Ratiometric SERS aptasensing for simultaneous quantitative detection of histamine and tyramine in fishes.

Herein, a SiO2@Ag NPs core/shell nanoparticles were synthesized to fabricate a surface-enhanced Raman spectroscopy (SERS) sensor for the simultaneous determination of histamine (HIS) and tyramine (TYR) based on specific aptamer recognition and ratiometric strategy. SiO2@Ag NPs with 4-thiosaminophenol (4-ATP) and Nile blue A (NBA) molecules were used as an internal standard (IS) and labeled with aptamers corresponding to HIS and TYR, respectively. Raman reporter molecules ROX and Cy5 labeled complementary DNA (cDNA) were then hybridized with aptamers to form rigid double-stranded DNA. After the HIS and TYR were captured by their aptamers, resulting in the dissociation of cDNA and separated from the SERS substrate. Therefore, the SERS signal intensity at 1503 cm-1 of ROX and 1358 cm-1 of Cy5 tagged on the terminal of cDNA decreased with the concentration of HIS and TYR increasing, while the SERS signal intensity at 1079 cm-1 of 4-APT and 592 cm-1 of NBA on the substrate remain stable. Thus, the concentrations of HIS and TYR can be determined by the I1503/I1079 and I1358/I592 values, respectively. This sensing strategy achieves a lower detection limit of 0.2 ng/mL for HIS and 0.05 ng/mL for TYR, respectively, demonstrating promising applications in sensitive detection of BAs in animal-derived foodstuff.

Effects of natural 24-epibrassinolide on inducing apoptosis and restricting metabolism in hepatocarcinoma cells.

BACKGROUND: 24-epibrassinolide (EBR) is a ubiquitous steroidal phytohormone with anticancer activity. Yet the cytotoxic effects and mechanism of EBR on hepatocarcinoma (HCC) cells remain elusive. METHODS: Cell counting kit-8 (CCK-8) assay was performed to evaluate cell viability. Real-time cell analysis (RTCA) technology and colony formation assays were used to evaluate cell proliferation. The apoptosis ratio was measured by flow cytometry. Seahorse XFe96 was applied to detect the effects of EBR on cellular bioenergetics. RNA-seq analysis was performed to investigate differences in gene expression profiles. Western blot and qRT-PCR were used to detect the changes in target molecules. RESULTS: EBR induced apoptosis and caused energy restriction in HCC, both of which were related to insulin-like growth factor-binding protein 1 (IGFBP1). EBR rapidly and massively induced IGBFP1, part of which was transcribed by activating transcription factor-4 (ATF4). The accumulation of secreted and cellular IGFBP1 had different important roles, in which secreted IGFBP1 affected cell energy metabolism by inhibiting the phosphorylation of Akt, while intracellular IGFBP1 acted as a pro-survival factor to resist apoptosis. Interestingly, the extracellular signal-regulated kinase (ERK) inhibitor SCH772984 and MAP/ERK kinase (MEK) inhibitor PD98059 not only attenuated the EBR-induced IGFBP1 expression but also the basal expression of IGFBP1. Thus, the treatment of cells with these inhibitors further enhances the cytotoxicity of EBR. CONCLUSION: Taken together, these findings suggested that EBR can be considered as a potential therapeutic compound for HCC due to its pro-apoptosis, restriction of energy metabolism, and other anti-cancer properties. Meanwhile, the high expression of IGFBP1 induced by EBR in HCC contributes to our understanding of the role of IGFBP1 in drug resistance.

Synthetic oleanane triterpenoid derivative CDDO-Me disrupts cellular bioenergetics to suppress pancreatic ductal adenocarcinoma via targeting SLC1A5.

To investigate the potential antitumor activity of synthetic triterpenoid, methyl-2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oate (CDDO-Me) in pancreatic ductal adenocarcinoma (PDAC), MTT cytotoxicity assay, and xenograft nude mice assay were performed to evaluate tumor growth in vitro and in vivo. Seahorse XFe96 bioenergetics analyzer was applied to determine aerobic glycolysis and mitochondrial respiration. Western blot and quantitative reverse transcription-polymerase chain reactions are used to detect protein and messenger RNA transcripts of SLC1A5 and metabolic enzymes. We confirmed the strong antitumor activity of CDDO-Me in suppressing PDAC growth. Mechanistically, we demonstrated CDDO-Me induced mitochondrial respiration and aerobic glycolysis dysfunction. We also verified CDDO-Me downregulated glutamine transporter SLC1A5, resulting in excessive reactive oxygen species (ROS) levels that suppressed tumor growth. Moreover, we confirmed that SLC1A5 depletion reduced the ratio of glutathione/oxidized glutathione. We also found CDDO-Me could inhibit N-linked glycosylation of SLC1A5, which promotes protease-mediated degradation. Finally, we confirmed SLC1A5 was significantly overexpressed in PDAC and closely correlated with the poor prognosis of PDAC patients. Our work uncovers CDDO-Me is effective at suppressing PDAC cell growth in vitro and in vivo and illuminates CDDO-Me caused excessive ROS and cellular bioenergetics disruption which contributed to CDDO-Me inhibited PDAC growth. Our data highlights CDDO-Me could be considered a potential compound for PDAC therapy, and SLC1A5 could be a novel biomarker for PDAC patients.

6-methoxydihydroavicine, the alkaloid extracted from Macleaya cordata (Willd.) R. Br. (Papaveraceae), triggers RIPK1/Caspase-dependent cell death in pancreatic cancer cells through the disruption of oxaloacetic acid metabolism and accumulation of reactive oxygen species.

BACKGROUND: Many extracts and purified alkaloids of M. cordata (Papaveraceae family) have been reported to display promising anti-tumor effects by inhibiting cancer cell growth and inducing apoptosis in many cancer types. However, no evidence currently exists for anti-pancreatic cancer activity of alkaloids extracted from M. cordata, including a novel alkaloid named 6­methoxy dihydrosphingosine (6-Methoxydihydroavicine, 6-ME) derived from M. cordata fruits. PURPOSE: The aim of this study was to investigate the anti-tumor effects of 6-ME on PC cells and the underlying mechanism. METHODS: CCK-8, RTCA, and colony-formation assays were used to analyze PC cell growth. Cell death ratios, changes in MMP and ROS levels were measured by flow cytometry within corresponding detection kits. A Seahorse XFe96 was employed to examine the effects of 6-ME on cellular bioenergetics. Western blot and q-RT-PCR were conducted to detect changes in target molecules. RESULTS: 6-ME effectively reduced the growth of PC cells and promoted PCD by activating RIPK1, caspases, and GSDME. Specifically, 6-ME treatment caused a disruption of OAA metabolism and increased ROS production, thereby affecting mitochondrial homeostasis and reducing aerobic glycolysis. These responses resulted in mitophagy and RIPK1-mediated cell death. CONCLUSION: 6-ME exhibited specific anti-tumor effects through interrupting OAA metabolic homeostasis to trigger ROS/RIPK1-dependent cell death and mitochondrial dysfunction, suggesting that 6-ME could be considered as a highly promising compound for PC intervention.

Signal amplification of SiO2 nanoparticle loaded horseradish peroxidase for colorimetric detection of lead ions in water.

In this work, we developed an aptamer-based optical assay for the analysis of Pb2+, a hazardous heavy metal that may be present in the food chain and harmful to human health. An aptamer targeted against Pb2+ was immobilized onto the microplate as the capture probe. SiO2 nanoparticles (NPs) were synthesized and used as carriers of the signaling horseradish peroxidase (HRP) to achieve amplification of the optical signal. Complementary DNA (cDNA) of the aptamer was also linked to the above mentioned SiO2 nanoparticle (NPs) as the signal probe. The aptamers were found to be able to capture Pb2+, and the unbound aptamers were subsequently hybridized with cDNA-HRP-SiO2 conjugates. As a result, the addition of TMB-H2O2 promoted the formation of blue products in the catalytic system. The assay adopting SiO2 NPs as an enhancer resulted in higher sensitivity with an LOD of 2.5 nM compared to normal procedures. The feasibility of the aptamer-based colorimetric assay was verified by successful detection of Pb2+ in water samples with recoveries in the range of 97.4-103.52%.

Plantago asiatica L. seeds extract protects against cardiomyocyte injury in isoproterenol- induced cardiac hypertrophy by inhibiting excessive autophagy and apoptosis in mice.

BACKGROUND: Cardiac hypertrophy is the early stage of many heart diseases, such as coronary heart disease, hypertension, valvular dysfunction and cardiomyopathy. Cardiomyocyte autophagy and apoptosis play an important role in the process of cardiac hypertrophic response. Plantago asiatica L. seeds extract (PASE) is prepared from a traditional herbal medicine in Asia with tremendous pharmacological activities. However, whether PASE could relieve cardiac hypertrophy has not been elucidated. The present study is aimed to investigate the effect of PASE on cardiac hypertrophy and explore its potential underlying mechanism. METHODS: Cardiac hypertrophy was induced in C57BL/6 mice by subcutaneous injection of isoproterenol (ISO) for two weeks. Meanwhile, the mice were intraperitoneally injected with PASE at dosages of 20, 40 and 80 mg/kg/day. Cardiac hypertrophy was evaluated by echocardiographic examination, haematoxylin and eosin staining and quantitative real-time polymerase chain reaction. Expressions of proteins involved in autophagy and apoptosis such as Beclin1, p62, LC3II, Bax, Bcl-2 and Cleaved-caspase-3 were detected by western blot analysis. Western blot, transient transfection, acridine orange staining, TUNEL staining and autophagy inducer were used to observe the effect and explore the mechanism of PASE on cardiomyocyte and H9c2 cells with excessive autophagy and apoptosis induced by ISO. RESULTS: ISO induction for two weeks disturbed the myocardial contractility and cardiac function of left ventricles of mice. PASE treated mice showed significantly improved cardiac function indexes, including EF, FS, SV and CO, compared with the ISO group. Treatment with PASE also decreased the heart weight/body weight ratio and cardiomyocyte size, and downregulated the mRNA and protein expressions of hypertrophic markers ANP, BNP, and ß-MHC. Furthermore, the changes of autophagy and apoptosis markers, such as LC3II, Beclin1, p62, Bcl-2, Bax and Cleaved-caspase-3 induced by ISO were resumed by PASE treatment. Consistently, PASE demonstrated similar effects on ISO-induced H9c2 cells as it did in vivo. In addition, PASE could counteract the increased autophagy induced by the autophagy inducer, rapamycin. CONCLUSION: PASE attenuated ISO-induced cardiac hypertrophy in mice by inhibiting excessive autophagy and apoptosis in cardiomyocytes. The novel findings may pave the way for the clinical usage of PASE for the prevention of heart diseases related with cardiac hypertrophy.

Chlorogenic acid depresses cellular bioenergetics to suppress pancreatic carcinoma through modulating c-Myc-TFR1 axis.

Pancreatic ductal adenocarcinoma (PDAC) is severe malignant tumor in human, the outcomes of PDAC is extremely poor. Here, we evaluated the potential anti-tumor activity of chlorogenic Acid (CA) in PDAC. Here, we found CA was effective to suppress PDAC cell growth in vitro and in vivo. Importantly, we found overall oxygen consumption rate was significantly decreased in CA dose-dependent manner. We also found glycolysis reverse was decreased in CA-treated cells, while basal glycolysis and glycolytic capacity were not significantly changed. Mechanistically, we demonstrated TFR1 could be a novel downstream target of CA, which is essential for PDAC cell growth and cellular bioenergetics maintenance. Furthermore, we validated that CA-reduced c-Myc resulted to down-regulation of TFR1, which contributes to mitochondrial respiration dysfunction and cell growth delay. Together, this study indicates that CA suppresses PDAC cell growth through targeting c-Myc-TFR1 axis and suggests CA could be considered as a promising compound for PDAC treatment.

A colorimetric aptamer-based method for detection of cadmium using the enhanced peroxidase-like activity of Au-MoS2 nanocomposites.

In this work, a colorimetric aptamer-based method for detection of cadmium using gold nanoparticles modified MoS2 nanocomposites as enzyme mimic is established. In short, biotinylated Cd2+ aptamers are immobilized by biotin-avidin binding on the bottoms of the microplate, the complementary strands of Cd2+ aptamers are connected to the Au-MoS2 nanocomposites which have the function of enhanced peroxidase-like activity. The csDNA-Au-MoS2 signal probe and target Cd2+ compete for binding Cd2+ aptamer, the color change can be observed by addition of chromogenic substrate, thereby realizing visual detection of Cd2+. The absorbance of the solution at 450 nm has a clear linear relationship with the Cd2+ concentration. The linear range is 1-500 ng/mL, and the limit of detection is 0.7 ng/mL. The assay was used to test white wine samples, the results are consistent with those of atomic absorption spectrometry; which prove that this method can be used for detection of Cd2+ in real samples.

Dummy template surface molecularly imprinted polymers based on silica gel for removing imidacloprid and acetamiprid in tea polyphenols.

Dummy template surface molecularly imprinted polymers based on silica gel were prepared through the surface molecular imprinting technique. Nonpoisonous nicotinamide, which is a structural analogue of imidacloprid and acetamidine, was chosen as the dummy template molecule. The obtained polymers were characterized using scanning electron microscopy, Fourier transform infrared spectroscopy, and X-ray diffraction. The results showed that the polymers exhibited high adsorption capacity and selectivity for imidacloprid and acetamiprid. The maximum adsorption capacities of the polymers toward imidacloprid and acetamiprid were 42.05 and 22.99 mg/g, and the adsorption could reach binding equilibrium within 150 min. The polymers were successfully applied as column-filling materials to extract imidacloprid and acetamiprid from tea polyphenols with a relatively high removal rate (92.36 and 95.20%). The polymers also showed great stability and reusability during the application. The obtained polymers possessed good application prospects for removing imidacloprid and acetamiprid in tea polyphenol production processes.

Colorimetric aptasensor for the detection of mercury based on signal intensification by rolling circle amplification.

Techniques that are sensitive to detect mercury ion (Hg2+) are very important, due to its serious threat to public health and food security. In this work, a colorimetric aptasensor was fabricated for the detection of Hg2+ based on rolling circle amplification (RCA). The aptamer was immobilized onto the microplate and hybridized with its complementary strand (cDNA1) which linked with a primer for triggering the RCA reaction of circular template. The successfully RCA process led to the formation of long ssDNA chains on the microplate, which created many hybridized DNA fragments for bio-cDNA2. The tagged amount of horseradish peroxidase (HRP) was enhanced through the avidin/biotin binding between avi-HRP and bio-cDNA2. In the addition of TMB-H2O2, HRP was catalyzed and generated an optical signal. However, in the presence of target, Hg2+ specifically and preferentially bound with aptamer and formed a strong and stable T-Hg2+-T complex, which led to the release of cDNA1 and HRP cluster. Consequently, the optical signal decreased. Our results showed that the limit of detection (LOD) of this system was 1.6 nM with excellent specificity, and that the detection signals were enhanced by up to 18 times under RCA conditions when compared with detections without RCA. This method has been successfully used to detect Hg2+ in water samples with a recovery of 98%-105.74%.

A novel bioassay based on aptamer-functionalized magnetic nanoparticle for the detection of zearalenone using time resolved-fluorescence NaYF4: Ce/Tb nanoparticles as signal probe.

Zearalenone (ZEN) is a non-steroidal estrogenic mycotoxin produced by fungi on stored grains. The earlier detection methods used for ZEN rely on expensive equipment, time-consuming sample preparation and temperature sensitive antibodies. The current work, proposed a novel strategy based on ZEN aptamer labeled with amine-functionalized magnetic nanoparticle (MNPs) as a capture probe and time-resolved fluorescence (TRFL) nanoparticles labeled with complementary DNA (cDNA) as a signal probe. Under the optimized conditions, TRFL intensity at 544 nm was used to measure ZEN (R2 = 0.9920) in the range of 0.001-10 ng mL-1 and limits of detection (LOD) for proposed method was 0.21 pg mL-1. The specificity of bioassay was also determined by using other mycotoxins (OTA, AFB2, DON and Patulin) and results showed that the aptamer are specific to recognize only ZEN. The analytical applications of the present bioassay in maize and wheat samples were also examined and results were compared with existing methods. Based on these findings, it is suggested to use current rapid and simple bioassay for the determination of ZEN in food and agricultural products.

Ultrasensitive SERS aptasensor for the detection of oxytetracycline based on a gold-enhanced nano-assembly.

This paper investigated a new detection method of oxytetracycline (OTC) in aquatic products with ultrasensitive detection limit. The method was constructed on the basis of raman hot spot between gold nanoparticles (AuNPs) (13nm and 80nm diameter respectively) linked by an DNA sequence. The DNA sequence combined with the OTC aptamer including its complementary sequence as well as a stem-loop structure. The raman signal molecule (4-MBA) was modified at the surface of 13nm AuNPs. After the exposure of OTC, the aptamer sequence was preferentially combined with OTC and partially dehybridized with its complementary sequence which led the 13nm AuNPs to get more closer to the 80nm AuNPs. The raman intensity was thus increased for the more enhanced hot spot generated. Under the optimal experimental conditions, the SERS signal was positively related to the OTC concentration with a wide working range of 4.60×10-2-4.60×102fg/mL and the limit of detection (LOD) was as low as 4.35×10-3fg/mL. The recovery rates of fishmeal ranged from 91.29-110.98%. The specificity of this method was further examined, and the results showed that the AuNPs based aptasensor was highly selective. This developed ultrasensitive aptamer-based SERS detection platform suggested that it may be a promising strategy for a variety of sensing applications.

Mn(2+)-doped NaYF4:Yb/Er upconversion nanoparticle-based electrochemiluminescent aptasensor for bisphenol A.

A novel aptasensor labeled with Mn(2+)-doped NaYF4:Yb/Er upconversion nanoparticles (NaYF4:Yb,Er/Mn UCNPs) was employed in electrogenerated chemiluminescence (ECL) for the sensitive detection of bisphenol A (BPA). The ECL aptasensor was assembled by immobilizing the thiolated aptamers of BPA covalently on a gold nanoparticle (AuNPs)-modified electrode and pairing with complementary DNA labeled with NaYF4:Yb,Er/Mn UCNPs. The ECL aptasensor can not only rapidly and accurately detect BPA concentrations from 0.05 to 100 ng/mL with a detection limit of 0.037 ng/mL but also provides a new platform for ECL applications based on the use of upconversion nanoparticles as a promising alternative material. Graphical Abstract The NaYF4:Yb,Er/Mn UCNPs combining with the BPA aptamer serving as recognition elements create a ECL platform for the sensitive detection of bisphenol A. The change in ECL signals induced by aptamer-target interactions was measured and a significant decrease in intensity was found on interaction with BPA in the concentration range of 0.05 to 100 ng/mL.

Chemiluminescent aptasensor for chloramphenicol based on N-(4-aminobutyl)-N-ethylisoluminol-functionalized flower-like gold nanostructures and magnetic nanoparticles.

A novel chemiluminescent aptasensor for the highly sensitive detection of chloramphenicol (CAP) in milk was successfully developed using biotinylated CAP aptamer-functionalized magnetic nanoparticles (MNPs) as capture probes and thiolated hybridized complementary strand-modified N-(4-aminobutyl)-N-ethylisoluminol (ABEI)-functionalized flower-like gold nanostructures (AuNFs) as signal probes. P-iodophenol (PIP) was also added to form an ABEI-H2O2-PIP steady-state chemiluminescence (CL) system. Based on a competitive format, the CL intensity was negatively correlated with the concentration of CAP in the range of 0.01-0.20 ng/mL and the detection limit was 0.01 ng/mL in buffer and 1 ng/mL in milk. The proposed method was successfully applied to measure CAP in milk samples and compared to a commercial ELISA method. The high sensitivity of AuNFs, excellent selectivity and stability of aptamers, and good overall stability of the chemiluminescent bioassay with magnetic separation make them a promising approach for the detection of small molecular illegal additives. Additionally, the high sensitivity, easy operation, and good reproducibility exhibited by the stable chemiluminescent bioassay demonstrate its applicability for the trace detection of CAP in applications, such as animal husbandry.

Upconversion luminescence resonance energy transfer-based aptasensor for the sensitive detection of oxytetracycline.

In this work, a biosensor based on luminescence resonance energy transfer (LRET) from NaYF4:Yb,Tm upconversion nanoparticles (UCNPs) to SYBR Green I has been developed. The aptamers are covalently linked to UCNPs and hybridized with their complementary strands. The subsequent addition of SYBR Green allows SYBR Green I to insert into the formed double-stranded DNA (dsDNA) duplex and brings the energy donor and acceptor into close proximity, leading to the fluorescence of UCNPs transferred to SYBR Green I. When excited at 980 nm, the UCNPs emit luminescence at 477 nm, and this energy is transferred to SYBR Green I, which emits luminescence at 530 nm. In the presence of oxytetracycline (OTC), the aptamers prefer to bind to its corresponding analyte and dehybridize with the complementary DNA. This dehybridization leads to the liberation of SYBR Green I, which distances SYBR Green I from the UCNPs and recovers the UCNPs' luminescence. Under optimal conditions, a linear calibration is obtained between the ratio of I530 to I477 nm (I530/I477) and the OTC concentration, which ranges from 0.1 to 10 ng/ml with a limit of detection (LOD) of 0.054 ng/ml.