Self-Internal Standard Fluorescence for Ultrasensitive Detecting of mtDNA to Evaluate Matrilineal Genetic Defect Levels.

Mitochondrial DNA (mtDNA) is a unique genetic material characterized by maternal inheritance. It possesses a circular structure devoid of histone protection and exhibits low cellular abundance, which poses great challenges for its sensitive and selective detection at the living cell level. Herein, we have designed three bis-naphthylimide probes with varying linker lengths (NANn-OH, n = 0, 2, 6), facilitating the formation of distinct twisted or folded molecular conformations in the free state. These probes emit the red fluorescence around 627 nm with different fluorescence quantum yields (ΦNAN0-OH = 0.0016, ΦNAN2-OH = 0.0136, and ΦNAN6-OH = 0.0125). When encountering mtDNA (0.4-3.4 µg/mL), these probes undergo conformational changes depending on the length of the attached C-strand and exhibit a gradually increasing fluorescence signal around 453 nm. The fluorescence intensity increased to 13.5-fold, 1.9-fold, and 8.2-fold, respectively. Notably, the red fluorescence intensities around 627 nm remain constant throughout this process, thus serving as an inherent correction mechanism for proportional fluorescence signal enhancement to improve selectivity and sensitivity. NAN0-OH, NAN2-OH, and NAN6-OH showed good linearity for mtDNA in the range of 0.4-3.4 µg/mL with detection limits of LODNAN0-OH = 1.04 µg/mL, LODNAN2-OH = 1.10 µg/mL, and LODNAN6-OH = 1.15 µg/mL. Cellular experiments reveal that NAN6-OH effectively monitors curcumin-induced mtDNA damage in HepG-2 cells while enabling monitoring of genetic mtDNA damage. We anticipate that this tool holds significant potential for the precise evaluation of maternal genetic defects, thereby enhancing hypersensitive assessment in clinical medicine.

Photochemical and biological dual-effects enhance the inhibition of photosensitizers for tumour growth.

Photosensitizers typically rely on a singular photochemical reaction to generate reactive oxygen species, which can then inhibit or eradicate lesions. However, photosensitizers often exhibit limited therapeutic efficiency due to their reliance on a single photochemical effect. Herein, we propose a new strategy that integrates the photochemical effect (type-I photochemical effect) with a biological effect (proton sponge effect). To test our strategy, we designed a series of photosensitizers (ZZ-sers) based on the naphthalimide molecule. ZZ-sers incorporate both a p-toluenesulfonyl moiety and weakly basic groups to activate the proton sponge effect while simultaneously strengthening the type-I photochemical effect, resulting in enhanced apoptosis and programmed cell death. Experiments confirmed near-complete eradication of the tumour burden after 14 days (Wlight/Wcontrol ≈ 0.18, W represents the tumour weight). These findings support the notion that the coupling of a type-I photochemical effect with a proton sponge effect can enhance the tumour inhibition by ZZ-sers, even if the basic molecular backbones of the photosensitizers exhibit nearly zero or minimal tumour inhibition ability. We anticipate that this strategy can be generalized to develop additional new photosensitizers with improved therapeutic efficacy while overcoming limitations associated with systems relying solely on single photochemical effects.

Photoinduced electron transfer (PeT) based fluorescent probes for cellular imaging and disease therapy.

Typical PeT-based fluorescent probes are multi-component systems where a fluorophore is connected to a recognition/activating group by an unconjugated linker. PeT-based fluorescent probes are powerful tools for cell imaging and disease diagnosis due to their low fluorescence background and significant fluorescence enhancement towards the target. This review provides research progress towards PeT-based fluorescent probes that target cell polarity, pH and biological species (reactive oxygen species, biothiols, biomacromolecules, etc.) over the last five years. In particular, we emphasise the molecular design strategies, mechanisms, and application of these probes. As such, this review aims to provide guidance and to enable researchers to develop new and improved PeT-based fluorescent probes, as well as promoting the use of PeT-based systems for sensing, imaging, and disease therapy.

Mitochondrial-Targeted Ratiometric Fluorescent Probe to Monitor ClO- Induced by Ferroptosis in Living Cells.

Ferroptosis is a type of iron-dependent programmed cell death. Once such kind of death occurs, an individual cell would undergo a series of changes related to reactive oxygen species (ROS) in mitochondria. A mitochondrial-targeted ratiometric fluorescent probe (MBI-OMe) was developed to specifically detect ferroptosis-induced ClO-, whose recognition group is p-methoxyphenol, and the mitochondrial-targeted group is benzimidazole. The fluorescence of MBI-OMe was first quenched by 30 µM of Fe3+, and then MBI-OMe appeared as a ratiometric signal at 477 nm and 392 nm in response to ferroptosis-induced ClO- in living cells. MBI-OMe was successfully used to evaluate changes in ClO- induced by ferroptosis.

mtDNA-Specific Ultrasensitive Near-Infrared Fluorescent Probe Enables the Differentiation of Healthy and Apoptotic Cells.

Mitochondrial DNA (mtDNA) as a class of important genetic material is easily damaged, which can result in a series of metabolic diseases, hereditary disease, and so on. mtDNA is an ultrasensitive indicator for the health of living cells due to the extremely short physiological response time of mtDNA toward damage (ca. 5.0 min). Therefore, the development of specific ultrasensitive fluorescent probes that can in real-time monitor mtDNA in vivo are of great value. With this research, we developed a near-infrared twisted intramolecular charge transfer (TICT) fluorescent probe YON. YON is a thread-like molecule with an A-π-D-π-A structure, based on the dicyanoisophorone fluorophore. The molecular design of YON enabled the specific binding with dsDNA (binding constant (K) = 8.5 × 105 M-1) within 1.3 min. And the appropriate water-oil amphiphilicity makes YON significantly accumulate in the mitochondria, enabling the specific binding to mtDNA. The fluorescence intensity at 640 nm of YON enhanced linearly with increasing concentrations of mtDNA. Dicyanoisophorone as the strong electron-withdrawing group that was introduced into both ends of the molecule resulted in YON being a classic quadrupole, so it could ultrasensitively detect trace mtDNA. The minimum detection limit was 71 ng/mL. Moreover, the large Stokes shift (λex = 435 nm, λem = 640 nm) makes YON suitable for "interference-free" imaging of mtDNA. Therefore, YON was used to monitor trace changes of mtDNA in living cells; more importantly, it could be used to evaluate the health of cells by monitoring microchanges of mtDNA, enabling the ultrasensitive evaluation of apoptosis.