Steric Hindrance-Mediated Enzymatic Reaction Enable Homogeneous Dual Fluorescence Indicators Aptasensing of Hepatocellular Carcinoma CTCs.

Circulating tumor cells (CTCs) serve as important biomarkers in the liquid biopsy of hepatocellular carcinoma (HCC). Herein, a homogeneous dual fluorescence indicators aptasensing strategy is described for CTCs in HCC, with the core assistance of a steric hindrance-mediated enzymatic reaction. CTCs in the sample could specifically bind to a 5'-biotin-modified glypican-3 (GPC3) aptamer and remove the steric hindrance formed by the biotin-streptavidin system. This influences the efficiency of the terminal deoxynucleotidyl transferase enzymatic reaction. Then, methylene blue (MB) was introduced to react with the main product poly cytosine (polyC) chain, and trivalent cerium ion (Ce3+) was added to react with the byproduct pyrophosphate to form fluorescent pyrophosphate cerium coordination polymeric nanoparticles. Finally, the CTCs were quantified by dual fluorescence indicators analysis. Under optimized conditions, the linear range was 5 to 104 cells/mL, and the limits of detection reached 2 cells/mL. Then, 40 clinical samples (15 healthy and 25 HCC patients) were analyzed. The receiver operating characteristic curve analysis revealed an area under the curve of 0.96, a sensitivity of 92%, and a specificity of 100%. Therefore, this study established a sensitive and accurate CTCs sensing system for clinical HCC patients, promoting early tumor diagnosis.

New Perylene-Based Chemiluminescent Polymer Nanoparticles for Highly Selective Detection of the Superoxide Anion In Vivo.

The superoxide anion (O2•-) is one of the primary reactive oxygen species in biological systems. Developing a determination system for O2•- in vivo has attracted much attention thanks to its complex biological function. Herein, we proposed a new perylene-based chemiluminescence (CL) probe, the SH-PDI polymer, which was capable of generating strong CL signals with O2•- in comparison with other ROS. The CL mechanism involved was proposed to be a kind of oxidation reaction induced by the breakage of the S-S and S-H bonds into sulfoxide bonds by O2•-. Subsequently, a nanoprecipitation method was introduced, using cumene-terminated poly(styrene-co-maleic anhydride) as the amphiphilic agent, to obtain water-soluble nanoparticles, SPPS NPs, which exhibited not only stronger CL intensity but also higher selectivity toward O2•- than the SH-PDI polymer. Moreover, the CL wavelength of the SPPS-O2•- system was found to be located at 580 and 710 nm, which was conducive to CL imaging. By virtue of these advantages, SPPS NPs were utilized to evaluate the O2•- level in vitro in the range of 0.25-60 µM at pH 7.0, with a detection limit of 8.2 × 10-8 M (S/N = 3). Moreover, SPPS NPs were also capable of imaging O2•- in an LPS-induced acute inflammation mice model and drug-induced acute kidney injury (AKI).

Novel Near-Infrared Iridium(III) Complex for Chemiluminescence Imaging of Hypochlorous Acid.

Chemiluminescence (CL) probes that possess near-infrared (NIR) emission are highly desirable for in vivo imaging due to their deeper tissue penetration ability and intrinsically high sensitivity. Herein, a novel iridium-based CL probe (NIRIr-CL-1) with direct NIR emission was reported as the result of hypochlorous acid (HClO)-initiated oxidative deoximation. To improve its biocompatibility and extend the CL time for in vivo imaging applications, this NIRIr-CL-1 was prepared as a CL nanoparticle probe (NIRIr-CL-1 dots) through encapsulation by an amphiphilic polymer Pluronic F127 (F127). All results demonstrate that the NIRIr-CL-1 dots have good selectivity and sensitivity for visualization of HClO even at the depth of 1.2 cm. Owing to these advantages, the CL imaging of exogenous and endogenous HClO in mice was achieved. This study could provide new insights into the construction of new NIR emission CL probes and expand their applications in biomedical imaging.

Endoplasmic Reticulum Peroxynitrite Fluctuations in Hypoxia-Induced Endothelial Injury and Sepsis with a Two-Photon Fluorescence Probe.

Sepsis is a serious systemic inflammatory disease that frequently results in death. Early diagnosis and timely targeted interventions could improve the therapeutic effect. Recent work has revealed that the reactive oxygen species (ROS) in the endoplasmic reticulum (ER) and hypoxia-induced endothelial injury play significant roles in sepsis. However, the relationship between the levels of peroxynitrite (ONOO-) and hypoxia-induced endothelial injury as well as different states of sepsis remain unexplored. Herein, we developed a unique two-photon fluorescent probe (ER-ONOO-) for detecting ONOO- in aqueous solution that has high sensitivity, high selectivity, and ultrafast response time. In addition, ER-ONOO- was successfully used to evaluate the levels of ONOO- at the ER with three kinds of methods in a hypoxia-induced endothelial injury model. Furthermore, ER-ONOO- is capable of monitoring the changes in organ fluorescence through ONOO- variation in different stages of a cecum ligation and puncture (CLP) mouse model. Moreover, we also confirmed that the endoplasmic reticulum stress and oxidative stress participated in the CLP model. Consequently, this research can provide a reliable tool for studying ONOO- fluctuation in sepsis and provide new insights into the pathogenic and therapeutic mechanisms involved.

Simplified Rapid Enrichment of CTCs and Selective Recognition Prereduction Enable a Homogeneous ICP-MS Liquid Biopsy Strategy of Lung Cancer.

The effective enrichment and hypersensitivity analysis of circulating tumor cells (CTCs) in clinical whole blood samples are highly significant for clinical tumor liquid biopsy. In this study, we established an easy operation and affordable CTCs extraction technique while simultaneously performing the homogeneous inductively coupled plasma mass spectrometry (ICP-MS) determination of CTCs in lung cancer clinical samples based on selective recognition reactions and prereduction phenomena. Our strategy allowed for the pretreatment of whole blood samples in less than 45 min after step-by-step centrifugation, which only required lymphocyte separation solution and erythrocyte lysate. Furthermore, a three-stage signal amplification system consisting of catalytic hairpin assembly (CHA), selective recognition for C-Ag+-C structures and Ag+ of copper sulfide nanoparticles (CuS NPs), and prereduction of Hg2+ through ascorbic acid (AA) was constructed by using mucin 1 as the CTCs marker and the aptamer for identification probes. In optimal conditions, the detection limits of ICP-MS were as low as 0.3 ag/mL for mucin 1 and 0.25 cells/mL for A549 cells. This method analyzed CTCs in 58 clinical samples quantitatively, and the results were consistent with clinical CT images and pathological findings. The area under the curve (AUC) value of the receiver operating characteristic (ROC) curve was 0.957, which provided a specificity of 100% and a sensitivity of 91.5% for the assay. Therefore, the simplicity of the extraction method, the accessibility, and the high sensitivity of the assay method make the strategies attractive for clinical CTCs testing applications.

Simultaneous Monitoring of HOCl and Viscosity with Drug-Induced Pyroptosis in Live Cells and Acute Lung Injury.

Pyroptosis is a newly identified form of cell death that is closely correlated with many diseases. Recent studies have indicated that the inflammation in pyroptosis would accelerate the generation of reactive oxygen species (ROS). In addition, intracellular viscosity is another key microenvironmental parameter that reflects many physiological and pathological states in the early stage, hypochlorous acid (HOCl), as an important ROS, also plays significant roles in a variety of pathologies. However, the fluctuation of viscosity and HOCl in the process of pyroptosis is still unknown. Herein, we present a dual-responsive fluorescent probe (Lyso-VH) for simultaneously detecting viscosity and HOCl. Lyso-VH was successfully used to image the fluctuation of HOCl and viscosity in the lysosome of three kinds of cells with dependent and independent channels. Moreover, Lyso-VH can be employed to investigate the changes of HOCl and viscosity during the process of pyroptosis in living cells and acute lung injury (ALI). Thus, this work can not only serve as a powerful tool to simultaneously visualize the fluctuation of HOCl and viscosity in lysosomes, but also provide a new insight into drug-induced pyroptosis in living cells and acute lung injury.

A Two-Photon Excited Near-Infrared Iridium(III) Complex for Multi-signal Detection and Multimodal Imaging of Hypochlorite.

Hypochlorite (ClO-), as a type of reactive oxygen species (ROS), plays a crucial role in the process of oxidative stress and is closely related to many diseases. Thus, developing a method for detecting and imaging of ClO- with high sensitivity and selectivity is of great significance. However, the applications of most luminescent probes are limited to the fact that the excitation and emission wavelengths of them are in the visible light region rather than in the near-infrared (NIR) region. Hence, an NIR iridium(III) complex (Mul-NIRIr) with two-photon excitation is designed for the detecting and imaging of ClO-. In the presence of ClO-, the luminescent intensity and lifetime of Mul-NIRIr are remarkably enhanced. Interestingly, Mul-NIRIr also exhibits excellent electrochemiluminescence (ECL) properties, and the ECL signal is significantly enhanced with the addition of ClO-. What is more, Mul-NIRIr is also suitable for the detection and analysis ClO- by flow cytometry. Therefore, Mul-NIRIr is developed to detect multiple signals and is successfully applied to detect exogenous and endogenous ClO- in living cells with one-photon, two-photon, and phosphorescence lifetime image microscopy (PLIM). In addition, Mul-NIRIr was successfully used for imaging of ClO- in tissues and inflammatory mouse models. All of the above results indicate that Mul-NIRIr is highly effective in detecting ClO- in living systems.

Multimodal Imaging Iridium(III) Complex for Hypochlorous Acid in Living Systems.

Biomolecule tracing with different imaging methods is of great significance for more accurately unravelling the fundamental processes in living systems. However, considering the different principles of each imaging method for probe design, it is still a great challenge to apply one molecular probe to achieve two or even more imaging analyses for biomarkers. In general, traditional oxime was reported as a recognition group for fluorescence imaging of HOCl. Herein, for the first time, we designed the oxime decorated iridium(III) complex, which can be directly used for chemiluminescence as well as two-photon luminescence and photoluminescence lifetime imaging of HOCl in living systems. Moreover, the novel chemiluminescence mechanism of Ir-CLFLPLIM for HOCl was also proposed and explored by continuously monitoring chemiluminescence peak shapes and mass spectra, inferring the reaction intermediate and calculating the chemical reaction energy range of the reaction process. This strategy could lead us to expand the chemiluminescence application of transition metal complexes and develop more multimodal imaging probes.

Visualization of Lung Inflammation to Pulmonary Fibrosis via Peroxynitrite Fluctuation.

Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive lung disease with short survival time. However, owing to the unknown etiology and the lack of sensitive and noninvasive methods, the diagnosis of IPF in the early stage is still full of challenges. Since the levels of oxidative stress in mitochondria are relevant to pulmonary fibrosis, we herein present a simultaneous near-infrared (NIR)-Ia window and ratiometic fluorescent probe, rTPONOO-1, with two-photon and mitochondria-targeting abilities to explore the potential biological roles of peroxynitrite (ONOO-) in different states of lung slices from healthy to lung inflammation and pulmonary fibrosis, and there is a good linear relationship between ratiometric fluorescence changes and the rate of pulmonary fibrosis from hematoxylin and eosin (H&E) and Masson staining. In addition, the therapeutic efficacy of aminoguanidine hemisulfate salt (AG) was also investigated. Thus, rTPONOO-1 has great potential in quickly predicting the progression of pulmonary fibrosis in the early stage and improving effective treatment.

Turn-on Fluorescent Probe for Exogenous and Endogenous Imaging of Hypochlorous Acid in Living Cells and Quantitative Application in Flow Cytometry.

Hypochlorous acid (HClO) acts as a dominant microbicidal mediator in the natural immune system, and the excess production of hypochlorites is related to a series of diseases. Thus, it is vitally important and necessary to develop a highly sensitive and selective method for HClO detection in living systems, and most of fluorescent probes are mainly focused on cells imaging. Besides, accurate HClO quantitative information about individual cells in a large cell population is extremely important for understanding inflammation and cellular apoptosis as well. In our work, a turn-on fluorescent probe has been synthesized, which can selectively and sensitively detect HClO with fast response time. The probe is almost nonfluorescent possibly due to both the spirolactam form of fluorescein and unbridged C═N bonds which can undergo a nonradiative decay process in the excited state. Upon the addition of ClO-, the probe was oxidized to ring-opened fluorescent form and the fluorescence intensity was greatly enhanced. In live cell experiments, the probe was successfully applied to image exogenous ClO- in HeLa cells and endogenous HClO in RAW 264.7 macrophage cells. In particular, the quantitative information on exogenous and endogenous HClO can also be acquired in flow cytometry. Therefore, the probe not only can image exogenous and endogenous HClO but also provides a new and promising platform to quantitatively detect HClO in flow cytometry.

Green synthesis of fluorescence carbon nanoparticles from yum and application in sensitive and selective detection of ATP.

Fluorescent carbon nanoparticles (CPs), a fascinating class of recently discovered nanocarbons, have been widely known as some of the most promising sensing probes in biological or chemical analysis. In this study, we demonstrate a green synthetic methodology for generating water-soluble CPs with a quantum yield of approximately 24% via a simple heating process using yum mucilage as a carbon source. The prepared carbon nanoparticles with an ~10 nm size possessed excellent fluorescence properties, and the fluorescence of the CPs was strongly quenched by Fe(3+), and recovered by adenosine triphosphate (ATP), thus, an 'off' and 'on' system can be easily established. This 'CPs-Fe(3+)-ATP' strategy was sensitive and selective at detecting ATP with the linear range of 0.5 µmol L(-1) to 50 µmol L(-1) and with a detection limit of 0.48 µmol L(-1).

Antiviral activity of Paulownia tomentosa against enterovirus 71 of hand, foot, and mouth disease.

The bark, leaves, and flowers of Paulownia trees have been used in traditional Chinese medicine to treat infectious and inflammatory diseases. We investigated the antiviral effects of Paulownia tomentosa flowers, an herbal medicine used in some provinces of P. R. China for the treatment of skin rashes and blisters. Dried flowers of P. tomentosa were extracted with methanol and tested for antiviral activity against enterovirus 71 (EV71) and coxsackievirus A16 (CAV16), the predominant etiologic agents of hand, foot, and mouth disease in P. R. China. The extract inhibited EV71 infection, although no effect was detected against CAV16 infection. Bioactivity-guided fractionation was performed to identify apigenin as an active component of the flowers. The EC50 value for apigenin to block EV71 infection was 11.0 µM, with a selectivity index of approximately 9.3. Although it is a common dietary flavonoid, only apigenin, and not similar compounds like naringenin and quercetin, were active against EV71 infection. As an RNA virus, the genome of EV71 has an internal ribosome entry site that interacts with heterogeneous nuclear ribonucleoproteins (hnRNPs) and regulates viral translation. Cross-linking followed by immunoprecipitation and reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that EV71 RNA was associated with hnRNPs A1 and A2. Apigenin treatment disrupted this association, indicating that apigenin suppressed EV71 replication through a novel mechanism by targeting the trans-acting factors. This study therefore validates the effects of Paulownia against EV71 infection. It also yielded mechanistic insights on apigenin as an active compound for the antiviral activity of P. tomentosa against EV71 infection.

Advancements in dual-target inhibitors of PI3K for tumor therapy: Clinical progress, development strategies, prospects.

Phosphoinositide 3-kinases (PI3Ks) modify lipids by the phosphorylation of inositol phospholipids at the 3'-OH position, thereby participating in signal transduction and exerting effects on various physiological processes such as cell growth, metabolism, and organism development. PI3K activation also drives cancer cell growth, survival, and metabolism, with genetic dysregulation of this pathway observed in diverse human cancers. Therefore, this target is considered a promising potential therapeutic target for various types of cancer. Currently, several selective PI3K inhibitors and one dual-target PI3K inhibitor have been approved and launched on the market. However, the majority of these inhibitors have faced revocation or voluntary withdrawal of indications due to concerns regarding their adverse effects. This article provides a comprehensive review of the structure and biological functions, and clinical status of PI3K inhibitors, with a specific emphasis on the development strategies and structure-activity relationships of dual-target PI3K inhibitors. The findings offer valuable insights and future directions for the development of highly promising dual-target drugs targeting PI3K.

Construction of a sequence activated fluorescence probe for simultaneous detection of γ-glutamyl transpeptidase and peroxynitrite in acute kidney injury.

Acute kidney injury (AKI) can result in a sudden decline in kidney function and, if not promptly diagnosed and treated, can lead to a high mortality rate. Therefore, there is a critical need for the development of a non-invasive and dependable early diagnostic method for AKI to prevent its progression and deterioration. To address the risk of misdiagnosis or overlooked diagnosis due to reliance on a single biomarker, we developed a novel molecular fluorescent probe (HX-GP) to simultaneously detect and image two biomarkers, γ-Glutamyl transpeptidase (γ-GGT) and Peroxynitrite (ONOO-), in the AKI process. HX-GP can specifically detect γ-GGT in the red fluorescence channel (λem = 613 nm) and ONOO- in the green fluorescence channel (λem = 518 nm). HX-GP demonstrated high sensitivity, selectivity, and rapid response, showing excellent biocompatibility and detection performance. In addition, HX-GP was successful in imaging experiments in a cell model of cisplatin-induced AKI, a result that highlights its potential application value in early diagnosis of AKI.

Development of novel focal adhesion kinase (FAK) inhibitors for targeting cancer: Structural insights and therapeutic potential.

Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase frequently overexpressed in various cancer cells, facilitating tumor growth through the regulation of cell adhesion, migration, and proliferation. Consequently, targeting FAK is considered a promising anti-tumor strategy, particularly for invasive cancers. Numerous potent small-molecule inhibitors have progressed to clinical trials. Among these, Defactinib is under evaluation for regulatory approval as a treatment for ovarian serous tumors. Furthermore, novel FAK inhibitors, including PROTACs, have emerged as key research focuses, anticipated to overcome the limitations of traditional inhibitors. In this Perspective, we highlight the protein structure, biological functions, relevant signaling pathways, and associations of FAK with cancer development. We also analyze the clinical status of FAK inhibitors, paying special attention to the various classes of FAK inhibitors, with detailed analyses of their chemical structures, structure-activity relationships (SARs), bioactivity profiles, selectivity profiles, and therapeutic potentials.

Fructose@histone synergistically improve the performance of DNA-templated Cu NPs: rapid analysis of LAM in tuberculosis urine samples using a handheld fluorometer and a smartphone RGB camera.

This study presented a nanoparticle-enhanced aptamer-recognizing homogeneous detection system combined with a portable instrument (NASPI) to quantify lipoarabinomannan (LAM). This system leveraged the high binding affinity of aptamers, the high sensitivity of nanoparticle cascade amplification, and the stabilization effect of dual stabilizers (fructose and histone), and used probe-Cu2+ to achieve LAM detection at concentrations ranging from 10 ag mL-1 to 100 fg mL-1, with a limit of detection of 3 ag mL-1 using a fluorometer. It can also be detected using an independently developed handheld fluorometer or the red-green-blue (RGB) camera of a smartphone, with a minimum detection concentration of 10 ag mL-1. We validated the clinical utility of the biosensor by testing the LAM in the urine of patients. Forty urine samples were tested, with positive LAM results in the urine of 18/20 tuberculosis (TB) cases and negative results in the urine of 6/10 latent tuberculosis infection cases and 10/10 non-TB cases. The assay results revealed a 100% specificity and a 90% sensitivity, with an area under the curve of 0.9. We believe that the NASPI biosensor can be a promising clinical tool with great potential to convert LAM into clinical indicators for TB patients.

Insight into small-molecule inhibitors targeting extracellular nucleotide pyrophosphatase/phosphodiesterase1 for potential multiple human diseases.

Extracellular nucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) has been identified as a type II transmembrane glycoprotein. It plays a crucial role in various biological processes, such as bone mineralization, cancer cell proliferation, and immune regulation. Consequently, ENPP1 has garnered attention as a promising target for pharmacological interventions. Despite its potential, the development of clinical-stage ENPP1 inhibitors for solid tumors, diabetes, and silent rickets remains limited. However, there are encouraging findings from preclinical trials involving small molecules exhibiting favorable therapeutic effects and safety profiles. This perspective aims to shed light on the structural properties, biological functions and the relationship between ENPP1 and diseases. Additionally, it focuses on the structure-activity relationship of ENPP1 inhibitors, with the intention of guiding the future development of new and effective ENPP1 inhibitors.

Target proteins-regulated DNA nanomachine-electroactive substance complexes enable separation-free electrochemical detection of clinical exosome.

Simple and reliable profiling of tumor-derived exosomes (TDEs) holds significant promise for the early detection of cancer. Nonetheless, this remains challenging owing to the substantial heterogeneity and low concentration of TDEs. Herein, we devised an accurate and highly sensitive electrochemical sensing strategy for TDEs via simultaneously targeting exosomal mucin 1 (MUC1) and programmed cell death ligand 1 (PD-L1). This approach employs high-affinity aptamers as specific recognition elements, utilizes rolling circle amplification and DNA nanospheres as effective bridges and signal amplifiers, and leverages methylene blue (MB) and doxorubicin (DOX) as robust signal reporters. The crux of this separation- and label-free method is the specific response of MB and DOX to G-quadruplex structures and DNA nanospheres, respectively. Quantifying TDEs using this strategy enabled precise discrimination of lung cancer patients (n = 25) from healthy donors (n = 12), showing 100% specificity (12/12), 92% sensitivity (23/25), and an overall accuracy of 94.6% (35/37), with an area under the receiver operating characteristic curve (AUC) of 0.97. Furthermore, the assay results strongly correlated with findings from computerized tomography and pathological analyses. Our approach could facilitate the early diagnosis of lung cancer through TDEs-based liquid biopsy.

Self-Assembled DNA Machine and Selective Complexation Recognition Enable Rapid Homogeneous Portable Quantification of Lung Cancer CTCs.

In this study, we systematically investigated the interactions between Cu2+ and various biomolecules, including double-stranded DNA, Y-shaped DNA nanospheres, the double strand of the hybridization chain reaction (HCR), the network structure of cross-linked HCR (cHCR), and small molecules (PPi and His), using Cu2+ as an illustrative example. Our research demonstrated that the coordination between Cu2+ and these biomolecules not only is suitable for modulating luminescent material signals through complexation reactions with Cu2+ but also enhances signal intensities in materials based on chemical reactions by increasing spatial site resistance and local concentration. Building upon these findings, we harnessed the potential for signal amplification in self-assembled DNA nanospheres and the selective complexation modulation of calcein in conjunction with the aptamer targeting mucin 1 as a recognition probe. We applied this approach to the analysis of circulating tumor cells, with the lung cancer cell line A549 serving as a representative model. Our assay, utilizing both a fluorometer and a handheld detector, achieved impressive detection limits of ag/ml and single-cell levels for mucin 1 and A549 cells, and this approach was successfully validated using 46 clinical samples, yielding 100% specificity and 86.5% sensitivity. Consequently, our strategy has paved the way for more portable and precise disease diagnosis.

Controllable self-assembled DNA nanomachine enable homogeneous rapid electrochemical one-pot assay of lung cancer circulating tumor cells.

A homogeneous rapid (45 min) one-pot electrochemical (EC) aptasensor was established to quantitatively detect circulating tumor cells (CTCs) in lung cancer patients using mucin 1 as a marker. The core of this study is that the three single-stranded DNA (Y1, Y2, and Y3) could be hybridized to form Y-shaped DNA (Y-DNA) and further self-assemble to form DNA nanosphere. The aptamer of mucin 1 could be complementary and paired with Y1, thus disrupting the conformation of the DNA nanosphere. When mucin 1 was present, the aptamer combined specifically with mucin 1, thus preserving the DNA nanosphere structure. Methylene blue (MB) acted as a signal reporter, which could be embedded between two base pairs in the DNA nanosphere to form a DNA nanosphere-MB complex, reducing free MB and resulting in a lower electrochemical signal. The results demonstrated that the linear ranges for mucin 1 and A549 cells were 1 ag/mL-1 fg/mL and 1-100 cells/mL, respectively, with minimum detectable concentrations were 1 ag/mL and 1 cell/mL, respectively. The quantitative analysis of CTCs in 44 clinical blood samples was performed, and the results were consistent with the computerized tomography (CT) images, pathological findings and folate receptor-polymerase chain reaction (FR-PCR) kits. The receiver operating characteristic (ROC) curve exhibited an area under the curve (AUC) value of 0.970. The assay revealed 100% specificity and 94.1% sensitivity. It is believed that this electrochemical aptasensor could provide a new approach to detect CTCs.