A smartphone-based fluorescent biosensor with metal-organic framework biocomposites and cotton swabs for the rapid determination of tetrodotoxin in seafood.

BACKGROUND: Tetrodotoxin (TTX) is a potent neurovirulent marine biotoxin that is present in puffer fish and certain marine animals. It is capable of causing severe neurotoxic symptoms and even death when consumed through contaminated seafood. Due to its high toxicity, developing an effective assay for TTX determination in seafood has significant benefits for food safety and human health. Currently, it remains challenging to achieve on-site determination of TTX in seafood. To facilitate mass on-site assays, more affordable technologies utilizing accessible equipment that require no skilled personnel are needed. RESULTS: A smartphone-based portable fluorescent biosensor is proposed for TTX determination by using metal-organic framework (MOF) biocomposites and cotton swabs. Oriented antibody (Ab)-decorated and fluorescent quantum dot (QD)-loaded MOF biocomposites (QD@MOF*Ab) are rapidly synthesized for binding targets and fluorescent responses by utilizing the tunability of zinc-based MOF. Moreover, facile Ab-immobilized household cotton swabs are utilized as TTX capture tools. TTX forms sandwich immune complexes with QD@MOF*Ab probes, achieving signal amplification. These probes are excited by a portable device to generate bright fluorescent signals, which can be detected by the naked eye, and TTX quantitative results are obtained using a smartphone. When observed with the naked eye, the limit of detection (LOD) is 0.4 ng/mL, while intelligent quantitation presents an LOD of 0.13 ng/mL at logarithmic concentrations of 0.2-400 ng/mL. SIGNIFICANCE: This biosensor is convenient to use, and an easy-to-operate analysis is completed within 15 min, thus demonstrating excellent performance in terms of detection speed and portability. Furthermore, it successfully determines TTX contents in puffer fish and clam samples, demonstrating its potential for monitoring seafood. Herein, this work provides a favorable rapid sensing platform that is easily portable.

A label-free ratiometric fluorescent aptasensor based on a peroxidase-mimetic multifunctional ZrFe-MOF for the determination of tetrodotoxin.

A Fe/Zr bimetal-organic framework (ZrFe-MOF) is utilized to establish a ratiometric fluorescent aptasensor for the determination of tetrodotoxin (TTX). The multifunctional ZrFe-MOF possesses inherent fluorescence at 445 nm wavelength, peroxidase-mimetic activity, and specific recognition and adsorption capabilities for aptamers, owing to its organic ligand, and Fe and Zr nodes. The peroxidation of o-phenylenediamine (OPD) substrate generates fluorescent 2,3-diaminophenazine (OPDox) at 555 nm wavelength, thus quenching the inherent fluorescence of ZrFe-MOF because of the fluorescence resonance energy transfer (FRET) effect. TTX aptamers, which are absorbed on the material surface without immobilization or fluorescent labeling, inhibit the peroxidase-mimetic activity of ZrFe-MOF. It causes the decreased OPDox fluorescence at 555 nm wavelength and the inverse restoration of ZrFe-MOF fluorescence at 445 nm wavelength. With TTX, the aptamers specifically bind to TTX, triggering rigid complex release from ZrFe-MOF surface and reactivating its peroxidase-mimetic activity. Consequently, the two fluorescence signals exhibit opposite changes. Employing this ratiometric strategy, the determination of TTX is achieved with a detection limit of 0.027 ng/mL and a linear range of 0.05-500 ng/mL. This aptasensor also successfully determines TTX concentrations in puffer fish and clam samples, demonstrating its promising application for monitoring trace TTX in food safety.

"Three-in-one" nanohybrids as synergistic nanozymes assisted with exonuclease I amplification to enhance colorimetric aptasensor for ultrasensitive detection of kanamycin.

Kanamycin (KAN) residues in animal-derived food can cause serious threats to human health. Herein, a colorimetric aptasensor was described using "three-in-one" nanohybrids (hemin@Fe-MIL-88NH2/PtNP) as synergistic nanozymes assisted with the exonuclease I (Exo I) signal amplification for the ultrasensitive detection of KAN. In the presence of KAN and Exo I, the KAN aptamer (APT) was specifically bound to KAN, and the remaining APT complementary strand (cDNA1) captured hemin@Fe-MIL-88NH2/PtNP labeled with the cDNA1 complementary strand (cDNA2). Due to the synergistic effect of hemin, Fe-MIL-88NH2 and platinum nanoparticles (PtNPs), hemin@Fe-MIL-88NH2/PtNP exhibited superior catalytic performance and could efficiently catalyze the 3,3',5,5'-tetramethylbenzidine (TMB)-H2O2 system for signal development. Moreover, Exo I could digest APT and release KAN into a new cycle for signal amplification. Based on multiple signal amplification effects, our proposed aptasensor achieved a wide detection range from 0.01 to 100 ng mL-1 with a low detection limit of 2 pg mL-1. This assay also successfully detected KAN-added milk and shrimp samples with added recovery ranges of 93.58%-106.08% and relative standard deviations (RSDs) of 2.20%-5.50%. The aptasensor enabled ultrasensitive, specific, and fast detection of KAN, and exhibited promising applications in the efficient detection of food pollutants.

A facile dual-mode aptasensor based on AuNPs@MIL-101 nanohybrids for ultrasensitive fluorescence and surface-enhanced Raman spectroscopy detection of tetrodotoxin.

The development of ultrasensitive, reliable, and facile detection technologies for trace tetrodotoxin (TTX) is challenging. We presented a facile dual-mode aptamer-based biosensor (aptasensor) for ultrasensitive fluorescence and surface-enhanced Raman spectroscopy (SERS) detection of TTX by using gold nanoparticles (AuNPs)-embedded metal-organic framework (MOF) nanohybrids (AuNPs@MIL-101) because of their superior properties. A TTX-specific aptamer labelled with fluorescence and Raman reporter cyanine-3 (Cy3-aptamer) was selected as the recognition element and signal probe. Without immobilisation processing steps, Cy3-aptamers were effectively adsorbed onto the surface of AuNPs@MIL-101, thereby generating both fluorescence quenching and SERS enhancement. The preferential binding of TTX towards the Cy3-aptamer triggered the release of rigid Cy3-aptamer-TTX complexes from the AuNPs@MIL-101 surface, which resulted in recovered fluorescence signals and weakened SERS signals. Switched fluorescence and SERS intensities exhibited excellent linear relationships with logarithms of TTX concentrations of 0.01-300 ng/mL, and ultrahigh detection sensitivities of 6 and 8 pg/mL, respectively, were obtained. Furthermore, two quantitative detection approaches for TTX-spiked puffer fish and clam samples obtained satisfactory spiked recoveries and coefficient of variation (CV) values. Notably, the dual-mode aptasensor also successfully determined natural TTX-contaminated samples, showing excellent practical applications. The results indicated that this dual-mode measurement not only was ultrasensitive and simple but also markedly boosted analysis reliability and precision. This study is the first to propose a dual-mechanism AuNPs@MIL-101-based aptasensor for detection of trace TTX and provides a favourable pathway for developing multimode sensing platforms for various applications.

Rapid and ultrasensitive detection of DNA and microRNA-21 using a zirconium porphyrin metal-organic framework-based switch fluorescence biosensor.

Sensitive and accurate detection of nucleic acid biomarkers is critical for early cancer diagnosis, disease monitoring, and clinical treatment. In this study, we developed a switch fluorescence biosensor for simple and high-efficient detection of nucleic acid biomarkers using 6-carboxyfluorescein (FAM)-modified single-stranded DNA (ssDNA) probes (FAM-P1/P2), and zirconium porphyrin metal-organic framework nanoparticles (ZrMOF) acted as fluorescence quencher. FAM-P1/P2 probes were adsorbed on ZrMOF surface because of π-π stacking, hydrogen bonding, and electrostatic interactions. Fluorescence quenching event occurred by fluorescence resonance energy transfer (FRET) and photo-induced electron transfer (PET) processes, thereby achieving the "off" fluorescence status. Once the specific binding was formed between the fluorescence probes and the targets, the rigid double-stranded DNA (dsDNA) structures were released from ZrMOF surface, resulting in the recovery of fluorescence and the "on" status. Because of the superior adsorption ability of ZrMOF toward ssDNA than dsDNA, the switch of fluorescence signals from "off" to "on" allowed rapid and ultrasensitive detection of ssDNA (T1) and microRNA-21 (miR-21) within 30 min. The limit of detection (signal-to-noise ratio = 3) for T1 and miR-21 were 2 fM and 11 aM, respectively. Moreover, the proposed strategy was very simple as it worked by the facile adsorption-quenching-recovery mechanism without difficult and complicated immobilization processes. Also, this biosensor showed an excellent analytical performance in the detection of miR-21 in human serum samples. Therefore, this biosensor might be considered a potential tool for the detection of DNA and miRNA biomarkers in clinical samples.

State-of-the-art progress of switch fluorescence biosensors based on metal-organic frameworks and nucleic acids.

Metal-organic frameworks (MOFs) have captured substantial attention of an increasing number of scientists working in sensing analysis fields, due to their large surface area, high porosity, and tunable structure. Recently, MOFs as attractive fluorescence quenchers have been extensively investigated. Given their high quenching efficiency toward the fluorescence intensity of dyes-labeled specific biological recognition molecules, such as nucleic acids, MOFs have been widely developed to switch fluorescence biosensors with low background fluorescence signal. These strategies not only lead to specificity, simplicity, and low cost of biosensors, but also possess advantages such as ultrasensitive, rapid, and multiple detection of switch fluorescence methods. At present, researches of the analysis of switch fluorescence biosensors based on MOFs and nucleic acids mainly focus on sensing of different types of in vitro and intracellular analytes, indicating their increasing potential. In this review, we briefly introduce the principle of switch fluorescence biosensor and the mechanism of fluorescence quenching of MOFs, and mainly discuss and summarize the state-of-the-art advances of MOFs and nucleic acids-based switch fluorescence biosensors over the years 2013 to 2020. Most of them have been proposed to the in vitro detection of different types of analytes, showing their wide scope and applicability, such as deoxyribonucleic acid (DNAs), ribonucleic acid (RNAs), proteins, enzymes, antibiotics, and heavy metal ions. Besides, some of them have also been applied to the bioimaging of intracellular analytes, emerging their potential for biomedical applications, for example, cellular adenosine triphosphate (ATP) and subcellular glutathione (GSH). Finally, the remaining challenges in this sensing field and prospects for future research trends are addressed. Graphical abstract.

Inhibitory effects of Lentinus edodes mycelia polysaccharide on α-glucosidase, glycation activity and high glucose-induced cell damage.

At present, diabetes and diabetic complications have become one of the serious diseases affecting human health. In this study, the inhibitory effects of Lentinus edodes mycelia polysaccharide (LMP) on α-glucosidase activity, the formation of advanced glycation end products (AGEs) and high glucose-induced human umbilical vein endothelial cells (HUVECs) damage were explored. The interaction between LMP and α-glucosidase and the inhibition against AGEs formation were investigated with spectroscopic techniques. The results revealed that LMP had a reversible inhibition on α-glucosidase activity in a mixed-type manner. When the concentration of LMP was 2.7 mM, the inhibition rate was 34.38 %. LMP quenched the fluorescence of α-glucosidase through the static quenching and formed the LMP-α-glucosidase complex. At 310 K, the number of binding sites (n) and binding constant (Kb) were 1.01 and 3.71 × 104 L mol-1, respectively. In addition, LMP could inhibit the formation of AGEs. Compared with 40 mM glucose treatment group, treatment with 0.05 mM LMP for 48 h increased the cell viability from 70.17% to 91.14% and decreased ROS production from 3.33-fold to 1.21-fold. LMP inhibited high glucose-induced activation of MAPK signaling pathways. These findings may promote the application of LMP in the functional food industry.

A study on the protective effects of taxifolin on human umbilical vein endothelial cells and THP-1 cells damaged by hexavalent chromium: a probable mechanism for preventing cardiovascular disease induced by heavy metals.

Hexavalent chromium [Cr(vi)] which is a kind of heavy metal with strong oxidizing ability can induce cardiovascular disease (CVD), while taxifolin can protect cells and organisms against suffering from oxidative stress. In this study, the inhibitory effects of taxifolin against Cr(vi)-induced cell damage in human umbilical vein endothelial cells (HUVECs) and THP-1 cells were investigated. Cr(vi) could increase the phosphorylation of p38 and JNK, regulate the expression of Bax and Bcl-2 in both cell lines. Meanwhile, the Cr(vi) stimulation led to an increase of the expression of ICAM-1 and VCAM-1, and upregulated the adhesion of THP-1 cells to HUVECs. Furthermore, Cr(vi) could induce the activation of the nuclear factor kappa B (NF-κB) signaling pathway, the accumulation of p65 in the nucleus, and the increase in the phosphorylation of IκB and the expression of cleaved caspase-1 and IL-1ß in THP-1 cells. However, taxifolin could reverse the effects by inhibiting the activation of mitogen-activated protein kinases (MAPKs) and NF-κB signaling pathways, regulating the expression of apoptosis-related proteins, and alleviating the adhesion of THP-1 cells to HUVECs. Our findings demonstrated that taxifolin was a potential agent to prevent endothelial dysfunction, monocyte inflammation and cell adhesion induced by Cr(vi).

Rapid and sensitive detection of prostate-specific antigen via label-free frequency shift Raman of sensing graphene.

The sensitive and accurate detection of cancer biomarkers is critically important to early clinical diagnosis, disease monitoring, and successful cancer treatment. Here, we first demonstrate an aptamer-based frequency shift Raman approach via sensing of graphene. This biosensor allows the rapid, sensitive, and label-free detection of the acknowledged protein cancer biomarker, prostate-specific antigen (PSA). Monolayer graphene is employed as the Raman substrate, which is highly sensitive to its electronic structure and interface properties. The PSA aptamer can be adsorbed strongly on the surface of substrates through π-π stacking interactions. The vibrational frequency of the G peak of graphene shifted upon the specific binding between the PSA and its aptamer. The corresponding frequency shifts of the G peak are directly correlated with PSA concentrations. The limit of detection is as low as 0.01 ng/mL, with a wide linear range from 0.05 ng/mL to 25 ng/mL. The analytic samples can be detected directly without any extensive preparation and label process. The whole detection is completed in only 30 min. Furthermore, excellent recoveries are acquired to validate the feasibility of this assay in human serum samples. The proposed technology could provide a selective, versatile, and user-friendly strategy for the early detection of cancer biomarkers.

A zirconium-porphyrin MOF-based ratiometric fluorescent biosensor for rapid and ultrasensitive detection of chloramphenicol.

An ultrasensitive and rapid detection of trace antibiotics is imperative for food safety and public health. Herein, we present a ratiometric fluorescent sensing strategy based on an aptamer labeled with a fluorescent dye and a highly stable zirconium-porphyrin MOF (PCN-222) as a fluorescence quencher for the high-efficiency detection of chloramphenicol (CAP). PCN-222 exhibits a strong adsorption ability toward the dye-labeled aptamer through π-π stacking, electrostatic, hydrogen bond, and coordination interactions. Experimental and simulation studies confirm that PCN-222 demonstrates a high quenching efficiency via fluorescence resonance energy transfer (FRET) and photoinduced electron transfer (PET) processes. In the presence of CAP, dye-labeled aptamers are released from the PCN-222 surface, resulting in the recovery of fluorescence. This proposed biosensor allows the complete detection of CAP within 26 min. For ratiometric measurement, its detection limit is as low as 0.08 pg mL-1 with a wide detection range from 0.1 pg mL-1 to 10 ng mL-1. It is successfully applied to analyze CAP in milk and shrimp samples, and its results are consistent with those of the commercial ELISA kit. This biosensor not only enables the rapid, ultrasensitive, and highly specific detection of CAP but also reveals excellent universality and multiplexed analysis performance.

Toxic effects of Cr(VI) on the bovine hemoglobin and human vascular endothelial cells: Molecular interaction and cell damage.

Hexavalent chromium [Cr(VI)] is the main harmful component in the atmosphere released by chemical industry. The study was conducted to assess Cr(VI) inducing cardiovascular diseases (CVDs) in vitro by investigating the effects of Cr(VI) on bovine hemoglobin (BHb) and human umbilical vein endothelial cells (HUVECs). Multi-spectroscopic techniques and molecular docking method were used to determine the interaction of Cr(VI) and BHb. Fluorescence spectra results showed that the quenching constant (Ksv) decreased with temperature raise, indicating that Cr(VI) quenches BHb fluorescence through static quenching mechanism. The number of binding sites was 1.14 (310 K), enthalpy and entropy changes revealed the interaction of Cr(VI) and BHb was driven by hydrogen bonds. The results of synchronous fluorescence and circular dichroism (CD) spectra suggested that Cr(VI) could change BHb conformation and influence the microenvironment of Trp and Tyr residues. Moreover, in order to study Cr(VI) induced HUVECs damage, inflammatory factors were detected at the mRNA level, JNK and p38 MAPK pathways were analyzed. The results shown that Cr(VI) could induce mRNA expression of NLRP3, ICAM-1, VCAM-1, TNF-α and IL-1ß, and increased intracellular ROS. Furthermore, Cr(VI) could induce oxidative stress in HUVECs, and then activate JNK and p38 MAPK pathways, ultimately lead to apoptosis of HUVECs by activating mitochondrial apoptosis pathways. These results suggested that Cr(VI) might bring about CVDs by both changing the BHb conformation and inducing HUVECs damage.

Alantolactone induces apoptosis and suppresses migration in MCF­7 human breast cancer cells via the p38 MAPK, NF­κB and Nrf2 signaling pathways.

Human breast cancer is a malignant type of cancer with high prevalence. In the present study, the anticancer effects of alantolactone, a sesquiterpene lactone, on the human breast cancer cell line MCF­7 were investigated in vitro. The MCF­7 cell morphology changed from diamond to round subsequent to treatment with alantolactone, and the cell viability reduced significantly compared with that of the control cells. Alantolactone induced apoptosis of MCF­7 cells by regulating the protein expression levels of B­cell lymphoma 2 (Bcl­2), Bcl­2­associated X protein, p53, caspase­3 and caspase­12, which are associated with the apoptotic pathway, and suppressed colony formation and migration by regulating the protein expression of matrix metalloproteinase (MMP)­2, MMP­7 and MMP­9. Cell signaling pathway analysis confirmed that alantolactone increased the phosphorylation of p38, and decreased the nuclear expression levels of p65 and nuclear factor erythroid 2­related factor 2 (Nrf2), suggesting that the apoptosis­promoting and migration­suppressing effect of alantolactone may partially depend on regulating the p38 MAPK, NF­κB and Nrf2 pathways. These results also suggested that alantolactone may become a potential therapeutic strategy for treating breast cancer.

Characterizing the binding interaction of astilbin with bovine serum albumin: a spectroscopic study in combination with molecular docking technology.

Astilbin (ASN) is a flavonoid compound isolated from the rhizome of Smilax china L. (Smilacaceae). It has many bioactivities, such as selective immunosuppression, antioxidant, anti-hepatic injury, etc., and is widely used in traditional Chinese medical treatments. The interaction of ASN with bovine serum albumin (BSA) was studied in a physiological buffer (pH = 7.40) using multi-spectroscopic techniques in combination with molecular docking methods. UV-vis absorption measurements proved that a ASN-BSA complex could be formed. Fluorescence data revealed that ASN could strongly quench the intrinsic fluorescence of BSA in terms of a static quenching procedure. The process of binding was spontaneous and the binding occurred mainly through hydrogen bonding and van der Waals forces. The distance r between donor (BSA) and acceptor (ASN) was calculated to be 4.80 nm based on Förster's non-radiative energy transfer theory. The binding constant (K a = 7.31 × 104 mol L-1) and the number of binding sites (n ≈ 1) at 298 K suggested that ASN only occupied one site in BSA with high affinity. Moreover, the results of molecular docking indicated that ASN was more likely to be located in site I (sub-domain IIA) of BSA. The results of synchronous fluorescence and three-dimensional fluorescence spectra showed that ASN induced conformational changes of BSA. The findings would be beneficial for research on the transportation, distribution and some important bioactivities of ASN in the human body.

A novel label-free fluorescence assay for one-step sensitive detection of Hg2+ in environmental drinking water samples.

A novel label-free fluorescence assay for detection of Hg2+ was developed based on the Hg2+-binding single-stranded DNA (ssDNA) and SYBR Green I (SG I). Differences from other assays, the designed rich-thymine (T) ssDNA probe without fluorescent labelling can be rapidly formed a T-Hg2+-T complex and folded into a stable hairpin structure in the presence of Hg2+ in environmental drinking water samples by facilitating fluorescence increase through intercalating with SG I in one-step. In the assay, the fluorescence signal can be directly obtained without additional incubation within 1 min. The dynamic quantitative working ranges was 5-1000 nM, the determination coefficients were satisfied by optimization of the reaction conditions. The lowest detection limit of Hg2+ was 3 nM which is well below the standard of U.S. Environmental Protection Agency. This method was highly specific for detecting of Hg2+ without being affected by other possible interfering ions from different background compositions of water samples. The recoveries of Hg2+ spiked in these samples were 95.05-103.51%. The proposed method is more viable, low-costing and simple for operation in field detection than the other methods with great potentials, such as emergency disposal, environmental monitoring, surveillance and supporting of ecological risk assessment and management.

A novel polysaccharide from Lentinus edodes Mycelia exhibits potential antitumor activity on laryngeal squamous cancer cell line Hep-2.

A novel polysaccharide [Lentinus edodes mycelia polysaccharide 2 (LMP2)] with a molecular weight of 2.27 × 10(4) Da was isolated from the Lentinus edodes mycelia and purified by Sephadex G-200 and diethylaminoethyl-32 column chromatography. HPLC results indicated that LMP2 contained mannose, arabinose, galactose, xylose, and rhamnose with a relative molar ratio of 1:0.74:3.23:1.18:10.98. Its antitumor activity was evaluated in vitro by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, colony formation assay, and transwell assay. LMP2 showed a remarkable inhibitory effect on the proliferation of Hep-2 cells, and at the concentration of 200 mg/ml, the inhibition ratio was 37.2 % after 72 h. Furthermore, colony formation of Hep-2 cells was reduced significantly after treatment with LMP2. Invasion of Hep-2 cells was inhibited significantly by LMP2. These results suggested that LMP2 could be explored as a potential antitumor material for laryngeal carcinoma.