Rational design of a near-infrared dual-emission fluorescent probe for ratiometric imaging of glutathione in cells.

Sensors for which the output signal is an intensity change for a single-emission peak are easily disturbed by many factors, such as the stability of the instrument, intensity of the excitation light, and biological background. However, for ratiometric fluorescence sensors, the output signal is a change in the intensity ratio of two or more emission peaks. The fluorescence intensity of these emission peaks is similarly affected by external factors; thus, these sensors have the ability to self-correct, which can greatly improve the accuracy and reliability of the detection results. To accurately image glutathione (GSH) in cells, gold nanoclusters (AuNCs) with intrinsic double emission at wavelengths of 606 nm and 794 nm were synthesized from chloroauric acid. With the emission peak at 606 nm as the recognition signal and the emission peak at 794 nm as the reference signal, a near-infrared dual-emission ratio fluorescence sensing platform was constructed to accurately detect changes in the GSH concentration in cells. In vitro and in vivo analyses showed that the ratiometric fluorescent probe specifically detects GSH and enables ultrasensitive imaging, providing a new platform for the accurate detection of active small molecules.

Enhancing intracellular mRNA precise imaging-guided photothermal therapy with a nucleic acid-based polydopamine nanoprobe.

Despite significant advancements in cancer research, real-time monitoring and effective treatment of cancer through non-invasive techniques remain a challenge. Herein, a novel polydopamine (PDA) nucleic acid nanoprobe has been developed for imaging signal amplification of intracellular mRNA and precise photothermal therapy guidance in cancer cells. The PDA nucleic acid nanoprobe (PDA@DNA) is constructed by assembling an aptamer hairpin (H1) labeled with the Cy5 fluorophore and another nucleic acid recognition hairpin (H2) onto PDA nanoparticles (PDA NPs), which have exceptionally high fluorescence quenching ability and excellent photothermal conversion properties. The nanoprobe could facilitate cellular uptake of DNA molecules and their protection from nuclease degradation. Upon recognition and binding to the intracellular mRNA target, a catalytic hairpin assembly (CHA) reaction occurs. The stem of H1 unfolds upon binding, allowing the exposed H1 to hybridize with H2, forming a flat and sturdy DNA double-stranded structure that detaches from the surface of PDA NPs. At the same time, the target mRNA is displaced and engages in a new cyclic reaction, resulting in the recovery and significant amplification of Cy5 fluorescence. Using thymidine kinase1 (TK1) mRNA as a model mRNA, this nanoprobe enables the analysis of TK1 mRNA with a detection limit of 9.34 pM, which is at least two orders of magnitude lower than that of a non-amplifying imaging nucleic acid probe. Moreover, with its outstanding performance for in vitro detection, this nanoprobe excels in precisely imaging tumor cells. Through live-cell TK1 mRNA imaging, it can accurately distinguish between tumor cells and normal cells. Furthermore, when exposed to 808-nm laser irradiation, the nanoprobe fully harnesses exceptional photothermal conversion properties of PDA NPs. This results in a localized temperature increase within tumor cells, which ultimately triggers apoptosis in these tumor cells. The integration of PDA@DNA presents innovative prospects for tumor diagnosis and image-guided tumor therapy, offering the potential for high-precision diagnosis and treatment of tumors.

Lysosomes Initiating and DNAzyme-Assisted Intracellular Signal Amplification Strategy for Quantification of Alpha-Fetoprotein in a Single Cell.

Cellular trace proteins are critical for maintaining normal cell functions, with their quantitative analysis in individual cells aiding our understanding of the role of cell proteins in biological processes. This study proposes a strategy for the quantitative analysis of alpha-fetoprotein in single cells, utilizing a lysosome microenvironment initiation and a DNAzyme-assisted intracellular signal amplification technique based on electrophoretic separation. A nanoprobe targeting lysosomes was prepared, facilitating the intracellular signal amplification of alpha-fetoprotein. Following intracellular signal amplification, the levels of alpha-fetoprotein (AFP) in 20 HepG2 hepatoma cells and 20 normal HL-7702 hepatocytes were individually evaluated using microchip electrophoresis with laser-induced fluorescence detection (MCE-LIF). Results demonstrated overexpression of alpha-fetoprotein in hepatocellular carcinoma cells. This strategy represents a novel technique for single-cell protein analysis and holds significant potential as a powerful tool for such analyses.

mRNA-activated DNAzyme nanoprobe for tumor cell precise imaging and gene therapy.

A novel Au-nucleic acid nanoprobe, catalyzed by mRNA, has been developed for live cell imaging and precise treatment of tumor cells. This nanoprobe exhibits the remarkable ability to differentiate between tumor cells and normal cells through live cell mRNA imaging, while selectively inducing apoptosis in tumor cells.

A S-substituted Nile Blue-derived bifunctional near-infrared fluorescent probe for in vivo carboxylesterase imaging-guided photodynamic therapy of hepatocellular carcinoma.

The development of theranostic probes that integrate both diagnostic and therapeutic functions still remains an intractable challenge in precise cancer treatment. Herein, a novel bifunctional near-infrared (NIR) fluorescent probe (CEP1) for carboxylesterase (CE) imaging and photodynamic therapy (PDT) of hepatocellular carcinoma (HCC) has been firstly developed and successfully applied in vitro and in vivo. The probe was constructed by introducing carbamate as both the recognition unit and the fluorescence quenching unit into the fluorophore S-substituted Nile Blue (ENBS) via a self-eliminating spacer with substituted chloride. It can be activated by CE and hydrolyzed into fluorescent ENBS, which recover fluorescence at about 700 nm, and can generate superoxide radical anions under NIR irradiation. Additionally, the probe could effectively distinguish tumor cells from normal cells by CE imaging of live cells. Furthermore, it could achieve CE imaging in vivo and significantly inhibits tumor growth by imaging-guided PDT. Therefore, this study offers a promising and attractive platform for activatable imaging-guided PDT of HCC.

Ginger (Zingiber officinale Roscoe) preparations for prophylaxis of postoperative nausea and vomiting: A Bayesian network meta-analysis.

ETHNOPHARMACOLOGICAL RELEVANCE: Ginger has been proposed for prevention of postoperative nausea and vomiting (PONV), however it remains equivocal whether ginger can be an alternative option and which certain preparation is optimal for PONV prophylaxis. AIM OF THE STUDY: We conducted a network meta-analysis (NMA) to compare and rank relative efficacy for PONV control among all available ginger preparations collected in the databases. METHODS: Eligible records were identified by retrieving Medline (via Pubmed), Embase, Web of Science, CENTRAL, CNKI, WHO ICTRP and ClinicalTrials.gov for randomized controlled trials that investigated the efficacy of ginger therapies for the prophylaxis of PONV. A bayesian NMA within random-effects models was implemented. Certainty of evidence for estimates was investigated following GRADE framework. We prospectively registered the protocol (CRD 42021246073) in PROSPERO. RESULTS: Eighteen publications comprising 2199 participants with PONV were identified. Ginger oil (RR [95%CI], 0.39 [0.16, 0.96]) appeared to have the highest probability of being ranked best to decrease the incidence of postoperative vomiting (POV), with statistical significance compared with placebo, based on high to moderate confidence in estimates. With regard to reducing postoperative nausea (PON), statistically superiority was not observed in ginger regimens compared with placebo based on moderate to low certainty of evidence. Reduction in antemetic use and nausea intensity were noticed in ginger powder and oil. Ginger was significantly associated with better efficacy for Asian, older age, higher dosage, preoperative administration, hepatobiliary and gastrointestinal surgery. CONCLUSIONS: Ginger oil appeared to be superior to other ginger treatments for the prophylaxis of POV. With regard to reducing PON, ginger preparations indicated no obvious advantages.

A Novel Fluorescent Aptamer Sensor with DNAzyme Signal Amplification for the Detection of CEA in Blood.

Carcinoembryonic antigen (CEA) is a tumor-specific biomarker; however, its low levels in the early stages of cancer make it difficult to detect. To address the need for analysis of ultra-low-level substances, we designed and synthesized a fluorescent aptamer sensor with DNAzyme signal amplification and used it for the detection of CEA in blood. In the presence of the target protein, the aptamer sequence in the recognition probe binds to the target protein and opens the hairpin structure, hybridizes with the primer and triggers a polymerization reaction in the presence of polymerase to generate double-stranded DNA with two restriction endonuclease Nb.BbvCl cleavage sites. At the same time, the target protein is displaced and continues to bind to another recognition probe, triggering a new round of polymerization reaction, forming a cyclic signal amplification triggered by the target. The experimental results show that the blood detection with CEA has a high sensitivity and a wide detection range. The detection range: 10 fg/mL~10 ng/mL, with a detection limit of 5.2 fg/mL. In addition, the sensor can be used for the analysis of complex biological samples such as blood.

A biodegradable and cofactor self-sufficient aptazyme nanoprobe for amplified imaging of low-abundance protein in living cells.

Despite the progress on the analysis of proteins either in vitro or in vivo, detection and imaging of low-abundance proteins in living cells still remains challenging. Herein, a novel biodegradable and cofactor self-sufficient DNAzyme nanoprobe has been deve-loped for catalytic imaging of protein in living cells with signal amplification capacity. This DNAzyme nanoprobe is constructed by assembling a DNAzyme subunit-containing aptamer hairpin (HP), another DNAzyme subunit strand (DS), and the molecular beacon (MB) substrate strand onto pH-sensitive ZnO@polydopamine nanorods (ZnO@PDA NRs) that work as DNAzyme cofactor suppliers. Such a nanoprobe can facilitate cellular uptake of DNA molecules and protection of them from nuclease degradation as well as release of them in cells by lysosomal acid-triggered dissolution of ZnO@PDA NRs into Zn2+ as DNAzyme cofactor. Upon recognition and binding with the intracellular protein target, the stem of HP is opened, after which the opened HP hybridizes with DS and generates activated DNAzymes. Each activated DNAzyme can catalyze the cleavage of many MB substrates through true enzymatic multiple turnovers, resulting in the separation of the quenched fluorophore/quencher pair labeled in MB and the generation of significantly amplified fluorescence. Using nucleolin (NCL) as a model protein, this nanoprobe enables the analysis of NCL with a detection limit of 1.8 pM, which are at least two orders of magnitude lower than that of non-catalytic imaging probe. Moreover, it could accurately distinguish tumor cells and normal cells by live cell NCL imaging. And the experimental results are also further verified by flow cytometry assays. The developed nanoprobe can be easily extended to detect other biomolecules by the change of their corresponding aptamer sequences, thus providing a promising tool for highly sensitive imaging of low-abundance biomolecules in living cells.

A MnOx-nucleic acid nanoprobe with enzyme-free cascade signal amplification for ultrasensitive intracellular microRNA imaging.

A novel MnOx-nucleic acid nanoprobe was constructed for catalytic imaging of microRNA in living cells based on the combination of catalytic hairpin assembly, hybridization chain reaction, and DNAzyme amplification. This nanoprobe exhibited ultrahigh sensitivity and specificity, and could distinguish tumor cells and normal cells by live cell microRNA imaging.

A colorimetric aptasensor based on a hemin/EpCAM aptamer DNAzyme for sensitive exosome detection.

Exosomes are considered as potential biomarkers that can reflect information from their parent cell-associated cancer microenvironment. Recently, aptasensors have been widely used for cancer and tumor exosome detection. Aptamers related to exosome surface proteins are usually used to introduce a sequence; the aptamer is used for exosome recognition, and the introduced sequence is used to form G-quadruplexes and for signal amplification. In this paper, we found that the EpCAM aptamer is rich in guanine and unimolecular G-quadruplex with a two-layer G-tetrad under acidic conditions, and we investigated its topology, thermal stability and dissociation constant with hemin. Based on this, our proposed colorimetric aptamer sensor combines the unmodified EpCAM aptamer with hemin to construct a hemin/G-quadruplex DNAzyme and catalyze the TMB-H2O2 system to generate a strong colorimetric signal. Therefore, colorimetric signal changes were negatively correlated with the exosome concentration. The linear range of the 1 h assay was 106-108 particles per mL, and the detection limit was 3.94 × 105 particles per mL. In addition, this method can detect exosomes in complex fetal bovine serum samples with good specificity and high sensitivity toward exosomes from breast, liver, and lung cancers with abnormal EpCAM protein expression.

MicroRNA-23a-3p promotes macrophage M1 polarization and aggravates lipopolysaccharide-induced acute lung injury by regulating PLK1/STAT1/STAT3 signalling.

Macrophage polarization is an important effector process in acute lung injury (ALI) induced by sepsis. MicroRNAs (miRNAs) have emerged as important players in regulating ALI process. Here, we showed that elevated microRNA-23a-3p (miR-23a-3p) promoted LPS-induced macrophage polarization and ALI in mice, while inhibition of miR-23a-3p led to reduced macrophage response and ameliorated ALI inflammation. Mechanically, miR-23a-3p regulated macrophage M1 polarization through targeting polo-like kinase 1 (PLK1). PLK1 was downregulated in LPS-treated macrophages and ALI mouse lung tissues. Knockdown of PLK1 increased macrophage M1 polarization through promoting STAT1/STAT3 activation, while overexpression of PLK1 reduced macrophage immune response. Collectively, our results reveal a key miRNA regulon that regulates macrophage polarization for LPS-induced immune response.

Mother's and Father's Migrating in China: Differing Relations to Mental Health and Risk Behaviors Among Left-Behind Children.

Background: In China, the figure for left-behind children (LBC) of migrants stood at 68. 77 million in 2015. Despite being seen as a whole in the last few decades, LBC today differ broadly in parental migrating status. This study focused on LBC with both parents migrating (BLBC), LBC with only mothers migrating (MLBC), LBC with only fathers migrating (FLBC), and previous LBC with one or both parents migrating (PLBC), separately. We aimed at exploring the extent to which LBC were being affected by each migrant parent on both mental health and risk behaviors. Methods: Data from 4,832 children were collected by a school-based survey in both rural and urban areas of China's Anhui province. Each participant anonymously completed a self-administered questionnaire containing the sociodemographics, the Strength and Difficulties Questionnaire (SDQ), the items from the Youth Risk Behavior Surveillance System (YRBSS), and Young's Internet Addiction Test for Chinese (YIAT-C). Data were analyzed using one-way ANOVA and the Chi-squared test. Associations were estimated by multiple linear regression and logistic regression analyses adjusted for several confounders. Results: The results suggested that BLBC (p < 0.001), MLBC (p < 0.05), FLBC (p < 0.01), and PLBC (p < 0.001) significantly scored higher for total difficulties along with emotional symptoms and conduct problems than never left-behind children (NLBC). Besides, BLBC, FLBC, and PLBC further reported a significantly higher rate of smoking (p < 0.001, p < 0.01, and p < 0.001, respectively) and drinking (p < 0.01, p < 0.05, and p < 0.01, respectively) than did NLBC. Also, MLBC appeared higher risks of smoking problems [OR = 2.31, 95% CI (1.45-3.69), p < 0.001] and the internet addiction [OR = 2.15, 95% CI (1.24-3.72), p < 0.01], when compared to NLBC. Conclusions: The findings provided insight into LBC within the different contexts of parental migrations and contributed to a better understanding of their specific and potentially persistent health risks. Correspondingly, the study highlighted the implications for differentiating LBC to capture the more vulnerable group and tailored interventions to prioritize.

An ultrasensitive multivariate signal amplification strategy based on microchip platform tailored for simultaneous quantification of multiple microRNAs in single cell.

MicroRNAs (miRNAs) play a very important regulatory role in life activities. Abnormal expression levels of miRNAs in cells are associated with various diseases, especially human cancer. Nevertheless, accurate detection of the copy numbers of various miRNA molecules in single cell is still a great challenge. In this study, an intracellular multivariate signal amplification strategy based on microchip platform was proposed, and an ultrasensitive single-cell analysis method was established for simultaneous quantification of absolute copy numbers of multiple miRNAs in a single cell. Using miRNA-21 and miRNA-141 as the analytical models of miRNAs, the detection limits of 1.0 and 2.0 fM were obtained. Based on the developed method, an analysis of 600 randomly acquired different types of cells was performed. The distribution of absolute copy numbers of miRNA-21 and miRNA-141 in six types of cells was obtained. It was found that the number of copies of miRNA-21 and miRNA-141 in different types of cancer cells showed different expression characteristics. The study results can help us more accurately understand cell-to-cell heterogeneity and the relationship between different miRNAs and different types of cancer at the single cell level.

A DNA-functionalized biomass nanoprobe for the targeted photodynamic therapy of tumor and ratiometric fluorescence imaging-based visual cancer cell identification/antitumor drug screening.

Survivin is widely expressed in tumor tissue, in which the in situ ratiometric fluorescence imaging of intracellular survivin mRNA can provide accurate information for the diagnosis and treatment of cancers, as well as the screening of antitumor drugs. However, the development of a nanoprobe that can be used simultaneously in the diagnosis and treatment of tumors and the screening of antitumor drugs remains a challenge. In an effort to address these requirements, a multifunctional biomass nanoprobe was developed for the photodynamic therapy (PDT) of tumors as well as cancer cell identification and antitumor drug screening based on the ratiometric fluorescence imaging of intracellular survivin mRNA. This nanoprobe was assembled from near-infrared (NIR) biomass quantum dots (BQDs), single-stranded DNA and NIR dye (dylight680) labeled single-stranded DNA. The BQDs contain a large number of chlorophyll molecules, meaning that they can produce a large amount of singlet oxygen under NIR light irradiation, thus realizing the PDT of a tumor. However, the specific binding of the nanoprobe to intracellular survivin mRNA causes the release of dylight680 and reduces the fluorescence resonance energy transfer (FRET) efficiency between the BQDs and dylight680 in the probe, thereby achieving the ratiometric fluorescence imaging of survivin mRNA. Therefore, the prepared nanoprobe can not only be used in the diagnosis of cancers, but also in the targeted PDT of tumors.

An aptamer-based four-color fluorometic method for simultaneous determination and imaging of alpha-fetoprotein, vascular endothelial growth factor-165, carcinoembryonic antigen and human epidermal growth factor receptor 2 in living cells.

The extraordinary fluorescence quenching capability of graphene oxide (GO) was coupled to the specific recognition capability of aptamers to design a four-color fluorescent nanoprobe for multiplexed detection and imaging of tumor-associated proteins in living cells. Specifically, alpha-fetoprotein (AFP), vascular endothelial growth factor-165 (VEGF165), carcinoembryonic antigen (CEA), and human epidermal growth factor receptor 2 (HER2) were detected. Due to strong π interaction, the fluorescence of labeled aptamers is quenched by GO. Four fluorophore-labeled aptamers that bind the tumor-associated proteins were adsorbed on GO to form the four-color nanoprobe with quenched fluorescence. The nanoprobes were internalized into cells via endocytosis, where the aptamer/GO nanoprobes bind the intracellular tumor-associated proteins. The aptamer-protein complexes thus formed detach from GO, and fluorescence recovers. Each analyte has its typical color (AFP: blue; VEGF165: green; CEA: yellow; HER2: red). As a result, simultaneous detection and imaging of multiple tumor-associated proteins in living cells were achieved. This nanoprobe has a fast response and is highly specific and biocompatible. The linear ranges for AFP, VEGF165, CEA, and HER2 are 0.8 nM-160 nM, 0.5 nM-100 nM, 1.0 nM-200 nM, and 1.2 nM-240 nM, respectively. Detection limits were 0.45 nM for AFP, 0.30 nM for VEGF165, 0.62 nM for CEA, and 0.96 nM for HER2. The probe allows for a fast distinction between tumor cells and normal cells via imaging. Graphical abstract Schematic presentation of the development of a four-color fluorometic method based on aptamer and graphene oxide for simultaneous detection and imaging of alpha-fetoprotein, vascular endothelial growth factor-165, carcinoembryonic antigen and human epidermal growth factor receptor 2 in living cells.

A peptide-based four-color fluorescent polydopamine nanoprobe for multiplexed sensing and imaging of proteases in living cells.

We develop a novel peptide-based four-color fluorescent polydopamine nanoprobe for multiplexed sensing and imaging of tumor-related proteases in living cells. This nanoprobe responds rapidly and selectively, enabling simultaneous and high-contrast imaging of multiple proteases in living cells. Furthermore, it could detect the changes of protease expression in living cells.

A fluorescent aptasensor based on single oligonucleotide-mediated isothermal quadratic amplification and graphene oxide fluorescence quenching for ultrasensitive protein detection.

In this work, we have developed a novel fluorescent aptasensor based on single oligonucleotide-mediated isothermal quadratic amplification (SOIQA) and graphene oxide (GO)-mediated fluorescence quenching for the ultrasensitive detection of proteins in a homogeneous solution. The SOIQA consists of a fluorophore-labeled aptamer hairpin probe containing T7 exonuclease (T7 Exo)-resistant 5'-protruding termini and a mismatch base at its 3'-end, DNA polymerase, T7 Exo and GO. The target analyte binds with the aptamer sequences and unfolds the fluorophore-labeled aptamer hairpin probe to form a new DNA hairpin, inducing the catalytic recycling of the target analyte (assisted by DNA polymerase) and DNA sequences (aided by T7 Exo) to achieve SOIQA, which results in the digestion of numerous fluorophore-labeled aptamer hairpin probes and the generation of a large amount of mononucleotides carrying the fluorophore. These mononucleotide products cannot be adsorbed onto the GO, leading to a dramatic increase in the fluorescence intensity for the amplified detection of the target molecules. In the absence of the target analyte, however, the SOIQA reaction is inhibited and the fluorophore-labeled aptamer hairpin probe is adsorbed onto the GO, leading to an extremely low fluorescence background signal. To test the feasibility of the SOIQA systems, a protein cancer marker, carcinoembryonic antigen (CEA) was used as the model analyte. The developed aptasensor could detect CEA with a detection limit of 28.5 fg mL-1 (∼142 aM), high specificity and a broad detection range of 6 orders of magnitude. And this one-step incubation can be completed in 60 min. In addition, the approach uses only one oligonucleotide strand, and is simple. Moreover, this SOIQA sensing method is suitable for rapid and direct quantification of proteins in complex biological samples such as clinical serum. Considering the simplicity and superior sensitivity/specificity, the developed sensing method provides a promising platform for the analysis of a variety of low-abundance biomolecules.