Dual-site recognition leads to a ratiometric fluorescent probe for norepinephrine: quantitative detection and visualization for in-depth understanding of hypertension

Abstract

Hypertension is closely associated with the activation of sympathetic nervous system (SNS). Norepinephrine (NE) belongs to the catecholamine family and is the primary neurotransmitter of SNS. Plasma and tissue NE levels serve as biomarkers of sympathetic overdrive in hypertension, providing measurable indicators to guide therapeutic approaches. However, there are only a few reliable tools to simultaneously quantify plasma NE and visualize tissue NE for comprehensive SNS assessment in hypertension. Herein, to selectively recognize the unique L-hydroxyethylamine and catechol moieties of NE, we designed and synthesized a dual-site fluorescent probe that can specifically bind to these two characteristic groups. The recognition process generates a macrocyclic-ring enhanced by silver bridging, accompanied by a ratiometric fluorescence change. The probe enables quantitative NE detection in both plasma and urine, as well as NE visualization in heart, kidney and adrenal gland tissues. Notably, our results demonstrated that spontaneously hypertensive rats (SHR) have higher circulating and tissue NE levels, indicating SNS overactivation. These findings provide a powerful molecular tool for the assessment of SNS activity and offer an in-depth understanding of hypertension.

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Introduction

Blood pressure has long been regarded as a typical cardiovascular function, which is closely associated to sympathetic nervous system (SNS) activity.^1,2 Norepinephrine (NE), a member of the catecholamine family (including dopamine, norepinephrine and epinephrine) (Fig. 1a), is the primary neurotransmitter of SNS.^3–8 Upon sympathetic activation, postganglionic neurons release NE, which acts on the target organs to modulate cardiovascular function and contribute to the blood pressure through adrenergic receptor signaling.^9–15 The release of NE is directly correlated with neural activity, making NE a reliable indicator for evaluating SNS activity.^16,17 Plasma and tissue NE levels serve as biomarkers of sympathetic overactivation in hypertension, and their quantification can guide the development of therapeutic strategies.^18–20 However, the low amount, easy oxidation and multiple interferences for NE make the accurate detection challenging. To date, high performance liquid chromatography (HPLC),^21,22 enzyme-linked immunosorbent assays (ELISA) and electrochemical methods^23–26 have been used for the quantification of NE, but they are limited by sensitivity and poor spatial resolution. In contrast, a promising alternative is fluorescence-based analysis which provides high sensitivity and superior spatiotemporal resolution with cellular precision. However, to our knowledge, there are only a few molecular tools capable of simultaneously quantifying plasma NE and visualizing tissue NE for comprehensive assessment of SNS in hypertension.

Fig. 1 General design of the fluorescent probe for specific quantification of NE. (a) Structures and metabolism of catecholamines. (b) Schematic illustration of the reported NE fluorescent probes based on single-site or dual-site recognition strategies. (c) Schematic illustration of the dual-site ratiometric NE fluorescent probe based on the cyclization recognition strategy in this work, as well as the application for quantifying plasma and urinary NE of hypertensive rats. (d) Design and construction of the PBN probes. (e) Proposed mechanism for the response of probe PBN-5 towards NE.

Currently available NE fluorescent probes are mostly based on the cascade nucleophilic reactions of the hydroxyethylamine moiety in NE.^27–30 Yin's group have used carbonate as the reactive site to recognize hydroxyethylamine which exhibited “off–on” fluorescence response with a detection limit of 64.3 μM.^29,30 Tian's group reported a fluorescent probe that can quickly respond to NE with “on–off” fluorescence and proposed an accelerated mechanism using folding and water-bridging.^27,28,31 Glass's group used an aldehyde group to recognize the hydroxyethylamine while using a boronic acid to bind with catechol to increase the binding efficiency (Fig. 1b).^32–36 These reported NE probes are based on using a single recognition site or a single-channel fluorescence change, from which it is easy to be interfered with by amino acids (10–500 μM) making it hard to achieve specific and quantitative detection due to a lack of self-calibration.³⁷ As such, it is of significant importance to develop highly-selective and quantifiable fluorescent probes for the precise and specific high-throughput detection of NE.

Ratiometric signal measurements can reduce the interference associated with probe concentration, excitation intensity and the environment through the calibration of two emission channels, thereby providing an ideal method for quantitative analysis.^38,39 In this respect, a probe for the specific and quantitative detection of NE should meet at least two prerequisites: (1) it should simultaneously recognize the two characteristic groups of NE: hydroxyethylamine and catechol, respectively. (2) The recognition preferably produces a ratiometric fluorescence signal which exhibits no crosstalk. The cyclization recognition strategy has been used for fluorescence recognition of amino acids, exhibiting advantages of high specificity and affinity.^40,41 The recognition process increases the structural rigidity of the macrocycle and suppresses the excited state isomerization, which facilitates fluorescence turn-on.⁴² Significantly, the cavities of macrocycles can also provide binding sites for ions and hydrogen bonds to improve the detection performance of such probes.⁴³

Here, we report a dual-site fluorescent probe, PBN-5, combining a cyclization recognition strategy with ratiometric signals to achieve selective quantification of NE (Fig. 1c). An aldehyde and boronic acid group were used as the two sites to bind with NE and form a macrocyclic-ring, a ratiometric signal was obtained to distinguish NE from other biological nucleophiles. Significantly, the addition of silver ions accelerated the recognition and amplified the signal by chelating with the as-formed macrocyclic-ring using silver bridging. Compared with the previously reported “off–on” probes, the ratiometric probe PBN-5 exhibits excellent anti-interference capability, thus providing higher accuracy for quantitative analysis. Notably, plasma NE and urinary NE of spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats were compared using our probe. Additionally, we investigated the NE levels in sympathetic nerve-rich cardiac, renal and adrenal gland tissues of both SHR and WKY rats to assess sympathetic-adrenal activity. This reliable probe serves as a valuable tool for the comprehensive study of the relationship between NE and SNS-associated diseases such as hypertension.

Results and discussion

Design and synthesis of PBN probes

To achieve the quantification of NE, the fluorescent probe needs to have both specificity and accuracy. For high specificity, a dual-site recognition strategy based on the unique structure of NE was used. While to achieve high accuracy, a ratiometric signal response was used for quantitative analysis, which is less influenced by environmental factors.

We first analyzed the chemical structure of NE and identified two active reactive groups: β-hydroxyethylamine and catechol. β-Hydroxyethylamine exhibits nucleophilic properties, as it can undergo a nucleophilic reaction with aldehyde groups. While boronic acid is a well-known functional group that can selectively recognize 1,2-diols or 1,3-diols to form five- or six-membered cyclic esters. Thus, we used a boronic acid group to bind with the two adjacent hydroxyl groups of catechol. Meanwhile, amino acids or other biological nucleophiles, as well as other catecholamine homologs, do not possess both groups simultaneously, allowing for the selective recognition to NE.

The two fluorophores need to be excited at the same wavelength and generate a fluorescence increase and decrease independently with low levels of crosstalk after recognition. The hydroxyethylamine of NE is in a L-configuration, which is also one of the structural characteristics. While, 1,1′-bi-2-naphthol (BINOL) has been widely investigated for the enantioselective recognition of amino acids.^44–46 Inspired by chiral recognition, we chose (S)-BINOL as a fluorophore containing an aldehyde as the hydroxyethylamine reaction site, which may exhibit significant fluorescence enhancement after binding and chiral matching with L-hydroxyethylamine. While naphthalimide was chosen as the other fluorophore due to the excellent matching of the excitation wavelength with that of (S)-BINOL, and the boronic acid group was chosen as the catechol reaction site to generate fluorescence quenching as induced by the electron-rich catechol of NE (Fig. 1c and e). As such, when the probe reacts with NE, the ratiometric fluorescence signal between the two fluorophores enables the quantification of NE.

The two recognition sites were connected by a linker, and the linker was varied to determine the most sensitive recognition system. Thus, we designed six compounds belonging to two series: PBN-1–3 contained flexible linkers; PBN-4–6 contained semi-rigid linkers with pyridine group to provide potential hydrogen bond interaction sites (Fig. 1d). The synthesis and characterization of the probes are given in the SI.

Spectral response and selectivity of the probe PBN-5

The response of the six probes designed above for NE was determined in PBS buffer (10 mM, pH = 7.4, 10% DMSO). To investigate the linker effect on the probes, we focused on the reaction kinetics. As shown in Fig. S1, the emission intensity at 400 nm decreased immediately, accompanied by a slower increase at 530 nm after mixing with NE within 50 min.

It is worth noting that the fluorescence of PBN-1–PBN-4 at 400 nm was suppressed, presumably due to the freedom of two fluorophores when using flexible linkers (Fig. S1a, c, e and g). The fluorescence at 526 nm occurred without the analytes in both PBN-1, PBN-2, PBN-4 and PBN-5 (Fig. S1a, c, g and i). It may be due to intramolecular hydrogen bonding interactions between the hydroxyl group of binaphthyl and boronic acid, which leads to the twisting of (S)-BINOL and the appearance of fluorescence. Thus, the results above indicated that semi-rigid linkers are more favorable for constructing dual-site probes when compared to fully flexible linkers, and PBN-5 was superior to other compounds. We speculated that the appropriate length and freedom of the linker made it easier for PBN-5 to combine with NE to form a cyclic compound. Thus, PBN-5 was subsequently evaluated using additional recognition and mechanism studies.

The fluorescence response of PBN-5 towards NE was evaluated in PBS buffer. A new emission peak at 526 nm appeared and gradually increased, accompanied by a fluorescence decrease at 400 nm with the addition of NE (Fig. 2a and b). The fluorescence intensity ratio of the dual channels (F₅₂₆/F₄₀₀) versus the concentration of NE was plotted in Fig. 2c, and exhibited good linearity within a concentration range of 0–50 μM NE. Given that complex biological environments might interfere with detection, the selectivity of PBN-5 towards NE was evaluated against other catecholamine homologues (DA, EP), catecholamine metabolites (HVA: the metabolite of DA, MHPG: the metabolite of NE, VMA: the metabolite of EP), neurotransmitters (5-HT, GABA), and amino acids in both PBS buffer and plasma. The results indicated that the fluorescence at 400 nm decreased with catecholamine homologues (DA, EP), while the signal at 526 nm was less affected by these analytes (Fig. S4). The ratio of the dual channels (F₅₂₆/F₄₀₀) exhibited high selectivity for NE in both PBS buffer and plasma (Fig. 2d and S5). The reaction was completed within 30 min according to the reaction kinetic experiments (Fig. 2e and f). These results confirmed that probe PBN-5 can effectively recognize NE with high sensitivity and selectivity.

Fig. 2 The photophysical properties and in vitro response to NE of probe PBN-5. (a) Fluorescence spectra of PBN-5 (10 μM) upon the addition of NE (0–50 μM) in PBS buffer (10 mM, pH = 7.4, 10% DMSO), excited at 350 nm. (b) Fluorescence intensity of PBN-5 at 400 nm and 526 nm upon the addition of NE in PBS buffer. Data are presented as mean ± S.D. Error bars: S.D., n = 5 independent experiments. (c) Plot and linear fitting of the fluorescence variation (F₅₂₆/F₄₀₀) as a function of the concentrations of NE (0–50 μM). Data are presented as mean ± S.D. Error bars: S.D., n = 5 independent experiments. (d) Selectivity of PBN-5 towards NE and other neurotransmitters (DA, EP, 5-HT, GABA), catecholamine metabolites (HVA, MHPG, VMA) and amino acids (Lys, Cys, Ser, Thr, Glu, Asp, Pro, Ala) (50 μM for each). Insert: color photograph under 365 nm excitation in the dark. (e) Time dependence of PBN-5 upon the addition of 50 μM NE for 0–50 min at 400 nm and 526 nm, respectively. (f) Time dependence of PBN-5 upon 10 μM, 25 μM, 50 μM NE for 0–50 min at F₅₂₆/F₄₀₀.

Response mechanism of the probe PBN-5 to NE

To further elucidate the recognition mechanism, ¹H NMR and MALDI-TOF MS for the reaction between PBN-5 and NE were conducted. As shown in Fig. 3a, PBN-5 exhibited a singlet at δ 10.33 ppm for the aldehyde proton. The aldehyde group formed an intramolecular hydrogen bond with o-hydroxyl group, which led to a highly downfield-shifted hydroxyl signal at δ 10.22 ppm. The addition of NE led to the disappearance of the aldehyde signal, and a new peak gradually appeared at δ 8.76 ppm. This new peak can be assigned to an imine proton formed from the condensation of the aldehyde group of PBN-5 and the amino group of NE. The intramolecular hydrogen bond between the aldehyde and hydroxyl group was also disrupted, resulting in a chemical shift of the hydroxyl signal to a higher field at δ 9.21 ppm.

Fig. 3 Response mechanism characterization of probe PBN-5 towards NE. (a) ¹H NMR spectra of the reaction mixture of PBN-5 with NE at various reaction times in DMSO-d₆. (b) MALDI-TOF MS of the reaction mixture of PBN-5 with NE. (c) Chemical structures of control molecules (S)-P1 and N1. (d) Fluorescence spectra of (S)-P1 (10 μM) upon addition of 1 mM NE in PBS buffer (10 mM, pH = 7.4, 10% DMSO), excited at 350 nm. (e) Fluorescence spectra of N1 (10 μM) upon addition of 1 mM NE in PBS buffer (10 mM, pH = 7.4, 10% DMSO), excited at 350 nm.

Meanwhile, the addition of NE led to the disappearance of the boronic acid protons (δ 8.58 ppm), accompanied by a chemical shift of the ortho aromatic proton signals to a higher field, indicating the condensation of the boronic acid of PBN-5 and the catechol of NE. Three new singlets at δ 6.38–6.48 ppm were observed in both experiments that belong to the benzene ring of NE. The mass spectra (MALDI-TOF) of the reaction mixture revealed the coexistence of multiple binding products (boronic acid binding complex PBN-NE-1, aldehyde binding complex PBN-NE-2, and dual-site macrocyclic binding complex PBN-NE-cyclic) in the reaction system (Fig. 3b). Among them, the signal observed at m/z = 934.3 exhibited the highest intensity, which could be attributed to the product PBN-NE-cyclic (calcd for [PBN-NE-cyclic + H]⁺, 934.3). These results confirmed that PBN-5 reacted with NE and formed a cyclic compound as expected.

In order to rationalize the ratiometric fluorescence singal, two control compounds ((S)-P1 and N1) containing the two recognition sites of PBN-5, respectively, were designed and synthesized (Fig. 3c). As we expected, both (S)-P1 and N1 exhibited a significant response with NE. An obvious fluorescence increase at 526 nm was observed after mixing probe (S)-P1 with NE (Fig. 3d), while probe N1, with a pK_a of 8.76 (Fig. S6), exhibited fluorescence quenching at 400 nm upon the incubation with NE (Fig. 3e), suggesting that the boronic acid group binds catechol in its neutral form rather than the hydroxylated form. More importantly, the binding affinity of N1 (with a binding constant K_a = 9634 M⁻¹) for NE is significantly higher than that of (S)-P1 (K_a = 1927 M⁻¹), which indicated the difference in NE-binding affinity between the two reactive sites of PBN-5 (Fig. S7). These results could account for the coexistence of multiple binding products in the MALDI-TOF MS (Fig. 3b), and it could contribute to the absence of an isosbestic point in the fluorescence titration curves (Fig. 2a). We also studied the fluorescence response of two enantiomers (S)-P1 and (R)-P1 towards NE under the same conditions, and (S)-P1 generated a higher fluorescence enhancement at 526 nm than (R)-P1 (Fig. S8). These results indicated that the boronic acid group of PBN-5 combined with catechol causing the fluorescence quenching of naphthalimide due to the electron-rich catechol. Meanwhile, the aldehyde group of PBN-5 combined with the amino group of NE to generate an imine, which enhanced the fluorescence at 526 nm by matching the chiral configuration to the twist of the binaphthyl plane. A ratiometric signal was observed after the recognition process which is consistent with our design presented in Fig. 1.

Effects of metal cations on the recognition of NE

Time-dependent experiments indicated that the fluorescence increase at 526 nm caused by the combination of aldehyde and the NE amino group is the rate-limiting process. As such, we wondered whether there was any way to accelerate the reaction in order to improve the detection efficiency. Metal cations, act as Lewis acids and can accept electron pairs. Therefore, they can form coordinative bonds with nucleophiles, such as amino groups, to enhance the nucleophilicity and accelerate imine formation with aldehydes. The addition of metal cations to BINOL-based chiral probe system is anticipated to enhance the fluorescence response through coordination with N/O atoms,^47,48 thereby promoting the recognition process.

The fluorescence intensity of PBN-5 remained unchanged after titrating with 11 metal cations (Fig. S9). Subsequently, the fluorescence response of PBN-5 towards NE was determined in the presence of the 11 metal cations in PBS buffer. The results indicated that certain metal cations (Na⁺, Ca²⁺, Fe²⁺, Mg²⁺, Fe³⁺) shortened the equilibrium time to approximately 20 min without enhancing the fluorescence signal (Fig. S10). Notably, after the addition of Ag⁺, the equilibration time was significantly reduced to 15 min, while the signal of the probe at F₅₂₆/F₄₀₀ was also amplified by 3.85-fold (Fig. 4a and b). The MALDI-TOF MS of the reaction mixture exhibited a predominate signal at m/z = 1040.2 (Fig. 4c), indicating that one equivalent of Ag⁺ was participating in the coordination.

Fig. 4 The response of probe PBN-5 towards NE in the presence of Ag⁺. (a) Fluorescence spectra of PBN-5 (10 μM) upon addition of NE (10 μM) in PBS buffer (10 mM, pH = 7.4, 10% DMSO) in the presence of Ag⁺ (10 μM) for 0–50 min, excited at 350 nm. (b) Fluorescence singal at F₅₂₆/F₄₀₀ of PBN-5 upon addition of NE (10 μM) in PBS buffer (10 mM, pH = 7.4, 10% DMSO) with or without Ag⁺. (c) MALDI-TOF MS of the reaction mixture of PBN-5 with NE in the presence of Ag⁺.

Heteroatoms such as N/O atoms can be used as electron-donating groups to coordinate with Ag⁺.⁴⁹ Multiple fluorescent probes have been reported to capture Ag⁺ using N/O-containing ligands as the chelating moieties,^50,51 such as pyridine.^52–56 Considering that Ag⁺ tends to form two coordination bonds, we speculated that it coordinated between the hydroxyl group of NE and the pyridyl nitrogen atom. The proposed structure of the recognition product PBN-NE-cyclic-Ag is illustrated in Fig. 4c, and its optimized structure was obtained by DFT calculation (B3LYP/def2TZVP), which reveals that the formation of a “silver-bridge” enhances the rigidity of the molecular system, and stabilizes the macrocyclic-ring (Fig. S11). The results suggested that Ag⁺ accelerated the binding process and amplified the signal. Therefore, 1 equivalent Ag⁺ was added to the system in subsequent experiments to improve the detection performance.

Quantification of NE in plasma and urine using probe PBN-5

Plasma NE is mainly released by sympathetic postganglionic neurons, serving as a sensitive indicator of SNS activity.^57–59 In contrast, urinary NE is derived from plasma NE through renal filtration, and reflects the integrated SNS activity over a period of time.^60,61 Plasma and urinary NE are both strongly associated with SNS activation: elevated levels indicate increased SNS outflow, while reduced levels may reflect sympathetic dysfunction.¹⁶ Combined analysis of NE in plasma and urine can comprehensively evaluate the sympathetic regulation status in hypertension.⁶²

Based on the high-performance of probe PBN-5, it was subsequently used to quantify plasma and urinary NE in SHR. To validate the efficacy of PBN-5 in trace NE quantification, PBS buffer (pH = 8.5, 50% DMSO) was utilized as the optimal detection system. The standard curves of the PBN-5 response to NE were obtained via adding different concentrations of NE and an equivalent of Ag⁺. In order to achieve high-throughput detection, the emission intensity at 526 nm and 400 nm were directly recorded using a microplate reader. As a result, the linear equation was obtained (F₅₂₆/F₄₀₀ = 0.04732 × [NE] (nM) + 0.4142, R² = 0.98) with NE concentration in the range of 0–40 nM (Fig. S12), and the limit of detection was calculated to be 1.8 nM, demonstrating its extraordinary sensitivity to actual trace NE in plasma and urine samples.

To compare NE levels between SHR and normotensive WKY rats, blood samples and 24 hour urine (total amount of urine over a 24 hours period) samples from both groups were separately collected. Due to the matrix effects caused by complex components in plasma and urine, the samples required pretreatment to prevent interference from cells and proteins in NE detection (Fig. 5a). Thus, acetonitrile was added to precipitate proteins, followed by centrifugation to collect the supernatant, which was further lyophilized and redissolved in PBS buffer containing Ag⁺. Following the addition of probe PBN-5 to react with NE, the reaction mixture was quantified using a microplate reader. To validate the detection method, a NE standard at concentrations of 2.5, 5, 10, 15 and 20 nM was spiked into both plasma and urine samples. The recovery rates (measured NE concentration/spiked NE concentration × 100%) were calculated using PBN-5 according to established sample pretreatment protocols and standard curve. As shown in Table S1, the measured concentration of NE showed good agreement with the spiked concentration, with the recovery rates ranged from 94.8% to 98.6% in plasma and the recovery rates ranged from 104.1% to 110.8% in urine, indicating that PBN-5 exhibited acceptable accuracy in detecting plasma and urinary NE.

Fig. 5 Quantitative detection of NE in plasma and urine from SHR and WKY rats using probe PBN-5. (a) Schematic diagram of sample pretreatment and NE quantification in rat plasma/urine using the probe PBN-5. (b) The fluorescence signals at F₅₂₆/F₄₀₀ obtained from NE detection in plasma of SHR and WKY rats using probe PBN-5. Data are presented as mean ± S.D. Error bars: S.D., n = 10 independent rats samples, black dots represent individual data points. Statistical significance is calculated with a two-tailed unpaired t test (**p < 0.01). (c) The fluorescence signals at F₅₂₆/F₄₀₀ obtained from NE detection in urine of SHR and WKY rats using probe PBN-5. Data are presented as mean ± S.D. Error bars: S.D., n = 4 independent rats samples, black dots represent individual data points. Statistical significance is calculated with a two-tailed unpaired t test (**p < 0.01). (d) The heatmap analysis of quantitative NE concentrations in plasma and urine between SHR and WKY rats.

Utilizing the established method, the quantitative analysis of NE was performed in blood and urine samples from SHR and control WKY rats. The signal of F₅₂₆/F₄₀₀ was significantly higher in both plasma (1.43-fold) and urine (1.38-fold) of SHR compared to WKY rats (Fig. 5b and c). The quantitative analysis results demonstrated that the plasma NE in WKY rats ranged from 4.24 nM to 15.36 nM, while the value measured in SHR ranged from 6.21 nM to 31.51 nM. Meanwhile, the urinary NE in WKY rats ranged from 6.76 nM to 8.68 nM, while the value measured in SHR ranged from 11.54 nM to 16.02 nM (Fig. 5d). The above results suggested that SHR have higher NE levels in plasma and urine, which indicated the SNS overactivation in spontaneous hypertension.

Fluorescence imaging of NE in living cells and tissues using probe PBN-5

The desirable characteristics of PBN-5 in vitro imply its potential for NE imaging in biological systems. To assess its biosafety, the cytotoxicity of the probe was evaluated using CCK-8 assay. As shown in Fig. S13, treatment with PBN-5 at varying concentrations (2, 4, 8, 10, 15, and 20 μM) for 24 h maintained cell viability above 80% in HepG2 (human hepatocellular carcinoma), 4T1 (mouse mammary carcinoma) and PC12 (rat adrenal medulla pheochromocytoma) cells, confirming its low cytotoxicity and excellent biocompatibility.

It has been reported that PC12 cells can synthesize and secrete NE.⁶³ Therefore, the incubation time was systematically optimized by treating PC12 cells with PBN-5 for varying durations (0, 15, 30, 60, 90, 120, 180 and 240 min). As shown in Fig. S14, the fluorescence intensity was observed to increase progressively over time until reaching a plateau at 60 min, establishing this incubation time as optimal for subsequent experiments. The capability of PBN-5 for visualizing endogenous NE was subsequently explored in different cells. PBN-5 was incubated with HepG2, 4T1 and PC12 cells for 60 min, and then the fluorescence imaging was performed. As shown in Fig. 6a and b, only PC12 cells exhibited obvious green fluorescence, and basically no emission intensity was observed in HepG2 and 4T1 cells. These results confirmed that PC12 cells contain a large amount of NE, which can be effectively visualized by probe PBN-5.

Fig. 6 Confocal imaging for NE in cells and tissues using probe PBN-5. (a) Confocal imaging for endogenous NE in HepG2 cells, 4T1 cells and PC12 cells treated with PBN-5 (10 μM), excited at 405 nm. (b) Quantitative analysis of the fluorescence intensity in HepG2 cells, 4T1 cells and PC12 cells. Data are presented as mean ± S.D. Error bars: S.D., n = 3 independent experiments. Statistical significance is calculated with a two-tailed unpaired t test (***p < 0.001). (c) Confocal imaging of NE at varying concentrations (0 mM, 0.1 mM, 0.5 mM, 1 mM) using PBN-5 in PC12 cells. (d) Quantitative analysis of fluorescence intensity of the probe in response to varying NE concentrations. Data are presented as mean ± S.D. Error bars: S.D., n = 3 independent experiments. (e–g) Comparative confocal imaging of NE and H&E staining in heart (e), kidney (f) and adrenal gland (g) tissues of SHR and control WKY rats, excited at 405 nm. (h) Quantitative analysis of the fluorescence intensity in heart, kidney and adrenal medulla of SHR and control WKY rats. Data are presented as mean ± S.D. Error bars: S.D., n = 3 rats. Statistical significance is calculated with a two-tailed unpaired t test (*p < 0.05, **p < 0.01).

Moreover, NE is primarily released from neuronal cells via exocytosis and acts on adrenergic receptors. Thus, the probe was further used to monitor the secretion process of NE stimulated by high-K⁺ solution (a mimic of electrical impulse). Upon high concentration K⁺ (1 mM) stimulation, a rapid decrease in fluorescence intensity was observed within 30 s (to 48.7% of the initial intensity), which may be due to the decrease of intracellular NE levels attributed to K⁺-induced exocytosis (Fig. S15). In contrast, when stimulated with PBS (without K⁺), negligible change in green channel fluorescence was observed. These results demonstrated that PBN-5 can reflect the endogenous NE changes and visualize the exocytosis process in living PC12 cells. Subsequently, PC12 cells pretreated with varying NE concentrations (0, 0.1, 0.5, 1 mM) were incubated with the probe for 60 min. As shown in Fig. 6c and d, the fluorescence intensity in cells increased proportionally with intracellular NE levels. The results further confirmed that probe PBN-5 can effectively monitor the change of intracellular NE levels.

Based on the performance of PBN-5 for NE visualization at the cellular level, we further applied the probe to tissue imaging of SHR and normotensive WKY rats. Sympathetic nerve enriched tissues (heart, kidney and adrenal medulla) were processed for fluorescence imaging. As shown in Fig. 6e and f, the fluorescence intensity in heart and kidney tissues of SHR was much higher than that of control group. We further performed the staining analysis of rat adrenal glands. H&E staining enables differentiation of the adrenal medulla from the outer cortex based on the distinct morphological characteristics (Fig. 6g). Then, the average fluorescence intensity of the medulla outlined by the white dashed line was calculated using H&E-stained images as a reference. Stronger fluorescence was observed in adrenal medulla than cortex in both SHR and normotensive WKY rats. These results indicated that NE is distributed in the medulla region, which was supported by the reported mass spectrometry imaging of adrenal NE.⁶⁴ Notably, the adrenal medulla of SHR exhibited slightly stronger fluorescence signals compared to the control group, suggesting marginally elevated NE concentration in adrenal gland of SHR (Fig. 6g and h). These results provided direct evidence for elevated tissue NE levels in hypertension. Combined with quantitative measurements of plasma and urinary NE, these findings demonstrated the activation of the sympathetic-adrenal axis in hypertension. The elevated NE may contribute to the blood pressure by activating α1/β1 receptors. As such, this study could provide key insights into the progression assessment and disease mechanisms of hypertension.

Conclusion

In summary, we have designed and synthesized a dual-site ratiometric fluorescent probe PBN-5 for precisely quantifying trace NE based on a macrocyclic recognition strategy. To specifically recognize the unique L-hydroxyethylamine and catechol moieties of NE, an aldehyde-substituted chiral binaphthyl and boronic acid groups were introduced to probe PBN-5. The detection process is based on a nucleophilic reaction between the aldehyde and amino group, and a condensation reaction between the boronic acid group and catechol, forming a macrocyclic-ring enhanced by silver bridging. By utilizing the probe PBN-5 for both NE quantification in plasma/urine and fluorescence imaging in tissue sections, we demonstrated the elevated circulating and tissue NE levels in spontaneous hypertension, indicating sympathetic nervous overexcitation. This study establishes a novel chemical approach for NE recognition, providing a molecular tool to deepen the understanding of NE levels and sympathetic activity in spontaneous hypertension. Future efforts will focus on applying the probe to clinical human samples, with particular attention to the more complex matrices compared to rodent models, as well as simplifying the pretreatment procedures. Meanwhile, a better matching of the reaction kinetics and binding constants between the two recognition sites is expected to further enhance the detection performance of the probe, while red-shifted wavelength is also crucial for improving the signal-to-noise ratio for in vivo imaging applications.

Author contributions

F. Wang carried out the experiments, analyzed the data, and wrote the original manuscript. J. Wang analyzed the data and participated in the discussion. W. Zhu and Y. Yang guided writing and edited the manuscript. Y. Xu, H. Li supervised the whole project & guided writing, reviewed, and edited the manuscript. T. D. James guided writing, reviewed, and edited the manuscript.

Conflicts of interest

There are no conflicts to declare.

Data availability

Reasonable requests for additional information can be made to the corresponding authors. The animal experiments were conducted under the protocol approved by the Scientific Investigation Board of East China University of Science and Technology (ECUST-2025-148).

The data supporting this article have been included as part of the SI. A detailed experimental methods, synthesis, structural characterizations, ¹H/¹³C NMR, HRMS spectra and additional experimental data supporting this article have been provided in the SI. See DOI: https://doi.org/10.1039/d5sc05925a.

Acknowledgements

This work is financially supported by National Key Research and Development Programs (2022YFD1700403 and 2023YFD1700303). The authors wish to thank Prof. Amilra Prasanna de Silva, for his valuable and thoughtful suggestions on this paper. TDJ wishes to thank the University of Bath and the Open Research Fund of the School of Chemistry and Chemical Engineering, Henan Normal University (2020ZD01) for support.

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