A novel molecular imaging probe [99mTc]Tc-HYNIC-FAPI targeting cancer-associated fibroblasts.

Fibroblast activation protein (FAP) is higher expressed on cancer-associated fibroblasts (CAFs) in most malignant epithelial neoplasms, which is lower expressed in normal tissues. As a promising small molecular probe, FAP inhibitor (FAPI) shows the specific binding to FAP. This study aimed to explore a novel molecular probe [99mTc]Tc-HYNIC-FAPI targeting CAFs. The in vitro characteristics of the probe were also evaluated. The FAPI targeting FAP was designed, synthesized and conjugated with the chelator 6-hydrazinylnicotinic acid (HYNIC) for radiolabeling with 99mTc. The radiolabeling yield, radiochemical purity and stability were evaluated by Instant thin-layer chromatography (ITLC) and High performance liquid chromatography (HPLC). Lipophilicity was performed by the distribution coefficient test. The binding and migration ability of the probe was assessed using the FAP transfected tumor cell line. The radiolabeling yield of [99mTc]Tc-HYNIC-FAPI was (97.29 ± 0.46) %. The radiochemical purity was more than 90% and kept stable until 6 h. The radioligand was shown as lower lipophilicity, of which logD7.4 value was - 2.38 [Formula: see text] 0.13. In vitro experiments, the results indicated that the probe showed binding properties, and inhibited the migration of tumor cells. The novel [99mTc]Tc-HYNIC-FAPI probe was successfully radiosynthesized and exhibited good radiochemical purity, stability and in vitro binding ability to tumor cells. The [99mTc]Tc-HYNIC-FAPI will be a promising SPECT/CT imaging probe.

Complementary DNA Significantly Enhancing Signal Response and Sensitivity of a Molecular Beacon Probe to Aflatoxin B1.

This paper reported an improved molecular beacon method for the rapid detection of aflatoxin B1 (AFB1), a natural mycotoxin with severe carcinogenicity. With the assistance of a complementary DNA (cDNA) chain, the molecular beacon which consists of a DNA aptamer flanked by FAM and BHQ1 displayed a larger fluorescent response to AFB1, contributing to the sensitive detection of AFB1. Upon optimization of some key experimental factors, rapid detection of AFB1 ranging from 1 nM to 3 µM, within 20 min, was realized by using this method. A limit of detection (LoD) of 1 nM was obtained, which was lower than the LoD (8 nM) obtained without cDNA assistance. This aptamer-based molecular beacon detection method showed advantages in easy operation, rapid analysis and larger signal response. Good specificity and anti-interference ability were demonstrated. This method showed potential in real-sample analysis.

A NIR fluorescent probe for the detection of renal damage based on overrepresentation of alanine aminopeptidase enzyme.

Kidney damage generates changes at the phenotypic and genotypic levels that allow its monitoring using different biomarkers in blood, urine or serum. Among these biomarkers, kidney failure causes the urine overrepresentation of the alanine aminopeptidase (APN) enzyme. Here, we describe the design of a molecular probe (NB-ALA) based on the Nile Blue fluorophore (NB), which can detect the APN enzyme in urine by simple fluorometric measurements.

Responsive Probes and Molecular Bioimaging.

This special collection highlights the recent state-of-the-art progress in the research of molecular probes, chemosensors, nanosensors, and applications in molecular recognition, sensing, and imaging. In their Guest Editorial, Run Zhang and Tony James provide an outline of the field and introduce the contributions to the special collection.

Recent advances in label-free imaging of cell-matrix adhesions.

Cell-matrix adhesions play an essential role in mediating and regulating many biological processes. The adhesion receptors, typically transmembrane integrins, provide dynamic correlations between intracellular environments and extracellular matrixes (ECMs) by bi-directional signaling. In-depth investigations of cell-matrix adhesion and integrin-mediated cell adhesive force are of great significance in biology and medicine. The emergence of advanced imaging techniques and principles has facilitated the understanding of the molecular composition and structure dynamics of cell-matrix adhesions, especially the label-free imaging methods that can be used to study living cell dynamics without immunofluorescence staining. This highlight article aims to give an overview of recent developments in imaging cell-matrix adhesions in a label-free manner. Electrochemiluminescence microscopy (ECLM) and surface plasmon resonance microscopy (SPRM) are briefly introduced and their applications in imaging analysis of cell-matrix adhesions are summarized. Then we highlight the advances in mapping cell-matrix adhesion force based on molecular tension probes and fluorescence microscopy (collectively termed as MTFM). The biomaterials including polyethylene glycol (PEG), peptides and DNA for constructing tension probes in MTFM are summarized. Finally, the outlook and perspectives on the further developments of cell-matrix adhesion imaging are presented.

Tracking endoplasmic reticulum viscosity during ferroptosis and autophagy using a molecular rotor probe.

Ferroptosis is a unique non-apoptotic cell death process associated with endoplasmic reticulum (ER) stress-related diseases. We have designed and synthesized a far-red emitting and ER targetable viscosity-sensitive fluorophore to track ER-phagy. Furthermore, the ER viscosity alteration during the ferroptosis process was investigated via intensity and lifetime-based spectroscopy and microscopy.

Menin "reads" H3K79me2 mark in a nucleosomal context.

Methylation of histone H3 lysine-79 (H3K79) is an epigenetic mark for gene regulation in development, cellular differentiation, and disease progression. However, how this histone mark is translated into downstream effects remains poorly understood owing to a lack of knowledge about its readers. We developed a nucleosome-based photoaffinity probe to capture proteins that recognize H3K79 dimethylation (H3K79me2) in a nucleosomal context. In combination with a quantitative proteomics approach, this probe identified menin as a H3K79me2 reader. A cryo-electron microscopy structure of menin bound to an H3K79me2 nucleosome revealed that menin engages with the nucleosome using its fingers and palm domains and recognizes the methylation mark through a π-cation interaction. In cells, menin is selectively associated with H3K79me2 on chromatin, particularly in gene bodies.

SNP Genotyping with Amplifluor-Like Method.

For SNP genotyping, amplification of fluorescence (Amplifluor) is a popular and actively developing method in the plant sciences. The "Amplifluor-like" is a "home-made" modification of the original commercial Amplifluor method. Amplifluor-like genotyping requires two essential components: (1) two allele-specific forward primers targeting the SNP site with one common reverse primer; and (2) a universal part with two non-allele-specific molecular probes containing one of the two used fluorophores and a quencher. Allele discrimination is based on the fluorescence score, where the dominance of one dye over the other confirms the presence of each specific SNP allele. The Amplifluor-like method is similar to commercial KASP and original Amplifluor methods but is much cheaper because all components can be ordered as regular and modified oligos. The easily adaptable Amplifluor-like method can be modified by any researcher to make it suitable for available instruments, reagents and conditions in low-budget laboratories for SNP genotyping of any plant species with identified genetic polymorphism.

Molecular Beacon Probe (MBP)-Based Real-Time PCR.

In the advancement of molecular biology techniques, several probe-based techniques, like molecular beacon probe (MBP) assay, TaqMan probe, and minor groove binder (MGB) probe assay, have been reported to identify specific sequences through real-time polymerase chain reaction (PCR). All probe-based methods are more sensitive than the conventional PCR for the detection and quantification of target genes. MBP is a hydrolysis probe that emits fluorescence when getting the specific sequences on the gene. Here, we describe the application of MBP for the identification of the motif sequences present in the promoters of differentially expressed genes.

Curcumin-based molecular probes for fluorescence imaging of fungi.

Fluorescence imaging is a powerful and widely used method to visualize and study living organisms. However, fungi are notoriously difficult to visualize using fluorescence microscopy, given that their cell wall represents a diffusion barrier, and the synthetic organic dyes available are very limited when compared to molecular probes available for other organisms. Moreover, these dyes are usually available in only one colour, preventing co-staining experiments. To fill this gap, curcumin-based molecular probes were designed based on the rationale that curcumin is fluorescent and has moderate toxicity toward fungi, implying its ability to cross the cell wall to reach targets in the intracellular compartments. A family of boron diketonate complexes was synthesized, based on a curcumin backbone, tuning their emission color from blue to red. These probes did not present noticeable toxicity to filamentous fungus and, when applied to their visualization, readily entered the cells and precisely localized in sub-cellular organelles, enabling their visualization.

Design and synthesis of a small molecular NIR-II chemiluminescence probe for in vivo-activated H2S imaging.

Chemiluminescence (CL) with the elimination of excitation light and minimal autofluorescence interference has been wieldy applied in biosensing and bioimaging. However, the traditional emission of CL probes was mainly in the range of 400 to 650 nm, leading to undesired resolution and penetration in a biological object. Therefore, it was urgent to develop CL molecules in the near-infrared window [NIR, including NIR-I (650 to 900 nm) and near-infrared-II (900 to 1,700 nm)], coupled with unique advantages of long-time imaging, sensitive response, and high resolution at depths of millimeters. However, no NIR-II CL unimolecular probe has been reported until now. Herein, we developed an H2S-activated NIR-II CL probe [chemiluminiscence donor 950, (CD-950)] by covalently connecting two Schaap's dioxetane donors with high chemical energy to a NIR-II fluorophore acceptor candidate via intramolecular CL resonance energy transfer strategy, thereby achieving high efficiency of 95%. CD-950 exhibited superior capacity including long-duration imaging (~60 min), deeper tissue penetration (~10 mm), and specific H2S response under physiological conditions. More importantly, CD-950 showed detection capability for metformin-induced hepatotoxicity with 2.5-fold higher signal-to-background ratios than that of NIR-II fluorescence mode. The unimolecular NIR-II CL probe holds great potential for the evaluation of drug-induced side effects by tracking its metabolites in vivo, further facilitating the rational design of novel NIR-II CL-based detection platforms.

Multi-stimuli-responsive molecular fluorescent probes for bioapplications.

Stimuli-responsive fluorescent probes have been widely utilized in detecting the physiological and pathological states of living systems. Numerous stimuli-responsive fluorescent probes have been developed due to their advantages of good sensitivity, high resolution, and high contrast fluorescent signals. In this feature article, the progress of multi-stimuli-responsive probes, including organic molecules and metal complexes, for the detection of various biomarkers for bio-applications is summarized. The feature article focuses on the applications of organic-molecule- and metal-complex-based molecular probes in biological systems for detecting different biomarkers of cancer or other diseases. The current challenges and potential future directions of these probes for applications in biological systems are also discussed.

CasSABER for Programmable In Situ Visualization of Low and Nonrepetitive Gene Loci.

Site-specific imaging of target genes using CRISPR probes is essential for understanding the molecular mechanisms of gene function and engineering tools to modulate its downstream pathways. Herein, we develop CRISPR/Cas9-mediated signal amplification by exchange reaction (CasSABER) for programmable in situ imaging of low and nonrepetitive regions of the target gene in the cell nucleus. The presynthesized primer-exchange reaction (PER) probe is able to hybridize multiple fluorophore-bearing imager strands to specifically light up dCas9/sgRNA target-bound gene loci, enabling in situ imaging of fixed cellular gene loci with high specificity and signal-to-noise ratio. In combination with a multiround branching strategy, we successfully detected nonrepetitive gene regions using a single sgRNA. As an intensity-codable and orthogonal probe system, CasSABER enables the adjustable amplification of local signals in fixed cells, resulting in the simultaneous visualization of multicopy and single-copy gene loci with similar fluorescence intensity. Owing to avoiding the complexity of controlling in situ mutistep enzymatic reactions, CasSABER shows good reliability, sensitivity, and ease of implementation, providing a rapid and cost-effective molecular toolkit for studying multigene interaction in fundamental research and gene diagnosis.

Isotope Exchange-Based 18F-Labeling Methods.

18F-Labeling methods for the preparation of 18F-labeled molecular probes can be classified into electrophilic fluorination, nucleophilic fluorination, metal-F coordination, and 18F/19F isotope exchange. Isotope exchange-based 18F-labeling methods demonstrate mild conditions featuring water resistance and facile high-performance liquid chromatography-free purification in direct 18F-labeling of substrates. This paper systematically reviews isotope exchange-based 18F-labeling methods sorted by the adjacent atom bonding with F, i.e., carbon and noncarbon atoms (Si, B, P, S, Ga, Fe, etc.). The respective isotope exchange mechanism, radiolabeling condition, radiochemical yield, molar activity, and stability of the 18F-product are mainly discussed for each isotope exchange-based 18F-labeling method as well as the cutting-edge application of the corresponding 18F-labeled molecular probes.

Radiosynthesis, optimization and pharmacokinetic study of the 99m Tc-labeled human epidermal growth factor receptor 2 affibody molecule probe 99m Tc-(HE) 3 Z HER2:V2.

OBJECTIVE: To prepare a single-photon molecular probe easily labeled with 99m Tc for evaluating the expression status of the human epidermal growth factor receptor 2 (HER2) receptor in ovarian cancer. MATERIALS AND METHODS: The HEHEHE tag was added to the amino terminus of the affibody Z HER2:V2 by the method of gene recombinant expression, and a new affibody was synthesized which was easy to be labeled with 99m Tc. The newly prepared affibody was labeled with 99m Tc, and pharmacokinetic studies were carried out. RESULTS: A new affibody (HE) 3 Z HER2:V2 was prepared by the method of gene recombination and expression, which is easy to be labeled with 99m Tc. The 99m Tc labeling of the affibody can reach about 95% at 90°C. The pharmacokinetic study has shown that the 99m Tc-labeled molecular probe has a fast clearance time in the blood and little side effect, which may be a promising single-photon emission computed tomography (SPECT) imaging agent. CONCLUSION: The affibody (HE) 3 Z HER2:V2 is easy to be labeled with 99m Tc, has a high yield and has a suitable half-life in vivo, which is suitable for the next step in ovarian cancer model imaging research.

A new organic molecular probe as a powerful tool for fluorescence imaging and biological study of lipid droplets.

Background: Lipid droplets (LDs) are critical organelles associated with many physiological processes in eukaryotic cells. To visualize and study LDs, fluorescence imaging techniques including the confocal imaging as well as the emerging super-resolution imaging of stimulated emission depletion (STED), have been regarded as the most useful methods. However, directly limited by the availability of advanced LDs fluorescent probes, the performances of LDs fluorescence imaging are increasingly unsatisfied with respect to the fast research progress of LDs. Methods: We herein newly developed a superior LDs fluorescent probe named Lipi-QA as a powerful tool for LDs fluorescence imaging and biological study. Colocalization imaging of Lipi-QA and LDs fluorescent probe Ph-Red was conducted in four cell lines. The LDs staining selectivity and the photostability of Lipi-QA were also evaluated by comparing with the commercial LDs probe Nile Red. The in-situ fluorescence lifetime of Lipi-QA in LDs was determined by time-gated detection. The cytotoxicity of Lipi-QA was assessed by MTT assay. The STED saturation intensity as well as the power- and gate time-dependent resolution were tested by Leica SP8 STED super-resolution nanoscopy. The time-lapse 3D confocal imaging and time-lapse STED super-resolution imaging were then designed to study the complex physiological functions of LDs. Results: Featuring with the advantages of the super-photostability, high LDs selectivity, long fluorescence lifetime and low STED saturation intensity, the fluorescent probe Lipi-QA was capable of the long-term time-lapse three-dimensional (3D) confocal imaging to in-situ monitor LDs in 3D space and the time-lapse STED super-resolution imaging (up to 500 STED frames) to track the dynamics of LDs with nanoscale resolution (37 nm). Conclusions: Based on the state-of-the-art fluorescence imaging results, some new biological insights into LDs have been successfully provided. For instance, the long-term time-lapse 3D confocal imaging has surely answered an important and controversial question that the number of LDs would significantly decrease rather than increase upon starvation stimulation; the time-lapse STED super-resolution imaging with the highest resolution has impressively uncovered the fission process of nanoscale LDs for the first time; the starvation-induced change of LDs in size and in speed has been further revealed at nanoscale by the STED super-resolution imaging. All of these results not only highlight the utility of the newly developed fluorescent probe but also significantly promote the biological study of LDs.

Generation of an Aptamer Targeting Receptor-Type Tyrosine-Protein Phosphatase F.

Cell-SELEX is a powerful tool to generate aptamers that specifically bind the native molecules on living cells. Here, we report an aptamer ZAJ4a generated by cell-SELEX. The molecular target of ZAJ4a was pulled down by the enriched cell-SELEX pool and identified to be the receptor-type tyrosine-protein phosphatase F (PTPRF) through a stable isotope labeling using amino acids in cell culture (SILAC)-based quantitative proteomic method. ZAJ4a showed high binding affinity with nanomolar range to cancer cells expressing PTPRF. Meanwhile, PTPRF was proven to highly express on several cancer cell lines using ZAJ4a as a molecular probe and to highly express in many kinds of cancer samples using gene expression profiling interactive analysis (GEPIA2) from the TCGA and GTEx databases. These results indicate that the aptamer generated by cell-SELEX showed good specificity at the molecular level. This cell-SELEX and target identification strategies show great potential for identifying biomarkers on the cell surface.

Fluorescent molecular probes for imaging and detection of oxidases and peroxidases in biological samples.

Oxidases and peroxidases are two subclasses of oxidoreductases. The abnormal expression of oxidases (such as tyrosinase, cytochrome P450 oxidases, and monoamine oxidases) and peroxidases (such as glutathione peroxidase, myeloperoxidase, and eosinophil peroxidase) is relative with some diseases. Therefore, the analysis of oxidases and peroxidases is great important for disease diagnosis and treatment. Fluorescent probes present simple protocol, high sensitivity and good stability in sensing field. Molecule fluorescent probes are constructed with chemical groups that tunes their fluorescence emission in response to binding events, chemical reactions, and the surrounding environment. A fluorescent probe is an efficient tool for visualizing the activity of enzymes in living organisms on the basis of its high specificity, sensitivity, and noninvasiveness characteristics. In this review, we focus on the sensing of oxidases and peroxidases by molecule fluorescent probes, and hope to bring new insight to wide researchers about oxidases and peroxidases in biological samples.

Optimization of an aptamer against Prorocentrum minimum - A common harmful algae by truncation and G-quadruplex-forming mutation.

Harmful algal blooms (HABs) caused by Prorocentrum minimum have seriously posed economic losses and ecological disasters. To reduce these losses, aptamers are used as a new molecular probe to establish rapid methods. Herein, to improve the affinity and application of aptamers in the detection of harmful algae, the optimization was performed on the previously reported aptamers against P. minimum. First, a total of seven candidate aptamers, including three truncated aptamers (TA1, TA2 and TA3) and four mutant aptamers (MA1, MA2, MA3 and MA4), were obtained by truncation and G-quadruplex (GQ)-forming mutation. Next, the specificity and affinity test by flow cytometry revealed that except for TA1 and TA2, all of the candidate aptamers are specific with the equilibrium dissociation constant of (40.4 ± 5.5) nM for TA3, (63.3 ± 24.0) nM for MA1, (71.7 ± 14.6) nM for MA2, (365.9 ± 74.4) nM for MA3, and (21.1 ± 0.5) nM for MA4, respectively. The circular dichroism analysis of the mutant aptamers demonstrated that the GQ structures formed by MA1/MA2, MA3 and MA4 were antiparallel, mixed parallel and parallel, respectively. The affinity of aptamers with various GQ is in the order of parallel structure > antiparallel structure > mixed parallel structure. In addition, to further improve binding ability, the binding conditions of MA4 were optimized as follows: binding time, 60 min; binding temperature, 37 °C; pH of the binding buffer, 7.5; and Na+/Mg2+ concentration in the binding buffer, 100 mM/0.5 mM. The binding examination by fluorescence microscopy showed that MA4 had a stronger binding ability to P. minimum than the original aptamer. Taken together, this study not only obtained an aptamer with higher affinity than the original aptamer, which laid a good foundation for subsequent application, but also may provide a feasible reference method for aptamer optimization.