Ariana A Arteaga-Castrejón

María R Trejo-Hernández

Yasmina Mekmouche

Agnes Amouric

Pierre Rousselot-Pailley

Viviane Robert

A Jalila Simaan

Thierry Tron
email: thierry.tron@univ-amu.fr

Fernando Martínez-Morales
email: fernandomm@uaem.mx

Relevance of surface exposed functionnalizable lysine in laccase for selective oxidation of substrates and future applications

Keywords:

Fungal laccases are oxidoreductases with wide applications due to their low substrate recognition specificity. Laccase LAC3 from Trametes sp C30 is our laccase model and since few years, its functionalization by external modules is studied to add new properties.

Surface-exposed Lys residues are usually chosen for functionalization by reductive amination. In this work, LAC3-K0, previously designed, was used as a platform to detect potential surface-exposed sites suitable to be replaced by a single lysine residue relevant for future functionalization studies. In a first approach, seven surface-exposed sites were selected on the LAC3-K0 3-D predicted model and the derivative single lysine mutants were obtained by site-directed mutagenesis (LAC3-UNIK 238 , etc). Moreover, additionally mutations were generated randomly from LAC3-K0 giving LAC3-UNIK N96D/V269K/A479T , that were all located on the laccase surface. All mutants were functionally expressed in Saccharomyces cerevisiae W303 as detected by oxidation of guaïacol or ABTS substrates.

The catalytic parameters of the variants showed that UNIK 238 , UNIK 269 and UNIK N96D/V269K/A479T have a high affinity for ABTS with a Km (mM) of 0.28 ± 0.006, 0.27 ± 0.0007 and 0.30 ± 0.004 respectively. The maximum speed Vmax (µM min -1 ) for these variants was 8.22 ± 0.291 for UNIK 269 and 11.98 ± 0.216 for UNIK N96D/V269K/A479T , respectively. All variants were stable at acidic pH and lose stability at neutral pH just like LAC3 y K0. The temperature stability was steady between 15 and 55 °C, at 70 °C most of the variants were almost completely inactivated. Furthermore, in the oxidation tests of phenolic compounds it was observed that all mutants were able to oxidize them, however, their activity varied greatly only by changing one or two residues on the surface of the laccase K0 regardless to the distance to the T1 site. Despite the differences observed in the catalytic parameters and in their ability to oxidize phenolic compounds, all the unique variants studied in this work seem to be appropriate sites for new functionalization of the laccase.

Introduction

Lacases (EC 1.10.3.2) are p-diphenol: oxidoreductases; they are multicopper blue enzymes characterized by the presence of four copper ions organized in 2 active centers. The type I copper site is involved in a cycle of the oxidation of phenolic substrates whereas the trinuclear copper center is involved in the concomitant O 2 reduction into water (Mate et al 2016; Stanzione et al 2019). Their low specificity enable these enzymes to catalyze the oxidation of a wide variety of phenolic compounds, aromatic amines and organic compounds. Laccases are considered as "green catalysts" due to their ability to use molecular O 2 as an electron acceptor generating water as the only by-product ( Since the past decades, laccases have received special attention thanks to their high electrocatalytic potential and environmentally friendly application. So their use has been extended in biotechnological processes including textile, cosmetic, food, pharmaceutical industry, biofuel industry, polymer synthesis and more recently in the nanobiotechnology industry [START_REF] Su | Laccase: a green catalyst for the biosynthesis of poly-phenols[END_REF]Pezzella et al 2015). This latter is emerging as a new frontier in the biotechnology field, where, by means of functionalization and immobilization techniques, enzymes can be grafted to diverse supports for the design of novel materials with enhanced characteristics and properties not yet discovered in nature or to mimetize nature´s abilities.

Additionally, these techniques are powerful tools to improve the properties of the enzyme since its application often faces production issues such as stability long term storage, operating, conditions at certain pH and temperature intervals, recovery and recycling of the catalyst.

Several research groups are developing immobilization processes via especially covalent bonding [START_REF] Mateo | Improvement of enzyme activity, stability and selectivity via immobilization techniques[END_REF]. As recently reviewed by Hoarau and colleagues that used chemically well-defined, self assembled monolayer (SAM) surfaces made of short ethylene glycol chains and cysteine-reactive maleimide groups that covalently attached the enzymes to the surface. This technique gives better control of the enzyme-support interaction, so which has been improved by the vectorial orientation of the enzyme leads also to an To achieve this, surface-exposed amino acid residues are usually targeted as binding sites to module groups. As an example, covalent binding between laccase and the surface of nanoparticles were used for the synthesis of nanomaterials through of specific reactions of covalent functionalization (Lalaoui et al 2016). It involved only one reactive functional group, the primary amino groups of a lysine residue for which the reactivity can be adjusted by careful pH monitoring (Robert et al 2017; Barbosa et al 2013) followed by reductive amination with an aldehyde functionalized pyrene module.

The molecular orientation of the enzyme on the electrode surface allowed to observe a direct electron transfer (DET) from the electrode to the enzyme (Lalaoui et al 2017;

Arrocha et al 2014). This DET was influenced by the controlled orientation of the protein increasing the number of "connected" enzymes (Lalaoui et al, 2016). As an other example, Martínez-Ortiz et al 2011 covalently coupled the laccase with the electrode surface through the T1 copper site and thus induced the direct transfer of electrons between the T1 and the graphite electrode surface. The DET of the laccase was successfully achieved, and the molecularly oriented laccase cathode showed 37% more power density and 43% more current density than the randomly bonded laccase cathode.

In conclusion, the modification of functional proteins by site-directed mutagenesis is an ideal approach to achieve module grafting to a protein [START_REF] Ying | Construction and direct electrochemistry of orientation controlled laccase electrode[END_REF]. Due to its wellknown topography some laccases are perfect candidates to gain insights for the modulation DET so that in the present work we focus on obtaining variants of laccase LAC3 in particular fron the variant K0 (Robert, V et al 2017) by site-directed mutagenesis with a single lysine, as UNIK 157 and UNIK 161 , the goal of this work is to gain insights of the role of a single lysine residue on the laccase surface without affecting the structure and functionality of the Lac3 enzyme and these can be implemented in processes in future works of covalent functionalization.

Materials and methods

Strains, media and plasmids

Escherichia coli strain DH5α (FΔ80lacZ ΔM15 Δ(lacZYAargF)U169 deoR recA1 endA1 hsdR17(rk, mk +) phoA supE44 thi1gyrA96 relA1 λ) was used for all DNA mutagenesis manipulations. Luria-Bertani (LB) broth was used for E. coli culture per litre (10 g tryptone, 5g yeast extract 10g NaCl). 100 ugmL -1 Ampicillin was added to LB medium when necessary. Plasmids and primers used in this work are listed in table 1. For the expression of recombinant laccases, the constitutive yeast expression plasmid pAKLAC3-K0 (Klonowska et al, 2005) was used as the parent plasmid to obtain the mutant derivatives from laccase. S. cerevisiae W303-1A (MATa{leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15}) was used as a host for laccase expression. S. cerevisiae transformants were routinely grown in selective medium (6.7 gL -1 YNB without amino acids,; 5 gL -1 casamino acids, 90 mgL-1adenine; 40 mgL -1 tryptophan; 50 mM succinate buffer, pH 5.3 ; 0.5% v/v guaiacol; 100 µM CuSO 4 and 20gL -1 of glucose (SD) as a carbon source.

Molecular model

A replacement 3-D model was generated from the Swiss Model server (swissmodel.expasy.org) using the laccase 2 crystal structure 1GYC from T. versicolor as template downloaded from the Protein Data Bank database (http://www.rcsb.org). This model was used to predict the surface-exposed amino acids residues on LAC3 (GenBank AAR00925.1) with the GetArea and PyMOL programs from expasy.

Mutants design and construction

Plasmid pAK145-LAC3-KO [START_REF] Robert | Probing the Surface of a Laccase for Clues towards the Design of Chemo-Enzymatic Catalysts[END_REF], a derivative of LAC3, was used as DNA template to obtain single lysine mutants (UNIK). The quick-change mutagenesis kit (Thermo Scientific) was used to obtain UNIK derivatives named LAC3-UNIK Q238K; D254K; V269K; T270K; D283K; A291K and D293K. The sequence of the mutagenic primers designed in this work is shown in Table 1. All mutant sequences were confirmed by DNA sequencing. In two cases, due to DNA polymerase error, spontaneous mutants were detected as in the case of UNIK N96D/V269K/A479T and UNIK DS89/A291K , were the unplanned mutations are indicated in bold (fig1). Because the spontaneous mutations mapped in the surface of the 3-D model of LAC3-KO, and are enzymatically active, we decided to include them in this study.

Yeast transformation and laccase activity assay

Plasmid transformation of S. cerevisiae W303-1A was performed with the lithium acetate/ ssDNA carrier/PEG protocol with a change in the time of incubation for 90 min (Gietz and Woods, 2002). After transformation, cells were spread on selective SD medium plates and were incubated at 28° C for 4 days. Qualitative laccase activity in the transformants was first detected on SD plates containing 100µM CuSO 4 and 0.5% v/v guaiacol. For laccase activity quantification, each clone was grow in microtiter plate cultures at 28° C on a rotary shaker at 120 rpm. Production of wild type LAC3 and LAC3-K0, used as controls in this study, was carried out as previously described in Zhou et al 2018. All the assays were performed in triplicate as follows: A 96-well microtiter plate was filled with 300μL of SD medium and each yeast colony was inoculated, the cells were grown for 3 days at 30°C. A 10μL aliquot of this cell suspension was taken and inoculated into 300μL of fresh SD medium, the cultures were grown for 24h at 30°C at 160 rpm [START_REF] Zhou | Production and manipulation of blue copper oxidases for technological applications[END_REF]. The cells were pelleted at 3000 rpm for 2 min, and the supernatant (SN) was transferred to a clean 96-well microtiter plate for measuring protein concentration by Bradford [START_REF] Bradford | A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[END_REF] and laccase activity. The supernatant was centrifuged at 3000 rpm for 2 min to remove any solid particles. Laccase activity from 5 µL of supernatant was determined at 25 °C by oxidation of 1 mM 2, 2´-azino-bis (3-ethylbenzothiazoline-6 sulphonic acid) (ABTS) in 100 mM sodium acetate buffer (pH 4) in a total volume of 1 ml. The oxidation of ABTS was determined at 436 nm (ɛ = 29,300 M -1 cm -1 ) using an UV/Vis spectrophotometer (DU 640 Beckman). Enzyme activities were expressed in international units (U). One unit of enzyme activity was defined as the amount of enzyme required to oxidize 1 mol of ABTS per min. The enzyme concentration was determined by the method of Bradford colorimetric assay on microtiter plates.

Production of laccase mutants

Yeast cultures transformed with the different mutants were cultivated for 60 hours with an initial OD 600 of 0.1 in 50 mL of SD medium in 250 mL flasks at 28 °C on a rotary shaker at 160 rpm. After centrifugation (4200 rpm, 10 min), the culture supernatants were filtered through filter paper (Whatman Nom. 1) then through glass-fibre filters (porosity 1.6 µM) and then concentrated in an ultrafiltration cell equipped (Amicon) with a membrane 10 kD (Millipore). The enzyme concentrate was stored at -80 °C in 30% (w/v) glycerol. Protein concentration was determined according to the method of Bradford, using BSA for the calibration curve.

2.6 Characterization of crude laccase kinetic parameters and stability conditions.

Kinetics parameters (Vmax and Km).

The kinetics parameters of the Michaelis-Menten equation were calculated according to Lineweaver-Burk plots [START_REF] Lineweaver | The determination of enzyme dissociation constants[END_REF]. The concentration used for ABTS was 0.25 mM, 0.5 mM, 1 mM, 2.5 mM, 3 mM and 5 mM. All the assays were conducted in triplicate in conditions previously described.

Temperature and pH stability of the laccase.

The thermostability of selected variants was investigated by incubating the crude supernatant from the growth culture of the variants at various temperatures (15, 25, 37, 55, and 70 °C) for 120 minutes and then cooling at 4°C. The laccase activity was then assayed at 25 °C in the conditions previously described, with 1 mM ABTS as substrate, 100 mM sodium acetate buffer (pH 4.4). The effect of pH on the laccase stability was studied by incubating the supernatants at distinct pH values in 100 mM acetate buffer (pH 3.5 -4.5) or 100 mM phosphate buffer (pH 5 -7.5) for 180 min at 25 °C, then, the supernatants were transferred to standard reaction conditions for laccase activity with 1 Mm of ABTS as the substrate.

Effect of temperature and pH on laccase activity

To study the effect of the temperature, supernatants were incubated at 15, 25, 37, 55, and 70 °C for 30 min, and laccase activity was then assayed at the corresponding temperature in standard conditions [START_REF] Zhou | Production and manipulation of blue copper oxidases for technological applications[END_REF]. To study the influence of pH, the laccase activity was assayed in the concentrated supernatant in 100 mM acetate buffer/100 mM phosphate buffer (pH 3.5 -7.5) at 25 °C with 1 mM of ABTS as the substrate.

Screening for the oxidation of phenolic compounds

For the oxidation assays with different phenolic compounds, the protocol described by Pardo et al 2015 was followed with some modifications, using catechol, vanillic acid, gallic acid, ferulic acid and vanillin were purchased from Sigma-Aldrich (Germany). The stock solutions for the different phenols were prepared at 10 mM in 20% ethanol except catechol which was dissolved in water, the reaction was performed for each mutant in 96-well plates by adding 10 µL of laccase concentrate extract, 180 µL of 100 mM acetate buffer, pH 5.0 and 20 µL of stock solution in each well and the control reaction without enzyme was included. The reactions were run in duplicate for each mutant in a Bio-Rad plate reader (Model 680 microplate reader) for each substrate at the appropriate wavelength (400-450 nm) and lectures were taken respectively at reaction times 0, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h, and 24 h. As described in Pardo et al 2015 (with a change in protein concentration units) average ΔAbs per µg of protein was calculated for each mutant and normalized against parent type (LAC3) and the results were represented in a heat map.

Results

Design and expression of laccase UNIK variants

To make some LAC3 functionnalizable tools by grafting modules which can be placed at different distance from the T1 or T2/3 copper sites on the LAC3 surface. In this goal, the eighty-five amino acid residues exposed on the surface of LAC3-K0 were identified and residue identity was analyzed. Surface exposed glycine and proline residues were not considered to avoid potential major changes in the structure-function relationship. Each of the other exposed residues could be replaced by a lysine (AAA or AAG codon) however seven of them were selected according to the distances with the T1 copper binding site. These distances were calculated from the laccase model of LAC3-K0 (see figure 1). Each of the mutants were constructed by site directed mutagenesis using pAKLAC3-UniK0 as template, a variant of LAC3 deprived of any lysine (by substitution of K40 (K40M) and K71 (K71H) (Lalaoui et al, 2017). Each of the K variants obtained where also called UNIK n where n indicate the position of the new lysine insertion on the LAC3 sequence.

The furthest K substitution from T1 was UNIK 283 (LAC3-K0-D283K) at 46.5 Å of distance and the closest was UNIK 269 (LAC3-K0-V269K) at 23 Å of distance. Table 3 shows the amino acids that were changed by lysine on the surface of K0, and a comparison of its characteristics was made, also shows the distance of such residue to the T1 copper junction site and the the theoric pI calculated for each variant, where no differences are found.

Detection of laccase activity in 96-well plate

Laccase UniKn variants were transformed into S. cerevisiae W303 and laccase activity was detected directly on a SD plate containing 0.5 v/v Guaïacol after 72 h incubation at 28 ° C. All LAC3-UniK variants tested positive for laccase activity. For quantification of laccase activity of yeast transformants 10 ul of culture supernatant of each variant was taken after 24 hours of growth in liquid medium and was performed in 96-well microtiter plate, the activity was measured under standard conditions in the presence of 1 mM ABTS, all assays were performed in triplicate compared to twild type LAC3 and LAC3-KO, Fig. 2A shows the laccase activity data; six out of nine variants (UNIK 238 , UNIK 254 , UNIK 270 , UNIK 283 , UNIK 291 and UNIK S89A/D291K showed a lower volumetric activity (0.01 to 0.017 U / mL) values compared to the K0 and LAC controls, 0.023 and 0.026 U / mL respectively; mutant UNIK293 does not present significantly differences in activity, with 0.025 U / mL. Two mutants, UNIK D96N/V269K/A479T and UNIK 269 , showed a higher activity compared to the controls. These results indicate that all these mutations on the surface do not affect the oxidation of ABTS and Guaïacol. Figure 2B shows that the specific activity of mutants UNIK 238 , UNIK 254 , UNIK 270 are two to trhee times below the values of the controls. In the case of mutants UNIK 269 , UNIK 283 , UNIK 291 no differences were observed compared to the controls. Notably, the values of specific activity of UNIK 293 , UNIK D96N/V269K/A479T and UNIK 269 were improved from 3 to 5 fold higher compared to the controls.

Laccase Activity from scaled-up cultures

Laccase activity of LAC3-Unik mutants was assayed in 200mL cultures for analysis of the biochemical characteristics, temperature and pH, stability experiments, range of substrates and kinetic parameters. Table 4 summarizes the values of the volumetric specific activity of the concentrated supernatants for each variant, where it can be seen that the variants UNIK 238 , UNIK 254 and UNIK S89A/D291K show a volumetric activity of 2.6 to 3.5 U / mL respectively, close to the K0 control with 3 U / mL, but even higher than the activity observed in LAC3 with 2.6 U / mL were obtained. Variants UNIK 270 , UNIK 283 , UNIK 293 and UNIK 291 showed values ranging from 1.98 to 2.6 U / mL; on the other hand, UNIK 269 and UNIK D96N/V269K/A479T attained values of two to three times than the controls, with 6.19 and 9.76 U / mL, respectively. However, in the results of specific activity it is observed that the majority of the mutants and even the controls had values between 2.42 to 4.44 U / mg. Is interesting to observe that mutants UNIK 293 , UNIK D96N/V269K/A479T and UNIK S89A/D291K 6.07, 18.58 and 21.02 U / mg presented values that are 2.4 to 8.3 times above that of K0 and 2.1 to 7.5 times with respect to LAC3.

Determination of kinetic parameters Vmax and Km

The kinetic parameters,Vmax and Km, from the differents variants, are shown in Table 3.

The kinetic constant was obtained using the Michaelis-Menten equation to evaluate the effect of the substrate concentration. The UNIK 238 , UNIK 269 and UNIK D96N/V269K/A479T variants showed a high affinity for the ABTS substrate with a Km of 0.28 ± 0.006 mM, 0.27 ± 0.0007 mM and 0.30 ± 0.004 mM respectively, the maximal velocity (Vmax) for these variants was 8.22 ± 0.291 (µM min -1 ) for the UNIK 269 variant and 11.98 ± 0.216 (µM min -1 ) for UNIK D96N/V269K/A479T. The other mutants did not show significant differences between them nor with LAC3.

pH and Temperature stability

To determine the effect of pH and temperature stability, the laccase activities were measured after several pretreatments respectively. As shown in figure 3A, as for LAC3 and LAC3-K0, all variants were stable at acidic pH with a top at pH 4.5. However, at neutral pH the stability was decreased until near zero at pH 7.5. The laccase stability at different temperatures was steady between 15 and 55 ° C, while at 70 °C and 120 minutes of incubation most of the variants were almost completely inactivated except for UNIK 238 , UNIK 270 (0.07 U/mL) and UNIK D96N/V269K/A479T (0.018 U/mL), figure 3B.

Effect of temperature and pH on laccase activity in the variants

The laccase activity of the variants is modified without major changes from pH 3.5 to pH 7.5 in ABTS as a substrate showing a slight increase to pH 6.5 for some variants among which the variant UNIK D96N/V269K/A479T and K0 variant, figure 4A. For the temperature, an optimal activity is shown between 15 and 55 °C, giving a remarkable decrease for all variants at 70 ° C, although, contrary to the stability tests in this test, all variants remained active, however, not all showed the same behavior, the UNIK S89A/D291 variant remains with it in the same ranges of activity up to 70 °C, as shown in figure 4B.

Oxidation of phenolic compounds

Fungi laccases are known for their ability to oxidize compounds derived from lignin (Wang et al 2018). Five phenolic compounds with different chemical structures (Table 2) were selected as substrates. In order to study the oxidation of phenolic compounds we used the enzyme extract concentrated obtained from each laccase variant (Table 4). All the variants, For all laccases variants, several common tendencies regarding their ability to oxidize different substrates can be observed. El acido galico y el catechol fueron de los compuestos oxidados con mayor facilidad para la mayoria de las enzimas, para el acido galico solo dos mutantes (UNIK 254 and UNIK 291 ) presentaron actividades relativas por debajo de LAC3 y para el catechol solo la mutante UNIK 283 y K0 la actividad fue la más baja. Para Vanillic acid solo una mutante presento dificultades para oxidar UNIK 270 , el resto de las mutantes fueron presentaron actividades relativas por encima de LAC3 siendo UNIK 238 la de mejor actividad. En el cuadro 5 ademas se puede observar como UNIK238, UNIK269 y su triple mutante UNIK D96N/V269K/A479T así como UNIK293 y UNIK S89A/D291K fueron capaces de oxidar la mayoría de los compustos con una actividad relativa superior a la de las otras mutantes.

  Bertrand et al 2017; Martínez et al 2017, Pardo, I et al 2018). Laccases are widely distributed in nature, however the most interesting, from a biotechnological point of view, are those from fungal origin. These enzymes perform a wide variety of biological functions including morphogenesis, stress defense , and lignin degradation (Su et al 2017). White-rot fungi are the most robust producers of these enzymes. Although the laccases produced by fungi have the greatest redox potential in nature, it is not yet enough to oxidize certain components of lignin and other substrates of higher redox potential, 730-790 mV (Pardo and Camarero, 2015; Yin, Q et al 2019).

  improvement in activity and selectivity (Hoarau et al, 2017; Shen et al, 2016; Liu et al 2013).

  of surface-directed enzyme function. Laccase LAC3 from Trametes sp. C30 has been studied for the oxidation of unnatural compounds (Klonowska et al 2005; Mekmouche et al 2015, Robert et al 2017) and their functionalization reactions have been shown to maximize

  K0 and the control LAC3 have the ability to oxidize all five compounds, The relative activity for each clone with respect to the control LAC3 was calculated according to the formula[START_REF] Pardo | Exploring the Oxidation of Lignin-Derived Phenols by a Library of Laccase Mutants[END_REF]:((∆Abs X -∆ Abs blank )/[protein] X )/((∆ Abs control -∆ Abs blank )/[protein] control )La vainillina fue uno de los compuestos más dificiles de oxidar ya que solo 4 clonas UNIK 238 , UNIK 293 , UNIK D96N/V269K/A479T and UNIK S89A/D291K presentan una actividad relativa muy por arriba de LAC3, dos de estas cuatro mutantes presentan la mayor cercanía de la K al T1 (UNIK 238 at 17 Å, and UNIK D96N/V269K/A479T at 20Å). Para el acido ferulico se observa que solo las mutantes UNIK 238, UNIK269, UNIK 270 y K0 fueron capaces de oxidarlo mejor que LAC3 y el resto de las mutantes, tres de las cuatro enzimas ya mencionadas presentan la mayor cercanía de K a T1 (UNIK 269 at 20Å y UNIK 270 at 27Å).


domain: chim phys sdv

text:
Xue Qiu
email: qiuxue@ouc.edu.cn

Jingyue Xu

Marcelina Cardoso

Dos Santos

Niko Hildebrandt
email: niko.hildebrandt@univ-rouen.fr

Dos Cardoso

Colin, I M Santos

Ribeiro Dos

Susumu G Santos

Multiplexed biosensing and bioimaging using lanthanide-based time-gated FRET

CONSPECTUS: The necessity to scrutinize more and more biological molecules and interactions both in solution and on the cellular level has led to an increasing demand for sensitive and specific multiplexed diagnostic analysis. Photoluminescence (PL) detection is ideally suited for multiplexed biosensing and bioimaging because it is rapid and sensitive and there is an almost unlimited choice of fluorophores that provide a large versatility of photophysical properties, including PL intensities, spectra, and lifetimes.

The most frequently used technique to detect multiple parameters from a single sample is spectral (or color) multiplexing with different fluorophores, such as organic dyes, fluorescent proteins (FPs), quantum dots (QDs), or lanthanide nanoparticles and complexes. In conventional PL biosensing approaches, each fluorophore requires a distinct detection channel and excitation wavelength. This drawback can be overcome by Förster resonance energy transfer (FRET) from lanthanide donors to other fluorophore acceptors. The lanthanides' multiple and spectrally narrow emission bands over a broad spectral range can overlap with several different acceptors at once, thereby allowing FRET from one donor to multiple acceptors. The lanthanides' extremely long PL lifetimes provide two important features. First, time-gated (TG) detection allows for efficient suppression of background fluorescence from the biological environment or directly excited acceptors. Second, temporal multiplexing, for which the PL lifetimes are adjusted by the interaction with the FRET acceptor, can be used to determine specific biomolecules and/or their conformation via distinct PL decays. The high signal-to-background ratios, reproducible and precise ratiometric and homogeneous (washing-free) sensing formats, and higher-order multiplexing capabilities of lanthanide-based TG-FRET have resulted in significant advances in the analysis of biomolecular recognition. Applications range from the fundamental analysis of biomolecular interactions and conformations, over high-throughput and point-of-care in-vitro diagnostics and DNA sequencing, to advanced optical encoding, using both liquid and solid samples and in-situ, in-vitro, and in-vivo detection with high sensitivity and selectivity.

In this Account, we discuss recent advances of lanthanide-based TG-FRET for the development and application of advanced immunoassays, nucleic acid sensing, and fluorescence imaging. In addition to the different spectral and temporal multiplexing approaches, we highlight the importance of a careful design and combination of different biological, organic, and inorganic molecules and nanomaterials for an adjustable FRET donor-acceptor distance that determines the ultimate performance of the diagnostic assays and conformational sensors in their physiological environment. We conclude by sharing our vision on how progress in the development of new sensing concepts, material combinations, and instrumentation can further advance TG-FRET multiplexing and accelerate a translation into routine clinical practice and the investigation of challenging biological systems.

INTRODUCTION

Although the origins of delayed luminescence detection and FRET date back over 100 years, [START_REF] Becquerel | Chapitre II: Durée et Intensité de La Lumière Émise[END_REF][START_REF] Cario | Über Zerlegung von Wasserstoffmolekülen durch angeregte Quecksilberatome[END_REF] the field of FRET biosensing started growing in the 1990s with the development of TG-FRET immunoassays, [START_REF] Morrison | Time-Resolved Detection of Energy Transfer: Theory and Application to Immunoassays[END_REF] commercial lanthanide-based TG-FRET immunoassays, [START_REF] Hemmila | Homogeneous Assay Platform for HTS[END_REF] FRET DNA sequencing, [START_REF] Ju | Fluorescence Energy Transfer Dye-Labeled Primers for DNA Sequencing and Analysis[END_REF] FRET with FPs, [START_REF] Heim | Engineering Green Fluorescent Protein for Improved Brightness, Longer Wavelengths and Fluorescence Resonance Energy Transfer[END_REF] and single-pair FRET, [START_REF] Ha | Probing the Interaction between Two Single Molecules: Fluorescence Resonance Energy Transfer between a Single Donor and a Single Acceptor[END_REF] to name only a few. Over the last 20 years, new fluorophores, such as QDs or other nanomaterials, [START_REF] Hildebrandt | Energy Transfer with Semiconductor Quantum Dot Bioconjugates: A Versatile Platform for Biosensing, Energy Harvesting, and Other Developing Applications[END_REF][START_REF] Algar | Photoluminescent Nanoparticles for Chemical and Biological Analysis and Imaging[END_REF] had a large influence on the development of advanced FRET sensing concepts, including their combination with lanthanides for TG-FRET. [START_REF] Hildebrandt | Energy Transfer with Semiconductor Quantum Dot Bioconjugates: A Versatile Platform for Biosensing, Energy Harvesting, and Other Developing Applications[END_REF][START_REF] Hildebrandt | Luminescent Terbium Complexes: Superior Förster Resonance Energy Transfer Donors for Flexible and Sensitive Multiplexed Biosensing[END_REF][START_REF] Cardoso Dos Santos | Quantum Dots for Förster Resonance Energy Transfer (FRET)[END_REF] The traditional advantages of TG-FRET are high spatial resolution, ratiometric and washing-free sensing formats, and efficient background-suppression. [START_REF] Zwier | Time-Gated FRET Detection for Multiplexed Biosensing[END_REF][START_REF] Qiu | A Clinical Role for Förster Resonance Energy Transfer in Molecular Diagnostics of Disease[END_REF][START_REF] Algar | FRET as a Biomolecular Research Tool -Understanding Its Potential While Avoiding Pitfalls[END_REF][START_REF] Zhang | Responsive Metal Complex Probes for Time-Gated Luminescence Biosensing and Imaging[END_REF] These unique features of TG-FRET result in high sensitivity, specificity, and simplicity, all of which are paramount for advanced biosensing and bioimaging. The versatility of combining various optically and biologically active materials and tuning their interaction via size, number, color, conformation, and proximity for the development of new multiplexing concepts, is a remarkable hallmark of TG-FRET, whose recent advances we discuss in this Account.

MULTIPLEXED TIME-GATED FRET

Lanthanide (e.g., Tb or Eu) complexes are interesting FRET donors because they possess relatively large PL quantum yields, extremely long PL lifetimes, and multiple narrow emission bands over a broad spectral range. [START_REF] Zhang | Responsive Metal Complex Probes for Time-Gated Luminescence Biosensing and Imaging[END_REF][START_REF] Sørensen | 5 Lanthanide Complexes Used for Optical Imaging[END_REF] Using FRET acceptors with specific fluorescence colors and placing them at controlled distances to the lanthanide FRET donor, gives access to both spectral (color) and temporal (lifetime) PL multiplexing, which can be combined into higher-order multiplexed and quantitative biosensing and bioimaging.

FRET distance control

Donor-acceptor distance is one of the most important parameters in FRET and a well-controlled distance is a prerequisite for efficient FRET. [START_REF] Hildebrandt | How to Apply FRET: From Experimental Design to Data Analysis[END_REF] The well-defined structures of biomolecules are natural resources for a specific donor-acceptor separation via controlled bioconjugation. [START_REF] Hermanson | Bioconjugate Techniques[END_REF] Moreover, they can also be used for the specific recognition of many biological and chemical targets and analytes. Modification of nucleic acids with fluorophores or functional bioconjugation groups at distinct positions is commercially available at many vendors. Proteins engineered with unnatural amino acids, [START_REF] Krall | Site-Selective Protein-Modification Chemistry for Basic Biology and Drug Development[END_REF] amino acid sequence recognized by enzymes, [START_REF] Rashidian | Enzymatic Labeling of Proteins: Techniques and Approaches[END_REF] or single cysteines [START_REF] Bernardim | Stoichiometric and Irreversible Cysteine-Selective Protein Modification Using Carbonylacrylic Reagents[END_REF] can also be site-specifically labeled with fluorophores. The much simpler structure of nucleic acids makes distance control significantly more straightforward compared to proteins. The same holds true for small fluorophores (e.g., dyes and lanthanide complexes) compared to relatively large nanoparticles (e.g., QDs). In addition to structure and size, shape and orientation of biomolecules and fluorophores must be considered for controlling FRET.

Spectral (PL color) multiplexing

The distinct PL spectra of different lanthanide complexes can be efficiently used for color multiplexing. [START_REF] Kovacs | Photophysics of Coumarin and Carbostyril-Sensitized Luminescent Lanthanide Complexes: Implications for Complex Design in Multiplex Detection[END_REF] For FRET multiplexing, Tb has the advantage of better-separated emission bands (compared to Eu), which are particularly well suited to be combined with differently colored acceptors, such as dyes or QDs (Figure 1A). [START_REF] Hildebrandt | Luminescent Terbium Complexes: Superior Förster Resonance Energy Transfer Donors for Flexible and Sensitive Multiplexed Biosensing[END_REF] The spectral range of acceptor fluorescence is mainly limited by the spectral overlap of acceptor absorption and lanthanide donor emission and starts around 500 nm (after the first emission band of Tb -cf. Figure 1A and1B). In particular, for QDs, orange to red emitting QDs are preferred because of their much broader spectral overlap (Figure 1A right). In a typical spectrally multiplexed TG-FRET measurement, TG PL intensities are collected in different donor and acceptor emission channels (Figure 1B). Spectral separation distinguishes the different FRET-sensitized acceptors and the FRET-quenched Tb donor. TG detection suppresses short-lived PL from sample autofluorescence or from acceptors that were directly excited by the excitation pulse. When the different acceptor PL spectra overlap within the distinct detection channels (e.g., due to the relatively broad PL spectra of dyes), so-called spectral crosstalk correction can be used to improve multiplexing specificity. [START_REF] Geißler | Six-Color Time-Resolved Forster Resonance Energy Transfer for Ultrasensitive Multiplexed Biosensing[END_REF] In multiplexed biosensing, each color detection channel is used for one specific target (analyte), which requires sequential measurements or multiple detectors.

Temporal (PL lifetime) multiplexing

The very long PL lifetimes of lanthanides are an excellent basis for FRET distance control. Doublestranded (ds)DNA is quite rigid and Tb-to-dye or Tb-to-QD distances can be adjusted with a resolution of circa 0.31 nm to 0.34 nm per base pair. [START_REF] Qiu | Multiplexed Nucleic Acid Hybridization Assays Using Single-FRET-Pair Distance-Tuning[END_REF][START_REF] Guo | Conformational Details of Quantum Dot-DNA Resolved by Forster Resonance Energy Transfer Lifetime Nanoruler[END_REF] Single-stranded (ss)DNA is less rigid and, depending on the environmental conditions, a denser conformation can be found, e.g., with ~0.15 nm per base when Tb is attached via ssDNA on QD surfaces. [START_REF] Guo | Conformational Details of Quantum Dot-DNA Resolved by Forster Resonance Energy Transfer Lifetime Nanoruler[END_REF] Despite their more complex structures, proteins can also be used to adjust Tb-to-QD donor acceptor distances. 1 QD surface coatings with defined thicknesses are another possibility to tune the distance between lanthanides and QDs. [START_REF] Chen | FRET-Modulated Multihybrid Nanoparticles for Brightness-Equalized Single-Wavelength Barcoding[END_REF][START_REF] Chen | Single-Nanoparticle Cell Barcoding by Tunable FRET from Lanthanides to Quantum Dots[END_REF] The shorter/longer the distance, the shorter/longer the PL lifetime of both the FRETquenched lanthanide donor and the FRET-sensitized dye or QD acceptor (Figure 1C). A temporally multiplexed biosensor must be designed, such that each target corresponds to a specific donor-acceptor distance and thus, a specific PL decay curve with a specific PL lifetime. In a typical measurement (Figure 1D), PL intensities of the FRET-sensitized acceptor are collected in multiple TG detection windows (e.g., 0.02 to 1.5 ms, 1.5 to 4.0 ms, and 4.0 to 8.0 ms after the excitation pulse) that can all be extracted from a single PL decay curve (one measurement for all TG detection windows). The multiplexed biosensor is calibrated by first quantifying the acceptor PL contributions for each single target within each TG detection window. For multiplexing, the PL contribution of each target within the different TG windows can be deconstructed by a straightforward matrix calculation. [START_REF] Qiu | Multiplexed Nucleic Acid Hybridization Assays Using Single-FRET-Pair Distance-Tuning[END_REF] The temporal widths of the detection windows must be adapted to the PL decay curves and the number of windows should be equal to that of the targets.

The method only uses TG PL intensities and does not require PL lifetime analysis. Because the complete PL decay curve is collected within one measurement, temporal multiplexing does not require sequential measurements or multiple detectors. Moreover, only a single FRET pair is required. On the downside, the complete PL intensity is divided into different fractions and there are contributions from multiple PL decay curves in the different TG detection windows. Therefore, biosensing with temporal multiplexing is usually less sensitive than with spectral multiplexing.

Spectrotemporal (combined color and lifetime) multiplexing

Spectral and temporal multiplexing can be combined into spectrotemporal multiplexing with different PL colors (several acceptors) and different PL lifetimes (different donor-acceptor distances) (Figure 2). [START_REF] Qiu | Three-Dimensional FRET Multiplexing for DNA Quantification with Attomolar Detection Limits[END_REF] In this so-called higher-order multiplexing, the number of distinguishable targets is the product ab when all targets are mixed within a sample or the exponentiation a b when targets can be spatially separated, with a and b being the number of distinguishable acceptors (color)

and PL decay curves (lifetime), respectively. Although theoretically a and b can be very large numbers, the practical limitations of both spectral and temporal multiplexing are also valid for their combination. Considering that efficient TG-FRET multiplexing was experimentally demonstrated with five different QD and dye acceptors and three different PL decay curves, [START_REF] Geißler | Six-Color Time-Resolved Forster Resonance Energy Transfer for Ultrasensitive Multiplexed Biosensing[END_REF][START_REF] Qiu | Multiplexed Nucleic Acid Hybridization Assays Using Single-FRET-Pair Distance-Tuning[END_REF][START_REF] Geißler | Quantum Dot Biosensors for Ultrasensitive Multiplexed Diagnostics[END_REF] spectrotemporal multiplexing with a = 5 and b = 3 should be practically feasible without significant additional complications when compared to spectral or temporal multiplexing alone. Notably, excitation-wavelength based differentiation of lanthanide complexes should be able to further C D extend the spectrotemporal multiplexing capability. [START_REF] Xiong | Excitation-and Emission-Wavelength-Based Multiplex Spectroscopy Using Red-Absorbing Near-Infrared-Emitting Lanthanide Complexes[END_REF][START_REF] Carro-Temboury | An Optical Authentication System Based on Imaging of Excitation-Selected Lanthanide Luminescence[END_REF] Technical implementation of spectrotemporal multiplexing into diagnostic assays should be relatively straightforward, considering that many fluorescence spectrometers and plate readers already provide multiple color channels and TG detection (often referred to as "TR-FRET" or "HTRF" by the different providers). 17

TG-FRET IMMUNOASSAYS

TG-FRET immunoassays usually apply two distinct antibodies (one conjugated with a lanthanidebased donor and the other with an appropriate fluorescent acceptor) that separately bind to different epitopes of a target antigen, such that they are in close proximity for FRET. Because TG-FRETsensitized acceptor PL can only occur upon specific antibody-target-antibody sandwich formation, high signal-to-background ratios can be accomplished without any separation steps. This unique advantage of lanthanide-based TG-FRET has been exploited for many years in highly sensitive and specific assays for in-vitro diagnostics, high-throughput drug screening, and the investigation of protein-protein interactions. 14,16-18

Multiplexed detection of tumor markers

The complexity of cancers with various sub-types most often requires the quantification of several tumor markers for specific diagnosis. Multiplexed TG-FRET immunoassay can detect such tumor marker panels from a single sample. As an example, the distinction between small cell and nonsmall cell lung carcinoma (SCLC and NSCLC) can be improved by quantifying neuron-specific enolase (NSE), carcinoembryonic antigen (CEA), the cytokeratin-19 fragment Cyfra21-1, squamous cell carcinoma antigen (SCC), and the carbohydrate antigen CA15.3 in human serum. [START_REF] Geißler | Six-Color Time-Resolved Forster Resonance Energy Transfer for Ultrasensitive Multiplexed Biosensing[END_REF] Two antibodies against each tumor marker were conjugated with Tb-dye FRET pairs, for which only the dye acceptors were different (Figure 3A). Thus, excitation of Tb resulted in FRET to only those dyes contained in antibody-tumor marker-antibody sandwich complexes and their TG PL could be used for specific tumor marker quantification via color multiplexing (Figure 3B).

For multiplexed quantification from a single sample, each target-specific (color or spectral) detection channel must first be calibrated to obtain signal (e.g., TG acceptor/donor intensity ratio: The same sandwich immunoassay principle can be used with Tb-to-QD FRET and the duplexed quantification of the epidermal growth factor receptors 1 and 2 (EGFR and HER2) [START_REF] Qiu | Nanobodies and Antibodies for Duplexed EGFR/HER2 Immunoassays Using Terbium-to-Quantum Dot FRET[END_REF] or the triplexed detection of CEA, NSE, and prostate specific antigen (PSA). [START_REF] Bhuckory | Triplexed CEA-NSE-PSA Immunoassay Using Time-Gated Terbium-to-Quantum Dot FRET[END_REF] Although the strongly reduced spectral crosstalk (Figure 1B) is an advantage for color multiplexing, the immunoassay design is more complex because the sizes of QDs are similar to that of the antibodies (Figure 3C).

Several antibodies can be attached to the surface of one QD, e.g., by covalent binding (with or without crosslinkers) between functional groups on the QD (e.g., maleimides or amines) and the antibody (e.g., free thiols or amino groups) or many other nanobioconjugation strategies. [START_REF] Bhuckory | Triplexed CEA-NSE-PSA Immunoassay Using Time-Gated Terbium-to-Quantum Dot FRET[END_REF][START_REF] Wegner | Quantum-Dot-Based Forster Resonance Energy Transfer Immunoassay for Sensitive Clinical Diagnostics of Low-Volume Serum Samples[END_REF][START_REF] Bhuckory | Evaluating Quantum Dot Performance in Homogeneous FRET Immunoassays for Prostate Specific Antigen[END_REF][START_REF] Mattera | Compact Quantum Dot-Antibody Conjugates for FRET Immunoassays with Subnanomolar Detection Limits[END_REF][START_REF] Sapsford | Functionalizing Nanoparticles with Biological Molecules: Developing Chemistries That Facilitate Nanotechnology[END_REF] Due to a certain variability in the position and flexibility of those functional groups, antibodies can assume different orientations on the QD surface. The donor-acceptor distances are usually larger than with dye-conjugated antibodies and also antigen binding sites may be blocked. As a rule of thumb, we found that complete immunoglobulin G (IgG) antibodies with many Tb donors combined with several smaller antibodies (e.g., reduced F(ab')2 or Fab') per QD acceptor provided the best sensing performance (Figure 3D). [START_REF] Wegner | Quantum-Dot-Based Forster Resonance Energy Transfer Immunoassay for Sensitive Clinical Diagnostics of Low-Volume Serum Samples[END_REF]

Size, orientation, conformation, and proximity for optimizing QD-based immunoassays.

FRET is only functional at donor-acceptor distances (R) below circa 10 nm and the FRET efficiency strongly depends those distances (~R -6 ). [START_REF] Hildebrandt | Energy Transfer with Semiconductor Quantum Dot Bioconjugates: A Versatile Platform for Biosensing, Energy Harvesting, and Other Developing Applications[END_REF][START_REF] Zwier | Time-Gated FRET Detection for Multiplexed Biosensing[END_REF][START_REF] Algar | FRET as a Biomolecular Research Tool -Understanding Its Potential While Avoiding Pitfalls[END_REF] Although Tb-to-QD FRET can extend the FRET distance range and Förster distances (R0, for 50% FRET efficiency R0 = R) can range from circa 5 to 11 nm, the sizes of both QDs and antibodies play an important role for TG-FRET assay development. Inorganic QDs are most often in a size range of 4 nm to 12 nm (diameters or ellipsoidal axes) and organic surface coatings can increase their diameters up to 20 nm or more. [START_REF] Mattera | Compact Quantum Dot-Antibody Conjugates for FRET Immunoassays with Subnanomolar Detection Limits[END_REF][START_REF] Morgner | A Quantum-Dot-Based Molecular Ruler for Multiplexed Optical Analysis[END_REF] Antibodies on the QD surface further increase R and thus, smaller alternatives to the relatively large IgGs (~159.56 nm [START_REF] Chen | FRET-Modulated Multihybrid Nanoparticles for Brightness-Equalized Single-Wavelength Barcoding[END_REF] ), [START_REF] Qiu | Nanobodies and Antibodies for Duplexed EGFR/HER2 Immunoassays Using Terbium-to-Quantum Dot FRET[END_REF] such as reduced IgGs (F(ab')2 or Fab'; ~4x5x8 nm 3 for Fab'), [START_REF] Qiu | Nanobodies and Antibodies for Duplexed EGFR/HER2 Immunoassays Using Terbium-to-Quantum Dot FRET[END_REF] nanobodies (VHH, ~42.52.5 nm 3 ), [START_REF] Qiu | Nanobodies and Antibodies for Duplexed EGFR/HER2 Immunoassays Using Terbium-to-Quantum Dot FRET[END_REF] repeat proteins (Reps; ~5.52.52.5 nm 3 ), 1 and albumin-binding domain-derived affinity proteins (ADAPTs; 11.52.5 nm 3 ), [START_REF] Wu | Quantum Dot-Based FRET Immunoassay for HER2 Using Ultrasmall Affinity Proteins[END_REF] can be attached to the surfaces of QDs (Figure 4A). 1,[START_REF] Qiu | Nanobodies and Antibodies for Duplexed EGFR/HER2 Immunoassays Using Terbium-to-Quantum Dot FRET[END_REF][START_REF] Wegner | Quantum-Dot-Based Forster Resonance Energy Transfer Immunoassay for Sensitive Clinical Diagnostics of Low-Volume Serum Samples[END_REF][START_REF] Wu | Quantum Dot-Based FRET Immunoassay for HER2 Using Ultrasmall Affinity Proteins[END_REF][START_REF] Wegner | Nanobodies and Nanocrystals: Highly Sensitive Quantum Dot-Based Homogeneous FRET Immunoassay for Serum-Based EGFR Detection[END_REF] The smaller the antibody, the more of them can be labeled on a QD, thereby providing more biological recognition elements per QD. [START_REF] Wegner | Quantum-Dot-Based Forster Resonance Energy Transfer Immunoassay for Sensitive Clinical Diagnostics of Low-Volume Serum Samples[END_REF][START_REF] Mattera | Compact Quantum Dot-Antibody Conjugates for FRET Immunoassays with Subnanomolar Detection Limits[END_REF][START_REF] Wu | Quantum Dot-Based FRET Immunoassay for HER2 Using Ultrasmall Affinity Proteins[END_REF][START_REF] Wegner | Nanobodies and Nanocrystals: Highly Sensitive Quantum Dot-Based Homogeneous FRET Immunoassay for Serum-Based EGFR Detection[END_REF] Because one QD can be sequentially FRET-sensitized by many lanthanide donors (due to the large difference between lanthanide and QD PL lifetimes), combination with large antibodies (e.g., IgG) labeled with many lanthanide donors can result in Tb-to-QD TG-FRET immunocomplexes with over 200

Tb donors per QD acceptor and a concomitant improvement of the assay performance. [START_REF] Wegner | Quantum-Dot-Based Forster Resonance Energy Transfer Immunoassay for Sensitive Clinical Diagnostics of Low-Volume Serum Samples[END_REF] FRET donor-acceptor distance is another important parameter that depends on both the sizes and orientations of the QD-antibody conjugates. For example, conjugation of antibodies via their endogenous disulfide groups on the ZnS shells of compact QDs can result in antigen-binding sites very close to the QD surface and improved TG-FRET immunoassay performance (Figure 4B).

Many parameters, including size, orientation, labeling ratio, environment, binding affinities, brightness, stability etc., need to be considered and optimized for Tb-to-QD TG-FRET immunoassay development, some of which are summarized in Supporting Table S1.

One can also use the target binding-dependent conformational change of a single protein bidomain for biosensing. When Rep-bidomains are conjugated with a Tb donor on one terminus and attached to a QD acceptor on the other terminus, target binding-induced conformational changes in the Rep-bidomain result in Tb-QD distance and concomitant FRET efficiency alterations (Figure 4C). 1 These conformational changes can be analyzed with sub-nanometer resolution via PL lifetimes (Figure 4D) and targets can be quantified at picomolar concentrations via TG-FRET ratios (Figure 4E), both using a single measurement at low concentrations in solution and under physiological conditions.

TG-FRET DNA/RNA HYBRIDIZATION ASSAYS

The structural simplicity of nucleic acids is ideal for designing FRET assays with specific donoracceptor distances. Therefore, spectral, temporal, and spectrotemporal TG-FRET multiplexing becomes significantly easier compared to protein-based systems. Base-by-base distance adjustment provides sub-nanometer spatial resolution to fine-tune PL lifetimes for TG-FRET temporal

A B

C D E multiplexing or for conformational analysis. [START_REF] Qiu | Multiplexed Nucleic Acid Hybridization Assays Using Single-FRET-Pair Distance-Tuning[END_REF][START_REF] Guo | Conformational Details of Quantum Dot-DNA Resolved by Forster Resonance Energy Transfer Lifetime Nanoruler[END_REF] Moreover, DNA can be amplified, which gives access to the quantification of extremely low concentrations of DNA or RNA targets even from clinical samples. 5A). [START_REF] Jin | Amplification-Free, and Sensitive Diagnostic Assay for Single-Step Multiplexed Fluorescence Detection of MicroRNA[END_REF] When attaching DNAs to QD, the valency (number of DNA strands per QD) must be well adjusted. Whereas a high valency is beneficial for many hybridization and FRET events per QD, too many DNAs in close distance may interfere with target hybridization. When polyhistidine-appended DNA is used for DNA-QD selfassembly, valency can be controlled by adjusting the respective DNA and QD concentrations. [START_REF] Qiu | Multiplexed Nucleic Acid Hybridization Assays Using Single-FRET-Pair Distance-Tuning[END_REF][START_REF] Guo | Conformational Details of Quantum Dot-DNA Resolved by Forster Resonance Energy Transfer Lifetime Nanoruler[END_REF] Another possibility is the use of biotinylated DNA and QDs with a limited amount of streptavidin on their surface. [START_REF] Qiu | Multiplexed Nucleic Acid Hybridization Assays Using Single-FRET-Pair Distance-Tuning[END_REF][START_REF] Qiu | Rapid and Multiplexed MicroRNA Diagnostic Assay Using Quantum Dot-Based Förster Resonance Energy Transfer[END_REF][START_REF] Xu | When Nanoworlds Collide: Implementing DNA Amplification, Nanoparticles, Molecules, and FRET into a Single MicroRNA Biosensor[END_REF] An enzyme-free multiplexed assay could be realized by attaching short DNA probes on the surfaces of different QDs and exploiting the combination of base-pairing and base-stacking, which allowed the Tb-DNA to hybridize to the QD-DNA only when the specific miR target was present in the sample. Although color multiplexing was more straightforward when using the narrow PL spectra of the QDs, the simpler assay format and the larger Tb-to-QD distances led to slightly lower sensitivities in the low nanomolar concentration range (Figure 5B). [START_REF] Qiu | Rapid and Multiplexed MicroRNA Diagnostic Assay Using Quantum Dot-Based Förster Resonance Energy Transfer[END_REF] Detection of much lower concentrations can be accomplished by isothermal DNA amplification, such as rolling circle amplification (RCA). [START_REF] Qiu | Three-Dimensional FRET Multiplexing for DNA Quantification with Attomolar Detection Limits[END_REF][START_REF] Qiu | Advanced MicroRNA-Based Cancer Diagnostics Using Amplified Time-Gated FRET[END_REF] RCA-TG-FRET could be used to distinguish wild-type (WT) and singlepoint mutated (MT) dsDNA in the BRAF gene for liquid biopsy cancer diagnostics (Figure 5C). [START_REF] Dekaliuk | Discrimination of the V600E Mutation in BRAF by Rolling Circle Amplification and Förster Resonance Energy Transfer[END_REF] Amplified Tb-to-dye TG-FRET duplexing could quantify the two WT and MT dsDNAs from a single sample in a 75 fM to 4.5 pM concentration range.

Spectrotemporally multiplexed DNA/RNA assays

Isothermal target-primed RCA-TG-FRET provides extremely high DNA and RNA target selectivity and femtomolar LODs for the application to a broader range of clinical samples, including cells, blood, and tissues. [START_REF] Qiu | Advanced MicroRNA-Based Cancer Diagnostics Using Amplified Time-Gated FRET[END_REF][START_REF] Dekaliuk | Discrimination of the V600E Mutation in BRAF by Rolling Circle Amplification and Förster Resonance Energy Transfer[END_REF] On the other hand, the relatively complicated sensing format

Optical barcoding

Cellular and molecular barcoding has become an important method for a better understanding of multiple time-dependent processes and interactions in developmental biology and gene function. 53- 55 Temporal TG-FRET multiplexing has the potential to increase the fluorescence multiplexing capability by combining lanthanides, dyes, and QDs within single hybrid nanoparticles. [START_REF] Chen | FRET-Modulated Multihybrid Nanoparticles for Brightness-Equalized Single-Wavelength Barcoding[END_REF][START_REF] Chen | Single-Nanoparticle Cell Barcoding by Tunable FRET from Lanthanides to Quantum Dots[END_REF] Attachment of lanthanide complexes and dyes on QDs with a SiO2 coating activates several different FRET channels between the various fluorophores, which can be adjusted by the SiO2 coating thickness, the photophysical properties of the fluorophores, and their density on the surface (Figure 8A). The different spectral overlaps and distances between the fluorophores determine the FRET efficiencies and the concomitant PL lifetimes of FRET donors and acceptors. For example, the attachment of Eu or Tb complexes on QDs with two different SiO2 coating thicknesses resulted in four distinct QD PL decays, all of which provided distinct intensities in three temporal detection windows that were encoded as red (R), green (G), and blue (B). Thus, the different intensity fractions in those three detection windows produced a unique RGB code for each of the four nanohybrids. When different cells were incubated with one type of nanohybrid, they could be efficiently distinguished in a mixture by recording a TG image in each detection window and overlaying them to an RGB image (Figure 8B). [START_REF] Chen | Single-Nanoparticle Cell Barcoding by Tunable FRET from Lanthanides to Quantum Dots[END_REF] The drawback of very different brightnesses of those purely temporally multiplexed codes could be overcome by co-assembling different amounts of Tb complexes and dyes on the same QD. While the Tb density mainly adjusted the QD PL intensity, the dye density influenced both PL decay time and intensity of the QDs. The combination of three nanohybrids (10Tb/QD, 30Tb/QD/10dye, and 60Tb/QD/40dye) with three TG detection windows (R: 0.05 -0.5 ms; G: 0.5 -1.0 ms; and B: 1.0 -4.0 ms) resulted in brightness-equalized codes that could be used to distinguish microbeads from a single overlay image (Figure 8C).

In-situ, in-vitro, and in-vivo imaging

TG-FRET amplification and nanohybrids can also be applied for imaging biomolecules and their interactions in living cells, tissues, and developing organisms, where FRET multiplexing at high signal-to-noise ratios and with high photostability is extremely important. [START_REF] Bunt | FRET from Single to Multiplexed Signaling Events[END_REF] For example, intracellular actin could be detected on the single protein level via in-situ RCA-TG-FRET coupled to immunochemistry (Figure 9A). [START_REF] Francés-Soriano | In Situ Rolling Circle Amplification Förster Resonance Energy Transfer (RCA-FRET) for Washing-Free Real-Time Single-Protein Imaging[END_REF] Because TG-FRET could only occur in the amplified RCP A B C (hybridization of both Tb-DNA and dye-DNA), the signal per actin was both strongly amplified and very specific, thereby not interfering with background signals and avoiding any washing steps (Figure 9B). Wash-free and autofluorescence-free imaging with simple sample preparation and the capability of higher-order multiplexing has the potential to become very useful for sensitive imaging of intra-and extracellular biomarkers in clinical diagnostics. The multiplexing advantages of TG-FRET nanohybrids can be further exploited for live-cell imaging and detecting intracellular biomolecular interactions. For such applications, peptides and proteins can be attached to QDs to induce various FRET processes between QDs, lanthanide complexes, dyes, and fluorescent proteins (FPs) (Figure 10A). [START_REF] Cardoso Dos Santos | Time-Gated FRET Nanoprobes for Autofluorescence-Free Long-Term In Vivo Imaging of Developing Zebrafish[END_REF][START_REF] Afsari | Time-Gated FRET Nanoassemblies for Rapid and Sensitive Intra-and Extracellular Fluorescence Imaging[END_REF] The versatility and multiplexing capability for autofluorescence-free imaging was demonstrated via i) triplexed intracellular TG-FRET between a Tb complex and three different QDs (Figure 10B), [START_REF] Cardoso Dos Santos | Time-Gated FRET Nanoprobes for Autofluorescence-Free Long-Term In Vivo Imaging of Developing Zebrafish[END_REF] ii) multistep intracellular TG-FRET from Tb-to-QD-to-dye (Figure 10C), [START_REF] Afsari | Time-Gated FRET Nanoassemblies for Rapid and Sensitive Intra-and Extracellular Fluorescence Imaging[END_REF] and iii) multistep TG-FRET from Tb-to-QD-to-FP to monitor the intracellular attachment of transiently expressed FP to the TG-FRET nanohybrids (Figure 10D). [START_REF] Cardoso Dos Santos | Time-Gated FRET Nanoprobes for Autofluorescence-Free Long-Term In Vivo Imaging of Developing Zebrafish[END_REF] Beyond multiplexing, the outstanding signal-to-noise ratios and photostability of TG-FRET nanohybrids could be used for long-term imaging in a developing organism. After microinjection of concentrated nanoprobes (few µM) in zebrafish embryos at the one-cell stage, autofluorescence-free in-vivo imaging could be performed for up to seven days (Figure 10E). This long-term imaging capability showed that even after an extreme increase in cell number and volume of the zebrafish larvae, the initially injected TG-FRET-nanohybrids still emitted bright PL.

CONCLUSIONS AND OUTLOOK

TG-FRET using lanthanide donors with long PL lifetimes and various acceptors, such as dyes, QDs, or FPs, can significantly improve multiplexing capabilities in biosensing and bioimaging. Spectral, temporal, and spectrotemporal multiplexing within a single sample, efficient suppression of sample However, TG-FRET is only one of the many available tools and its benefits must always be weighed against its limitations (e.g., relatively low photon flux, few available probes, specialized setups for imaging), some of which present important challenges for future TG-FRET research. A perfect method with the best performance for all bioanalytical parameters does unfortunately not exist. Both analytical priority (e.g., sensitivity, specificity, multiplexing, simplicity) and available sensing technologies (e.g., optical, electrochemical, mass-based) for a specific application must be carefully considered during biosensor development. The discovery and understanding of more and more complicated biological systems comprising multiple parallel and sequential biomolecular interactions in clinical, molecular, developmental, and neuro biology will make multiplexed detection and imaging even more demanding and important in the future. Technical advances, new and better materials, smart multihybrid nanosystems, and interdisciplinary studies with the potential to implement sophisticated TG-FRET multiplexing probes into sophisticated biological systems will be important drivers for future applications and the translation of TG-FRET multiplexing into a standard technology for multi-level analysis in biosensing and bioimaging.

Figure 1 .

 1 Figure 1. Spectral and temporal FRET multiplexing. (A) Spectral overlap between the emission of one Tb donor (black) and the absorption of five different dye (left) or QD (right) acceptors (colored). (B) The spectral separation of PL from Tb donors (black) and from dye (left) or QD (right) acceptors permits multiplexed PL color detection via different bandpass filters (striped transmission spectra). A and B reproduced with permission from ref. 14. Copyright 2014 Elsevier. (C) Tuning the donor-acceptor (D-A) distance by the number of DNA base pairs (left), results in a D-A distance-dependent FRET efficiency (center) and a concomitant D-A distance-dependent PL lifetime of the FRET donor and acceptor (right). (D) PL decay curves of FRET-sensitized Cy5.5 dyes separated by 10, 13, or 18 base pairs (bps) from the Tb donor. The different TG detection windows (1 -blue; 2 -green; 3 -red) can be simultaneously extracted from each single PL decay curve. Different intensities represent the target concentrations (0 to 3 nM from black to brown). C and D adapted with permission from ref. 28. Copyright 2017 Wiley.

Figure 2 .

 2 Figure 2. Spectrotemporal FRET multiplexing. Two different PL colors and two different PL decays measured in two temporal detection windows can be combined to quantify the concentrations (via the PL intensity) of four different DNA targets in solution. Reproduced with permission from ref. 2. Copyright 2018 American Chemical Society.

Figure 3 .

 3 Figure 3. TG-FRET color multiplexing for tumor marker immunoassays. (A) In the absence of tumor markers, Tb excitation results only in Tb PL (no FRET). (B) Formation of specific immunological sandwich complexes results in tumor marker-specific Tb-to-dye TG-FRET. A and B adapted with permission from ref. 27. Copyright 2013 American Chemical Society. (C) Tb-to-QD TG-FRET immunoassays, for which the relatively large sizes of QDs change the acceptor-antibody conjugate. For clarity, only one antibody per QD is shown. Schemes are not to scale. (D) Calibration of the three Tb-QD FRET immunoassays can be used for triplexed tumor marker quantification from a single serum sample. Reproduced with permission from ref. 34. Copyright 2020 The Authors, some rights reserved; exclusive licensee MDPI, Basel, Switzerland. Distributed under a Creative Commons Attribution License 4.0 (CC BY) https://creativecommons.org/licenses/by/4.0/.

Figure 4 .

 4 Figure 4. Size and orientation of QD-antibody conjugates. (A) Schematic presentation of biological recognition proteins with various sizes attached to QD surfaces. Protein structures adapted from the RCSB Protein Data Bank (rcsb.org) PDB IDs "1TGT" (for IgG, F(ab')2, and Fab'), "5IVO" (for VHH), "3LTJ" (Rep), and "1GJT" (ADAPT). (B) Mild reduction of antibody disulfides (green and orange circles) for direct conjugation to QDs. The different orientations result in surface-near and surface-far antigen binding sites (red and blue circles). The smaller the antibody, the better the immunoassay detection performance (assay calibration curve in the center). Adapted with permission from ref. 49. Copyright 2016 The Royal Society of Chemistry. (C) An Rep bidomain (A3_gGFPD) with a Tb donor and QD acceptor leads to Tb-to-QD FRET upon Tb excitation. Depending on target binding of bA3-2 (top), BFP (bottom), or both (right), the bidomain conformation changes. (D) PL decay time analysis can quantify the conformational changes shown in C. (E) TG-FRET ratios of QD and Tb PL can quantify the target concentrations during sequential target-bidomain binding. C to E reproduced with permission from ref. 1. Copyright 2020 American Chemical Society.

Figure 5 .

 5 Figure 5. TG-FRET color multiplexing for RNA/DNA hybridization assays. (A) Specific hybridization of two adaptor oligos on different miRNA targets followed by ligation brings specific FRET DNA probes in close proximity for multiplexed TG-FRET quantification of the three miRNAs (graph shows the analysis of nine samples with different miRNA concentrations). Reproduced with permission from ref. 35. Copyright 2015 Wiley. (B) Target-specific Tb-DNA/QD-DNA hybridization resulted in TG-FRET, which could be used for triplexed miRNA quantification (graph shows the analysis of nine samples with different miRNA concentrations). Reproduced with permission from ref. 37. Copyright 2015 American Chemical Society. (C) After denaturation of dsDNA WT and MT targets, specific hybridization with a padlock probe, ligation, and target-primed RCA results in a long RCA product (RCP). Tb-dye FRET DNA-probes can hybridize to specific positions in the RCP to efficiently distinguish WT and MT dsDNA (graph shows the analysis of six samples with different DNA target concentrations). Reproduced with permission from ref. 38. Copyright 2019 American Chemical Society.

4 . 2

 42 Figure6A; catalytic hairpin assembly, CHA -Figure6B) to quantify miR-20a and miR-21 from a single sample.[START_REF] Guo | Amplified, and Multiplexed Detection of MicroRNAs Using Time-Gated FRET and Hybridization Chain Reaction[END_REF][START_REF] Xu | Single-Measurement Multiplexed Quantification of MicroRNAs from Human Tissue Using Catalytic Hairpin Assembly and Förster Resonance Energy Transfer[END_REF] Using hairpin probes with Tb and Cy5.5 labeled at different sequence positions, the amplified HCR or CHA products resulted in Tb-Cy5.5 distances of ~4.3 nm and ~5.2 nm for HCR and ~4.2 nm and ~5.1 nm for CHA, as determined by PL lifetime analysis, taking advantage of the FRET molecular ruler. Both techniques could perform duplexed miR quantification in a circa 30 pM to 400 pM concentration range (Figures6C and 6D) and CHA could quantify both miRs at low picomolar concentrations from total RNA extracts of five different human tissue samples, demonstrating the actual applicability for clinical testing.

Figure 6 .

 6 Figure 6. TG-FRET temporal multiplexing for RNA/DNA hybridization assays. Both HCR (A) and CHA (B) are based on two pairs of hairpin probes that contain Tb donors and Cy5.5 acceptors at distinct positions. The targetspecifically amplified products result in multiple FRET pairs at two distinct distances. Thus, short and long distance FRET with a single FRET pair can be used for duplexed miR quantification (C for HCR and D for CHA). A and reproduced with permission from ref. 39. Copyright 2019 American Chemical Society. B and D reproduced with permission from ref. 40. Copyright 2019 American Chemical Society.

  adapted to the sensing conditions, in particular when RCA-TG-FRET is combined with QDs.[START_REF] Xu | When Nanoworlds Collide: Implementing DNA Amplification, Nanoparticles, Molecules, and FRET into a Single MicroRNA Biosensor[END_REF] In terms of spectral and temporal TG-FRET multiplexing, the repetitive sequence of the target-specific RCA product (RCP) can be used to hybridize many fluorescent DNA probes with different sequence lengths and fluorophores at distinct positions. Thus, amplification (many FRET pairs within one RCP), spectral multiplexing (one Tb donor combined with different dye acceptors), and temporal multiplexing (different Tb-dye distances) can be implemented in a single RCA-TG-FRET sensor (Figure7). The combination of two FRET pairs with two temporal detection channels each was used for quadruplexed detection of four ssDNA targets at sub-picomolar concentrations.[START_REF] Qiu | Three-Dimensional FRET Multiplexing for DNA Quantification with Attomolar Detection Limits[END_REF]

Figure 7 .

 7 Figure 7. TG-FRET spectrotemporal multiplexing for RNA/DNA hybridization assays. (A) Each DNA target is hybridized by a specific padlock probe followed by RCA. The RCP is hybridized by Tb-donor and dye-acceptor DNA probes. (B) Spectra of Tb absorption (black) and emission (green) that partly overlaps with the absorption of Cy3.5 (orange) and Cy5.5 (red). (C) Emission spectra of Tb, Cy3.5, and Cy5.5. Optical bandpass filter transmission spectra (color detection channels) are shown in gray. (D) Two FRET pairs (Tb-Cy3.5 and Tb-Cy5.5) and two different donoracceptor distances (shown in bps) provide four distinguishable signals (TG1 to TG4: two distinct TG detection windows for each color). (E) Recovery of varying target concentrations from 10 different samples using spectrotemporal TG-FRET multiplexing. Dotted lines represent known concentrations; data points represent RCA-TG-FRET measurements. Reproduced with permission from ref. 2. Copyright 2018 American Chemical Society.

3

 3

Figure 8 .

 8 Figure 8. Optical barcoding via TG-FRET multiplexing. (A) Adjusting the density of Tb/Eu complexes and dyes on the surface of SiO2 coated QDs (x: adjustable thickness) results in various tunable FRET processes. (B) PL decay curves of four TG-FRET nanoparticles (Tb-QD and Eu-QD with x = 6 nm or 12 nm) in three TG-detection windows give rise to RGB ratios specific to each type of nanoprobe. The four RGB codes were used to distinguish cells containing one of the four nanoprobes within one field of view by overlaying the three RGB images. (C) Co-attaching specific amounts of Tb complexes and dyes on a QD was used to produce temporally multiplexed nanohybrids with similar brightnesses, such that all luminescent barcodes could be imaged (microbeads with three different nanohybrids in R, G, B, and overlay images) with a single measurement without contrast adjustment. A and C adapted with permission from ref. 30. Copyright 2018 Wiley. A and C adapted with permission from ref. 3. Copyright 2019 American Chemical Society.

Figure 9 .

 9 Figure 9. In-situ RCA-TG-FRET for washing-free imaging of single actin proteins. (A) Schematic assay protocol, including immunochemistry, RCA, and incubation with Tb-DNA and dye-DNA FRET probes. (B) -actin expressed in HaCaT cells could be detected via TG-FRET after permeabilization, RCA protocol, and few minutes of incubation with the FRET DNA probes. TG-FRET provided more specific and background-free signals compared to TG Tb PL and continuous wave (CW) Cy3.5 dye PL without washing the cells after probe incubation. Scale bars: 10 µm. Reproduced with permission from ref. 57. Copyright 2020 American Chemical Society.

Figure 10 .

 10 Figure 10. Live-cell and in-vivo TG-FRET imaging. (A) TG-FRET nanohybrids consist of a central QD, to which different peptides, proteins, and FRET donors/acceptors can be attached for multiplexed sensing design. (B) Fourcolor TG-FRET imaging of COS-7 cells microinjected with Tb-QD nanoprobes composed of turquoise (tQD), orange (oQD), and red (rQD) QDs. (C) Three-color imaging of intracellular multistep TG-FRET from Tb-to-QD-to-dye (Alexa Fluor 647: AF647). (D) Three-color imaging of multistep TG-FRET from Tb-to-QD-to-FP (mCherry: mCh) upon intracellular assembly of transiently expressed FPs to Tb-QD nanohybrids. (E) Long-term Tb-to-QD TG-FRET imaging in developing zebrafish embryos. Scale bars 10 µm (B, C, D) and 100 µm (E). C adapted with permission from ref. 58. Copyright 2016 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. Distributed under a Creative Commons Attribution License 4.0 (CC BY) https://creativecommons.org/licenses/by/4.0/. A, B, D, E adapted with permission from ref. 4. Copyright 2020 Wiley.

  -free assay formats, and high photostability provide many opportunities for implementing TG-FRET into clinical diagnostics and bioanalytical research. TG-FRET can perform in-vitro, in-situ, and in-vivo analysis of low volume serum samples, living cells, tissues, and developing organisms with high spatial resolution, high selectivity, and high sensitivity.

ACKNOWLEDGEMENTS

This work has been supported by the Korean National Research Foundation, Seoul National University, the National Natural Science Foundation of China (No. 82104120), Université Paris-Saclay, Université de Rouen Normandie, INSA Rouen, CNRS, European Regional Development Fund, Labex SynOrg (ANR-11-LABX-0029), Carnot Institute I2C, XL-Chem graduate school (ANR-18-EURE-0020 XL CHEM), the Region Normandie, Institut Universitaire de France, and the China Scholarship Council.

SUPPORTING INFORMATION

The Supporting Information is available free of charge at … Additional Information concerning size, orientation, conformation, and proximity for optimizing