Quantum dots for Förster Resonance Energy Transfer (FRET)

Journal Pre-proof Quantum Dots for Förster Resonance Energy Transfer (FRET) Marcelina Cardoso Dos Santos, W. Russ Algar, Igor L. Medintz, Niko Hildebrandt PII:

S0165-9936(19)30661-2

DOI:

https://doi.org/10.1016/j.trac.2020.115819

Reference:

TRAC 115819

To appear in:

Trends in Analytical Chemistry

Received Date: 3 December 2019 Accepted Date: 22 January 2020

Please cite this article as: M.C. Dos Santos, W.R. Algar, I.L. Medintz, N. Hildebrandt, Quantum Dots for Förster Resonance Energy Transfer (FRET), Trends in Analytical Chemistry, https://doi.org/10.1016/ j.trac.2020.115819. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Crown Copyright © 2020 Published by Elsevier B.V. All rights reserved.

Quantum Dots for Förster Resonance Energy Transfer (FRET) Marcelina Cardoso Dos Santos1,2*, W. Russ Algar3, Igor L. Medintz4, and Niko Hildebrandt1,5*

1

NanoBioPhotonics Institute for Integrative Biology of the Cell (I2BC) Université Paris-Saclay, Université Paris-Sud, CNRS, CEA 91400 Orsay France 2

Team Cytoskeleton Dynamics and Motility Institute for Integrative Biology of the Cell (I2BC) Université Paris-Saclay, Université Paris-Sud, CNRS, CEA 91198 Gif-sur-Yvette Cedex France

3

Department of Chemistry University of British Columbia Vancouver, BC V6T 1Z1 Canada 4

Center for Bio/Molecular Science and Engineering Code 6900, U.S. Naval Research Laboratory Washington, DC 20375 United States 5

nanoFRET.com Laboratoire COBRA (Chimie Organique, Bioorganique, Réactivité et Analyse) Université de Rouen Normandie, CNRS, INSA 76821 Mont-Saint-Aignan France

*To whom correspondence should be addressed: [email protected]; [email protected]

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TABLE OF CONTENTS 1. Introduction 2. Quantum Dot Bioconjugation 3. FRET Theory and Formalism 3.1. General FRET Theory 3.2. QDs as FRET Donors 3.3. QDs as FRET Acceptors 4. QD-FRET Biosensing 4.1. Molecular Rulers 4.2. FRET Assays 4.2.1. Immunoassays 4.2.2. Enzyme Assays 4.2.3. Hybridization (Nucleic Acid) Assays 4.3. Multistep FRET or Higher Order Multiplexing 4.4. FRET Imaging 4.4.1. In vitro imaging 4.4.2. In vivo imaging 5. Conclusions and Perspective 6. References

ABSTRACT: The analysis of biomolecular interactions using quantum dots (QDs) as both FRET donors and acceptors has become an established technique in the life sciences. This development has been driven by the unique properties of QDs, which include large surfaces for the attachment of biomolecules, high brightness and photostability, strong and spectrally broad absorption, and color-tunability via QD size, shape, and material. Applications include molecular rulers for structural analysis, small-molecule sensors, immunoassays, enzyme assays, nucleic acid assays, fluorescence imaging in-vitro and in-vivo, and molecular logic gates. Here, we will explain the theory of QD-based FRET, review some aspects of QD surface functionalization that are important for FRET, and highlight and discuss the advantages and disadvantages of QDs in FRET-biosensing using both spectroscopy and imaging techniques.

KEYWORDS: quantum dots, FRET, biosensors, immunoassays, imaging, multiplexing 2

1. Introduction Due to their unparalleled properties, since their first application in biological systems [1], [2], core-shell semiconductor quantum dot (QD) nanocrystals have become important fluorophores for the biosensing community. Their structure provides them with unique photophysical properties, such as high brightness (due to high quantum yields and absorption coefficients), chemo- and photostability, broad absorption and narrow emission spectra, wide spectral coverage from the visible to near-infrared (NIR), and color-tunability by varying their physicochemical characteristics (size, shape, and core-shell composition). Moreover, QDs acquire new properties when coupled with other organic or inorganic materials. In the field of nanophotonics, this highly coveted versatility makes QD probes superior to conventional fluorophores. The aim of this review is to highlight and discuss the ability of QDs to take part in and advance Förster Resonance Energy Transfer (FRET) for analytical applications. QDs have been shown to be both excellent energy donors, for a large variety of organic and inorganic molecules (dyes, fluorescent proteins), polymers, and metal nanoparticles (NPs) [3]–[5], and acceptors, for lanthanide-based compounds (e.g., chelates, cryptates, nanoparticles), other QDs, and fluorescent polymers [6]–[8]. Many methods of biomolecular detection, including FRET, continue to use standard fluorescent probes, such as dyes or fluorescent proteins [8], [9]. Despite their photophysical drawbacks, including rapid photobleaching, self-quenching at high concentrations, or instability in biological media, these probes are very functional and exist in almost any desired color. Rather than replacing dyes or fluorescent proteins, QDs should be regarded and used as an alternative fluorescent nanoprobe that can provide several advantages over standard fluorophores. In particular, the longer photoluminescence (PL) lifetimes (in the tens to hundreds of nanoseconds), the broader excitability (nearly arbitrary choice of excitation wavelength and one excitation source for different QDs), and the narrower emission bands (significantly better multiplexing capabilities) of QDs present unique benefits for FRET. In 3

this review, we mainly focus on the bioanalytical properties of QDs as versatile FRET platforms. We will discuss different QD bioconjugation methods and explain the basic concept of FRET and the most important FRET parameters (e.g., Förster distance, FRET efficiency, FRET ratio) useful for performing and analyzing FRET experiments. We will also give an overview of how fast QDs revolutionized the field of FRET biosensing and their adaption to various bioanalytical applications ranging from clinical assays to imaging in cells or tissues.

2. Quantum Dot Bioconjugation The use of QDs in FRET-based biosensors requires significant preparation efforts in order to transform them into efficient bioconjugates. A very important point concerns the biocompatibility of the QD material, which is most often accomplished by an appropriate surface coating, which can range from thin capping ligands to thick polymer, lipid, or silica shells. The many surface coating strategies have been described in detail elsewhere [10], [11] and here we will restrain ourselves to discussing the requirements and consequences of surface functionalization for FRET biosensing. The quality of functionalization depends on the coating material, its quantity, charge, affinity, and organization on the QD surface. Although in some cases, it may be sufficient to provide negative surface charges to prevent QDs from aggregating into the cytosol or sticking to the cell membrane, a well-optimized QD surface functionalization is crucial to avoid undesirable reactions within often challenging biological environments [12], [13]. Although QD toxicity is still an ongoing discussion, welldesigned core-shell QDs most often effectively prevent heavy metals from leaching and from contaminating the biological sample [12]–[15]. Some recent in-vivo studies have also shown the absence of a long-term toxicity [16]–[20]. Nevertheless, an application of QDs in-vivo in humans is quite improbable to be realized within the near future.

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Analysis of biological interactions is most often based on targeting the biomolecule of interest by a second (or several other) biomolecule that is (are) capable of recognizing the target. To ensure a luminescent signal transducing the biological recognition, fluorescent probes must be attached to the recognition biomolecule, which is usually termed “bioconjugation”. QDs have been successfully conjugated to many biomolecules (e.g., proteins, peptides, nucleic acids, aptamers) and biologically-active small molecules (e.g., drugs) [11], [21], [22] [23]. In order to target cellular events, such as signaling cascades, QDs were applied to sense ligand-receptor interactions [24], [25]. Sensing of antigen-antibody interactions was extensively explored in solution [26]–[30]. Drug delivery studies could adapt FRET technology to monitor an intracellular drug release from a binding site on QDbioconjugates [31]. The most often used QD-bioconjugates are QD-DNA or QD-RNA systems used in different hybridization studies [32]–[35]. Another example are peptides, which were designed to facilitate cellular uptake (so-called cell-penetrating peptides) or targeting of QDs [36], or to enable FRET-based sensing of enzymes at the QD surface [24], [37]–[39]. Different QD-bioconjugates are schematically shown in Figure 1A. In order to obtain the most suitable system for a given application, some experimental conditions should be taken into account [40]. Arguably, the six most important criteria for functional QD bioconjugates (Figure 1B) are [22] (i) valence (ratio of a biomolecules per QD), (ii) orientation of biomolecules on the QD, (iii) distance between biomolecules and QD, (iv) affinity between biomolecules and QD, (v) organization of biomolecules around the QD, and (vi) controlled chemistry for all QD-bioconjugates. First, the ratio of biomolecules per QD can vary from below one to a few dozen. This valence depends mainly on the sizes of both the biomolecule and the QD and, if possible, it is optimized toward the detection technique (e.g., single molecule detection, wide-field imaging, immunoassay). Second, the orientation of the

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biomolecules on the QD surface needs to allow access to the recognition site specific for the target molecule (e.g., binding site for antibodies). The third point (distance) is highly important for FRET, which is a distance dependent energy transfer, as will be described in section 3. Thus, the QD-biomolecule distance should be controlled as precisely as possible. Fourth, the affinity between biomolecules and QD should be as strong as possible to guarantee the stability of the assembly. However, in some cases the biosensing application may require electrostatic binding, which can be too weak or condition-dependent (e.g., pH, ionic strength) to reliably control the valence. Fifth, the biomolecules should be homogeneously distributed over the QD surface to prevent problems of steric hindrance and non-specific binding. Sixth, all biomolecules should be connected to QD by using controlled bioconjugate chemistry. Uncontrolled or different chemistries can lead to non-reproducible and different distances, which will strongly influence FRET and therefore the biosensing performance. Respecting these six criteria is a good route to optimal analytical conditions, expressed through significant and reproducible FRET signals. To these six cardinal points, one can add the size of the biomolecule, the size of the QD, and the availability of binding sites on the biomolecules (among many other criteria) that can influence the six criteria mentioned above and modify the biosensing performance.

Figure 1. (A) Schematic representation of possible QD-bioconjugates with DNA, RNA, peptides, or proteins (including fluorescent proteins, antibodies etc.). (B) Illustration of six important criteria for QDbioconjugates. Adapted and reprinted with permission from [22]. Copyright 2006 Macmillan Publishers Ltd.: Nature Materials.

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Naturally, all bioconjugate properties are determined, in part, by the chemistry used for conjugation. Here, we briefly describe some techniques commonly used for QD bioconjugation. These techniques can be classified by the strength and specificity of the interaction. The least specific and least stable is electrostatic interaction. A charged QD surface can be directly connected with any oppositely charged biomolecule. For example, the highly negatively charged phosphate backbone of DNA enables its attachment to QDs with positively charged surface coatings [41], [42]. There are also several examples of QD–protein conjugates and protein coronas in biological media are often formed by electrostatic interactions between QDs and proteins. The same strategy can be used to attach proteins (e.g., maltose binding protein, antibodies) to a negatively charged QD surface functionalized with DHLA (dihydrolipoic acid) ligands [43]–[46]. This approach is very simple to perform and bioconjugation is instantaneous and results in high biomolecular loading. Nevertheless, there is very little control over the conjugation ratio and protein orientation. Also, binding can be disrupted when working in physiological media where high ionic strength screens electrostatic interactions. The second most stable conjugation is self-assembly through the affinity of thiols, polyhistidines, or phosphorothioates for the inorganic shell material of the QD. Frequently, cysteine residues on proteins expose thiol functions, which can coordinate with Zn from the QD shell, a strategy that was also used for FRET biosensing [25], [28], [38]. For the development of QD-based immunoassays, free thiol groups can be obtained by reducing disulfide bonds in cysteines, which connect two paired domains of antibodies (e.g., in the antibody hinge region). Another popular self-assembly chemistry is based on the metalaffinity of polyhistidines (Hisn). The imidazole side chains on the Hisn residues can coordinate to the Zn-rich QD surface. His-tags are relatively easy to express through recombinant proteins or synthetic peptides and their relatively small size usually does not

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significantly influence protein functionality. Regarding FRET applications, photonic wires or enzyme activity sensors were produced by His6-QD affinity [47], [48]. A major drawback of self-assembly interactions is their instability at high dilutions (i.e. low concentrations). For example, thiolated biologicals can desorb and also reform disulfide bonds between each other. Also, cysteines need to be reduced to expose thiols for QD attachment, which narrows the experimental pH range. Nevertheless, bioconjugation protocols are relatively facile to implement and conjugation times are rather short. Third interaction in term of affinity strength is biotin-streptavidin binding, which is one of the strongest non-covalent interactions known in biology. It is also one of the most frequently used bioconjugation strategies and many fluorophores (including QDs) are commercially available with biotin or streptavidin functions. Biotinylated biomolecules can be simply attached to QD surfaces containing one or multiple streptavidins. One drawback may be the organization of streptavidin on the QD surface in terms of accessibility of biotin binding sites. Also, the strength of conjugation will depend on the attachment procedure of streptavidin to the QD surface (e.g., covalent, electrostatic etc. – vide supra). Nevertheless, because of ease of use, such bioconjugates have been applied for energy transfer between QDs and gold nanoparticles or for nucleic acid biosensing [32], [34], [49] to name only a few of the many applications. Very high affinity can be obtained through covalent bonds between QD surface ligands and biomolecules. Again, the conjug

ation strength also depends on the strength

of ligand surface attachment (e.g., monothiol ligands have lower affinity than multithiol ligands such as DHLA). QDs can be coated with ligands exposing various functions, such as carboxylic acids, amines, thiols, or azides. For example, carboxy-functionalized QDs can be attached to the N-terminal amines of peptides by creating a covalent amide bond [48].

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3. FRET Theory and Formalism 3.1. General FRET Theory

FRET can be defined as an energy transfer process between two molecules, one of which is an energy donor and the other an energy acceptor. The energy transfer does not occur due to donor emission and acceptor absorption but by charge-charge interaction between oscillating donor and acceptor dipoles in close proximity (ca. 1 to 10 nm) [50]–[54]. In addition to the proximity condition, the transition dipole moments of donor and acceptor must have favorable orientation with respect to each other. The orientation factor between two dipole moments is known as κ2. For many biosensing applications that measure an ensemble of many FRET pairs (in contrast to single molecule FRET, which measures single FRET pairs), dynamic averaging of donor and acceptor dipole orientations gives rise to an orientation κ2 =2/3. Fast isotropic rotation can be verified through unpolarized emission of QDs, which is often the case and makes estimation of κ2 less complicated than for dyes, which may take on certain orientations and allow for κ2 values between 0 and 4 [50]. A third condition that needs to be fulfilled for FRET is energetic resonance between donor emission and acceptor absorption, as expressed by the spectral overlap integral: = where

(1)

is the extinction coefficient of acceptor and the PL intensity of the donor needs to be

normalized to unity over the entire wavelength integration range ( ). Distance (R), orientation (κ2), and energetic resonance (J) in combination with PL parameters of the donor (PL quantum yield

and PL lifetime τD), the refractive index of the surrounding medium

(n), and Avogadro’s number (NAV) define the FRET rate:

9

)

=

!"# $% &

(2)

'

At 50 % FRET efficiency, the FRET rate equals the sum of the rates of radiative and nonradiative decay, which corresponds to the inverse PL lifetime (kFRET =

+ ! = τD-1). The

donor-acceptor distance at 50 % FRET efficiency is defined as the so-called Förster distance (R0). Replacing R by R0 and kFRET by τD-1 in equation 2, leads to the equation for R0:

)

( =)

!"#

%$*

/,

(3)

The FRET efficiency is defined by rate constants or distances:

-

=

./012

./012 3. 0 3. 40

=

./012

./012 3&56

=

3

0 ' ) 07

=

' 7

' ' 73

(4)

EFRET can also be defined by PL intensities, lifetimes, or quantum yields of the donor in the presence (subscript DA) or in the absence (subscript D) of the acceptor:

-

=1−

: " :

=1−

& " &

=1−

"

(5)

Equations 3 to 5 can be used to calculate donor-acceptor distances or FRET efficiencies for a give FRET pair. This data, in turn, can be used for extracting conformational information from a spectroscopic measurement or to evaluate if the FRET pair can be used for efficient biosensing. Although EFRET is usually calculated by measuring the donor PL in the absence (Figure 2A) and in the presence (Figure 2B) of the acceptor, it is also important to measure the PL of

10

the acceptor in the presence of the donor upon excitation of the donor (Figure 2C). Sensitization of the acceptor upon donor excitation provides good evidence that donor quenching was caused by FRET to the acceptor and not due to other quenching mechanisms.

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Figure 2. (A) Observation of direct emission of the donor in the absence of the acceptor. (B) Observation of the donor quenching in the presence of the acceptor as a result of FRET. (C) Observation of the acceptor sensitization in the presence of the donor as a result of FRET.

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Biosensing experiments often aim at quantifying concentrations of FRET partners (in their associated and dissociated form) at fixed donor-acceptor distances (R = constant). Such measurements often use a FRET ratio (not the FRET efficiency) to quantify FRET:

;(-< =>?@A = where

and

∗ :" B:"

(6)

: "

are the respective acceptor and donor PL intensities of the FRET pair upon

excitation of the donor.

∗

is the acceptor background PL (acceptor PL intensity in the

absence of donor upon excitation at the donor excitation wavelength). The population of interacting biomolecules will be detected through acceptor sensitization ( quenching (

) over donor

). Such a ratiometric measurement is very precise (low coefficients of

variation) because both numerator and denominator are equally influenced by perturbations or fluctuations during the measurement or from one measurement to another. Often, biosensing systems that use organic dyes or fluorescent proteins are exposed to a risk of spectral crosstalk (or bleedthrough), which can occur when performing multichannel detection (Figure 3A). Because of their broad spectra, the acceptor may be directly excited when its absorption spectrum overlaps with the donor's excitation wavelength range (selected by bandpass filters). Such spectral crosstalk requires background subtraction (

∗

in equation

6) and therefore an additional measurement (acceptor alone with donor excitation wavelength). When QDs are used, there is often no need of such background subtraction because their broad absorption spectra allow for selection of an excitation wavelength that does not co-excite the FRET acceptor (Figure 3B). Thus, only two instead of three measurements are required and these can be performed on the same sample (donor and acceptor detection channel when FRET is present). In addition to spectral crosstalk for excitation, there can also be spectral crosstalk for emission. As shown in Figure 3, the emission filter of the dye Alexa Fluor 555 contains a significant contribution of Alexa Fluor

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488 PL intensity, which again requires background subtraction (PL of donor in the absence of the acceptor measured in the acceptor detection channel). The narrow PL emission spectra of QDs can also prevent or significantly decrease such emission spectral crosstalk (Figure 3B). In addition to less experimental efforts, reduced spectral crosstalk is also beneficial for spectral multiplexing because one can distinguish more different QDs than different dyes.

Figure 3. Example of spectral crosstalk. (A) Alexa Fluor 488 / Alexa Fluor 555 and (B) QD 525 / Alexa Fluor 555 donor/acceptor FRET pairs. Excitation (Ex) and emission (Em) peaks were selected using an exemplary filter sets: 365/12 and 525/15; 485/20 and 520/20; 550/15 and 572/15 nm for QD, Alexa 488, Alexa 555 excitation and emission, respectively. Dotted curves are absorption spectra and continuous curves emission spectra. Excitation spectral crosstalk (in red) is due to acceptor absorption in the donor excitation wavelength range (blue (A) or grey (B) area). Emission spectral crosstalk (in brown) occurs when the emission filter for one fluorophore overlaps with the PL spectra of both of them. E.g., Alexa Fluor 555 Em filter (yellow) detects the maximum of Alexa 555 PL intensity but has a significant contribution from Alexa Fluor 488 PL (green spectrum). Image adapted from AAT Bioquest Spectra Viewer.

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3.2. QDs as FRET Donors QDs are versatile materials that can function as both donors and acceptors in FRET. Application of QDs as FRET donors is relatively simple. The most important photophysical points to consider when choosing an appropriate acceptor are the spectral overlap of its absorption with the QD emission and its quantum yield for efficient detection of FRET sensitized acceptor PL. Naturally, chemical (e.g., stability), physical (e.g., distance), biological (e.g., environment), and biochemical (e.g., bioconjugation) properties also require some consideration when selecting the best acceptor. The main benefits of using QDs as FRET donors are: High extinction coefficients. QDs have high extinction coefficients, which are frequently larger than 106 M-1 cm-1 over a broad spectral range. Organic dyes usually reach extinction coefficients of only a few 105 M-1 cm-1 over a limited spectral range [55], [56]. These high extinction coefficients increase the brightness of the entire FRET system and thereby facilitate FRET detection at very low concentrations and down to the single molecule level. Broad excitation spectra. QDs are characterized by extremely broad absorption spectra starting at approximately the wavelength of maximum PL intensity and increasing steadily toward the UV. This broadband excitation allows for using almost any wavelength below the PL emission wavelength range and to excite multiple different QDs with the same excitation source.

Excitation wavelengths can be separated by hundreds of nanometers from the

emission wavelengths (large “effective” Stokes shift), which is important for an efficient separation of excitation and emission light. The flexible choice of excitation wavelength also circumvents the problem of direct acceptor excitation. Color tunability. Due to the quantum confinement effect, QD PL color can be continuously tuned by the size of the core material. The composition of the semiconductor material also

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defines the spectral range that is accessible by quantum confinement and thus any color between UV and NIR can be obtained by composition and size adaption. Accordingly, the spectral overlap between QD donor PL and acceptor absorption can be optimized and, in this way, the Förster distances can also be adjusted for specific sensing applications.

Narrow emission spectra. The narrow and symmetric emission bands of QDs can be very efficiently resolved. In combination with their size-tunability, QD colors can be adjusted to match emission filters (Figure 4).

Figure 4. Example of spectral selection of 5 different QDs emitting in the visible range. Bandpass filters (dotted lines) were selected to optimize detection of QDs having emission peaks at 451 (blue), 484 (cyan), 545 (green), 599 (orange) and 637 (red) nm. The small sidebands (blue, cyan, and green spectra) are caused by impurities (small fractions of differently sized QDs). All QDs were synthetized at the U.S. Naval Research Laboratory, Washington, DC.

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Spectral crosstalk close to zero. The narrow and symmetric QD donor emission spectra also minimize crosstalk with acceptor fluorescence spectra. The absence of a red tail, which is very characteristic of organic dyes, is another great asset. As a consequence, QD donor PL does not appear in the acceptor detection channel (compare Figures 3 and 4).

Multiplexing. The color-tunable broad absorption and narrow emission spectra are ideal for multicolor detection. This unique multiplexing capability allows for the simultaneous detection of several different QD donors (in combination with different or the same acceptors) from the same sample. Large surface-to-volume ratio. The surface areas of QDs represent several nm2 that can serve as biosensing interface. This general benefit of nanoparticles allows for the attachment of several biomolecules and/or several (n) FRET acceptors on the QD surface resulting in enhanced biological interaction and FRET efficiency (EFRET = nR06/(nR06+R6)). Modification with several and/or different biomolecules (e.g., proteins, peptides, nucleic acids) creates new opportunities in biosensing design, which are not accessible with small organic dyes as FRET donors. Long excited-state lifetime. QDs possess PL lifetimes in the tens to hundreds of ns, which is approximately 10 to 100 times longer compared to the most organic dyes (few ns). This difference can be used to distinguish acceptor emission from FRET sensitization via QDs (longer PL decay) and from direct excitation (shorter PL decay). In time-gated detection, the longer PL decays can be used to reduce the background PL (from directly excited acceptors and sample autofluorescence) and increase signal-to-noise ratios. No fluorophore is perfect and even QDs have disadvantages that often have the same origin than their advantages. The large sizes of QDs (compared to molecular fluorophores) can have a strong influence on the conjugated biomolecule or biomolecules. Thus, the QD17

bioconjugate can have significantly altered binding functions and kinetics compared to the biomolecule alone. Because QDs do not emit from the surface but rather from the core, too large of a QD can significantly reduce the useable distance range for FRET because acceptors and biomolecules can only be attached at the surface (outside) of the QD. The large surface area can also be disadvantageous because it provides a large space for non-specific interactions. In FRET experiments, the exact number of acceptors on the surface may be difficult to control and non-spherical shapes of QDs can lead to different donor-acceptor distances even if the same conjugation chemistry was used. These effects lead to a distribution of the overall FRET efficiency. QDs are also not ideal candidates for a single molecule application because they are prone to blinking (dark states for extended time periods), which also influences the overall quantum yield and FRET efficiency in an ensemble experiment. The photophysical properties of QDs can be significantly influenced by the environment (water, biomolecules, pH, temperature etc.) and optimization of QD surfaces concerning biocompatibility and colloidal stability is crucial for biosensing [15], [48], [57]. Although PL quenching is most often found for interaction with the environment, PL can also be enhanced by surface biomolecules [58], [59]. This increase may arise from better passivation of the QD surface and a concomitant reduction in surface trap states. In addition to the photophysical properties, precipitation in aqueous or aggregation in biological media can be another disadvantage of QDs. This can happen, for example, when striving to prepare water soluble QDs coated with silanol groups [60]. According to in-vitro and drug delivery studies, QDs can spontaneously enter into cells by endocytosis [61]. This cellular uptake can be further improved by so-called cell-penetrating peptides [14], [15], [37]. However, endocytosis requires QD design for efficient endosomal or lysosomal release, which is not a trivial endeavor and often results in mixed populations of trapped (in endosomes or lysosomes) and released (in the cytosol) QDs [12], [18], [62]. Direct introduction into the

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cytosol may be accomplished through electroporation or microinjection [31], [57], [63]; however, these techniques are considered to be rather invasive and can compromise cell integrity and viability [64]. QD-based FRET biosensing is mainly 21st century research. One of the earliest contributions (from 2001) investigated FRET from QDs to dye-labeled biomolecule [65]. In this study, the authors functionalized QD donor surfaces with biotinylated bovine serum albumin (BSA) and conjugated dye acceptors to streptavidin to observe both acceptor sensitization and donor quenching. The major problem of this early QD-FRET work was uncontrolled aggregation, which prevented precise control over the assemblies and FRET processes. In 2003, two independent studies demonstrated the first DNA- and protein-based QD FRET sensors, respectively [66], [67]. Dynamics of DNA replication were studied by FRET when a polymerase incorporated dye-labeled nucleotides into a nascent strand hybridized to the thiolated-DNA on the QD surface. The protein-based QD-FRET sensing was demonstrated using dye-labeled cyclodextrin binding to surface-immobilized labeled or unlabeled maltose-binding protein (MBP). The use of the MBP terminal His-tags to directly assemble the proteins on the QD surface enabled precise control over both their number and orientation. Other applications have quickly emerged, including multiplexed protein sensing or nanoantennas for photodynamic therapy (PDT) [59], [68], [69]. 3.3. QDs as FRET Acceptors Although most of the QD advantages and disadvantages discussed above also count for QD acceptors, the requirements for appropriate FRET donors to efficiently transfer energy to QD acceptors are more demanding. The aim of exciting only the donor by light excitation (chemically or biologically excited FRET, i.e., CRET or BRET, will not be discussed in this review) and avoiding direct excitation of the acceptor becomes almost impossible for QD acceptors because of their spectrally broad and strong absorption. Because the donor emission 19

needs to overlap with the acceptor absorption and the excitation of the donor is usually lower in wavelength (higher in energy) than its emission (except for upconversion materials), QD acceptors will always receive significant direct excitation. Due to their high extinction coefficients, in most cases this excitation will be more efficient than the one of the donor. Thus, most of QD acceptors will be in an excited state, which makes FRET almost impossible and at least very inefficient. Moreover, most fluorophores have a PL lifetime shorter than QDs, which makes the situation even more complicated because the directly excited QD acceptors will remain longer in their excited states than the donors. Long-lifetime donors (longer than the QD lifetime) and pulsed excitation are able to overcome this problem because even if both donor and QD acceptor get excited by the light pulse, the QD acceptors will decay faster and there will be a sufficient amount of excited donors and ground state acceptors after an appropriate delay following the excitation pulse. This long-lifetime donor FRET concept was proposed in 2005, in a study that showed the inexistence of FRET in both steady-state and time-resolved spectroscopy configurations when using organic dyes as FRET donors and QDs as FRET acceptors [70]. Using a long-lived ruthenium-complex as donor, the authors could observe its lifetime quenching. However, they could not observe any QD sensitization and thus the intuitive suggestion was the use of even longer lifetime donors, which was demonstrated to be successful in the same year by using terbium chelates with millisecond excited-state lifetimes as donors [71]. Since then, many studies have used lanthanide (Ln)-based FRET donors for QD FRET acceptors in biosensing [25], [72], [73]. This unique FRET combination provides several interesting advantages for FRET: Extended PL decay time. Due to the large difference in Ln (~ms) and QD (~ns) excited-state lifetimes, FRET-sensitized QD acceptors decay with the same lifetime than the FRETquenched Ln donors because excitation is received from the long-lived excited states of Ln 20

and the additional and comparatively short intrinsic PL lifetime of the QD can be neglected. Therefore, both Ln donor and QD acceptor can be detected by time-gated PL measurements (Figure 5), which consists of sample excitation with a pulsed light source and PL detection in a temporal window that can be adjusted and optimized to the specific PL decay properties of the FRET pair.

Figure 5. Principle of time-gated FRET detection. PL intensity is plotted as a function of time (in logarithmic scales). FRET-quenching of a long-lived Ln donor (green line) results in a reduced decay time (green dots) and FRET-sensitization of the QD acceptor results in an increased decay time (red dots) compared to emission from direct QD excitation (red area). A time-delay of 10µs allows for suppressing most of the sample autofluorescence (blue area). Adapted with permission from reference [93]. Copyright 2016 Trends in Analytical Chemistry.

Autofluorescence-free PL intensity signal. Time-gated detection allows for selection of a time-delay and, consequently, the detector can collect only “useful” photons arriving from either the quenched donor or the sensitized acceptor but not from non-specific short-lived

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fluorescence, such as sample autofluorescence. For example, a delay of 10 µs can efficiently suppress almost all autofluorescence background. No PL from directly excited acceptors. Another advantage of time-gated detection is that the emission from directly excited QDs, although very strong in intensity, is short-lived and will therefore not be present in the delayed detection window. This is advantageous because IA* (equation 6) becomes zero and no acceptor fluorescence correction is necessary. Moreover, the signal-to-background ratio will be higher. Acceptor multiplexing. If QDs are used as acceptors, their FRET-sensitized PL (instead of their FRET-quenched PL in case of QD donors) can be used for multiplexing. Due to their broad absorption spectra, different QDs can overlap with the same Ln donor and thus a single donor (and single excitation source) can be used to FRET-sensitize different QD acceptors. More details can be found in the application section 4. Extremely large Förster distances. The broad overlap of Ln emission with QD absorption combined with the extremely high extinction coefficients of QDs, allows for very large Förster distances of R0 > 10 nm. Such large R0 values can significantly extend the FRET range to distances well beyond the typical 10 nm maximum distance. The application of QD-acceptors for time-gated FRET biosensing has been demonstrated for both spectroscopy and imaging [24], [26], [29], [74], [75], and spectral time-gated FRET multiplexing was realized for up to five different QD colors [76].

4. QD-FRET Biosensing The first applications of QD-FRET were demonstrated by Kagan et al. on QD-QD FRET and date from 1996 [77], [78]. They could observe acceptor sensitization of large QD

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acceptors as well as quenching of small QD donors in a mixed close-packed CdSe solid and showed that the FRET mechanism (dipole-dipole interaction) can be applied to energy transfer between QDs. A few years later, Willard et al. used biotin-streptavidin as biological binding model between QD donors and dye acceptors (vide supra) [65]. In these sections, we will describe representative examples, including FRET-based molecular or spectroscopic rulers and FRET bioassays, multiplexed and multistep FRET, and FRET imaging, which highlight the very versatile use of QDs in FRET biosensing and imaging over the last 20 years. Several recent reviews have shown applicability and significant advantages of FRET for different bioanalytical communities [79]–[81]. 4.1. Molecular rulers Because FRET is a near-field interaction between donor and acceptor, there is no limitation due to light diffraction and FRET can be used to measure biomolecular interactions in a sub-nanometer to few nanometer distance range. Determination of R0 (equation 3) and EFRET (equation 5) by spectroscopic measurements can be used to calculate the distance R between donor and acceptor (equation 4). Stryer and Haugland first applied FRET to measure short distances between molecules and coined this technique a “spectroscopic ruler” (“molecular ruler” is also used) [82]. This first application consisted of quantifying distances between a α-naphthyl (donor)-dansyl (acceptor) FRET pair using a well-defined peptide (poly-L-proline) spacer. Due to the outstanding optical properties of QDs, the possibility of coupling several acceptors to one QD, and the use of QDs as acceptors with lanthanide donors, the measurable QD-based FRET distance range is longer than for conventional (e.g., dye-dye) FRET pairs, and can in exceptional cases extend up to ca. 20 nm [79]. In addition to QD-QD FRET, QDs have been applied as energy transfer donors for gold NPs (in this case the nanometal surface energy transfer or NSET shows a R-4 distance

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dependence), dyes, or fluorescent proteins [63], [83], [84]. By studying structural changes of Cy3-conjugated polyproline attached to the surface of a QD, Boeneman-Gemmil et al. could confirm Stryer’s FRET spectroscopic ruler application also for QD donors (Figure 6) [83]. Due to the nontrivial size of the QD (FRET is calculated from the QD center but the molecules are attached to the QD surface, usually via linkers that add further separation) the longest measurable donor-acceptor distance was estimated to ca. 10 nm. Combining QD acceptors with lanthanide-based donors allows for FRET over longer distances. The exceptional overlap of lanthanide PL emission and QD absorption spectra and the millisecond-long PL lifetimes of the lanthanide donors can be used to measure donor-acceptor distances of up to ca. 19 nm [85]. However, also in this case, the QD radius must be subtracted from the center-to-center donor-acceptor distance. Hildebrandt et al.

have

demonstrated the application of multiplexed Ln-to-QD FRET as molecular ruler using terbium complexes in combination with commercial QDs and biotin-streptavidin or polyhistidine-mediated Tb-QD donor-acceptor assembly [74], [85], [86]. These studies revealed different morphological characteristics of QDs, such as shape, size, and bioconjugation ratio, under physiological conditions and within a single FRET experiment. These molecular ruler results were correlated to those of dynamic light scattering (DLS) or transmission electron microscopy (TEM). Moreover, FRET is well complementary to these standard techniques because it can provide effective sizes of the particle without hydration layers (as in DLS) or the missing contrast of organic coatings (as in TEM).

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Figure 6. Illustration of a QD-dye FRET molecular ruler investigating polyproline conformation on a QD surface by a 530 nm emitting QD donor and a Cy3 dye acceptor. The Cy3-peptide is assembled on the QD surface using polyhistidine metal-affinity coordination. (A) helical conformation and (B) helical conformation with a cis bond of polyproline. Figure reproduced from American Chemical Society [83], copyright 2015.

4.2. FRET Assays 4.2.1. Immunoassays FRET-based immunoassays between a donor-labeled antibody and an acceptor-labeled antibody, both binding to the same target (antigen), are important diagnostic tools because they are homogeneous assays. In contrast to heterogeneous assays, in which the antibodies are immobilized to a solid support and require several incubation and separation steps, homogeneous assays are mix-and-measure tests that do not require any washing steps. This simplicity results from the FRET signal that is only present if both antibodies (donor and acceptor) bind to the same antigen and distinct from the fluorescence signals of the single (unbound) antibodies. Moreover, the use of two different primary antibodies to bind the target, increases the specificity compared to an immunoassay based on a single target-specific antibody [87]. Although QD-FRET has been used in various diagnostic applications [88], FRET immunoassays are rather complicated when QDs are used as FRET donors. The reason is the large size of the donor-antibody-antigen-antibody-acceptor assembly. A full IgG antibody is already quite large (ca. 150 kDa molecular weight and 14.5 nm × 8.5 nm × 4 nm dimension)

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[89] and attachment to a QD of several nm in diameter, binding to the antigen (also a few nanometers in size, exact size depends on the antigen to be detected) and binding to the second acceptor antibody can easily lead to donor-acceptor distances beyond 10 nm and therefore very inefficient FRET or no FRET at all.

In 2005 Goldman et al. attached

polyhistidine-appended antibody fragments against 2,4,6-trinitrotoluene (TNT) on QDs and addition of a TNT analogue (TNB) labeled with a black hole quencher (BHQ) resulted in antibody-TNB recognition and quenching of the QD PL by FRET to the BHQ. TNT efficiently competed with TNB for the antibody binding sites and an increasing concentration of TNT led to an increasing QD PL intensity, which was used for TNT quantification [90]. In 2006, Nikiforov and Beechem used a slightly different strategy by attaching the antigen to the QD surface. This cortisol antigen was recognized by an Alexa Fluor dye (AF647)-conjugated anti-cortisol antibody, which resulted in FRET from QD to AF647. Addition of free cortisol led to a competition with QD-cortisol for the AF647-antibody and a concomitant decrease of AF647 PL (at 670 nm) and increase of QD PL (at 605 nm). The ratio of PL intensities at 605 and 670 nm was used for cortisol quantification [91]. The limitations associated with the large QD-based FRET immunoassay complexes can be overcome by using QDs as acceptors and lanthanide complexes as donors. This approach allowed for a longer FRET range and time-gated detection for efficient autofluorescence suppression (vide supra), and thus for using actual donor-antibody-antigenantibody-acceptor sandwich immunoassays [25], [92], [93]. These time-gated Ln-to-QD immunoassays presented an advancement of well-established commercial technologies that use Ln-donors and dye-based acceptors (e.g., PerkinElmer: LANCE Technology, CisBio: HTRF Technology, ThermoFisher: TRACE Technology) and could therefore be performed on commercial time-resolved fluorescence plate reader systems (e.g., Thermo Fisher Scientific, PerkinElmer, Edinburgh Instruments, Tecan, BMG Labtech, BioTek, SAFAS,

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Berthold Technologies, Hidex, Molecular Devices). In addition to the demonstration of enhanced multiplexing capability compared to dyes [76], Tb-to-QD FRET immunoassays have been developed for duplexed detection of epidermal growth factor receptors [30], highly sensitive detection of total prostate specific antigen in serum samples [26], [28], [94], [95], and with various types of antigen binders, including reduced antibodies [26], [28], [94], [95], nanobodies [30][96], and engineered scaffold affinity proteins [97].

4.2.2. Enzyme Assays Enzyme assays are among the most popular QD-based FRET assays and several enzyme assays have been developed to detect the proteolytic activity of different model proteases [98]–[105], nucleases [99], [106], [107] as well as kinases [105], [108]–[111] and phosphatases [110][112]. Proteases catalyze the hydrolysis of chemical bonds between amino acids. Depending on their specificity, these enzymes can precisely target a specific sequence of amino acids or in an unrestricted manner cleave any α-peptide bond [113]. Proteases play a critical role in maintaining cellular homeostasis via the regulation of activity of various proteins (other enzymes, antibodies, and hormones). As proteases are very widespread throughout the human genome they are more frequently exposed to genetic mutations [114]. Thus, the deregulation of their activity contributes to a variety of pathological conditions, including cancer, cardiovascular, neurodegenerative, or inflammatory diseases [115]–[118]. The easiest way to monitor protease activity by FRET is to use donor and acceptor linked to the termini of a peptide. This peptide must contain a specific sequence of amino acids that will be cleaved by the protease of interest, thereby separating donor and acceptor. Thus, the decreasing FRET signal will be a function of enzyme activity. Many QD-FRET assays have 27

been used to measure enzyme activity of different types of proteases (e.g., caspase [100], [102]–[104], collagenase [100], [101], [104], trypsin and chymotrypsin [100], [104], [105], thrombin [100], [103], [104], and metalloproteinase [101], [103]). Most of these enzyme sensors consist of a QD donor conjugated with a short-sequence peptide carrying the acceptor (Figure 7A). In order to increase the FRET efficiency, several acceptor peptides can be conjugated to the same QD [99]. In this configuration, proteolytic activity can then be monitored without any washing or separation steps by simple PL intensity detection. Similar to immunoassays, the acceptor can be fluorescent or a fluorescence quencher, which determines the mode of detection (acceptor sensitization or donor quenching). Medintz et al. developed such QD-FRET sensing approaches to target caspase-I, thrombin, collagenase, and chymotrypsin by tuning the modular peptide substrates [100]. These peptides were designed to have four distinct functionalities: (i) self-assembly on the QD surface via a Nterminal His6 domain, (ii) cysteine dye-attachment domain at the C-terminus, (iii) donoracceptor rigid helix spacer region, and (iv) protease cleavage site. To measure enzymes activities, FRET pairs were composed of Cy3 acceptors and DHLA-coated QD538 donor in case of caspase-I and of a fluorescence quencher (QXL-520) as acceptor and the same donor in case of three other proteases (thrombin, collagenase, and chymotrypsin). Proteolysis was readily detectable by cleavage of a single peptide, which resulted in significant differences in FRET efficiencies. To standardize the study, quantitative data for proteolytic activity were determined by the analysis of QD PL recovery kinetics as a function of protease concentration (Michaelis constant (KM), the maximum proteolytic velocity (Vmax), turnover number (kcat), and kcat/KM ratio). These studies demonstrated that QD-based FRET enzyme assays can be applied to a panel of various enzymes with lower substrate concentrations ([QD]=200nM and [peptide]=0.2–1.0µM) compared to conventional assays. Moreover, substrates exposed on the

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QD surface were easily accessible to proteases and provided high sensitivities to the enzyme assays. Targeting other types of enzymes often requires more complex strategies. Kinase and phosphatase activities are mainly measured via indirect approaches. These enzymes are involved in nearly all signaling pathways, which means that they have the power to activate or deactivate any cellular process from proliferation to cell death. For this reason, different works were mainly focused on developing therapeutic kinase inhibitors [119]. Basically, the presence or absence of a phosphoryl group on a serine, threonine, or tyrosine residue side chain is subject to sensing. The phosphorylated side chain can be recognized using specific antibodies. Acceptor-dye labeled antibodies can bind to a modified substrate displayed on the QD to indirectly determine their activity. Usually, this substrate is a short peptide carrying the sequence to be phosphorylated by a specific kinase. Ghadiali et al. investigated QD-based kinase activity FRET assays for the development of kinase-mediated phosphorylation of pharmaceutical inhibitors [108]. Their strategy was based on recognition of short peptides attached to QD donors by anti-phosphotyrosine antibodies conjugated with a dye acceptor (Figure 7B). QD-peptide conjugates were incubated with tyrosine kinases in excess of ATP and dye(Alexa647)-phosphotyrosine antibody. Concomitant QD quenching and dye sensitization was observed using steady-state and confirmed by time-resolved spectroscopy. With a similar idea of developing therapeutic kinase inhibitors, different groups investigated QD-based FRET enzyme activity configurations. For example, Freeman et al. sensed the activity of casein kinase (CK2) [110], whereas Yildiz et al. monitored the activity of phosphoinositide-dependent protein kinase-1 (PDK1) [111].

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Figure 7. Schematic representation of QD-based FRET enzyme assays. (A) Sensing of protease activity via peptide linker cleavage, which interrupts FRET from a QD to an adequate acceptor (A). Figure adapted from Ivyspring International Publisher ref. [104], copyright 2012. (B) Sensing of kinasemediated phosphorylation of peptide-QD conjugates, antibody recognition of phosphopeptide, and FRET detection. Figure adapted from American Chemical Society [108], copyright 2010.

QD-based FRET sensing of enzyme activity was also extended to other enzymes, such as telomerase and polymerase [66] or histone acetyltransferase (HAT) [120]. The unique photophysical properties of QDs have also been exploited to simultaneously monitor the activity of several enzymes using multiplexed QD-to-dye FRET or NSET from QDs to gold nanoparticles (e.g., for protease and kinase [121] and trypsin and chymotrypsin [122]) or multistep Ln-to-QD-to-dye FRET relays (e.g., for trypsin and chymotrypsin [123], [124]).

4.2.3. (Nucleic Acid) Hybridization Assays

In addition to proteins, nucleic acids are frequently used targets and recognition molecules for QD-based FRET biosensing. Nucleic acids are very versatile biological tools because they are easily manageable in terms of their length or secondary structure. FRET30

biosensing capabilities are most often exploited through simple hybridization of two complementary DNA strand. However, other DNA/RNA properties, such as polymerization [66], [99], [125], [126], ligation [127], [128], and cleavage [99], [106] were also used to develop and/or optimize assays. The simplest configuration consists of donor and acceptor labeled oligonucleotides with complementary sequences. FRET is turned on when donor and acceptor are brought in close proximity by base pairing between those two oligonucleotides. In 2005, two studies demonstrated that this well-known concept can be implemented by FRET between QD donors and dye acceptors [106], [129]. Since then, many more or less complex configurations of QD-FRET nucleic acid biosensors have been designed and optimized toward enhanced sensitivity and specificity. A dual hybridization system was developed by Algar et al. in order to explore the potential of QD-FRET in diagnostics of genetic disorders (spinal muscular atrophy) or applied to pathogen detection (E. coli) [130]. This strategy was taking advantage of the multiplexing capabilities of QDs to study two DNA targets from the same sample using two-color detection. The FRET pairs were composed of two different DNA-probes with QD donors and two different DNA-targets with different dye acceptors (Figure 8A). The major improvement was single-color excitation of the different QDs (due to their broad absorption spectra), whereas most multiplexing diagnostics require multiple excitation sources.

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Figure 8. (A) Double-FRET pair hybridization assay for simultaneous detection of two different target oligonucleotides. Figure extracted from ref [130]. Copyright 2007 Elsevier. (B) Tb-to-QD FRET sensor design for the specific quantification of two micro-RNAs. QDs conjugated with two target-specific DNA adapters using hexahistidine (His6) or streptavidin-biotin (sAv-biot) are mixed with Tb-conjugated reporter DNAs (I). Addition or two micro-RNAs to the solution (II) leads to the formation of stable double-stranded configurations that allow for Tb-to-QD FRET (III). Figure extracted from ref [34]. Copyright 2017 John Wiley and Sons.

Hybridization systems became more and more sophisticated and PL lifetime was also shown to be a powerful tool to extend optical multiplexing in the temporal dimension. Recently, Qiu et al. could demonstrate that DNA-based distance tuning between donor and acceptor could be used for temporally multiplexed FRET (Figure 8B) [34]. Two different 32

sensing approaches for duplexed detection of microRNA and DNA were designed by using Tb-dye or Tb-QD FRET pairs. Controlled tuning of the distance between donor and acceptor (from 10 to 25 nucleotides) resulted in modification of the PL decay curves and time-gated detection in different temporal windows was used for multiplexed target quantification. This strategy can be very useful to simultaneously differentiate multiple nucleic acid biomarkers at low nM concentration. Recently, a similar strategy was applied to understand the conformational organization of single and double-stranded DNA and to possibly extend Tbto-QD FRET to higher order temporal multiplexing [131]. As an alternative to hybridization of two separate DNA strands, intrastrand base pairing can lead to “loop patterns” also known as hairpins. Such 3D hairpin motifs are frequently used in so-called molecular beacon assays [132]–[134]. Donor and acceptor are conjugated to the both ends of the DNA strand, which leads to very efficient FRET in the case of a closed hairpin configuration. A complementary target DNA can open the hairpin, which increases the distance between donor and acceptor, leading to a decreased FRET efficiency. Usually, molecular beacon probes contain a fluorescent donor and a quenching (non-luminescent) acceptor, such as Iowa Black (IaB) [135], [136], DABCYL [137], or gold nanoparticles [135], [138]. An example of QD-based molecular beacons is shown in Figure 9. The designed hairpin structure was conjugated with a biotin and an acceptor dye (6-carboxy-X-rhodamine ROX) on its 3’ and 5’ termini, respectively [139]. Streptavidin coated QDs bound to the biotin end, which resulted in FRET-quenching of the QD and FRET-sensitization of ROX. Addition of complementary target DNA led to a strong recovery of QD PL and decrease of ROX PL, which was used for target quantification. This study also demonstrated two-photon excitation FRET, because a wavelength of 800 nm was used to excite the QD donors. Such NIR excitation is very beneficial for reduced background fluorescence, usually originating from direct excitation of acceptors or from sample autofluorescence.

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Figure 9. Schematic illustration of a two-photon excited molecular beacon (MB) with QDs as FRET donors and 6-carboxy-X-rhodamine (ROX) as FRET acceptors assembled by biotin-streptavidin (SA) interaction. Recognition of target DNA with MB results in an open MB configuration and FRET inhibition. Reproduced with permission from Figure extracted from [139]. Copyright 2011 Royal Society of Chemistry.

4.3. Multistep FRET or Higher Order Multiplexing QDs are not limited to single-step FRET but can play a dual role of acceptor and donor, in so-called FRET relays. In such configurations, QDs are acting as central assembly platforms of energy harvesting and transfer. Multistep FRET relays allow for monitoring the interaction of at least three FRET partners simultaneously, which can significantly improve the versatility of the detection system. Initially, QDs were used in multistep FRET cascades (photonic wires) of up to four sequential dye/fluorescent protein acceptors [140]. Thereafter, QD-based multistep FRET was extended to various configurations with luciferase as primary energy donor, [47], [141] or gold nanoparticles [142] or dye [122] as final energy acceptor. An original case of a multistep relay using lanthanide complexes as initial donors was developed by Algar et al. [124]. The main advantage of this configuration is the introduction of the PL lifetime as an important parameter to the multistep system. This system contained

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long-lifetime (lanthanides) and short-lifetime (QDs and dyes) components (Figure 10). When FRET was initiated, the long-lifetime was transferred from lanthanide to QD and then from QD to dye. For the simultaneous detection of two DNA markers from a single sample, Tbdonors and dye-acceptors were co-assembled onto the same QD surface. Two different ssDNA probes were conjugated using polyhistidine-binding to the QD surface (PRB A and PRB B). These probes hybridized with their complementary target strands (TGT A and B), which were conjugated with Alexa Fluor 647 dyes and Tb, respectively. The presence of both targets could be distinguished by the two different FRET configurations (long-lifetime Tb-toQD-to-dye and short-lifetime QD-to-dye). Thanks to time-gated detection and an appropriate time delay (here a few tens of µs), the entire multistep FRET could be registered (FRET1 from Tb-to-QD followed by FRET2 from QD-to-AF647). The same system was later successfully applied to enzyme activity tests [123] and the design of molecular logic devices [143], [144].

Figure 10. Tb-to-QD-to-dye FRET relay. (A) Two different single stranded PRB DNAs are coassembled on the QD surface allowing hybridization of two target (TGT) DNAs. Both of them can be detected by combining Tb-to-QD FRET1 and QD-to-dye FRET2. (B) Immediately after pulsed excitation the excited-states (*) of the FRET partners allow for FRET2 only (B1 and B2). After a 55 µs time-delay only Tb remains excited and sequential FRET1 + FRET2 becomes possible (B3 and B4). Figure adapted with permission from ref [124]. Copyright 2012 American Chemical Society.

4.4. FRET Imaging Similar to spectroscopy, QD-based FRET imaging can be performed in steady-state, time-resolved, and time-gated detection modes. Steady-state FRET imaging usually consists

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in choosing a FRET pair whose PL emission signals (donor and acceptor) can be resolved in two distinct detection channels without or with minimal spectral cross talk. This method does not require any special technical implementation and the fluorescence signal can be registered by standard CMOS or CCD cameras or, in case of low photon flux, EMCCD cameras. Timeresolved FRET imaging applies fluorescence lifetime imaging microscopy (FLIM) to investigate FRET by donor PL lifetime quenching [145][146]. Such experiments require more sophisticated imaging setups and most often photon counting detectors (e.g., photomultipliers, avalanche photo diodes) are used and the sample is scanned pixel per pixel instead of the full detection of an image by a camera. Time-gated imaging uses a long PL lifetime donor and a short PL lifetime acceptor. A pulsed light source excites the donor, which, in turn, can transfer its energy to a proximate acceptor. The FRET-sensitized acceptor then adapts the same PL lifetime as the FRET quenched donor and the FRET-sensitized acceptor PL intensity is measured in a specific temporal window (Figure 5). The time-delay after excitation efficiently suppresses direct short-lifetime emission from unpaired acceptors and sample autofluorescence background. Such time-gated FRET setups require time-resolved cameras (e.g., intensified CCD) or optical shutters that can gate both excitation and detection. In this section, we will discuss a few examples of in-vitro and in-vivo QD-FRET applications. 4.4.1. In vitro imaging The intracellular concentration of various ions can be considered as an indicator of a physiological state of the cell and numerous studies have investigated QD-based FRET sensing configurations for the detection of different ion species. The pH-sensitive fluorescent protein (FP) mOrange was used in a QD-FRET-based pH sensing study [147]. This FP, capable to detect pH~7, was used to analyze intracellular pH differences near the physiological pH range. Carbodiimide chemistry was used to conjugate mOrange to QDs and the nanoassemblies were delivered to HeLa cells using cell penetrating peptides. FRET

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efficiencies were found to be high in the neutral pH of extracellular environment and in early endosomes but decreased progressively when approaching the nucleus. This pH-dependent FRET behavior could be correlated to endosomal maturation states resulting in decreasing pH. In this study, QDs were used in order to enhance the photostability of the probe and to increase the imaging time up to ~30 min. Similar results were found when mCherry FPs expressed in the cytosol were brought to self-assembly with microinjected QDs [63]. Dyes are another common acceptor for QD-based FRET. This FRET pair was already used in drug delivery applications to address molecules at specific locations in the cell. Boeneman et al. designed a peptide that was conjugated via a histidine-tag to QDs, such that they were specifically binding to the outer leaflet of the plasma membrane lipids in PC12 Adh cells [14]. This specific sequence allowed the QDs to reside on the cell membrane for more than 48 hours. Co-staining with a rhodamine dye led to efficient FRET and thus rhodamine PL via QD excitation (Figure 11A). Winckler et al. also used QD-to-dye FRET to study cell adhesion [148]. In this study, a DiD probe (acceptor) was used to label the plasma membrane and QD FortOrange (donor) to stain the cell adhesion surface. In order to obtain a homogenous layer of QDs, they were spin-coated in a thin film of PMMA. Appearance of focal adhesion points were followed by FRET when the membrane extensions (filopodia) were the closest to the substrate (Figure 11B). Many studies also described quenching of QD PL in the presence of metal nanoparticles. This configuration was for example used for sensing of enzyme kinetics [149] or of mammalian cell types and states using nanoparticlebased sensor arrays [150]. Some studies were interested in fluorescence recovery of those nanoprobes as a result of biosensing. A QD-gold nanoparticle (AuNP) sensing probe dedicated to detect µM-range changes of intracellular fluoride anions was designed by Xue et al. [151]. Adherent RAW264.7 macrophages were incubated with the nanoprobe for 1 h at 37°C, which resulted in significant quenching of the QD PL due to the proximity of the AuNP

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acceptors. In contrast, supplementing cells with 50 µM fluoride before the addition of the QD-AuNP probe resulted in a marked increase of intracellular QD fluorescence (Figure 11C).

Figure 11. (A) PC12 Adh cells labeled sequentially with 550 nm emitting QD-peptide (JB858) conjugates and Rhodamine B phosphoethanolamine (Rhod-PE). The image shows Rhod-PE PL FRET-sensitized by the QD donor. Adapted from ref.[14]. Copyright 2013 American Chemical Society. (B) Live MCF7 cell in adhesion. The plasma membrane was labeled with DiD and cell surface adhesion with QDs in PMMA. Merged image of FRET channel (in red) and DIC (grey). The red spots represent adhesion points between the cell and the surface. Adapted from ref. [148] Copyright 2010 American Chemical Society. (C) Live RAW264.7 macrophages stained with a QD-AuNP nanoprobe and supplemented with 50 µM of fluoride anions. Merged image of donor channel (in green) and DIC (grey). Adapted from ref. [151]. Copyright 2012 Elsevier.

Imaging of QDs as FRET acceptors was realized by lanthanide acceptors and time-gated detection. A first study introducing lanthanide as FRET donors for QD acceptors, demonstrated the possibility of imaging microbeads [75]. Time-gated microscopy showed efficient FRET between terbium-labeled streptavidin and biotin-conjugated QDs. This concept of Tb-to-QD FRET imaging could also be transferred to the cellular context [24], [152], [153]. Faklaris et al. investigated the dimerization of plasma membrane receptors (dopamine, SNAP-D2) using tag technologies [152]. HEK 293 cell were transfected with SNAP-tagged D2 receptors. SNAP could be recognized by its specific benzylguanine (BG) ligand, which was conjugated with a Tb complex (Lumi4-Tb) as FRET donor and with biotin. This biotin-tag allowed for QD-streptavidin binding, which resulted in efficient FRET from Tb to QD. This work provided an important proof of feasibility of in-vitro Ln-to-QD FRET using microscopy detection. To further advance toward protein-protein interaction studies

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without the use of tagging technology, Afsari et al. examined the performance of Tb-to-QD FRET in extracellular and intracellular environment (Figure 12A) [24]. Extracellularly, two different domains of the epidermal growth factor receptor (EGFR) overexpressed on A431 cells were targeted by two specific antibodies conjugated with Tb and to the surface of QDs, respectively. Time-gated microscopy was used to identify the binding of both antibodies to EGFR via Tb-to-QD FRET (Figure12B). Intracellular sensing can be more complex, since QDs need to penetrate the cell membrane and in the ideal case enter the cytosol to enable sensing. Intracellular Tb-to-QD FRET and Tb-to-QD-to-dye FRET relays was realized by microinjection (Figure 12C), whereas cell penetrating peptide (CPP)-mediated endocytosis was used for Tb-to-QD FRET imaging in endosomes and lysosomes (Figure 12D). Although not yet experimentally demonstrated, lanthanide-to-QD time-gated FRET imaging has a large potential for tissue or small animal imaging, because it can efficiently suppress any autofluorescence background. Very recently, multidonor-multiacceptor FRET networks were designed to be used in optical barcoding [154]. Chen et al. reported that these multihybrid nanoparticles, made of central QDs and surface-bound Tb-donors and Cy-acceptors, were characterized by donor-independent and acceptor-dependent FRET efficiencies. This property enabled a straight forward tuning of the nanoparticle’s PL intensity and PL lifetime. As a consequence, brightness-equalized and lifetime distinguishable nanoparticles were developed with a strong potential to be applied in single measurement multiplexed imaging via optical barcoding.

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Figure 12. (A) Schematic presentation of FRET imaging approaches with extracellular FRET between Tb- and QD-functionalized antibodies that bind to different epitopes of EGFR on the cell membrane (image B), cytosolic FRET from Tb-to-QD (image C1) and FRET relays from Tb-to-QD-to-dye (image C2) using microinjection, and CPP-mediated endocytic FRET from Tb-to-QD (image D). Scale bars, 20 µm. Adapted from ref. [24] Copyright 2016 American Association for the Advancement of Science.

4.4.2. In vivo imaging Beyond applications for in-vitro sensing, QD-based FRET is quite promising for tissue or even in-vivo studies. Fluorescence (or fluorescence quenching) in-vivo imaging is very challenging due to high autofluorescence and low penetration depths (absorption) and scattering for tissues. Several groups have managed to partly overcome these problems by using QD-based FRET sensors. For example, Wang et al. have design nanoassemblies of QDs with PL quenchers bioconjugated by heparin-protamine self-assembly [155]. Injection into mice led to disassembly of the nanoassemblies by linker cleavage via endogenous proteases

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(legumain). Enhanced protease activity near the tumor led to enhanced QD PL signal (PL recovery due to reduced FRET). Another example of enzyme activity in vivo imaging was demonstrated by Moquin et al. [39] Real-time caspase-1 activity was measured following the induction of systemic inflammation by lipopolysaccharide (LPS). The caspase-1 nanosensor was composed of QD donors and rhodamine B acceptors, connected through a short peptide, cleavable by caspase-1, which is one of the most involved enzymes in inflammation (Figure 13A1). The immune response could be monitored when LPS was intraperitoneally co-injected with the nanosensors. In this configuration, the linkage between donor and acceptor was disrupted, which resulted in significantly increased QD donor PL, measured in live animals and dissected brains 2 h after LPS injection, when compared to animals treated with the nanosensor alone (Figure 13A2). Ingram et al. studied brain tissue behavior during onset of drug-induced seizure using FRET between red-emitting QD donors coupled with oxygensensitive luminescence quenching dye (platinum(II)-octaethylporphine ketone) acceptors (Figure 13B1) [156]. FRET complexes were embedded within a thin film (40 nm) of polymer (polyvinyl chloride with bis(2-ethylhexyl)sebacate). This optical sensor enabled visualization of O2 gradients across the brain slices. Simultaneous tissue perfusion and sensor excitation allowed to following in real-time extracellular changing of O2 concentrations in living tissues by FRET signal enhancement when oxygen was consumed (Figure 13B2, 20 s) or quenching when [O2] increased (Figure 13B2, 70 s).

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Figure 13. (A1) Principle of a caspase-1 nanosensor. Nanosensor is composed of QD donors and rhodamine B acceptors, connected through a short peptide, cleavable by caspase-1. In the absence of caspase-1, acceptor signal can be detected. In the presence of caspase-1 the acceptor molecules are detached from the QD, which in turn, will recover their PL. (A2) Fluorescence images of live mice and dissected brain following injection of the nanosensors alone or in the presence of LPS. (B1) Principle of O2 nanosensor. Nanosensor is composed of a QD-dye FRET pair. In the presence/absence of O2 dye is quenched/emitting. (B2) Optical sensor localizing changes in O2 dynamics across brain tissue. Adapted from ref. [39] (A) and [156] (B) Copyright 2013 American Chemical Society.

5. Conclusions and Perspective QD-based FRET has become a versatile and powerful tool for the analysis of many different biomolecules and biomolecular interactions. Many studies have used QDs as donors and/or acceptors in spectrally and temporally resolved multiplexing for both spectroscopy and imaging in-vitro and in-vivo. The physical and chemical properties of the nanoparticles, as well as their unique photophysics with broad absorption and narrow emission spectra that can be tuned by material composition and size, have significantly advanced optical biosensing beyond the possibilities of conventional fluorophores. Although stable and biocompatible

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QDs with high brightness and colors ranging from the blue to the NIR have been developed, the production at larger scales of biocompatible QDs is still an issue because of reduced quality in both physical and chemical properties compared to small scale production. QDs will most probably never replace other fluorophores, such as dyes or fluorescent proteins, because these have their own advantages. Over the last 20 years, QDs have become irreplaceable fluorophores in the fluorescence and FRET toolboxes. In particular for FRET, the combination of nanoparticle properties with bright and colorful fluorescence results in many benefits that cannot be provided by any other optical probe. QDs will continue to bring up completely new scientific ideas and applications in multiplexed and sensitive FRET biosensing, and will also be implemented as standard nanoparticle fluorophores to improve and advance existing research fields and technologies. 6. References [1] [2] [3] [4]

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Quantum Dots for Förster Resonance Energy Transfer (FRET) Marcelina Cardoso Dos Santos, W. Russ Algar, Igor L. Medintz, and Niko Hildebrandt

Highlights • Quantum dot bioconjugation strategies • FRET theory for quantum dot donors and acceptors • Biosensing using quantum dots and FRET • Quantum dot molecular/spectroscopic rulers • Quantum dot immunoassays, enzyme assays, and nucleic acid assays • Multistep FRET and higher order multiplexing • Imaging using quantum dots and FRET