Diagnosis of Bacterial Plant Diseases via a Nitroreductase-Activated Fluorescent Sensor.

Plant diseases caused by bacteria have become one of the serious problems that threaten human food security, which led to the remarkable reduction of agricultural yields and economic loss. Nitroreductase (NTR), as an important biomarker, is highly expressed in bacteria, and the level of NTR is closely related to the progression of pathogen infection. Therefore, the design of small-molecule fluorescent sensors targeting NTR is of great significance for the detection and diagnosis of plant pathogenic bacteria. In this study, a new fluorescent sensor targeting NTR was discovered and then successfully applied to the imaging of zebrafish and pathogenic bacteria. Most importantly, the developed sensor achieved the real-time diagnosis of Brassica napus L. infected with bacteria, which provides a promising tool for examining the temporal and spatial infection of plant pathogens in precision agriculture.

Development of Small-Molecule Fluorescent Probes Targeting Enzymes.

As biological catalysts, enzymes are vital in controlling numerous metabolic reactions. The regulation of enzymes in living cells and the amount present are indicators of the metabolic status of cell, whether in normal condition or disease. The small-molecule fluorescent probes are of interest because of their high sensitivity and selectivity, as well as their potential for automated detection. Fluorescent probes have been useful in targeting particular enzymes of interest such as proteases and caspases. However, it is difficult to develop an ideal fluorescent probe for versatile purposes. In the future, the design and synthesis of enzyme-targeting fluorescent probes will focus more on improving the selectivity, sensitivity, penetration ability and to couple the fluorescent probes with other available imaging molecules/technologies.

Pyroglutamate Aminopeptidase I Promotes Hepatocellular Carcinoma via IL-6/STAT3 Activation as Revealed by a Specific Biosensor.

As a global health challenge, hepatocellular carcinoma (HCC) is strongly associated with chronic inflammation. Targeting inflammation, particularly inflammatory factors, is regarded as an important strategy for HCC diagnosis and treatment. Pyroglutamic aminopeptidase I (PGP-I), a common exopeptidase, was recently identified as a novel inflammatory cytokine in cells. However, whether PGP-I is involved in HCC development and can be regarded as a biomarker remains unclear. To address this issue, endogenous PGP-I was imaged in live cells and in vivo, and the related biochemical and pathological processes were analyzed accordingly with a newly developed fluorogenic PGP-I biosensor. Bioimaging with the specific biosensor demonstrated the aberrant expression of PGP-I in HCC cell lines and tumor-bearing nude mice. Moreover, overexpression of PGP-I in HCC cells promoted tumor progression, whereas knockdown of PGP-I significantly suppressed tumor cell growth and migration. The activity of PGP-I was further identified to be highly related to the phosphorylation of STAT3, which could be impeded by the natural product parthenolide. Collectively, these findings suggest that PGP-I, which can promote hepatocellular tumor progression through the classical inflammation-/tumor-related IL-6/STAT3 pathway, may serve as a potential HCC biomarker and therapeutic target.

Multienzyme-Targeted Fluorescent Probe as a Biosensing Platform for Broad Detection of Pesticide Residues.

Pesticide residues, significantly hampering the overall environmental and human health, have become an increasingly severe issue. Thus, developing rapid, cost-effective, and sensitive tools for monitoring the pesticide residues in food and water is extremely important. Compared to the conventional and chromatographic techniques, enzyme inhibition-based biosensors conjugated with the fluorogenic probes provide effective alternative methods for detecting pesticide residues due to the inherent advantages including high selectivity and sensitivity, simple operation, and capability of providing in situ and real-time information. However, the detection efficiency of a single enzyme-targeted biosensor in practical samples is strongly impeded by the structural diversity of pesticides and their distinct targets. In this work, we developed a strategy of multienzyme-targeted fluorescent probe design and accordingly obtained a novel fluorescent probe (named as 3CP) for detecting the presence of wide variety of pesticides. The designed probe 3CP, targeting cholinesterases, carboxylesterases, and chymotrypsin simultaneously, yielded intense fluorescence in the solid state upon the enzyme-catalyzed hydrolysis. It showed excellent sensitivity against organophosphorus and carbamate pesticides, and the detection limit for dichlorvos achieved 1.14 pg/L. Moreover, it allowed for the diffusion-resistant in situ visualization of pesticides in live cells and zebrafish and the sensitive measurement of organophosphorus pesticides in fresh vegetables, demonstrating the promising potential for tracking the pesticide residues in environment and biological systems.

Rational Design of Esterase-Insensitive Fluorogenic Probes for In Vivo Imaging.

Small-molecule fluorogenic probes are indispensable tools for performing research in biomedical fields and chemical biology. Although numerous cleavable fluorogenic probes have been developed to investigate various bioanalytes, few of them meet the baseline requirements for in vivo biosensing for disease diagnosis due to their insufficient specificity resulted from the remarkable esterase interferences. To address this critical issue, we developed a general approach called fragment-based fluorogenic probe discovery (FBFPD) to design esterase-insensitive probes for in vitro and in vivo applications. With the designed esterase-insensitive fluorogenic probe, we successfully achieved light-up in vivo imaging and quantitative analysis of cysteine. This strategy was further extended to design highly specific fluorogenic probes for other representative targets, sulfites, and chymotrypsin. The present study expands the bioanalytical toolboxes available and offers a promising platform to develop esterase-insensitive cleavable fluorogenic probes for in vivo biosensing and bioimaging for the early diagnosis of diseases.

In vivo fluorescent screening for HPPD-targeted herbicide discovery.

BACKGROUND: 4-Hydroxyphenylpyruvate dioxygenase (HPPD), playing a critical role in vitamin E and plastoquinone biosynthesis in plants, has been recognized as one of the most important targets for herbicide discovery for over 30 years. Structure-based rational design of HPPD inhibitors has received more and more research interest. However, a critical challenge in the discovery of new HPPD inhibitors is the common inconsistency between molecular-level HPPD-based bioevaluation and the weed control efficiency in fields, due to the unpredictable biological processes of absorption, distribution, metabolism, and excretion. RESULTS: In this study, we developed a fluorescent-sensing platform of efficient in vivo screening for HPPD-targeted herbicide discovery. The refined sensor has good capability of in situ real-time fluorescence imaging of HPPD in living cells and zebrafish. More importantly, it enabled the direct visible monitoring of HPPD inhibition in plants in a real-time manner. CONCLUSION: We developed a highly efficient in vivo fluorescent screening method for HPPD-targeted herbicide discovery. This discovery not only offers a promising tool to advance HPPD-targeted herbicide discovery, but it also demonstrates a general path to develop the highly efficient, target-based, in vivo screening for pesticide discovery. © 2022 Society of Chemical Industry.

Review on the recent progress in the development of fluorescent probes targeting enzymes.

Enzymes are very important for biological processes in a living being, performing similar or multiple tasks in and out of cells, tissues and other organisms at a particular location. The abnormal activity of particular enzyme usually caused serious diseases such as Alzheimer's disease, Parkinson's disease, cancers, diabetes, cardiovascular diseases, arthritis etc. Hence, nondestructive and real-time visualization for certain enzyme is very important for understanding the biological issues, as well as the drug administration and drug metabolism. Fluorescent cellular probe-based enzyme detectionin vitroandin vivohas become broad interest for human disease diagnostics and therapeutics. This review highlights the recent findings and designs of highly sensitive and selective fluorescent cellular probes targeting enzymes for quantitative analysis and bioimaging.

Human Neutrophil Elastase Activated Fluorescent Probe for Pulmonary Diseases Based on Fluorescence Resonance Energy Transfer Using CdSe/ZnS Quantum Dots.

There is an increasing demand for effective noninvasive diagnosis against common pulmonary diseases, which are rising sharply due to the serious air pollution. Human neutrophil elastase (HNE), a typical protease highly involved in pulmonary inflammatory diseases and lung cancer, is a potential predictor for disease progression. Currently, few of the HNE-targeting probes are applicable in vivo due to the limitation in sensitivity and biocompatibility. Herein, we reported the achievement of in vitro detection and in vivo imaging of HNE by incorporating the HNE-specific peptide substrate, quantum dots (QDs), and organic dyes into the fluorescence resonance energy transfer (FRET) system. The refined nanoprobe, termed QDP, could specifically measure the HNE with excellent sensitivity of 7.15 pM in aqueous solution and successfully image the endogenous and exogenous HNE in living cells. In addition, this nanoprobe enabled HNE imaging in mouse models of lung cancer and acute lung injury, and the HNE activity at high temporal and spatial resolution was continuously monitored. Most importantly, QDP successfully discriminated the serums of patients with lung diseases from those of the healthy controls based on the HNE activity determination. Overall, this study demonstrates the advantages of a FRET-system-based nanoprobe in imaging performance and provides an applicable tool for in vivo HNE detection and pulmonary disease diagnosis.