Response strategies and biological applications of organic fluorescent thermometry: cell- and mitochondrion-level detection.

Temperature homeostasis is critical for cells to perform their physiological functions. Among the diverse methods for temperature detection, fluorescent temperature probes stand out as a proven and effective tool, especially for monitoring temperature in cells and suborganelles, with a specific emphasis on mitochondria. The utilization of these probes provides a new opportunity to enhance our understanding of the mechanisms and interconnections underlying various physiological activities related to temperature homeostasis. However, the complexity and variability of cells and suborganelles necessitate fluorescent temperature probes with high resolution and sensitivity. To meet the demanding requirements for intracellular/subcellular temperature detection, several strategies have been developed, offering a range of options to address this challenge. This review examines four fundamental temperature-response strategies employed by small molecule and polymer probes, including intramolecular rotation, polarity sensitivity, Förster resonance energy transfer, and structural changes. The primary emphasis was placed on elucidating molecular design and biological applications specific to each type of probe. Furthermore, this review provides an insightful discussion on factors that may affect fluorescent thermometry, providing valuable perspectives for future development in the field. Finally, the review concludes by presenting cutting-edge response strategies and research insights for mitigating biases in temperature sensing.

Ultrafast Detection of Monoamine Oxidase A in Live Cells and Clinical Glioma Tissues Using an Affinity Binding-Based Two-Photon Fluorogenic Probe.

Abnormal expression of monoamine oxidase A (MAO-A) has been implicated in the development of human glioma, making MAO-A a promising target for therapy. Therefore, a rapid determination of MAO-A is critical for diagnosis. Through in silico screening of two-photon fluorophores, we discovered that a derivative of N,N-dimethyl-naphthalenamine (pre-mito) can effectively fit into the entrance of the MAO-A cavity. Substitutions on the N-pyridine not only further explore the MAO-A cavity, but also enable mitochondrial targeting ability. The aminopropyl substituted molecule, CD1, showed the fastest MAO-A detection (within 20 s), high MAO-A affinity and selectivity. It was also used for in situ imaging of MAO-A in living cells, enabling a comparison of the MAO-A content in human glioma and paracancerous tissues. Our results demonstrate that optimizing the affinity binding-based fluorogenic probes significantly improves their detection rate, providing a general approach for rapid detection probe design and optimization.

Intramolecular charge transfer enhancing strategy based MAO-A specific two-photon fluorescent probes for glioma cell/tissue imaging.

MAO-A promotes the proliferation of human glioma cells. Herein, we report a series of MAO-A specific two-photon small molecular fluorescent probes (A1-5) based on an intramolecular charge transfer enhancing strategy. The activity of endogenous MAO-A can be selectively imaged using A3 as a representative probe in different biological samples including human glioma cells/tissues via two-photon fluorescence microscopy. The study provides new tools for the visual detection of glioma.

Recent advances in activity-based probes (ABPs) and affinity-based probes (AfBPs) for profiling of enzymes.

Activity-based protein profiling (ABPP) is a technique that uses highly selective active-site targeted chemical probes to label and monitor the state of proteins. ABPP integrates the strengths of both chemical and biological disciplines. By utilizing chemically synthesized or modified bioactive molecules, ABPP is able to reveal complex physiological and pathological enzyme-substrate interactions at molecular and cellular levels. It is also able to provide critical information of the catalytic activity changes of enzymes, annotate new functions of enzymes, discover new substrates of enzymes, and allow real-time monitoring of the cellular location of enzymes. Based on the mechanism of probe-enzyme interaction, two types of probes that have been used in ABPP are activity-based probes (ABPs) and affinity-based probes (AfBPs). This review highlights the recent advances in the use of ABPs and AfBPs, and summarizes their design strategies (based on inhibitors and substrates) and detection approaches.

Simultaneously Detecting Monoamine Oxidase A and B in Disease Cell/Tissue Samples Using Paper-Based Devices.

As enzymes in the outer membrane of the mitochondrion, monoamine oxidases (MAOs) can catalyze the oxidative deamination of monoamines in the human body. According to different substrates, MAOs can be divided into MAO-A and MAO-B. The imbalance of the MAO-A is associated with neurological degeneration, while excess MAO-B activity is closely connected with Parkinson's disease (PD) and Alzheimer's disease (AD); therefore, detection of MAOs is of great significance for the diagnosis and treatment of these diseases. This work reports the multiplexed detection of MAO-A and MAO-B using paper-based devices based on chemiluminescence (CL). The detection limits were 5.01 pg/mL for MAO-A and 8.50 pg/mL for MAO-B in human serum. In addition, we used paper-based devices to detect MAOs in human cells and tissue samples and found that the results of paper-based detection and Western blotting (WB) showed the same trend. While only one antibody can be incubated on the same membrane by WB, multiple antibodies incubated on the same paper enabled simultaneous detection of MAO-A and MAO-B by paper-based devices. The paper-based assay could be used for preliminary early screening of clinical samples for MAOs and can be extended as an alternative to WB for multiplexed detection of various proteins in disease cell or tissue samples.

Topochemical assembly of levodopa nanoparticles network as a high-performance biosensing platform coupling with π-π stacking and electrostatic repulsion interactions.

Topochemical assembly carbon materials with unique structure rarely investigated and reported. Herein, we demonstrate the feasibility of using topochemical assembly levodopa nanoparticles with dendritic structure (LNDS) network as a new high-performance biosensing platform based on noncovalent functionalization of LNDS with a fluorescent oligonucleotide. The proposed platform is dependent on the competition of π-π stacking and electrostatic repulsion interactions between LNDS and fluorescent oligonucleotide. The obtained LNDS with 96.1% quenching efficiency is synthesized by using levodopa as the single precursor by natural oxidation or microwave irradiation. The constructed platform can be used for simple and efficient probing single-nucleotide polymorphisms (SNPs) and cDNA by fluorescence restoration with a highly sensitivity and selectivity, remarkably superior to those based on graphenes. Additionally, an aptasensor is further constructed for small molecule ATP detention in serum with a low detection limit of 4 µM. To the best of our knowledge, this is the first attempt to use LNDS to design a biosensing platform, and therefore opens possibilities for new types of nanoparticle-based molecule approaches, and sequencing technologies.

Rational Design of a Two-Photon Fluorogenic Probe for Visualizing Monoamine Oxidase†A Activity in Human Glioma Tissues.

Monoamine oxidases have two functionally distinct but structurally similar isoforms (MAO-A and MAO-B). The ability to differentiate them by using fluorescence detection/imaging technology is of significant biological relevance, but highly challenging with available chemical tools. Herein, we report the first MAO-A-specific two-photon fluorogenic probe (F1), capable of selective imaging of endogenous MAO-A enzymatic activities from a variety of biological samples, including MAO-A-expressing neuronal SY-SY5Y cells, the brain of tumor-bearing mice and human Glioma tissues by using two-photon fluorescence microscopy (TPFM) with minimal cytotoxicity.

Two-component ratiometric sensor for Cu2+ detection on paper-based device.

The copper(II) ion (Cu2+) has played an indispensable role in diverse kinds of functional physiological processes of organisms, which has become of growing interest. Despite the fact that numerous Cu2+ test papers using fluorescent probes have been fabricated, sensors featuring the ratiometric property that integrates quenched probes and an inner standard dye are rarely reported. Herein, a two-component ratiometric sensor in a paper-based device is proposed to realize highly selective Cu2+ detection. To overcome shortcomings such as low signal-to-noise ratio and incorrect response of the quenching probe, a novel BODIPY-based turn-off probe (P2017) is designed and introduced into the paper-based device with better water solubility and selectivity for Cu2+ detection. Furthermore, a reference dye (B001), exhibiting an emission at 690 nm when the excitation wavelength is 480 nm, is also introduced into the paper-based device. These two components can enhance the quality of the signal as P2017 is sensitively quenched by Cu2+, while B001 with a photostable property, serving as an internal benchmark, is unable to react with Cu2+. The results indicated that the two components provided a new concept for optimizing paper-based device fabrication and developing accurate, simple, and inexpensive Cu2+ detection methods, which could be potentially applied to monitor human health and the environment in remote areas. Graphical abstract.