Organic probes for NO-activatable biomedical imaging: NIR fluorescence, self-luminescence, and photoacoustic imaging

Abstract

Nitric oxide (NO) is a crucial signaling molecule involved in diverse physiological and pathological processes, making its precise detection essential for exploring its biological roles. Optical imaging is particularly attractive for NO detection due to its non-invasive nature, high sensitivity, and excellent spatial resolution. However, it suffers from limited tissue penetration and low signal-to-background ratios resulting from strong light scattering and autofluorescence. To overcome these challenges, several advanced imaging strategies have been developed, including near-infrared (NIR) fluorescence imaging that leverages optical regions with less light-tissue interactions, self-luminescence imaging that avoids the need for real-time light excitation, and photoacoustic imaging that detects acoustic signals with minimal attenuation. This review systematically summarizes recent advances in organic molecular probes for NO detection using these imaging modalities, focusing on their design strategies, recognition mechanisms, and biological applications. Finally, current challenges and future directions are discussed to guide the development of next-generation NO probes for both fundamental research and clinical translation.

Scheme 1. Design strategies for NO-activatable organic probes. Cyclization of o-phenylenediamine based on PET (a) and ICT (b) mechanisms. (c) N-Nitrosation of aromatic secondary amine. (d) Deamination of aromatic primary amine. (e) Formation of the Se–NO bond. (f) Conversion of thiosemicarbazide into oxadiazole. (g) Ratiometric fluorescence probe. (h) Tandem activatable probe. (i) Tandem bioluminescence probe.

Scheme 2. Chemical structures of NO-activated NIR-I fluorescent probes.

Fig. 1. (a) Chemical structure of TAMP_M1 and its activation process in the presence of tumour and lymphocyte biomarkers. (b) NIR Fluorescence (NIRF) spectra of TAMP_M1 in the presence of individual biomarkers or combined biomarkers in corresponding buffers at 37 °C for 2 h. (c) In vitro selectivity of TAMP_M1. (d) NIRF image of the LPS-inflamed and CT26 tumour tissues after local injection of probe (0.1 mM, 10 μL) for 30 min. Reproduced from ref. with permission from Springer Nature, copyright 2023.

Fig. 2. (a) Schematic diagram of the reaction mechanism between BPTQ and NO. (b) Fluorescence (FL) spectra of BPTQ (10 μM) incubated with varying NO concentrations (0–100 μM). (c) Linear correlation between BPTQ fluorescence intensity and NO concentration. Reproduced from ref. with permission from Elsevier, copyright 2024.

Fig. 3. (a) Reaction mechanism of CS–Se with NO. (b) UV-vis and (c) fluorescence spectra of CS–Se before and after the reaction with NO. Reproduced from ref. with permission from Elsevier, copyright 2023.

Scheme 3. Chemical structures of NO-activated NIR-II fluorescent probes.

Fig. 4. (a) Reaction mechanism of NRP@M-PHCQ with NO. Absorbance (b) and fluorescence (c) of NRP@M-PHCQ after the addition of NO concentrations (5–30 μM). The insets show the photos of NRP@M-PHCQ before (left) and after (right) the addition of NO (30 μM). Reproduced from ref. with permission from American Chemical Society, copyright 2023.

Fig. 5. (a) The general strategies for constructing NO-responsive ratiometric fluorescent probes. (b) Ratiometric probe NFL-NH₂. (c) Fluorescence intensity of NFL-NH₂ after adding the NO donor (0–60 μM) in ethanol solution. (d) Linear relationship between fluorescence intensity ratio (F₇₀₅/F₇₈₀) and NO concentration (0–10 μM). Reproduced from ref. with permission from American Chemical Society, copyright 2024. (e) The response mechanism of IRNO toward NO and the preparation of the NO-responsive nanoprobe DCNP@MPS@IRNO. (f) Illustration of the NO-responsive nanoprobe DCNP@MPS@IRNO for in vivo monitoring of liver injury induced by drug overdose. Reproduced from ref. with permission from American Chemical Society, copyright 2021.

Scheme 4. Chemical structures of NO-responsive ratiometric fluorescent probes.

Fig. 6. (a) Schematic diagram showing the BioLeT mechanism. (b) Molecular design of DAL based on the BioLeT mechanism. Reproduced from ref. and with permission from American Chemical Society, copyright 2015.

Scheme 5. Chemical structures of NO-responsive self-luminescence probes.

Fig. 7. (a) Schematic illustration of the sequential chemiluminescence activation mechanism. (b) Chemiluminescence spectra of DPD_GN (30 μM) in the presence of individual biomarkers or combined biomarkers in PBS (10 mM, pH 7.4) at 37 °C. (c) Real-time chemiluminescence imaging in orthotopic lung tumour after intratracheal injection of DPD_GN. The inhibitor GGsTop was intraperitoneally injected 12 h before chemiluminescence imaging. (d) Quantified chemiluminescence signal from the lung area of 4T1-bearing mice in (b) (n = 3 independent mice, mean ± s.d.). Reproduced from ref. and with permission from Wiley, copyright 2024.

Fig. 8. (a) Schematic illustration of ARET-based ratiometric nanoplatform. (b) Schematic diagram of the probable responsive mechanism of afterglow probes. AF1 and AF2 represent the afterglow for afterglow substrate (MEHPPV) and responsive molecules, respectively. A12RET represents the afterglow resonance energy transfer from AF1 to AF2. (c) Structural change of the responsive molecule NRM before and after response to NO. (d) Afterglow images of RAN1 treated with different NO concentrations. (e) Representative afterglow images, as a function of post-injection time of RAN1 (10 μg mL⁻¹) in LPS or PBS-pretreated mice. Reproduced from ref. and with permission from Springer Nature, copyright 2022.

Scheme 6. Chemical structures of NO-activated photoacoustic probes.

Fig. 9. (a) Absorbance ratio (Abs₇₀₅/Abs₇₈₅) of PA_NO–2 at different NO concentrations in PBS buffer. Insets: photographs of PA_NO–2 solutions in the absence and presence of NO. (b) PA images of the PA_NO–2 solution recorded at different NO concentrations upon excitation at 690 and 790 nm, respectively, and the corresponding ratiometric PA images (PA₆₉₀/PA₇₉₀). (c) The PA ratios (PA₆₉₀/PA₇₉₀) of PA_NO–2 at different NO concentrations and the linear fitting (red). Reproduced from ref. with permission from American Chemical Society, copyright 2024. (d) NO-reactive mechanism of DTP-BTDA and construction of RAPNP. (e) Schematic showing the activation of the fluorescence signal at 940 nm and the PA signal at 720 nm of RAPNP in IBD mice by the endogenous NO. Reproduced from ref. with permission from Elsevier, copyright 2023.

Fig. 10. (a) Schematic showing the reaction between APNO-1080 and NO. (b) Representative PA images of a 4T1-Luc tumour and the tumour-less control after treatment with APNO-1080 (50 μM). (c) Representative PA images of a heterotopic A549-Luc2 tumour and the tumour-less control after treatment with APNO-1080. Reproduced from ref. with permission from American Chemical Society, copyright 2021. (d) Ratiometric PA images of tumours in different treatment groups (SNP, sodium nitroprusside; CDDP, cisplatin). (e) PA ratios of tumours from (d). (f) Line graph: NO concentration levels of tumours were calculated from the average PA ratio in (e). Reproduced from ref. and with permission from Wiley, copyright 2024.