–7]. Well-designed ligand structures (e.g., chelates or cryptates) coordinate a central lanthanide ion and serve as both a light absorbing “antenna” and a “cage” to protect against PL quenching from the outer environment. The antenna structure should be a chromophoric unit with strong absorption and the ability to efficiently transfer the absorbed energy to the central lanthanide ion. The antenna can be incorporated into the cage structure, bind to the cage, or coordinate directly to the lanthanide ion in parallel with the cage. In most cases, the ligand is designed to occupy all of the coordination sites of the lanthanide ion in order to suppress PL quenching from water molecules. However, when a separate antenna unit needs to coordinate to the lanthanide ion, or when coordination of an analyte to the lanthanide ion produces a signal change suitable for sensing, less than full coordination is more appropriate. Once sensitized by the antenna, lanthanide ions provide extremely long excited-state lifetimes (up to milliseconds) and multiple narrow emission bands in the visible region of the spectrum. The long PL decay times allow for efficient suppression of short-lived autofluorescence background from the sample or from other fluorophores by using pulsed excitation and time-gated detection (i.e., measuring the long-lived PL intensity decay in a time-window that opens after the other fluorescent components have already decayed). The emission spectra of lanthanide ions are usually red-shifted by more than a hundred nanometers from the ligand absorption (a large effective Stokes shift), which, when combined with time-gating, allows detection of lanthanide emission with almost no background from excitation light. These features enable the very high sensitivity of luminescent lanthanide biosensors. To exploit luminescent lanthanide complexes for biosensing, Förster resonance energy transfer (FRET) has frequently been utilized and provides two important capabilities. First, when lanthanide complexes are paired as FRET donors with different fluorophores as FRET acceptors, nanometric distance information can be obtained from luminescence measurements [8–13]. Second, FRET biosensors can offer on/off signaling for quantitative analysis of biological processes such as cellular signaling, ligand–receptor binding, protein–protein interactions, DNA/RNA hybridization, and enzymatic reactions [14–30]. Although lanthanide-based FRET biosensors have been reported and applied for decades [5,31–38], and despite the more recent development of lanthanide-based nanoparticles [39], increasing demand for the simultaneous measurement of multiple biological parameters (e.g., concentrations or distances) in a single sample, so-called multiplexing, has stimulated the renewed interest in novel lanthanide-based FRET biosensing approaches. In preference to other lanthanide ions, luminescent Tb complexes (LTCs) have been the major players in this field because of their large PL quantum yields, extremely long PL lifetimes, and multiple spectrally resolved emission bands. Although Eu complexes are also very bright emitters in the visible spectrum, and have arguably been applied for biosensing more often than Tb complexes (because of fewer problems with back-energy transfer from the ion to ligand), the emission bands of Tb are much better separated and therefore superior for spectral multiplexing. In this article, we review recent developments and applications of multiplexed Tb-based FRET biosensors, emphasizing our own

research program that focuses on the development of multiplexed molecular recognition and interaction assays. Although there are several very interesting optical imaging applications using LTCbased biosensors and FRET [40–48], this topic has been recently reviewed elsewhere [49] and will not be discussed here. Nevertheless, the recent advances in LTC-based FRET probes and their many advantages for biosensing provide good evidence that multiplexed LTC-based FRET imaging techniques will be available in the near future. 2. LTCs and FRET Many recent reviews and textbooks cover the general theory and applications of FRET [8,9,11,13–17,19,30,32], including the details for lanthanide complexes in particular [31,32,49]. In order to understand the theoretical background of the multiplexed Tb-based FRET applications presented in this article, we briefly review the most important concepts in this section. FRET is a non-radiative energy transfer process between two molecules or particles. The energy donor (D) is a luminophore (e.g., organic dye, fluorescent protein (FP), quantum dot (QD), lanthanide complex) in an electronically excited state. FRET can occur if the electronic transitions from this excited state to lower-lying states are in energetic resonance with the electronic transitions, from the ground state to higher-lying states, of a suitable acceptor (A) in close proximity to D (ca. 1–20 nm). This resonance condition requires spectral overlap between D emission and A absorption. A can be a luminophore or a non-luminescent FRET quencher (e.g., BlackHole quencher or gold nanoparticle). The FRET efficiency (FRET ) is usually determined by the D–A distance, r, and the Förster distance or Förster radius, R0 (which can be calculated by the D–A orientation, the emission and absorption spectra of D and A, and the luminescence quantum yield of D), or by luminescence intensities (I) or decay times () of D in the absence (subscript D) and in the presence (subscript DA) of A (Eq. (1)). FRET =

1 1 + (r/R0 )

6

=

R06 R06

+ r6

=1−

IDA DA =1− ID D

(1)

The Förster distance R0 (or Förster radius) can be calculated by Eq. (2). R0 = 0.02108(2 ˚D n−4 J)

1/6

(2)

2

is the orientation factor between the emission and where absorption transition dipole moments of D and A, respectively, ˚D is the donor PL quantum yield, n is the refractive index of the solvent, and J (in M−1 cm−1 nm4 ) defines the spectral overlap of D emission and A absorption using  the intensity normalized emission spectrum of D (I¯D () where I¯D () = 1) with and the molar absorptivity (or extinction coefficient) spectrum of A (εA ()) as defined by Eq. (3).



J=

I¯D ()εA ()4 d

(3)

LTCs such as the macrocyclic ligand-based Lumi4–Tb complex developed in Raymond’s group (Fig. 1) [50] are usually applied as FRET donors. There are four major advantages that distinguish LTCs significantly from other FRET donors. 2.1. Unpolarized emission Tb possesses multiple emission transition dipole moments (unpolarized emission) and therefore a dynamic averaging can be applied for the orientation factor 2 . Thus, even with a FRET acceptor having a fixed transition dipole moment, 2 is limited to values between 1/3 and 4/3. In many biosensing applications, the acceptor (labeled to a biomolecule) has fast isotropic rotation and the

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Fig. 1. LTC-properties. Lumi4–Tb complex absorption and emission spectra (left), structure (center), and PL decay (right).

frequently applied averaging condition of 2 = 2/3 becomes a good approximation. Detailed studies of all possible 2 values can be found in the literature [51–53]. 2.2. Long-lived excited states Similar to other lanthanide-based luminophores, LTCs possess extremely long luminescence decay times of up to a few milliseconds [4,5,7,34]. These PL “lifetimes” are ca. 106 -fold longer than those of fluorophores such as organic dyes, FPs, or QDs (nanosecond decay times), and at least 103 -fold longer than the auto-fluorescence of biological samples (usually in the nanosecond to microsecond range). The exceptionally long excited-state lifetimes of LTCs have three advantages for FRET-based biosensing: (i) The large difference in the excited-state lifetimes of D and A leads to approximately equal luminescence decay times for FRET-quenched D and FRET-sensitized A. As a consequence, the decay time of D in the presence of A ( DA in Eq. (1)) can be replaced by the decay time of A in the presence of D ( AD = ␶DA) [11,54], and time-resolved PL from both D and A can be used for FRET analysis. (ii) FRET from multiple LTCs to a single A is possible because the short-lived excited state of A will be immediately de-excited after FRET-sensitization from one LTC, and thus the same A can be FRET-sensitized again by another LTC that is still in its excited state due to the long lifetime. If sufficient LTCs are available (and can all be excited) for one A, then the probability of FRET-sensitization (P) will increase with the number of LTCs (m) as shown in Eq. (4).



P = 1 − (1 − FRET )

m

=1−

r6 R06 + r 6

m

(4)

(iii) Sample auto-fluorescence and emission from directly excited acceptors can be very efficiently suppressed by pulsed excitation and time-gated detection (e.g., PL intensity integration in a time-window from 0.05 to 1 ms after excitation pulse). Delayed detection of FRET-sensitized acceptor PL will be void of any background PL arising from auto-fluorescence, direct acceptor PL, or donor PL (assuming no spectral crosstalk) and is therefore insensitive to concentration effects (e.g., excessively high concentrations of D or A) and incomplete bioconjugation (unlabeled biomolecules and/or free D or A). 2.3. Antenna ligand absorption Light absorption by the Tb3+ -coordinating ligand and subsequent energy transfer to the Tb3+ -ion has two advantages. First, ligand excitation wavelengths are well separated from Tb emission wavelengths (often called the effective Stokes shift), which allows facile blocking of stray excitation light by filters and/or dichroic

mirrors. Second, the PL quantum yield of D (˚D in Eq. (2)) is the quantum yield of the Tb3+ -ion and not the quantum yield of the complete LTC, which is the product of sensitization efficiency by the ligand and the Tb3+ PL quantum yield: ˚LTC = ˚sens × ˚Tb . Thus, the FRET-efficiency, FRET , is only dependent on the Tb3+ PL quantum yield and not on the Tb3+ -sensitization by the ligand, and LTCs with high ˚Tb = ˚D can be designed for efficient FRET.

2.4. Emission spectra The multiple narrow and well-separated emission bands in the PL spectra of LTCs are the reason that one LTC donor can be multiplexed with several different acceptor fluorophores (Fig. 2). These LTC emission bands are in a spectral range where many fluorophores are excellent absorbers and therefore relatively large spectral overlap integrals can be achieved. Förster distances of up to 11 nm are possible with LTC-QD D–A pairs [49]. By choosing acceptors that emit in wavelength regions in between or beyond the LTC emission bands, multiplexed (simultaneous) PL detection of the LTC donor and the different acceptors becomes possible. Spectral separation becomes more efficient as the acceptor PL spectra become narrower and better separated. In the event of spectral crosstalk, where several acceptors emit in the same wavelength region of the optical transmission filter, a correction is necessary in order to achieve efficient multiplexing. An example of such crosstalk correction is presented in Section 3. LTCs have been used as donors in combination with organic dyes, fluorescent proteins, or other natural fluorophores such as allophycocyanin, and semiconductor quantum dots in order to realize optical biosensors [33,55–101]. It should be noted that many of these studies have also used Eu complexes as donors; however, these FRET systems are beyond the scope of this review. Vouthier et al. recently developed a homogeneous binding assay for leptin detection using Tb-to-dye FRET for high-throughput screening (HTS) [85]. Leptin is a hormone that regulates diverse biological functions as a signal transmitter for the body energy status, immunity, and reproduction. Gene mutations in leptin and its associated receptor can cause obesity and type II diabetes. Additionally it has been shown that there is a correlation between leptin–receptor binding and tumor size. Robust and cost-efficient HTS assays for finding antagonist molecules, which could block the activity of the receptor, is of great interest for the development of efficient therapies. The authors developed a new binding assay based on Cisbio’s Tag-lite technology, for which the leptin receptor was fused with a SNAP-tag and expressed in HEK293 cells. The LTClabeled ligand bound covalently to the SNAP-tag, which enabled FRET to a dye-leptin conjugate upon leptin–receptor binding. The FRET-sensitized time-gated dye emission was used for sensitive quantification of the leptin–receptor interaction. This assay is the first non-radioactive assay allowing for reliable pharmacological

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Fig. 2. LTC-donor-based multiplexed FRET. Top: Broad spectral overlap between LTC emission (relative PL intensity ILum. ) and the absorption (molar absorptivity ε) of several different acceptors. High ε values lead to large overlap integrals (Eq. (2)). Bottom: Separation of the single Tb emission bands allows the measurement of different acceptors in wavelength ranges with very low Tb intensity (represented by the shaded bandpass filter transmission spectra). LTC-PL-spectra in black. Left: organic dyes OregonGreen (blue), AlexaFluor555 (green), AlexaFluor568 (orange), Cy5 (red) and AlexaFluor700 (brown); right: QDs Qdot525 (blue), Qdot565 (green), Qdot605 (orange), Qdot655 (red), and QDot705 (brown).

study of leptin–receptor binding toward HTS for molecules or drugs that could act as leptin agonists or antagonists. An important application of FPs is monitoring of cellular activity after and during manipulation of different cell compartments in order to understand their functionality and to develop efficient disease therapies. Two of the main challenges in fluorescence-based cellular investigations are strong autofluorescence and scattering from cell compartments. LTC-based time-resolved PL measurements can efficiently overcome these problems [55–65]. Zeng et al. developed a HTS-compatible, homogeneous cellular assay for monitoring coactivator-associated arginine methyltransferase 1 (CARM1) cellular activity [65]. CARM1 plays an important role as a coactivator of specific factors that are responsible for the etiology and the progression of cancer. Enhancement or inhibition of CARM1 activity with modulators is important for a better understanding of the physiological and pathological processes associated with CARM1. The authors followed the methylation of poly(A) binding protein 1 (PABP1) by CARM1 as an indicator for its activity. MCF7 cells expressing a green fluorescent protein (GFP)-PABP1 fusion protein, which can act as FRET acceptor for a LTC donor, were used for these experiments. After incubation with different modulators, the cells were lysed and incubated with specific primary antibodies for the methylated PABP1 and LTC-labeled secondary antibodies. Methylated GFP-PABP1 could then be detected by time-resolved Tb-to-GFP FRET. Using this technique, the authors performed HTS of 320 compounds of the National Institute of Health Clinical Collection Library as possible CARM1 modulators. Apart from measuring the PL of FRET-sensitized acceptors for analyte quantification, another biosensing strategy is the analysis of FRET-quenched LTC PL caused by plasmonic coupling between LTCs

and metal surfaces [102–104] or by Black-Hole quenchers (BHQ) [105–107]. Li et al. investigated an aptamer-based Tb-to-BHQ FRET sensor for the detection of adenosine in unprocessed and undiluted serum [107]. The biosensor consisted of three nucleic acid strands: the linker DNA, which provided two segments for hybridization of the LTC-labeled strand and the BHQ-labeled strand, and one segment for recognition of the adenosine by the formation of a specific aptamer structure. In the absence of adenosine, the BHQ- and LTClabeled oligonucleotides are both hybridized on the linker strand and the BHQ efficiently FRET-quenches the LTC PL. The addition of adenosine leads to the formation of the adenosine-binding aptamer structure and the displacement of the BHQ strand. This BHQ dissociation results in an increase of LTC PL intensity. Time-gated PL measurements allowed the suppression of autofluorescence and other short PL-lifetime background signals, which are significant in serum samples. A limit of detection (LOD) of 60 ␮M adenosine (similar to other homogeneous adenosine aptamer-based sensors in buffer solution) was realized in 100% serum samples. Although the advantages of LTCs for multiplexed FRET are numerous (vide supra), and a theoretical construction of a multiplexed LTC-based FRET sensor is relatively easy, the practical development of such biosensors is much more challenging. Pulsed excitation and time-gated or time-resolved detection must be combined with different wavelength separation technologies (e.g., dichroic mirrors, bandpass filters, mono/polychromators), biomolecules must be conjugated with the LTC donors or the different acceptors, and these bioconjugates must retain their biological and photophysical functionalities in order to establish both biological and optical multiplexing. In recent years, different multiplexed LTC-based FRET biosensors have been developed, all of which have

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3-intensity optical probe with 315 (14,348,907-fold multiplexing) distinguishable PL signal combinations (Scheme 2) would appear to be technically feasible. In the following sections, we will outline some recent single-step and double-step LTC-based multiplexed applications using dyes and quantum dots as acceptors. 3. Multiplexed FRET from LTCs to dyes

Scheme 1. LTC-FRET-based multiplexing on the color and time scale. FRET 1 occurs from one type of LTC to differently colored acceptor fluorophores and is detected with time-gating (delayed). FRET 2 occurs between different types of fluorophores and is detected immediately (after excitation or FRET-sensitization of the first fluorophore).

used either the principle of one-step FRET from one type of LTC (in an equal or higher concentration to the acceptors) to different acceptor molecules or particles (spectral multiplexing), or doublestep FRET from one LTC to one acceptor and from there to another acceptor (spectrotemporal multiplexing). The principle of such LTC-based FRET multiplexing is presented in Scheme 1 for 5-fold spectral and 2-fold spectrotemporal multiplexing. Although this could theoretically lead to 10-fold multiplexing by very careful spectral separation of the 10 acceptors, this multiplexing combination is limited in practice because the FRET 2 step is immediate (compared to FRET 1) and therefore similar colors of FRET 1 and FRET 2 acceptors cannot be distinguished. If different PL decay times can be distinguished (i.e., different  DA =  AD due to different FRET efficiencies resulting from different D–A distances for the same D–A pair; for example,  DA1 = 0.1 ms,  DA2 = 0.5 ms and  DA3 = 2.0 ms), then temporal and spectral multiplexing could be reliably combined. When different acceptor PL intensities can also be distinguished and controlled (e.g., for optical barcoding) [108], this parameter can be used as a third multiplexing dimension. A measurement in x color channels (spectral distinction of different PL emission bands by bandpass filters), each providing y timechannels (temporal distinction of different PL decay curves by timegating), which can distinguish z PL intensity levels (photon count distinction of different fluorophore concentrations), would lead to zxy distinguishable codes as illustrated in Scheme 2. Although such multiplexing has never been realized, a 5-color, 3-decay time, and

The large variety and wide commercial availability of organic dyes has made them the most frequently applied class of fluorophores for luminescence-based biosensing, especially for FRET [10,17,109]. The combination of LTC donors with organic dye acceptors has also been used for multiplexed biosensing [70,72–74,77,79,99]. In an initial study, Kupcho et al. used one LTC in combination with two acceptors, fluorescein and AlexFluor 633 (AF633), for the time-gated spectroscopic investigation of ligand–receptor binding [70]. A nuclear receptor was bound by a specific LTC-antibody and fluorescein and AF633 were conjugated with peptides, which were derived from a coactivator (coregulatory proteins enhancing transcriptional activity) and a corepressor (coregulatory proteins repressing transcriptional activity), respectively. Addition of a receptor antagonist to a solution containing all of the aforementioned reagents led to increased FRET-sensitization of fluorescein (association of the coactivator peptide) and decreased FRET-sensitization of AF633 (disruption of the corepressor peptide). The opposite behavior was observed using an antagonist for the nuclear receptor. This duplexed LTCto-dye FRET system allowed a simultaneous investigation of the orthogonal ligand–receptor binding-effects. Based on this first publication, Kokko et al. applied duplexed LTC-to-dye FRET to a homogeneous immunoassay for prostate specific antigen, which exists in serum in both free and bound forms [72]. The ratio of total PSA (free plus bound) and free PSA is an important measure for prostate cancer diagnostics. The authors used LTC-antibody conjugates and AlexaFluor 680 (AF680)-antibody conjugates targeting both free and bound PSA, and AlexaFluor 488 (AF488)-antibody conjugates targeting only free PSA (the three different antibodies were specific for different epitopes of free or bound PSA). Time-gated PL intensities from FRET-sensitization of AF488 and AF680 were used for the determination of free and bound PSA. The obtained LODs of the duplexed homogeneous immunoassay were 0.74 ng/mL for free PSA and 0.6 ng/mL for total PSA. Kim et al. extended the LTC-to-dye FRET system to a triplexed approach

Scheme 2. Photoluminescence multiplexing in three dimensions. The combination of x PL wavelengths (e.g., from different QD sizes) with y PL decay times (e.g., from different LTC-donor to QD-acceptor distances) and z PL intensities (e.g., from microbeads doped with different concentrations of the LTC-QD hybrid nanoparticles) can offer zxy different distinguishable signals for multiplexed sensing or optical barcoding. The example of 5 colors, 3 decay times and 3 intensities leads to 14,348,907 different codes.

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using fluorescein, semi-naphthalene fluorescein (SNAPFL) and Cy5 as simultaneous acceptors for the investigation of the estrogen receptor (ER) [79]. ER is a cellular regulatory system involving four components for gene transcription in target tissue cells. ERagonist binding leads to a conformational change and enables the recruitment of coactivator proteins, which are responsible for the upregulation of gene transcription. In contrast, antagonist binding leads to prevention of recruitment. Using the four-color LTC-to-dye FRET system, the authors investigated if the displacement of an antagonist with an agonist leads to instant binding of the coactivator or if an intermediary agonist-liganded state without coactivator exists. The LTC was attached to ER via biotin–streptavidin binding and acted as one donor for all acceptors. The three acceptors were the estrogen agonist-fluorescein, the antagonist-SNAPFL, and the coactivator-Cy5 conjugates. The results clearly demonstrated the possibility of simultaneous detection of three independent FRET signals when fluorophores, measurement settings, and filters are carefully chosen. Importantly, the authors showed that ligand displacement from an antagonist to an agonist is directly connected with a recruitment of the coactivator. In a recent study, we demonstrated that even five-fold multiplexed LTC-to-dye FRET provides very sensitive diagnostics [99]. Our FRET biosensor used one LTC as donor and the organic dyes OregonGreen 488 (QG488), AlexaFluor 555 (AF555), AlexaFluor 568 (AF568), Cy5, and AlexaFluor 700 (AF700) as acceptors. The absorption and emission spectra of these fluorophores are shown in Fig. 2. The five LTC-dye combinations provided Förster distances between 4.4 and 6.0 nm. The different optical bandpass

filters used for wavelength separation of the single detection channels (Fig. 2) clearly show transmission for several of the dye emission spectra, which leads to so-called spectral crosstalk of various dyes to different detection channels. This crosstalk could be efficiently corrected by multiplying the measured time-gated PL intensities in the five detection channels with an inverted matrix, which was acquired in single LTC-dye measurements before the multiplexed measurement and accounted for the contributions of the five dyes in the five detection channels. In addition to the spectral crosstalk, the quintuplexed biosensor was also very challenging from a biological point of view. As shown in Scheme 3, a homogeneous immunoassay (liquid phase assay without any washing or separation steps) for five different lung cancer tumor markers in a single 50 ␮L serum sample was realized. This setup required 15 different biological components—5 tumor markers and 10 antibodies—for multiplexed biological recognition. Despite this challenging immunoassay system, the spectroscopic crosstalk correction allowed for efficient discrimination between normal and elevated tumor marker concentrations, and precise marker quantification, under very challenging conditions with all the markers at different biologically relevant concentrations ranging from 0.5fold to 10-fold the clinical cut-off values (Fig. 3). The multiplexed homogeneous immunoassay was performed on a clinical diagnostic plate reader and provided low picomolar detection limits that were below the clinical cut-off values for all five tumor markers. Such examples demonstrate the very high sensitivity and direct applicability of multiplexed LTC-to-dye biosensors for facile integration into real-life in vitro diagnostics.

Scheme 3. 15-Component multiplexed LTC-to-dye FRET immunoassay for the simultaneous detection of the five lung cancer tumor markers NSE, SCC, CEA, Cyfra21-1 and CA15.3. The top images show the different antibody (AB) pairs labeled with LTCs (Tb) and five different organic dyes (D1–D5) before (left) and after (right) addition of the tumor markers (TMs). TM-AB recognition leads to the formation of (Tb-AB)-TM-(dye-AB) sandwich complexes and FRET from LTCs to dyes. Star-shaped molecules indicate luminescence, whereas the black spheres indicate dark states. The bottom graphs indicate the resulting time-gated PL intensities, which are proportional to the different TM concentrations. Reprinted with permission from [99]. Copyright 2013 The American Chemical Society.

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Fig. 3. Five-fold LTC-to-dye FRET multiplexed lung cancer immunoassay with simultaneously increasing (left) and different tumor marker concentrations (right) in a single 50 ␮L serum sample. Left: Measured tumor marker concentration as a function of known marker concentrations. Normal (0.5–1× the highest concentration found in healthy persons) and elevated concentrations (>1× the highest concentration) are indicated by the green and red area, respectively. Right: In order to generate very challenging assay conditions, the concentrations of the different tumor markers are held at constant high or low levels, or changed from high to low (or low to high) concentrations within six different human serum samples. Symbols represent measured concentrations, and the dashed lines indicate known concentrations within the six samples. The different colors indicate the different tumor markers: blue: NSE; green: SCC; orange: CEA; red: Cyfra21-1; brown: CA15.3. Reprinted with permission from [99]. Copyright 2013 The American Chemical Society.

4. Multiplexed FRET from LTCs to QDs Although multiplexed LTC-to-dye FRET is possible and can still provide high sensitivity when an appropriate spectral crosstalk correction is applied, multiple acceptors with less emission overlap, higher brightness, and better stability (less photobleaching) are highly desirable. Such photophysical properties can be implemented by using semiconductor quantum dots as FRET acceptors. Comparing the left-hand side (dyes) and the right-hand side (QDs) of Fig. 2 shows the main advantages of QDs over organic dyes. Assuming similar PL quantum yields, the much higher molar absorptivities of QDs provide better brightness (PL quantum yield multiplied by molar absorptivity). The large molar absorptivity values over a wide spectral range are also extremely important for spectral overlap with the LTC emission, which is an important requirement for efficient FRET (cf. Eqs. (1)–(3)). Förster distances

of more than 10 nm can be achieved for LTC-to-QD FRET pairs [49], which is significantly larger than the R0 -values for conventional D–A pairs [110,111] and permits non-trivial energy transfer beyond the often cited FRET limit of 10 nm [9,11]. Considering PL properties, the much narrower emission bands of QDs significantly reduce the spectral crosstalk compared to the dye emission spectra, which all show an additional red-shifted shoulder beyond their maximum emission peak. Another advantage is the size- and material-tunability of QDs, which means that any desired emission wavelength can be generated by changing the size and/or material composition of the QDs. Several reviews about the photophysical properties of QDs and their advantages concerning biosensing and FRET can be found in the recent literature [20,24,26,112,113]. In two initial studies on the feasibility of LTC-to-QD FRET for multiplexed diagnostics (Scheme 4, left) and as a multiplexed spectroscopic ruler (Scheme 4, right), we utilized the well-known

Scheme 4. Multiplexed LTC-to-QD FRET for ultra-sensitive diagnostics (left) and spectroscopic ruler measurements (right) based on biotin–streptavidin for proof-of-principle studies. The multiplexed detection provides sub-picomolar (sub-100 attomol) detection limits of five different FRET pairs in a single 150 ␮L sample using time-gated FRET detection. The molecular ruler can determine sizes, shapes and organic coating thicknesses of five different QDs inside a single 150 ␮L sample at sub-nanomolar concentrations under physiological conditions using time-resolved PL decay analysis of LTC donors and QD acceptors. Reprinted with permission from [90] (left) and [91] (right). Copyright 2010 Wiley-VCH Verlag GmbH & Co. KGaA.

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biotin–streptavidin biological binding system to create five different LTC-QD FRET pairs (absorption and emission spectra are shown in Fig. 2). In both cases, pulsed UV excitation (337.1 nm nitrogen laser with 20 Hz repetition rate) was used to excite the LTC donors. At such UV wavelengths, all of the QDs were excited much more efficiently than the LTC, leading to excited states that were counterproductive for FRET because only QDs in their ground state can act as FRET acceptors. It is here that the long excited state lifetime of LTC donors (almost 106 -fold longer than the excited state lifetimes of QDs) is essential. After some hundreds of nanoseconds, the probability that the QD acceptor has decayed back into its ground state is close to unity, whereas the probability that the LTC donor has decayed back into its ground state is close to zero, such that FRET between an excited-state LTC donor and ground-state QD acceptor becomes possible and can be very efficient when the LTC and QD are in close proximity (e.g., by biotin–streptavidin binding). Another advantage of this large difference in excited-state lifetimes is the possibility of FRET from many LTCs to a single QD. One QD can accept the energy from one LTC, lose this energy within nanoseconds by photon emission, and can then accept the energy from another LTC, which is still in an excited state that can persist over milliseconds. This excited state lifetime mismatch increases the probability of QD-FRET-sensitization (cf. Eq. (4)) and can lead to brighter time-gated QD PL since the excitation of many LTC donors can lead to many FRET-excitations of one QD. In the case of the quintuplexed LTC-to-QD FRET assay (Scheme 4, left) [90], the ratio of the time-gated PL from the QDs and LTCs was used to quantify the different LTC/QD FRET pairs on a commercial diagnostic fluorescence plate reader (KRYPTOR, Cezanne/BRAHMS/ThermoFisher). Sub-picomolar LODs were achieved for all five FRET-pairs in a single 150 ␮L sample. These multiplexed LODs were only slightly larger than for corresponding assays where there was only a single FRET-pair per sample, and corresponded to a 40–240-fold improvement (depending on the different QDs) compared to the “gold standard” single FRET-pair assay using different donor and acceptor fluorophores, namely europium trisbipyridine cryptate (Eu-TBP) as the donor and crosslinked allophycocyanin (APC) as the acceptor. The EuTBP/APC system is used in many commercial assays for fluorescence plate readers under the brand names HTRF (Cisbio) [114] or TRACE (Cezanne/BRAHMS/ThermoFisher) [115]. The quintuplexed spectroscopic ruler took advantage of the very high spatial resolution of FRET (cf. Eq. (1)) in the 1–20 nm range. Considering that Förster’s contributions relating FRET theory and spectroscopy were published in the 1940s [116–120], and given the nanometric D–A distance range accessible with FRET, which is far below the diffraction limit of light [121,122], FRET was arguably the first optical superresolution technique for spatial analysis. In contrast to the diagnostic approach (timegated detection), the LTC-to-QD FRET multiplexed molecular ruler analyzed the complete PL decay curves of the five different LTCstreptavidin–biotin–QD FRET systems [91]. Through determination of the different decay time components of the overall multiexponential PL decays, it was possible to estimate, with nanometer precision at sub-nanomolar concentrations under physiological conditions, the size and shape of five commercial QDs as well as the thickness of their polyethylene glycol (PEG)-based coatings. Further comparison with data from transmission electron microscopy (TEM) and dynamic light scattering (DLS), and an improvement in the algorithm for analyzing multiplexed LTC donor and QD acceptor decay curves, showed that the multiplexed LTC molecular nanoruler is very well suited to accurately measuring the properties of biocompatible QDs (with organic surface coatings) at concentrations and conditions suitable for optical biosensing [97]. Recent efforts concerning LTC-to-QD FRET have focused on the integration of this promising technology into clinical diagnostic

immunoassays, which require the use of biomarker-specific antibodies instead of biotin–streptavidin recognition. Liu et al. developed a homogeneous time-gated LTC-to-QD FRET immunoassays against alpha-fetoprotein (AFP) and carcinoembryonic antigen (CEA) [95,96]. Although the estimated LODs were quite promising, these two papers had limited information about the photophysical and FRET properties of the assays and their stability. These assays were also performed in buffer, whereas immunoassays require the measurement of serum or plasma samples. In a recent study, we presented a homogeneous time-gated LTC-to-QD FRET immunoassay for CEA, which used commercial QDs (Qdots, Life Technologies) with an emission maximum around 655 nm as acceptors [49]. This assay provided a LOD of 2.6 ng/mL CEA in 70 ␮L serum samples, measured on the KRYPTOR time-resolved fluoroimmunoassay (TR-FIA) plate reader. In order to demonstrate the multiplexing feasibility of LTC-to-QD FRET immunoassays, and to investigate the influence of different antibody sizes, we performed a detailed study using two different commercial QDs (eFluorNCs, eBioscience), emitting around 605 nm (QD605) and 650 nm (QD650), with either full IgG, reduced F(ab )2 , or F(ab) antibodies targeting prostate specific antigen (PSA). These different antibodies can lead to different D–A distances and orientations (Scheme 5), and also have different affinities for the antigen [usually Ka (IgG) > Ka (F(ab )2 ) > Ka (F(ab))] [101]. Apart from these possible variations, the three types of antibodies also offer different conjugation conditions for the relatively small LTCs and the relatively large QDs (more LTCs can be conjugated to larger antibodies and a greater number of smaller antibodies can be conjugated to QDs). This aspect is especially important for LTC-to-QD FRET because many LTCs can transfer their energy to a single QD (vide supra). A combination of LTC-conjugated IgG and F(ab)-conjugated QDs led to 210 LTC donors per QD acceptor (taking into account only the amount of D and A and not the possible variations in distances and orientations). Although the complete sandwich immunoassay system (two antibodies, one antigen, and the thick PEG-based QD coating) imposed a relatively large D–A distance, all combinations of the TR-FIAs against PSA with both the QD650 and the QD605 acceptor led to significant LTC FRET-quenching and QD FRET-sensitization as shown in Fig. 4 for the LTC-F(ab )2 -PSAF(ab )2 -QD650 system. As expected from the size estimates, the combination of LTC-IgG with the F(ab)-QD led to the highest sensitivities (LOD of 0.05 nM or 1.6 ng/mL PSA in 50 ␮L serum samples), whereas the difference in LODs between the F(ab )2 -QD and the F(ab)-QD systems was only marginal. Importantly, this LTC-to-QD FRET immunoassay combined all the necessary attributes necessary for a real-life application in clinical diagnostics: homogeneous, serum-based, sensitive, specific, fast, small, reproducible, robust, flexible, multiplexed, and stable. In an attempt to further decrease the D–A distance, we also applied single-domain antibodies (VH H) in a LTC-to-QD FRET-based homogeneous immunoassay against serum-soluble epidermal growth factor receptor (EGFR) [123]. In fact, the conjugation of multiple VH H per QD, in combination with a shorter possible D–A distance compared to the aforementioned antibody systems, allowed the first demonstration of a VH H-based homogeneous FRET immunoassay (Scheme 6). Although a timeresolved analysis revealed relatively strong non-specific binding of serum albumin (causing significant background FRET without any EGFR in the samples), and the quenching of LTC PL was relatively low (most probably due to the large excess of LTCs in the sample, which did not participate in FRET), very low concentrations of EGFR could still be determined both in buffer and serum samples because significant QD-FRET sensitization was observed when EGFR was added to the LTC-VH H and QD-VH H conjugates (Fig. 5). The relatively low dynamic range of approximately one order of magnitude could possibly be improved by optimizing the concentrations of LTC-VH H and QD-VH H conjugates, or by automated

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Scheme 5. Homogeneous TR-FIA against PSA based on LTC-to-QD FRET. LTC-antibody conjugates (top) and QD-antibody conjugates (bottom) each contain a different primary antibody (which are further reduced to F(ab )2 and F(ab) fragments) against PSA. Reprinted with permission from [101]. Copyright 2013 The American Chemical Society.

Fig. 4. PL decay curves for a representative LTC-to-QD FRET TR-FIA against PSA (cf. Scheme 5). The addition of 50 ␮L serum samples containing increasing PSA concentrations (from black to blue) to a 100 ␮L solution of LTC- and QD-antibody conjugates (constant concentrations) leads to increasing FRET-sensitization of the QD PL (QD channel) and increasing FRET-quenching of the LTC PL (Tb channel). Reprinted with permission from [101]. Copyright 2013 The American Chemical Society.

Scheme 6. Homogeneous VH H-based TR-FIA against EGFR using LTC-to-QD FRET. The combination of LTC-labeled VH H and VH H-labeled QDs allows the formation of sandwich immunocomplexes, which results in FRET from several LTCs to a central QD (right). Time-gated detection of FRET-sensitized QD PL is used for a sensitive quantification of soluble EGFR in buffer or serum (cf. Fig. 5). Reprinted with permission from [123]. Copyright 2013 Wiley-VCH Verlag GmbH & Co. KGaA.

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dilution from clinical plate reader systems [101]. The developed LTC-to-QD FRET immunoassays and the extremely high sensitivity of the LTC-to-QD FRET multiplexing demonstrate the significant potential of this technology for multiplexed in vitro diagnostics. Duplexed immunoassays against different biomarkers in a single serum sample have already been developed in our laboratories, and these, as well as higher multiplexing assays, are expected to be published in the near future.

5. Multiplexed FRET from LTCs to QDs to dyes (multistep FRET)

Fig. 5. Calibration curves for the VH H-based LTC-to-QD immunoassays (cf. Scheme 6) using 50 ␮L buffer (diamonds) or serum (circles) samples. Time-gated PL intensity ratios (QD PL divided by LTC PL) increase with increasing EGFR concentration until the EGFR-VH H binding is saturated and no further FRET sensitization occurs. The LODs (within the 50 ␮L samples) were 0.12 nM (23 ng/mL or 6 fmol) and 0.18 nM (34 ng/mL or 9 fmol) for buffer and serum samples, respectively. Reprinted with permission from [123]. Copyright 2013 Wiley-VCH Verlag GmbH & Co. KGaA.

A more sophisticated LTC-based FRET system using multistep LTC-to-QD-to-dye FRET has been recently developed to further increase the flexibility and dimensionality of multiplexed sensing [93,94]. In this approach, LTCs and dyes are conjugated to the same central QD (e.g., via peptides or oligonucleotides) and are therefore in close proximity to the QD. Similar to the previously noted LTC-to-QD FRET systems, pulsed UV excitation excites both the LTC and QD, whereas the dye is not excited and remains in its ground state. This initial or prompt situation (and the fact that the dyes were chosen to be efficient FRET acceptors for the QD) does not lead to FRET from the LTC to the QD (FRET1), but rather leads to efficient FRET from the QD to the dye (FRET2). This sit-

Fig. 6. (A) One QD is used as a nanoscaffold for the controlled assembly (via His6 ) of two different types of probe oligonucleotide (PRB A and PRB B). The addition of LTC and AF647-labeled target oligonucleotides (AF647-TGT A and LTC-TGT B) leads to DNA hybridization. (B) Prompt FRET2 from QD-to-AF647 (no delay: 0 ␮s) is only dependent on TGT A hybridization. (C) Time-gated FRET1 (55 ␮s delay) is only dependent on TGT B hybridization. As FRET1 can be followed by FRET2, both FRET1 + FRET2 need to be taken into account. (D) Detection of FRET1 is used to quantify TGT A independent of TGT B. (E) FRET1 and FRET2 are used to quantify TGT B independent of TGT A. Reprinted with permission from [94]. Copyright 2012 The Amercian Chemical Society.

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Fig. 7. LTC-to-QD-to-dye FRET relays for molecular logic devices (MLDs). (a) The initial input state (0,0) contains only QDs. (b) The other three input states (0,1), (1,0), and (1,1) contain distinct valences of LTC (Input A) and AF647 (Input B). The corresponding PL spectra are time-gated for FRET1 + FRET2 (orange) and immediate for FRET2 only (blue). For time-gated monitoring (orange), the LTC is excited at 339 nm while for immediate PL monitoring (blue) the QD is excited at 400 nm, which excludes the LTC. Reprinted with permission from [124]. Copyright 2013 The Royal Society of Chemistry.

uation changes after a delay of several microseconds following the excitation pulse because the QDs and dyes have decayed back into their ground states, whereas a large majority of LTCs are still in their excited states. Similar to the LTC-to-QD FRET, this

time-delayed or time-gated situation allows efficient FRET from Tbto-QD (FRET1) followed by another QD-to-dye FRET step (FRET2) from the re-excited QD. The detailed principle and several applications (e.g., duplexed protease activity and inhibition assays as

Fig. 8. (a) Steady-state PL intensity ratios of A647(at 670 nm)/QD(at 625 nm) for different LTC and AF647 valences per QD. PL intensity ratios above/below 1 are defined as ON/OFF output states. (b) Time-gated (delay of 55 ␮s) PL intensity ratios of A647(at 670 nm)/LTC(at 550 nm) for different LTC and AF647 valences per QD. PL intensity ratios above/below 0.50 are defined as ON/OFF output states. (c) INHIBIT (INH) logic gate time-gated PL intensity ratio output created by the inputs (0,0): only QD, (1,0): 12 LTC and 8 AF647 per QD, (0,1): 4 LTC and 25 AF647 per QD and (1,1): 25 LTC and 25 AF647 per QD. (d) XOR logic gate time-gated PL intensity ratio output created by the inputs (0,0): only QD, (1,0): 12 LTC and 8 AF647 per QD, (0,1): 4 LTC and 12 AF647 per QD and (1,1): 25 LTC and 25 AF647 per QD. Reprinted with permission from [124]. Copyright 2013 The Royal Society of Chemistry.

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well as a kinetic analysis) of this technology have been recently reviewed [112], and will not be mentioned in detail again here. As a brief example of such a multiplexed LTC-to-QD-to-dye FRET relay, we review a duplexed DNA-hybridization assay here (Fig. 6). Two different single-stranded (ss) DNA oligonucleotide probes (PRB A and PRB B) were self-assembled to the Zn-rich surface of the QD via polyhistidine tags. Two target ssDNA oligonucleotides were labeled with either LTC (TGT B) or AF647 (TGT A) and hybridized to the complimentary probe ssDNA, bringing the LTC and AF647 in close proximity to the QDs (Fig. 6A). Prompt detection after pulsed excitation measured FRET2 (QD-to-AF647 FRET, Fig. 6B) whereas time-gated detection after 55 ␮s measured FRET1 + FRET2 (LTCto-QD-to-AF647, Fig. 6C). As a result of the two orthogonal FRET processes (FRET1 only depended on LTC-assembly and FRET2 only depended on AF647-assembly), prompt detection of the AF647/QD PL ratio could be used to quantify TGT A independent of TGT B concentration (Fig. 6D), and time-gated detection of QD + AF647 PL sensitization could be used to quantify TGT B independent of TGT A (Fig. 6E). Thus, duplexed detection was achieved with only a single QD color by using the spectrotemporal multiplexed LTC-to-QD-todye FRET relay approach. In another recent study, we extended the FRET relay approach to the design of molecular logic devices (MLDs), which are potentially useful tools for integration into various biosensing applications [124]. Controlled assembly of LTCs and AF647 dyes to a QD surface via polyhistidine-functionalized peptides provided distinct valences of LTC and AF647, which were used as the two inputs for the different logic gates. Time-gated and prompt detection of FRET1 and FRET2 and only FRET2 using the defined LTC and AF647 valences led to the four necessary input states (0,0), (1,0), (0,1) and (1,1) for two-input-single-output photonic logic gates (Fig. 7). Prompt measurement of the AF647/QD PL ratio led to an almost linear increase with increasing AF647 valence (almost independent of the LTC valence) and PL ratios below and above 1 were defined as the output states OFF and ON, respectively (Fig. 8a). Time-gated detection of the AF647/LTC PL ratio led to an initial increase with AF647 valence followed by a decrease with higher AF647 valence values. Thus, the output state OFF could be realized by low and high AF647 valences (PL ratios below 0.5) whereas the output state ON was reached for medium AF647 valences (Fig. 8b). This very interesting feature could be used for the creation of a photonic INHIBIT gate, for which only the input state (1,0) generates an ON output whereas all the other input states lead to an OFF output (Fig. 8c), and also for the creation of a XOR gate, for which (1,0) and (0,1) inputs lead to ON and (0,0) and (1,1) lead to OFF (Fig. 8d). Apart from further logic gates such as AND, OR, NAND, NOR, YES, and NOT, another very unique property of the LTC-to-QD-to-dye FRET-relay MLDs was their set-reset capability over several regeneration cycles by using enzymes for peptide cleavage and enzyme inhibitors to permit re-assembly of new LTC and AF647-labeled peptides. Recently, other Boolean logic states for the creation of circuits such as half-adders, half-subtractors, multiplexers, demultiplexers, transfer gates, and keypad locks have been realized [125].

6. Summary and perspective In this review, we have described a variety of LTC-based FRET systems for multiplexed optical biosensing. The unique photophysical properties of LTCs, including their narrow and well-separated emission bands, their stability in biological media, and especially their extremely long excited-state lifetimes up to several milliseconds, make these terbium complexes very special donors for sensitive multiplexed FRET biosensing. Their ability to be combined with FRET acceptors such as organic dyes, fluorescent proteins, and semiconductor quantum dots offers high flexibility and enables

the creation of otherwise impossible FRET-pairs with Förster distances that permit energy transfer beyond the often cited FRET range of 1–10 nm. Although the combination of lanthanides and FRET has been known for a long time, optical multiplexing of different biological interactions inside a single sample for the sensitive quantification of multiple concentrations or distances by LTC-based FRET biosensors is a relatively young field of research. The examples presented here included multiplexed cancer diagnostics using LTCto-dye FRET, homogeneous LTC-to-QD FRET immunoassays against different tumor markers, and LTC-to-QD-to-dye FRET relays for DNA hybridization, enzyme kinetics assays, and molecular logic devices. Although the orders of multiplexing and the sensitivities of these assays are very impressive, further improvements and completely new concepts for multiplexed optical sensing are feasible and will be important for sophisticated biosensing applications in the near future. Another very promising field for multiplexed LTC-based FRET is fluorescence imaging, especially for cellular and tissue imaging applications where the simultaneous detection of several biological interactions with highly reduced autofluorescence background is possible using time-gated PL detection. Although this review has described many of the unique advantages of LTCs for FRET-based biosensing, one should always keep in mind that every probe has its disadvantages. The lower amount of emitted photons per unit time from LTCs (due to their long excited-state lifetimes) is an important aspect that should be carefully taken into account when choosing the FRET-pair for a specific biosensing application. The most appropriate methods and technology should always be selected for a particular diagnostic test, and, in the case of immunoassays, there are many different techniques available [126]. Specific economic and regulatory issues notwithstanding, selection of a technology for biosensing applications is usually governed by analytical figures of merit (e.g., sensitivity and specificity toward the target analyte), sample properties (e.g., volume and nature of the biological fluid), the probe material (e.g., stability and safety), the instrument (e.g., footprint and cost) and the experimental details (e.g., analysis time and level of training required). LTC-FRET is but one of the many possible technologies that may have the best combination of features to address a particular challenge in biosensing. In any case, LTCs have an important place in the colorful and versatile toolbox of luminescent FRET probes. We are looking forward to many more interesting developments in LTC-based multiplexed FRET techniques and applications in the future. Acknowledgements We thank the Agence National de la Recherche France (project NanoFRET) and the Natural Sciences and Engineering Research Council of Canada for financial support. References [1] K.B. Cederquist, S.O. Kelley, Curr. Opin. Chem. Biol. 16 (2012) 415–421. [2] M. Fakruddin, Z. Hossain, H. Afroz, J. Nanobiotechnol. 10 (2012), http://dx.doi.org/10.1186/1477-3155-1110-1131. [3] E. Zahavy, A. Ordentlich, S. Yitzhaki, A. Shaffermann, Nano-Biotechnology for Biomedical and Diagnostic Research, Springer, Netherlands, 2012. [4] J.-C.G. Bünzli, Lanthanide Probes in Life, Chemical and Earth Science: Theory and Practice, Elsevier, Amsterdam/New York, 1989. [5] J.-C.G. Bünzli, Chem. Rev. 110 (2010) 2729–2755. [6] J.-C.G. Bünzli, S.V. Eliseeva, Chem. Sci. 4 (2013) 1939–1949. [7] S.V. Eliseeva, J.-C.G. Bünzli, Chem. Soc. Rev. 39 (2010) 189–227. [8] R.M. Clegg, in: T.W.J. Gadella (Ed.), Laboratory Techniques in Biochemistry and Molecular, Elsevier B.V., 2009. [9] J.R. Lakowicz, Principles of Fluorescence Spectroscopy, third ed., Springer, New York, 2006. [10] I.L. Medintz, N. Hildebrandt, FRET-Förster Resonance Energy Transfer. From Theory to Applications, Wiley-VCH, Weinheim, Germany, 2013. [11] B. Valeur, Molecular Fluorescence: Principles and Applications, Wiley-VCH, Weinheim/New York, 2002.

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