# Fluorescent Artificial Receptor for Dopamine based on Molecular Recognition-driven Dynamic Covalent Chemistry in a Lipid Nanoreactor

Bohdan Kozibroda, Jean-Marie Lehn, Andrey S. Klymchenko

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Bohdan Kozibroda, Jean-Marie Lehn, Andrey S. Klymchenko. Fluorescent Artificial Receptor for Dopamine based on Molecular Recognition-driven Dynamic Covalent Chemistry in a Lipid Nanoreactor. Angewandte Chemie International Edition, 2025, 64 (14), 10.1002/anie.202419905. hal-05079365

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# Fluorescent Artificial Receptor for Dopamine based on Molecular Recognition-driven Dynamic Covalent Chemistry in a Lipid Nanoreactor

Bohdan Kozibroda, Jean-Marie Lehn, and Andrey S. Klymchenko*

<figure><img src="image_2.png" /></figure>

**Angewandte**
International Edition Chemie
**Abstract:** Molecular recognition and detection of small bioactive molecules, like neurotransmitters, remain a challenge for chemists, whereas nature found an elegant solution in the form of protein receptors. Here, we introduce a concept of a dynamic artificial receptor that synergically combines molecular recognition with dynamic imine bond formation inside a lipid nanoreactor, inducing a fluorescence response. The designed supramolecular system combines a lipophilic recognition ligand derived from a boronic acid, a fluorescent aldehyde based on push-pull styryl pyridine and a phenol-based catalyst. The recognition ligand specifically captures dopamine inside lipid nanodroplets and thus triggers imine bond formation with the aldehyde, producing the emission color change. The rational design of the fluorescent aldehyde, the catalyst, and the recognition ligand allows dramatic acceleration of the imine bond formation required for rapid sensing of dopamine. The nanoprobe enables dopamine detection with micromolar sensitivity and single-nanoprobe imaging of dopamine gradients through its robust two-color ratiometric response. It displays remarkable selectivity without the interference of competing biogenic primary amines and biological media: blood serum, plasma, urine and cell lysate. The proposed concept of a dynamic artificial receptor offers a solution to the long-standing problem of molecular recognition and sensing of small molecules in complex biological media.

## Introduction

Molecular recognition and detection of small bioactive molecules by fluorescent probes is a challenging problem, which attracts significant interest.[1] In order to achieve high affinity and specificity for a target molecule, the probe needs to present a specific recognition unit(s) and high structural match with the target, which is not an easy task. Particularly important targets are neurotransmitters (NTs) such as dopamine, norepinephrine, serotonin, glutamate, GABA, etc.[2] These are small organic molecules playing a role of messengers within synapses connecting a neuron to another neuron, muscle and organ. They are essential in the

functioning of the central and peripheral neural systems, involving memory, attention, learning, emotion, movement, sensory functions, etc.[3] NTs are present in biological fluids, such as serum and urine, and can serve as biomarkers of neural disorders, such as depression,[4] Alzheimer's disease,[5] epilepsy,[6] etc. Therefore, their quantitative detection is important for both fundamental research and clinical diagnostics.[2a,7] However, it remains a challenge to design probes for NTs, because these are present in biological systems in relatively small concentrations (from sub-nanomolar to micromolar), being mixed with a variety of other molecules of similar structure.[2a] A number of molecular and nanoscale probes were developed for the detection of NTs,[2a,8] which include molecular probes bearing multiple recognition ligands,[9] specifically cleavable probes,[10] supramolecular probes based on macrocycles,[11] nanoparticles bearing recognition ligands[12] and molecular imprinted polymers.[13] However, generally, these methods suffer from complex protocols of preparation and lack of affinity and specificity to the target NT. These limitations concern also probes for dopamine, which is a key NT, playing a central role in cognitive and motor functions.[2a,3, 14] Despite significant research efforts in the development of fluorescent probes for dopamine, commercial dopamine sensors are not yet available on the market.[15]

An emerging solution is to exploit dynamic covalent chemistry (DCvC),[16] based on reversible covalent bonds of imines,[17] hydrazones, boronic acid esters,[18] dithiols,[19] etc. DCvC can generate constitutional libraries, which allows finding the key (active molecule) complementary to the lock (biomolecule).[20][21] Combining DCvC with molecular recognition allows shifting the equilibrium towards the formation of artificial receptors that could match the target of interest,[22] in particular inorganic anions[23] and cations.[24] Environment-sensitivity of dynamic covalent bonds opens the route to stimuli-responsive systems.[25][26] So far, examples of using DCvC for the detection of NTs are mainly limited to molecular probes.[2a,8] Indeed, an elegant design of fluorescent probes for dopamine was proposed, where a fluorescence dye was functionalized with both boronic acid and aldehyde groups that dynamically react with catechol and amino groups of dopamine, respectively.[9,27] However, this type of molecular probes presents poor affinity to the target. The use of DCvC with molecular probes in an aqueous environment is made difficult by the sensitivity of dynamic covalent bonds to hydrolysis, especially in the case of imines.[17] On the other hand, imines are particularly suitable for the recognition and detection of NTs, because most of them contain amino group.

The solution can be inspired by natural systems, which usually generate imine bonds in specific binding sites of proteins, such as rhodopsins,[28] retinol-binding protein,[29] and pyridoxal enzymes,[30] providing shielding effect from water. In synthetic systems, it could be realized using nanoreactors,[31] which are nanoscale compartments in the form of micelles,[32] emulsions,[33] polymerosomes,[34] etc. They confine the molecules of interest at high local concentration and isolate them from the external aqueous environment.[31] However, the idea of combining molecular

<footnote>[*] B. Kozibroda, A. S. Klymchenko
UMR 7021 CNRS, Laboratoire de Bioimagerie et Pathologies, ITI
SysChem-Chimie des Systèmes Complexes, Université de Strasbourg,
74 route du Rhin, 67401 Illkirch (France)
E-mail: andrey.klymchenko@unistra.fr</footnote>

<footnote>B. Kozibroda, J.-M. Lehn
Laboratoire de Chimie Supramoléculaire, Institut de Science et d'Ingénierie Supramoléculaires (ISIS), Université de Strasbourg, 8 allée Gaspard Monge, 67000 Strasbourg (France)</footnote>

<footnote>© 2025 The Author(s). Angewandte Chemie International Edition published by Wiley-VCH GmbH. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.</footnote>
recognition with DCvC in a synthetic nanoreactor to obtain an optically responsive system has not been explored so far. Particularly attractive here are lipid nanoemulsions (NEs), i.e. nanodroplets composed of oil core and surfactant shell.[33,35] They can be readily prepared by nanoemulsification from components that are generally recognized as safe (GRAS), which makes them particularly suitable for biological applications. Recently, we showed that dynamic covalent bonds such as hydrazone[36] and imine[37] can be effectively formed because the reactive components are concentrated in the non-aqueous environment, thus favoring dynamic bond formation. Moreover, we showed that a fluorescent aldehyde could change its emission color due to imine formation,[37] which could be an attractive analytical signal for NT detection. However, NTs are polar molecules and, therefore, are not expected to accumulate and form imine bonds inside the apolar core of lipid droplets. Here, we hypothesize that a molecular recognition ligand confined inside the droplet core can trigger effective capture of NTs, driving the imine bond formation. This implies that the imine bond would form in the droplet core only if the amine of interest matches the recognition ligand, which provides the basis for specific detection of NTs.

In the present work, we develop the concept of dynamic artificial receptor for NT capture and detection, which couples molecular recognition with imine bond formation inside a lipid nanoreactor (lipid droplet of NEs), driving the chromogenic (ratiometric) fluorescence response. It was realized for dopamine, which remains a challenging NT analyte for detection by fluorescent probes. The present DCvC nanoreactor is composed of a highly apolar fluorescent aldehyde and a boronic acid derivative encapsulated inside the oil core of lipid droplets. The fluorescent aldehyde was based on styryl pyridine, which displayed fast imine bond formation coupled with fluorescence response. We found that being loaded inside lipid droplets of NEs it displays a fluorescence response to dopamine only when the hydrophobic boronic acid derivative is present, indicating that the imine bond formation is triggered by molecular recognition of dopamine. Remarkably, the obtained nanoprobe is selective to dopamine without significant response to other NTs, including analogous catechol norepinephrine. The nanoprobe showed micromolar sensitivity to dopamine and remained fully functional in the presence of an excess of complex mixture of different NTs as well as in a variety of biological media: blood serum, plasma, urine, and cell lysate. The developed concept opens a new route to nanoscale molecular recognition and detection systems for small molecules.

## Results and Discussion

### Design Concept

The idea is to combine the recognition ligand (RL) that can specifically bind to the target NT (dopamine) and fluorescent aldehyde (FA) that would serve as a reporting unit that can form imine with the NT. Placing both components

within a lipid nanoreactor (lipid droplet of NEs) would confine them at high concentrations, thus ensuring their close proximity in an apolar environment. In this case, the presence of the target NT is expected to trigger a synergetic phenomenon: the recognition ligand could capture a relatively polar dopamine molecule into the apolar core of the nanoreactor, which would allow the imine bond formation with the fluorescent aldehyde required for the fluorescence response. The apolar core of the nanoreactor is expected to favor the imine formation and exclude any other amines (like peptides, proteins, or other biogenic amines), which are generally highly polar. Previously, imine bond formation was exploited in a dopamine molecular probe, which operated by a photoinduced electron transfer (PET) mechanism, providing fluorescence intensity response to catecholamines.[9a,b] We followed a different strategy: the aldehyde group plays a role of an electron acceptor in a push-pull fluorophore. After imine bond formation, its acceptor property would decrease and thus change the emission color of the fluorophore. This mechanism would allow designing a ratiometric probe for dopamine, something that was not reported before. This idea was supported by our previous observation that push-pull dye PA (Figure 1) shifted to blue its absorption and emission maximum on imine formation with a lipophilic amine loaded inside lipid nanodroplets.[37–38] Therefore, PA was selected for the present study. Moreover, we designed a styryl-pyridine aldehyde derivative (FA1, Figure 1), bearing long alkyl chains, in order to improve the response to the amines (see below). On the other hand, recognition of dopamine can be provided by boronic acid derivatives, as shown in some examples of molecular probes for dopamine.[9, 39] Here, we designed a recognition ligand (RL1, Figure 1) – a highly lipophilic boronic acid derivative, bearing orthodialkylamino-methyl group. The latter group is expected to ensure the tetrahedral configuration of boron, which favors its interaction with the catechol moiety of dopamine.[40]

### Fluorescent Aldehydes for DCvC with Amines

First, we tested a previously reported fluorescent aldehyde PA in a reaction with a model apolar amine (dodecylamine) in lipid droplets of NEs. The latter were formulated using Labrafac oil and Cremophor ELP surfactant by nanoemulsification method,[33a] which yielded NEs droplets of ~50-nm hydrodynamic diameter, according to dynamic light scattering (DLS, Table S3). The imine formation within NEs led to a blue shift of the emission (Figure 2a,c), in line with the previous report.[37] Even though the progress of the reaction was observed on the scale of hours, its completion required more than 24 hours, which is too slow to be used in sensing applications. As the imine formation can be catalyzed by H-bond donors, we studied a series of known and newly designed potential catalysts (Figures 2 and S1): aliphatic alcohol (catalyst C1), fluorinated aliphatic alcohol (catalyst C2), apolar phenol (catalyst C3); fatty acid (catalyst C4) and highly hydrophobic carboxylic acid (catalyst C5). Remarkably, catalysts C3 and C5 provided strong
<figure><img src="image_3.png" /><figcaption>**Figure 1.** Combining DCvC with molecular recognition inside a lipid nanoreactor (nanoemulsion droplet). (top). Chemical structures of fluorescent aldehydes (PA and FA1), recognition ligand (RL1), and neurotransmitter (dopamine) used in this work (bottom).</figcaption></figure>

<figure><img src="image_4.png" /><figcaption>**Figure 2.** Imine formation between fluorescent aldehyde (PA) and a model amine (dodecylamine) in situ inside lipid nanoreactor: effect of catalysts. (a) Scheme of the reactions of PA with dodecylamine inside NEs. (b) Structures of tested catalysts for the imine formation inside NEs. (c,d) Fluorescence spectra of PA inside NEs in the presence of dodecylamine for different incubation times without (c) and with catalyst C3 (d). (e) Comparison of reaction kinetics for the imine formation from PA and dodecylamine in NEs without and with tested catalysts. Kinetics curves reflecting blue/green intensity ratio in the fluorescence spectra vs incubation time. Dashed lines connect data points for guiding the eye, not for data fitting. Formulations of NEs (1 µL) without or loaded with catalysts C1-C5 (10 wt% vs the lipid core) were each diluted into 1 mL of PBS (pH = 7.4, no Ca²⁺, Mg²⁺), PA and dodecylamine were added from a stock solution in DMSO to a final concentration of 5 and 100 µM, respectively.</figcaption></figure>

acceleration of the reaction of the imine formation, as one could see in the evolution of the fluorescence spectrum (Figure 2d and S1f for catalysts C3 and C5, respectively) and the kinetics curves (Figure 2e). On the other hand, both tested alcohols (catalysts C1 and C2) showed little catalytic effect on the reaction, probably because their H-bond donor acidity was not sufficient. Thus, the presence of strong H-bond donors can dramatically improve the kinetics of imine formation in the low polar core of NEs. For the next steps,

we selected catalyst C3, because we preferred avoiding carboxylic acids (the case of catalysts C4 and C5), which shift thermodynamic equilibrium away from imine due to amine protonation.

We considered that the kinetics of imine formation could be further improved by designing an alternative fluorescent aldehyde. To this end, we placed the aldehyde group in *ortho*-position to the pyridine unit, which is expected to enhance the imine formation, bio-inspired from a pyridoxal
cofactor.[41] This pyridine-aldehyde unit was conjugated to a *para*-dialkylamino-styrene unit in order to generate a push-pull dye, where carbonyl and dialkylamino groups serve as electron acceptor and donor, respectively. The push-pull fluorophores with a 5-pyridine-2-carboxaldehyde acceptor unit have not been explored to date. The synthesis of FA1 involved four steps, starting from the alkylation of aniline with dodecyliodide (Figure 3a). The obtained tertiary amine was then reacted under Vilsmeier–Haack conditions to yield an aldehyde. The latter was then converted into a styrene derivative by a Wittig reaction. Finally, the styrene component was coupled with a 5-bromo-2-pyridinecarboxaldehyde by a Heck reaction to yield the target fluorescent aldehyde FA1.

In organic solvents of varied polarity, FA1 showed an absorption band centered around 414–442 nm with good extinction coefficient 41,200–42,700 m⁻¹cm⁻¹, without clear dependence on the solvent polarity (Figure S2, Table S1). In contrast, the emission band showed a strong bathochromic shift on increase in the solvent polarity from 454 nm in apolar cyclohexane to 665 nm in polar DMSO (Figure 3b). A linear correlation of band maximum (expressed in wave-numbers) vs empirical polarity index E<sub>T</sub>(30)<sup>[42]</sup> was observed (Figure 3c). These data suggest strong solvatochromism of FA1, which is clearly linked to its push-pull character, generally associated with an excited state-charge transfer state, highly sensitive to solvent polarity.[43] Moreover, FA1 displayed good quantum yields in the studied solvents ranging from 10 to 73 %, with the highest values in solvents of medium polarity (Table S1). In Labrafac oil, the quantum yield and the emission maximum of FA1 were close to that in apolar 1,4-dioxane. Based on the linear fit (Figure 3c), the estimated E<sub>T</sub>(30) index of Labrafac was 36.1, the corre-sponding low polarity of this medium-chain triglyceride oil. Importantly, NEs encapsulating FA1 showed good fluorescence quantum yield (52 %) and emission maximum close to that in neat Labrafac (Figure 3b). The estimated low value of polarity index (E<sub>T</sub>(30)=36.9. Figure 3c) suggested that FA1 is well encapsulated inside the apolar oil core of NEs. In vitamin E acetate (VEA) oil, the emission spectra of FA1 were blue shifted by 37 nm compared to Labrafac (Figure S3), clearly due to its lower polarity

(E<sub>T</sub>(30)=32.9, Figure 3c). In NEs with VEA oil core, the emission of FA1 displayed nearly the same blue shift (36 nm), which confirmed that FA1 is rather located in the oil core of NEs, revealing its low polar environment with E<sub>T</sub>(30)=33.9 (Figure 3c). Finally, in micelles made exclu-sively of surfactant Cremophor ELP (12 nm sized, according to DLS, Table S3), the emission of FA1 was significantly red-shifted compared to NEs containing Labrafac and VEA (by 14 and 50 nm, respectively, Figure S3), indicating much higher local polarity (E<sub>T</sub>(30)=38.0). This result provides additional confirmation that FA1 is mainly located in the apolar oil core rather than in a more polar interface of the surfactant. This was expected given that FA1 was designed with two highly lipophilic dodecyl chains.

Next, we studied the reactivity of FA1 encapsulated inside NEs with dodecylamine. As for PA dye, the presence of amine led to the blue-shifted emission of FA1 (Figure 4a), indicating the formation of the imine that reduces the electron acceptor character of the aldehyde. Importantly, without any catalyst, the reaction proceeded much faster, so that it was completed within ~1 h (Figure 4a,c). This dramatically faster reaction kinetics can be explained by the presence of pyridine nitrogen in *ortho*-position to the aldehyde group. The addition of catalyst C3 further accelerated the reaction so that it was completed within nearly 20 min (Figure 4b,c). Overall, the rational design of the fluorescent aldehyde and appropriate catalyst allowed us to improve the timescale of the imine formation from ~24 h to ~20 min, which corresponds to >60-fold improvement in the reactivity. Given that in the case of FA1-loaded NEs, the dodecylamine concentration used was 5 times lower compared to that used for PA-loaded NEs, the improvement in the reactivity of FA1 vs PA is even higher.

To understand better the role of the lipid nanoreactor, we tested the reactivity of our FA1 encapsulated into NEs with primary amines of different lipophilicity (alkyl chain length). Remarkably, the color response clearly depended on lipophilicity of the amines (calculated LogP, CLogP): in the concentration range from 1 to 1000 µM, polar butyl-amine (ClogP=0.923) did not induce any color response of FA1, octylamine (ClogP=3.039) – medium response, while dodecylamine (ClogP=5.155) induced the strongest re-

<figure><img src="image_5.png" /><figcaption>**Figure 3.** Synthesis of new fluorescent aldehyde (FA1) and its solvatochromic properties. (a) Scheme of synthesis of the FA1. (b) Normalized fluorescence spectra of FA1 in solvents of different polarity and in Labrafac oil and Labrafac NEs. (c) Calibration of the band maximum position (expressed in wavenumbers) of FA1 vs empirical polarity parameter E<sub>T</sub>(30). Data points for FA1 in vitamin E acetate (VEA) oil, VEA NEs, Labrafac, Labrafac NEs, and Cremophor ELP micelles were placed on the curve.</figcaption></figure>
<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /><figcaption>**Figure 4.** Reactivity of FA1 with amines inside NEs. (a,b) Fluorescence spectra of FA1 inside NEs in the presence of dodecylamine for different incubation times without (a) and with catalyst C3 (b). (c) Comparison of reaction kinetics for the imine formation from FA1 and dodecylamine in NEs with and without catalyst. Kinetics curves reflecting blue/green intensity ratio in the fluorescence spectra vs incubation time. Dashed lines connect data points for guiding the eye, not for data fitting. Formulations of NEs (1 µL) loaded with FA1, with or without catalyst C3 (9 wt% vs the lipid core) were each diluted into 1 mL of PBS (pH = 7.4, no Ca²⁺, Mg²⁺). FA1 loading was 1.8 and 1.7 wt% vs the lipid core for formulations without and with catalyst C3. The final concentration of FA1 in a mixture was 5 µM. Dodecylamine was added from a stock solution in DMSO to a final concentration of 20 µM. (d-f) Fluorescence spectra of NEs loaded with FA1 (0.17 wt% vs the oil core) with catalyst C3 (9 wt% vs the lipid core) in the presence of primary amines of different lipophilicity: butylamine (d), octylamine (e) and dodecylamine (f). NEs were incubated with or without the amines (1, 10, 100, 1000 µM) for 2 h at rt.</figcaption></figure>

sponse, observed as a blue-shifted emission (Figure 4d-f). Thus, the lipid nanoreactor not only favors imine bond formation, but also prevents polar amines with ClogP < 1 from interaction with FA1. Importantly, the vast majority of amines naturally present in biological systems, including NTs, are polar species (Figure 5b) and should be systemati-cally excluded from the lipid nanoreactor, as it is shown for butylamine. Therefore, we expect that implementing a capture mechanism through molecular recognition will trigger the phase transfer of a given polar NT into the oil core of NEs and, thus, drive NT-specific fluorescence response.

Combining DCvC with Molecular Recognition in Lipid Nanoreactor

In the final step, we designed the recognition ligand RL1 (Figure 1), which contains a boronic acid for reaction with the catechol group of dopamine and two alkyl chains to ensure its localization inside the oil core of the lipid nanoreactor. It was synthesized in one step by reductive amination by reacting 2-formylphenylboronic acid with didodecylamine in the presence of sodium triacetetoxyborohydride (see SI). RL1 was co-encapsulated with FA1 and

catalyst C3 inside the lipid nanoreactor (0.17 wt% of FA1, 27 wt% of RL1, 9 wt% of catalyst C3 vs oil core) and the obtained supramolecular probe was studied in the presence of biologically relevant molecules bearing primary amino group (Figure 5). In the presence of dopamine, the resulted nano-formulation displayed a characteristic blue shift in the emission spectrum of FA1, which significantly evolved on the time scale of 1 h (Figure 5d). In sharp contrast, control conditions without RL1 led to no spectral shifts in the presence of dopamine. These results suggest that recognition ligand RL1 captures dopamine into the core of the lipid nanoreactor, which triggers the reaction with FA1. To verify whether this system provides specific recognition of dop-amine, we studied the optical response to other amines, including NTs GABA, serotonin, histamine, noradrenaline, and two biogenic primary amines structurally similar to dopamine: tyramine, 4-hydroxy-3-methoxyphenetylamine. Remarkably, most of them did not provide a significant optical response, at the same studied conditions, suggesting specificity of our system to the target dopamine (Figures 5e and S4). The only exception was noradrenaline, which displayed a small optical response (Figures 5e and S4f). The latter can be explained by the presence of a catechol unit, which could be also recognized by RL1. However, the spectral response was much weaker compared to dopamine,
<figure><img src="image_12.png" /><figcaption>**Figure 5.** Supramolecular nanoprobe for dopamine combining new fluorescent aldehyde (FA1), catalyst C3, and the recognition ligand (RL1) inside lipid droplet nanoreactor. (a) Principle of the nanoprobe for dopamine, which recognizes the catechol unit of dopamine and changes the emission color due to the formation of an imine bond with the fluorescent aldehyde. (b) The set of biogenic primary amines tested in this work. CLogP is shown below the names of the compounds. (c, d) Fluorescence spectra of NEs loaded with fluorescent aldehyde without (c) and with (d) RL1 (27 wt% vs the oil core) in the presence of dopamine (1 mM) for different incubation times. (e) Ratiometric response of the nanoprobe to dopamine and other tested biogenic primary amines vs incubation time, showing molecular specificity to the former. Dashed lines connect data points for guiding the eye, not for data fitting. Formulations of NEs (10 µL) loaded with FA1 (0.17 wt% vs the oil core), catalyst C3 (9 wt% vs the oil core), without (control) or with RL1 (27 wt% vs the oil core) were each diluted into 1 mL of PBS (pH = 7.4, no Ca²⁺, Mg²⁺). Tested amines were added from stock solutions in milli-Q water to 1 mM final concentration. (f) Spectral response of the nanoprobe to dopamine and a series of 16 bioactive molecules. NEs were diluted (200x) into PBS (pH = 7.4, no Ca²⁺, Mg²⁺, + 0.5 mM Na₂SO₃) and incubated with a bioactive molecule (1 mM) for two hours at room temperature.</figcaption></figure>

which is probably because this molecule is more polar than dopamine, so its capture by RL1 could be less efficient. In addition, RL1 could possibly complex the ethanolamine fragment of noradrenaline, and thus inhibit the imine formation. Building on these results, we tested 10 additional bioactive molecules: other known NTs, like amino acids and acetylcholine, the precursor of dopamine – DOPA, and some diols that could react with boronic acid. None of these species induced a detectable spectral response of our dopamine-sensing system, confirming its remarkable selectivity. One should note that ClogP for all tested biogenic amines is systematically below 1 (Figure 5b), which excludes them from the NEs oil core, and explains the absence of the optical response. Thus, our supramolecular system, combining DCvC with molecular recognition in the lipid nanoreactor, operates as a dynamic artificial receptor and fluorescent nanosensor (nanoprobe) that provides specific recognition to the target dopamine.

To understand better the spectral response of our nanoprobe to dopamine, we performed additional spectroscopy analysis. The absorption spectra of the nanoprobe also showed blue shifts in the presence of dopamine, suggesting a chemical transformation of the FA1 dye (Figure S5). Moreover, in the presence of dopamine, the excitation spectrum recorded at the maximum corresponding to the imine was blue-shifted compared to that recorded

at the emission of FA1 (Figure S5). The emission spectra showed excitation dependence, where blue-shifted excitation (corresponding to the imine of FA1) led to blue-shifted emission (Figure S5). Overall, the optical spectroscopy analysis suggested that the imine formation between FA1 and dopamine caused the observed spectral shifts. The chemical transformation of FA1 inside the nanoprobe in the presence of dopamine was confirmed by thin-layer chromatography. The reaction mixture of the nanoprobe with dopamine reduced with sodium borohydride (to convert unstable imine into stable amine) showed the formation of new emissive species with lower Rf (Figure S6), in contrast to the control NEs without RL1.

To provide direct proof that this reaction corresponds to the imine formation, we studied NMR spectra of the FA1 alone, FA1 with dopamine, and FA1 with RL1 and dopamine in a d6-DMSO and CDCl3 solvent mixture. To ensure a reaction between FA1 and dopamine similar to that in the lipid nanoreactor, high concentrations (5 mM) of all components were used, compatible with NMR measurements. While FA1 showed a characteristic peak of aldehyde proton at 9.97 ppm (peak Ha, Figure 6a), the presence of dopamine led to the appearance of a new peak at 8.22 ppm, characteristic of imine (peak Hd, Figure 6a). On the other hand, the peak corresponding to the proton in the aromatic pyridine also showed a shift from 8.81 to 8.63 ppm (peaks
<figure><img src="image_13.png" /><figcaption>**Figure 6.** NMR and optical characterization of imine formation between FA1 and amines. (a) ¹H NMR of FA1, FA1 with dopamine and FA1 with dopamine and RL1 in a mixture of d6-DMSO and CDCl3 (1/2, v/v) at 5 mM concentration. Absorption (b) and fluorescence (c) spectra of the FA1 and corresponding reaction mixtures with dopamine after their rapid dilution in dioxane to 5 µM. The excitation wavelength was 400 nm. (d) Fluorescence spectra of the reaction mixture of FA1 (2.5 µM) with biogenic amines (1 mM) in 1,4-dioxane with 10 vol% of DMSO in presence of catalyst C3 (0.16 mM), RL1 (0.46 mM), and trimethylamine (3 mM), corresponding to the final concentrations of components (FA1, RL1, and catalyst C3) in the 200-fold diluted NEs.</figcaption></figure>

Hb and Hc, respectively, Figure 6a). When the integral of the reference TMS was normalized to 100, the integrals of the aldehyde (Ha) and aromatic (Hb) protons decreased from 27 and 28, respectively, in neat FA1 to 3 in the presence of dopamine, while the new imine peaks (Hc) and (Hd) displayed the integral of 28 (Figure 6a). This result suggested that aldehyde FA1 was converted to an imine in the presence of dopamine. Very similar NMR spectral changes were observed in the presence of both dopamine and RL1, indicating that the recognition ligands do not influence the imine formation when studied in homogeneous solvent conditions (Figure 6a). Moreover, the methylene proton of RL1 displayed a shift from 3.65 to 4.09 ppm in the presence of dopamine, which could be explained by the formation of the boronic acid ester with the catechol (Figure S7). Importantly, the absorption and fluorescence spectra of rapidly diluted solutions from the reaction mixtures showed significantly blue-shifted maxima for both conditions where dopamine was present (Figure 6b,c). To make a systematic study of the optical properties of the imine of FA1 in different solvents, we incubated FA1 with a 2-fold mole excess of dodecylamine in deuterated chloroform. ¹H NMR confirmed the complete transformation of FA1 into the imine: the integrals of the characteristic proton peaks of the imine matched that of the remaining one mole equivalent of dodecylamine, whereas the corresponding peaks of the FA1 were absent (Figure S8). This stock was further used to prepare diluted solutions in nine solvents of varied polarity, which were studied by absorption and fluorescence spectroscopy immediately after preparation. We found that the absorption and emission spectra of the FA1 imine were systematically blue-shifted compared to those of FA1 aldehyde for all studied solvents (Table S4 vs Table S1). The emission maximum of the FA1 imine showed red shifts with an increase in the solvent polarity, indicating that it preserved solvatochromic properties (Figure S9, Table S4). However, this red shift was weaker compared to FA1 aldehyde: from apolar cyclohexane to polar DMSO, the red shift was 139 nm (5730 cm⁻¹) for FA1 imine vs 211 nm (6990 cm⁻¹) for FA1 aldehyde. Moreover, for the most polar solvents (e.g. acetonitrile and DMSO), FA1

showed a decrease in the fluorescence quantum yield (Table S1), typical for push-pull dyes,[43] whereas FA1 imine did not show this effect (Table S4). The observed blue shifts, the weaker solvatochromism, and no quenching in polar solvents indicate the lower push-pull character of the imine, clearly because the imine group is a weaker electron acceptor than the aldehyde group.

Thus, our data provide direct proof that the imine formation between FA1 and dopamine, identified by NMR, is responsible for the blue-shifted emission. In solution, this imine formation does not require the presence of RL1 because both FA1 and dopamine are present in a water-free medium, which ensures the reaction. We further confirmed that by studying 7 biogenic amines in the organic solvent mixture (1,4-dioxane with 10% of DMSO) in the presence of RL1. All studied amines, except GABA showed blue-shifted emission (Figure S10), suggesting that selectivity to dopamine was totally lost in a homogeneous solvent medium despite the presence of the recognition ligand RL1. The absence of response in the case of GABA is probably because this amino acid is present in a zwitterion form that deactivates its amino group, while in the case of histamine, the stronger shift is probably because imidazole favors imine bond formation in solution. In sharp contrast to the results in organic solvents, in the lipid nanoreactor with RL1, the reaction of FA1 is only possible with dopamine (but not other tested bioactive amines), because RL1 ensures specific capture of rather polar dopamine into the oil core from the aqueous medium. Thus, the present supramolecular system combining molecular recognition and DCvC within a lipid nanoreactor displays a synergistic process, where the capture of dopamine by RL1 leads to the optical response of FA1.

To validate the capacity of the nanoprobe to detect quantitatively dopamine, we first performed the titration of the nanoprobe with increasing concentrations of dopamine from 1 to 1000 µM. Sodium sulfite was added to a physiological medium (PBS buffer) to minimize the oxidation of dopamine by dissolved oxygen. Remarkably, we observed a clear dose effect, where at small concentrations of dopamine a shoulder at around 450 nm appeared and grew up to become a dominating band at higher concen-
trations (Figure 7a). The quantification of the nanoprobe response was done by measuring the ratio of the blue to red parts of the spectra, which correspond to emission maxima of imine and aldehyde forms of FA1, respectively (Figure 7b). We observed a linear relation of the blue/red intensity ratio vs dopamine concentration range 0–100 µM (Figure S11). The estimated limit of detection (LoD) was 11 µM. Then, the titration was performed in the presence of all mentioned amines mixed together (100 µM each) resulting in a very similar titration curve (Figures 7b and S11), indicating that the detection of target dopamine can be done in a complex mixture of other amines of close structure. Moreover, in the presence of 10% fetal bovine serum (FBS), which is a complex mixture of biomolecules, including proteins and small molecular metabolites derived from blood, the nanoprobe worked well showing similar titration curve vs dopamine concentration (Figures 7b and S11). The obtained LoD values were 4 and 12 µM for the mixture of competitor amines and with 10% FBS, respectively. This indicates that a highly complex biological medium does not really interfere with the detection of dopamine by our supramolecular nanoprobe. Moreover, this result also suggests that the nanoprobe resists to the presence of proteins and other components of FBS. Indeed, a comparison of the nanoprobe in a buffer with and without 10% FBS revealed no changes after 2 h of incubation, confirming that components of serum do not produce any non-specific response of the nanoprobe (Figure S12). Moreover, free FA1 dye in 10% FBS displayed weak and strongly redshifted emission compared to the nanoprobe in the same medium (Figure S12). Therefore, we can also conclude that the encapsulated dye FA1 does not leak from the nanoprobe into FBS, otherwise it would lead to significant changes in its emission maximum. The observed resistance to dye

leakage in FBS is expected because of the very high lipophilicity of this dye (ClogP = 15.06), which, according to our previous studies on NEs, ensures stable encapsulation of lipophilic dyes inside the oil core without dye leakage in biological media.[44]

Ultimately, to further challenge the nanoprobe, we extended the pallet of tested biological liquids to bovine plasma, bovine urine, porcine urine, and cell lysate (obtained by lysis of HEK293T cells). Moreover, this time the measurements were done on an automated plate reader using much smaller volumes (100 µL vs 1 mL) in a 96-well plate, which allowed fast high-throughput measurements and smaller sample consumption. Remarkably, according to seven replicates for each studied biological fluid, the nanoprobe showed a practically linear response to dopamine in the concentration range from 1 to 100 µM of dopamine (Figure 7c). At higher concentrations (from 100 µM to 1 mM) the probe showed deviation from linearity downwards, probably due to saturation effects (Figure S13). One should also note that in bovine urine (and to a lower extent in porcine urine), the dependence on the dopamine concentration was steeper, which could be explained by the favorable effects of some components of urine. In any case, none of the tested complex biological fluids inhibited the response of the nanoprobe to dopamine, although quantitative detection of dopamine in these media would require a dedicated titration in each case. Indeed, the obtained LoD values in these media were similar to that in the control PBS: 1.1, 2.8, 2.3, 1.8, and 0.9 µM for fetal bovine serum, bovine plasma, bovine urine, porcine urine, and cell lysate, respectively, compared to 1.5 µM for control PBS. One should stress that the probe worked well even in cell lysate (concentration of the total protein was 0.061 mg/mL after dilution from the cell lysate stock, Figure S14), which is a

<figure><img src="image_14.png" /><figcaption>**Figure 7.** The titration of the supramolecular nanoprobe with dopamine and its operation in different biological media. (a) Fluorescence spectra of the nanoprobe titration with increasing concentrations of dopamine (2 h incubation), (b) The titration curves obtained by a spectrofluorometer reflecting blue/green intensity ratio in the fluorescence spectra vs dopamine concentration in three different media in PBS (pH 7.4) with: (1) 0.5 mM sodium sulfite, (2) 0.5 mM sodium sulfite and a mixture of studied biogenic primary amines as competitors (GABA, serotonin, histamine, noradrenaline, tyramine, 4-hydroxy-3-methoxy phenethylamine at 100 µM concentration each), and (3) 10% of fetal bovine serum. (c) The titration curves obtained by a plate reader in 96-well plate reflecting blue/green fluorescence intensity ratio vs dopamine concentration in different PBS containing (pH 7.4) media with: (1) 0.5 mM sodium sulfite, (2) 10% of fetal bovine serum (3) 10% of bovine plasma, (4) 50% of porcine urine, (5) 50% of bovine urine, (6) 5% of cell lysate (final concentration of the total protein: 0.061 mg/mL). Formulations of NEs (44 µL) loaded with FA1 (0.17 wt% vs the oil core), RL1 (27 wt% vs the oil core), and catalyst C3 (9 wt% vs the lipid core) were each diluted into 8.8 mL of the corresponding medium. The final concentration of FA1 was 2.5 µM. Resulting solutions were deposited into (a, b) cuvettes (1 mL) or (c) wells (100 µL) of the 96-well plate. Dopamine was added from stock solutions in milli-Q water to obtain a varied final concentration. Error bars correspond to standard deviation based on three (b) or seven (c) replicates.</figcaption></figure>
particularly complex mixture of biomolecules, reflecting the composition of the cell interior. These results highlight the exquisite selectivity of the nanoprobe to dopamine with minimal interferences even in highly complex biological fluids.

## Optimizations of the Nanoprobe

Next, we investigated whether the developed nanoprobe has the potential to be improved in terms of sensitivity and response speed, which is important for biological applications in the NTs detection.[1-2] To this end, we varied the following parameters: the size of the nanoprobe, its dilution, the external pH, and the nature of the recognition ligand. To compare the response values of the nanoprobes, we performed all experiments on a plate reader and analyzed change in their emission ratio as (R-R₀)/R₀, where R and R₀ are blue/green ratios in the presence and absence of dopamine, respectively. We found that variation of the hydrodynamic diameter from 48 to 148 nm did not affect the nanoprobe response to dopamine (Figure S15a), while the increase in its dilution produced a systematic increase in the response (Figure S15b). The latter could be explained by the increase in the ratio of dopamine to FA1, pushing the reaction towards the imine formation. The most dramatic effects were observed as a function of pH: the response to dopamine increased >3-fold at pH 9.4, but was almost completely lost at pH 6.4 (Figure S15c). We assume that higher pH increases the fraction of non-protonated dopamine in solution, which shifts the equilibrium towards the imine formation, leading to a stronger optical response. The lower pH produces exactly the opposite effect, favoring the protonation of dopamine and decreasing the stability of the pH-sensitive imine bond.[17] To improve the capture of dopamine, we also designed an analogue of boronic acid, without an amino group forming the intramolecular coordination bond with boron. Surprisingly it produced no ratio response to dopamine, whereas in the presence of lipophilic tertiary amine (N,N-dimethyldodecylamine) a rapid increase in the ratio value was observed in the presence of dopamine (Figure S16). The latter confirms that amine is important to activate boronic acid towards catechol.[18,40] Remarkably, the kinetics of the response was much faster compared to nanoprobe based on RL1 (without or with lipophilic tertiary amine, Figure S16). The response of the nanoprobe with RL2 was already detected after <10 s of addition of dopamine, which showed that nanoprobe response speed can be largely improved. Importantly, we confirmed that this faster nanoprobe preserved selectivity to dopamine in comparison to other biogenic amines (Figure S17). Then, we tested the sensitivity of our original and two improved nanoprobes using the plate reader. For all three nanoprobes, the titration gave linear correction of the intensity ratio vs dopamine concentration in the range until 50 µM (Figure S18). The obtained LoD for the original nanoprobe was 2.4 µM (average value from curves in Figures 7c and S18), whereas at pH 9.4 the LoD improved to 0.6 µM. The nanoprobe with RL2 displayed a LoD of 5.4 µM, which was

close to that of the original one, but it allowed much faster detection (20 min incubation vs 2 h). Thus, higher pH enhanced the sensitivity, and changing the recognition ligand improved kinetics while keeping a similar sensitivity.

## Imaging Dopamine Gradients at the Single Nanoprobe Level

Finally, we explored the potential of these nanoprobes to image the distribution of dopamine by fluorescence microscopy. In order to access imaging at the single droplet level, we tested the original nanoprobe of 48-nm hydrodynamic diameter as well as larger nanoprobes (106 and 148 nm). The use of larger particles is expected to improve their brightness, proportional to the cube of their radius,[45] and to decrease their diffusion constant, both important for single-particle detection. They were placed in solutions with and without dopamine and imaged using two detection channels, corresponding to the blue and green parts of their emission spectrum. All tested nanoprobes showed a strong response to dopamine according to the imaging experiments, with emission color changing from red to blue (Figure S19), corresponding to ~3-fold change in the detected blue/green ratio. However, for small nanoprobes, the solutions appeared homogeneous, while for the largest ones the particles could be detected, clearly because they are brighter and they diffuse slower. To further minimize diffusion of the nanoprobes, we used 2% agarose gel. Importantly, after 4000-fold dilution from the original formulation, individual particles could be observed, presenting a similar blue/green emission ratio corresponding to the orange pseudo-color (Figure S20). In the presence of dopamine, all particles switched to a blue pseudo-color, which reflected a much higher blue/green ratio (Figure S20). Thus, despite this strong dilution, the nanoprobes remained fully functional, allowing dopamine detection at the single nanodroplet level. Then, we artificially created a gradient of dopamine by injecting its 1 mM solution into one end of a microfluidic channel filled with nanoprobes in a gel of 1% agarose (Figure 8a). To verify the presence of the gradient, a reference solution of rhodamine B was used in the neighbor channel, where we could see that the color density decayed progressively and disappeared before the middle of the channel (Figure 8a). Then, we imaged the nanoprobes by the same two-color ratiometric method every 2 mm along the channel. In the area where dopamine was deposited, we observed blue NPs corresponding to the maximal response of the probe to dopamine (Figure 8b). Then, shifting towards decreasing concentrations of dopamine resulted in a gradual change of the emission color to blue-green, green, yellow, and then orange (Figure 8b). The distribution of the blue/green emission ratio obtained from these images confirmed the gradual ratio decrease on moving from the point of dopamine addition towards the areas of lower dopamine concentrations within the microfluidic channel (Figure S21). This gradual change corresponded well to the decay of color density for the reference dye rhodamine B (Figure 8a), suggesting that we can follow the gradient of dopamine using the ratiometric response of our nanoprobes.
<figure><img src="image_15.png" /><figcaption>**Figure 8.** Imaging of the dopamine concentration gradient by the nanoprobes (148 nm diameter) immobilized in an agarose gel. (a) Photo of a microfluidic device with created gradients of dopamine and Rhodamine B (model). The nanoprobes were added (2000-fold dilution) to agarose solution (1% of agarose in PBS (pH = 7.4, no Ca²⁺, Mg²⁺ + 0.5 mM Na₂SO₃) at 50°C, after which it was deposited into separate microfluidic channels (60 µL into each). The nanoprobe composition: 0.17 wt% of FA1, 27 wt% of RL1, 9 wt% of catalyst C3 vs oil core. After jellification at room temperature, dopamine solution (1 mM) in the PBS buffer or rhodamine B (1 mM) solution in the PBS buffer (40 µL each) was added to reservoirs on one side of the channels. (b) Confocal microscopy images, representing the blue/green intensity ratio of nanoprobes, recorded each 2 mm along dopamine concentration gradient after two hours at room temperature. 0 mm point corresponds to the center of the channel inlet where dopamine was deposited.</figcaption></figure>

## Conclusions

In the present work, we introduce a concept of capture and detection of a small molecule (neurotransmitter dopamine), which couples molecular recognition with imine bond formation inside a lipid nanoreactor (lipid nanodroplet), driving the ratiometric fluorescence response. To this end, we designed a nanoreactor that contains highly lipophilic fluorescent aldehyde and boronic acid derivative encapsulated inside the oil core of lipid nanodroplets. The fluorescent aldehyde, based on push-pull styryl pyridine dye (FA1), displayed fast imine bond formation inside the nanoreactor coupled with fluorescence response, in contrast to previously reported pyrene-based push-pull aldehyde displaying much lower reactivity. Moreover, the imine bond formation with these fluorescent aldehydes can be accelerated by co-encapsulated lipophilic H-bond donor (acidic) catalysts, in particular, a phenol derivative. Due to the strong solvatochromism of the push-pull dye FA1, we identified a very low local polarity inside the nanoreactor. The latter was crucial for excluding polar amines (like model butylamine and all tested biogenic amines) and favoring imine bond formation with amines capable to partition into the low-polar oil core of the nanoreactor. The lipophilic boronic acid derivative was designed as a recognition ligand (RL1) to capture dopamine in the oil core of the nanoreactor through the formation of boronic ester bonds with the catechol unit. We found that the fluorescent aldehyde FA1 and the recognition ligand RL1 loaded together inside the lipid nanodroplets display a fluorescence response to dopamine (emission blue shift), whereas the control system without the recognition ligand does not sense dopamine. This result indicates that the imine bond formation is triggered by the molecular recognition of dopamine inside the lipid nanoreactor by RL1 that ensures the transfer of dopamine into its oil core. The imine bond formation, triggering the blue shift of FA1, was confirmed by ¹H NMR,

while the systematic optical spectroscopy analysis of the FA1 imine suggested that the blue shift is linked to a weaker acceptor capacity of the imine group vs the aldehyde group. Thus, using a lipid nanoreactor in an aqueous medium, we achieved a control of dynamic imine chemistry through an encapsulated catalyst, molecular recognition ligand, and an analyte (dopamine). Remarkably, the obtained supramolecular nanoprobe was selective for dopamine without significant response to other NTs, including analogous catechol norepinephrine, and a large panel of other relevant small molecules. Therefore, our supramolecular system operates as a dynamic artificial receptor and a nanoprobe with a specific optical response to dopamine. The nanoprobe showed micromolar sensitivity to dopamine and remained fully functional in the presence of an excess of a complex mixture of different NTs. Moreover, the nanoprobe remained fully functional with preserved micromolar sensitivity in a variety of complex biological media: fetal bovine serum, bovine plasma, bovine urine, porcine urine, and cell lysate. The achieved remarkable selectivity could be explained by the synergic effects of molecular recognition with dynamic imine bond formation, conditioned by the hydrophobic oil core of the nanoreactor. The latter prevents other polar molecules with amino groups (including NTs, amino acids, and proteins with CLogP below 1) from entering the nanoreactor and forming imine bonds with the fluorescent aldehyde. Thus, based on this phase-transfer molecular selection, only dopamine, specifically captured by the recognition ligand inside the nanoreactor, could produce the fluorescence response of the nanoprobe. This finding points to the great potential of the present approach to recognize and sense small molecules in complex biological media. To explore the potential of the nanoprobe, we also varied its size, concentration, external pH, and the recognition ligand. We found that higher pH can significantly improve the sensitivity of the nanoprobe (with LoD = 0.6 µM), while the rational design of the recognition ligand can further improve
the response speed to the seconds time scale. Finally, fluorescence microscopy of immobilized nanoprobes revealed their capacity to detect dopamine at the single-particle level by robust ratiometric detection method and image gradients of dopamine created in a microfluidic channel. The reported concept of dynamic artificial receptor is expected to yield advanced nanoprobes for detection and imaging of NTs, hormones, metabolites, and other bioactive molecules in vitro and in vivo. However, prior to challenging this type of nanoprobe with other biological applications, further work on improving the sensitivity of the nanoprobes towards the nanomolar range will be needed, which could be realized by designing improved recognition ligands in combination with fluorescent aldehydes and tuning properties of the nanoreactor. Such research is ongoing and will be reported in due course. Overall, this work addresses the fundamental challenges in the field of molecular recognition and sensing of small molecules. It shows the route to “Lego”-like nanoprobes built from individual recognition and fluorescent units with programmable selectivity and sensitivity to bioactive molecules.

## Acknowledgements

This work was supported by the USIAS (University of Strasbourg Institute for Advanced Study), the Fondation Jean-Marie Lehn as well as the Interdisciplinary Thematic Institute SysChem, via the IdEx Unistra (ANR-10-IDEX-0002), the CSC Graduate School (CSC-IGS ANR-17-EURE-0016) within the French Investments for the Future Program and Agence Nationale de la Recherche (ANR) AmpliSens ANR-21-CE42-0019-01 and SenEmul ANR-22-CE18-0034. We acknowledge IRCAD (France) for providing one of the biological fluids and Rémi Pelletier for help with the cell lysate preparation.

## Conflict of Interest

The authors declare no conflict of interest.

## Data Availability Statement

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

**Keywords:** dynamic artificial receptor • dynamic imine chemistry • fluorescent probes • molecular recognition • neurotransmitter detection

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Manuscript received: October 15, 2024

Accepted manuscript online: January 13, 2025

Version of record online: February 3, 2025