Multisignal Biosensors Based on Mn Paramagnetic Relaxation and Nanocatalysis for Norovirus Detection.

A multisignal method for the sensitive detection of norovirus based on Mn paramagnetic relaxation and nanocatalysis was developed. This dual-modality sensing platform was based on the strong relaxation generated by cracked Au@MnO2 nanoparticles (NPs) and their intrinsic enzyme-like activity. Ascorbic acid rapidly cracked the MnO2 layer of Au@MnO2 NPs to release Mn(II), resulting in the relaxation modality being in a "switch-on" state. Under the optimal conditions, the relaxation modality exhibited a wide working range (6.02 × 103-3.01 × 107 copies/µL) and a limit of detection (LOD) of 2.29 × 103 copies/µL. Using 4,4',4″,4″'-(porphine-5,10,15,20-tetrayl) tetrakis (benzenesulfonic acid) (tpps)-ß-cyclodextrin (tpps-ß-CD) as a T1 relaxation signal amplification reagent, a lower LOD was obtained. The colorimetric modality exploited the "peroxidase/oxidase-like" activity of Au@MnO2 NPs, which catalyzed the oxidation of colorless 3,3',5,5'-tetramethylbenzidine (TMB) to blue oxidized TMB, which exhibited a working range (6.02 × 104-6.02 × 106 copies/µL) and an LOD of 2.6 × 104 copies/µL. In addition, the rapid amplification reaction of recombinase polymerase enabled the detection of low norovirus levels in food samples and obtained a working range of 101-106 copies/mL and LOD of 101 copies/mL (relaxation modality). The accuracy of the sensor in the analysis of spiked samples was consistent with that of the real-time quantitative reverse transcription polymerase chain reaction, demonstrating the high accuracy and practical utility of the sensor.

Synthetic biology for the food industry: advances and challenges.

As global environmental pollution increases, climate change worsens, and population growth continues, the challenges of securing a safe, nutritious, and sustainable food supply have become enormous. This has led to new requirements for future food supply methods and functions. The use of synthetic biology technology to create cell factories suitable for food industry production and renewable raw material conversion into: important food components, functional food additives, and nutritional chemicals, represents an important method of solving the problems faced by the food industry. Here, we review the recent progress and applications of synthetic biology in the food industry, including alternatives to: traditional (artificial pigments, meat, starch, and milk), functional (sweeteners, sugar substitutes, nutrients, flavoring agents), and green (green fiber, degradable packing materials, green packaging materials and food traceability) foods. Furthermore, we discuss the future prospects of synthetic biology-based applications in the food industry. Thus, this review may serve as a reference for research on synthetic biology in the: food safety, food nutrition, public health, and health-related fields.

A universal CRISPR-Cas14a responsive triple-sensitized upconversion photoelectrochemical sensor.

It has recently been discovered that, like other members of the Cas family (12a and 13a), the clustered regularly interspaced short palindrome repeat CRISPR-Cas14a system not only mediates high-sensitivity detection with exceptionally strong gene editing ability but is also generally useful for DNA detection via fluorescence. Photoelectrochemical (PEC) sensors have been widely applied as efficient analytical tools. Measuring electrical signals is more cost-effective and the necessary equipment is more easily portable than fluorescence signal detectors, but their stability still needs to be improved. The high base resolution of CRISPR-Cas14a can compensate for such shortcomings. Therefore, electrical signals and fluorescence signals were combined, and the development of a universal CRISPR-Cas14a-responsive ultrasensitive upconversion PEC sensor is described in this paper. Moreover, strand displacement amplification (SDA) and a near-infrared (NIR) light source were utilized to further improve the stability and sensitivity of the photoelectric signals. At the same time, the modified working electrode (UCNPs-ssDNA-CdS@Au/ITO) on the three-electrode disposable sensor was used as the reporter probe, which cooperates with the trans-cleavage activity of Cas14a endonuclease. To verify the universality of this sensor, the UCNPs-Cas14a-based PEC sensor was applied for the detection of the small-molecule toxin T2 and protein kinase PTK7. Here, we report that the limit of detection of this reagent was within the fg range, successfully applied to the detection of T2 in oats and PTK7 in human serum. We propose that by combining PEC and CRISPR-14a, UCNPs-Cas14a-based PEC sensors could become powerful drivers for the extensive development of ultrasensitive, accurate and cost-effective universal sensors for detection and diagnosis.

Fabrication of Magnetic Al-Based Fe3O4@MIL-53 Metal Organic Framework for Capture of Multi-Pollutants Residue in Milk Followed by HPLC-UV.

The efficient capture of multi-pollutant residues in food is vital for food safety monitoring. In this study, in-situ-fabricated magnetic MIL-53(Al) metal organic frameworks (MOFs), with good magnetic responsiveness, were synthesized and applied for the magnetic solid-phase extraction (MSPE) of chloramphenicol, bisphenol A, estradiol, and diethylstilbestrol. Terephthalic acid (H2BDC) organic ligands were pre-coupled on the surface of amino-Fe3O4 composites (H2BDC@Fe3O4). Fe3O4@MIL-53(Al) MOF was fabricated by in-situ hydrothermal polymerization of H2BDC, Al (NO3)3, and H2BDC@Fe3O4. This approach highly increased the stability of the material. The magnetic Fe3O4@MIL-53(Al) MOF-based MSPE was combined with high-performance liquid chromatography-photo diode array detection, to establish a novel sensitive method for analyzing multi-pollutant residues in milk. This method showed good linear correlations, in the range of 0.05-5.00 µg/mL, with good reproducibility. The limit of detection was 0.004-0.108 µg/mL. The presented method was verified using a milk sample, spiked with four pollutants, which enabled high-throughput detection and the accuracies of 88.17-107.58% confirmed its applicability, in real sample analysis.

Critical role for cholesterol in Lassa fever virus entry identified by a novel small molecule inhibitor targeting the viral receptor LAMP1.

Lassa fever virus (LASV) is endemic in West Africa and causes severe hemorrhagic fever and sensorineural hearing loss. We identified a small molecule inhibitor of LASV and used it to analyze the mechanism of entry. Using a photo-reactive analog that retains antiviral activity as a probe, we identified the inhibitor target as lysosome-associated membrane protein 1 (LAMP1), a host factor that binds to the LASV glycoprotein (GP) during infection. We found that LAMP1 binding to LASV GP is cholesterol-dependent, and that the inhibitor blocks infection by competing with cholesterol in LAMP1. Mutational analysis of a docking-based model identified a putative inhibitor binding site in the cholesterol-binding pocket within the LAMP1 domain that binds GP. These findings identify a critical role for cholesterol in LASV entry and a potential target for therapeutic intervention.

Development and application of magnetic solid phase extraction in tandem with liquid-liquid extraction method for determination of four tetracyclines by HPLC with UV detection.

A novel HPLC-UV method was developed for the determination of four tetracyclines based on magnetic solid phase extraction in tandem with liquid-liquid extraction. The water-soluble amino functionalized magnetite nanoparticle (MNP-NH2) was used as an adsorbent for extraction/preconcentration of tetracycline, oxytetracycline, chlortetracycline, and doxycycline from bovine milk samples. Fourier transform infrared spectrometer, transmission electron microscope, X-ray diffraction, and elemental analyze techniques were used to characterize the material. Some key parameters which influence liquid-liquid extraction and magnetic dispersive solid-phase extraction procedure, including volume of extraction solvent, the amount of adsorbent, the pH, extraction and desorption time, the composition of the eluent, and elution frequency were investigated. The proposed method exhibited a linear range of 50.0-2500.0 µg L-1 (r2 = 0.9941) with and good reproducibility (RSD < 2.2%, n = 3). The limit of detection and quantification were 40.0 and 50.0 µg L-1. This method was verified using milk sample spiked with four tetracyclines (100.0-200.0 µg L-1), and accuracies of 87.8-107.5%, which confirmed its applicability in real-sample analysis. The proposed method also shows potential application prospects for other water-soluble adsorbents.

Graphene oxide composites for magnetic solid-phase extraction of twelve quinolones in water samples followed by MALDI-TOF MS.

Antibiotic compounds in natural waters are normally present at low concentrations. In this paper, an easy and highly sensitive screening method using graphene oxide-functionalized magnetic composites (GO@NH2@Fe3O4) combined with matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) was established for twelve quinolone antibiotics. GO@NH2@Fe3O4 composites were utilized as adsorbents for magnetic solid-phase extraction. This method combines the advantages of magnetic solid-phase extraction and MALDI-TOF MS, which allows for fast detection of quinolones at low concentrations. To improve absorption efficiency, the following parameters were individually optimized: sample acidity, extraction time, amount of adsorbent used, eluent used, and desorption time. Under the optimum conditions, the established method gave a low detection limit of 0.010 mg/L and allowed the high-throughput screening of twelve quinolone antibiotics (enoxacin, norfloxacin, ciprofloxacin, pefloxacin, fleroxacin, gatifloxacin, enrofloxacin, levofloxacin, sparfloxacin, danofloxacin, difloxacin, and lomefloxacin). The proposed method, having an easily prepared sorbent with a high affinity for quinolones and a convenient, high-throughput detection step, has been shown to have merit for the detection of antibiotics in water samples. Graphical abstract Schematic illustration of the (A) preparation of GO@NH2@Fe3O4 and (B) operating procedure for the MSPE and MALDI-TOF MS detection of QNs.

Highly Selective, Aptamer-Based, Ultrasensitive Nanogold Colorimetric Smartphone Readout for Detection of Cd(II).

A highly selective and sensitive method for Cd(II) detection was developed based on aptamer and gold nanoparticles (AuNPs) combined with a colorimetric smartphone readout. The experimental conditions such as reaction time of polydiene dimethyl ammonium chloride (PDDA) and AuNPs, PDDA dose, time of aptamer and PDDA incubation, and aptamer concentration were optimized. Under the optimized conditions, the color and red(R) value of the solution was concentration-dependent on Cd(II). The proposed method exhibited a linear range of 1-400 ng/mL (r2 = 0.9794) with a limit of detection (LOD) of 1 ng/mL. This method had been successfully applied to test and quantify Cd(II) in water and rice samples, and the results were in full agreement with those from the atomic absorption spectrometer. Therefore, low-cost colorimetry demonstrated its potential for practical application in visual or quantitative detection with a smartphone. This approach can be readily applied to other analytes.

The Calcium Channel Blocker Bepridil Demonstrates Efficacy in the Murine Model of Marburg Virus Disease.

No therapeutics are approved for the treatment of filovirus infections. Bepridil, a calcium channel blocker developed for treating angina, was identified as a potent inhibitor of filoviruses in vitro, including Ebola and Marburg viruses, and Ebola virus in vivo. We evaluated the efficacy of bepridil in a lethal mouse model of Marburg virus disease. A dose of 12 mg/kg bepridil once or twice daily resulted in 80% or 90% survival, respectively. These data confirm bepridil's broad-spectrum anti-filovirus activity warranting further investigation of bepridil, or improved compounds with a similar mechanism, as a pan-filovirus therapeutic agent.

In Vitro and In Vivo Activity of Amiodarone Against Ebola Virus.

At the onset of the 2013-2016 epidemic of Ebola virus disease (EVD), no vaccine or antiviral medication was approved for treatment. Therefore, considerable efforts were directed towards the concept of drug repurposing or repositioning. Amiodarone, an approved multi-ion channel blocker for the treatment of cardiac arrhythmia, was reported to inhibit filovirus entry in vitro. Compassionate use of amiodarone in EVD patients indicated a possible survival benefit. In support of further clinical testing, we confirmed anti-Ebola virus activity of amiodarone in different cell types. Despite promising in vitro results, amiodarone failed to protect guinea pigs from a lethal dose of Ebola virus.

Identification of Combinations of Approved Drugs With Synergistic Activity Against Ebola Virus in Cell Cultures.

Background: A need to develop therapeutics to treat Ebola virus disease patients in remote and resource-challenged settings remains in the wake of the 2013-2016 epidemic in West Africa. Toward this goal, we screened drugs under consideration as treatment options and other drugs of interest, most being small molecules approved by the Food and Drug Administration. Drugs demonstrating in vitro antiviral activity were advanced for evaluation in combinations because of advantages often provided by drug cocktails. Methods: Drugs were screened for blockade of Ebola virus infection in cultured cells. Twelve drugs were tested in all (78 pair-wise) combinations, and 3 were tested in a subset of combinations. Results: Multiple synergistic drug pairs emerged, with the majority comprising 2 entry inhibitors. For the pairs of entry inhibitors studied, synergy was demonstrated at the level of virus entry into host cells. Highly synergistic pairs included aripiprazole/piperacetazine, sertraline/toremifene, sertraline/bepridil, and amodiaquine/clomiphene. Conclusions: Our study shows the feasibility of identifying pairs of approved drugs that synergistically block Ebola virus infection in cell cultures. We discuss our findings in terms of the theoretic ability of these or alternate combinations to reach therapeutic levels. Future research will assess selected combinations in small-animal models of Ebola virus disease.

Interferon-ß and Interferon-γ Are Weak Inhibitors of Ebola Virus in Cell-Based Assays.

Previous studies have demonstrated little efficacy of interferons (IFNs) in animal models of Ebola virus disease. However, these studies were limited to a small number of type I IFNs and, during the most recent outbreak of Ebola virus, questions regarding the suitability of the animal models to evaluate IFNs were raised. To address the potential that anti-Ebola virus activity was overlooked, type I and type II IFNs (α-2a, α-2b, -ß, -γ, and -universal) were tested in a variety of cell types (Vero E6, Huh 7 cells, and human macrophages). IFNs are weak inhibitors of Ebola virus Makona in these cell lines.

Analysis of an electro-optic modulator based on a graphene-silicon hybrid 1D photonic crystal nanobeam cavity.

We propose and numerically study an on-chip graphene-silicon hybrid electro-optic (EO) modulator operating at the telecommunication band, which is implemented by a compact 1D photonic crystal nanobeam (PCN) cavity coupled to a bus waveguide with a graphene sheet on top. Through electrically tuning the Fermi level of the graphene, both the quality factor and the resonance wavelength can be significantly changed, thus the in-plane lightwave can be efficiently modulated. Based on finite-difference time-domain (FDTD) simulation results, the proposed modulator can provide a large free spectral range (FSR) of 125.6 nm, a high modulation speed of 133 GHz, and a large modulation depth of ~12.5 dB in a small modal volume, promising a high performance EO modulator for wavelength-division multiplexed (WDM) optical communication systems.

Broadband photodetection in a microfiber-graphene device.

We propose and experimentally demonstrate a microfiber-graphene device. Owing to the interaction between the graphene film and the evanescent field leaked from the microfiber, the hybrid photoconductive device exhibits a high photoresponse. A maximum photocurrent responsivity of ~2.81 mA/W is achieved in the telecommunication band. A nearly flat photoresponse spectrum within broad operational band ranging from 1500 nm to 1600 nm is also obtained as a consequence of the dispersionless and flat absorption of graphene. These results show that the proposed photocurrent generation device could provide an effective solution for broadband photodetection.

Sensitive detection of atrazine in tap water using TELISA.

A highly sensitive flow injection analysis (FIA)-based thermal enzyme-linked immunoassay, TELISA, was developed for the rapid detection of atrazine (ATZ) in tap water. ATZ and ß-lactamase-labeled ATZ were employed in a competitive immunoassay using a monoclonal antibody (mAb). After the off-column liquid-phase competition, the mAb was captured on the Protein G Sepharose™ 4 Fast Flow (PGSFF) column support material. Injected ß-lactamase substrate ampicillin was degraded by the column-bound ATZ-ß-lactamase, generating a detectable heat signal. Several assay parameters were optimized, including substrate concentration, flow rates and regeneration conditions, as well as the mAb and ATZ-ß dilution ratios and concentrations. The assay linear range was 0.73-4.83 ng mL(-1) with a detection limit of 0.66 ng mL(-1). An entire heat signal requires 10 min for generation, and the cycle time is less than 40 min. The results were reproducible and stable. ATZ-spiked tap water samples exhibited a recovery rate of 103%-116%, which correlated with the UHPLC-MS/MS measurements. We attributed this significant increase in sensitivity over our previously published work to the following factors: (i) the capture of already-formed immune complexes on the column via immobilized Protein G, which eliminated chemical immobilization of the antibody; (ii) off-column preincubation allows the formation of immune complexes under nearly ideal conditions; and (iii) multiple buffers can be used to, in one case, enhance immune-complex formation and in the other to maximize enzymatic activity. Furthermore, the scheme creates a universal assay platform in which sensing is performed in the off-column incubation and detection after capture in the enzyme thermistor (ET) detector, which opens up the possibility of detecting any antigen for which antibodies were available.

Interferon-ß and mycophenolic acid are potent inhibitors of Middle East respiratory syndrome coronavirus in cell-based assays.

The Middle East respiratory syndrome coronavirus (MERS-CoV) presents a novel emerging threat to public health worldwide. Several treatments for infected individuals have been suggested including IFN, ribavirin and passive immunotherapy with convalescent plasma. Administration of IFN-α2b and ribavirin has improved outcomes of MERS-CoV infection in rhesus macaques when administered within 8 h post-challenge. However, detailed and systematic evidence on the activity of other clinically available drugs is limited. Here we compared the susceptibility of MERS-CoV with different IFN products (IFN-α2b, IFN-γ, IFN-universal, IFN-α2a and IFN-ß), as well as with two antivirals, ribavirin and mycophenolic acid (MPA), against MERS-CoV (Hu/Jordan-N3/2012) in vitro. Of all the IFNs tested, IFN-ß showed the strongst inhibition of MERS-CoV in vitro, with an IC50 of 1.37 U ml(-1), 41 times lower than the previously reported IC50 (56.08 U ml(-1)) of IFN-α2b. IFN-ß inhibition was confirmed in the virus yield reduction assay, with an IC90 of 38.8 U ml(-1). Ribavirin did not inhibit viral replication in vitro at a dose that would be applicable to current treatment protocols in humans. In contrast, MPA showed strong inhibition, with an IC50 of 2.87 µM. This drug has not been previously tested against MERS-CoV and may provide an alternative to ribavirin for treatment of MERS-CoV. In conclusion, IFN-ß, MPA or a combination of the two may be beneficial in the treatment of MERS-CoV or as a post-exposure intervention in high-risk patients with known exposures to MERS-CoV.

Micro-/nanostructures for surface-enhanced Raman spectroscopy: Recent advances and perspectives.

Surface-enhanced Raman spectroscopy (SERS) has great potential for the analysis of molecules adsorbed on metals with rough surfaces or substrates with micro-/nanostructures. Plasmonic coupling between metal nanoparticles and the morphology of the rough metal surface can produce "hot spots" that enhance Raman scattering by adsorbed molecules, typically at micro- to nanomolar concentrations, although high enhancement factors can also facilitate single-molecule detection. This phenomenon is widely applicable for chemical analysis and sensing in various fields. In this review, the latest research progress on SERS micro-/nanosensors is evaluated, and the sensors are classified according to their individual functions. Furthermore, the design principles and working mechanisms of reported SERS-active micro-/nanostructured substrates are analyzed, and the design features adopted to overcome the difficulties associated with precision detection are explored. Finally, challenges and directions for future development in this field are discussed. This review serves as a design guide for novel SERS-active substrates.

Programmable DNA tweezers-SDA for ultra-sensitive signal amplification fluorescence sensing strategy.

BACKGROUND: DNA tweezers, classified as DNA nanomachines, have gained prominence as multifunctional biosensors due to their advantages, including a straightforward structure, response mechanism, and high programmability. While the DNA tweezers demonstrate simultaneous, rapid, and stable responses to different targets, their detection sensitivity requires enhancement. Some small molecules, such as mycotoxins, often require more sensitive detection due to their extremely high toxicity. Therefore, more effective signal amplification strategies are needed to further enhance the sensitivity of DNA tweezers in biosensing. RESULTS: We designed programmable DNA tweezers that detect small-molecule mycotoxins and miRNAs through simple sequence substitution. While the DNA tweezers demonstrate simultaneous, rapid, and stable responses to different targets, their detection sensitivity requires enhancement. We introduced the Strand Displacement Amplification (SDA) technique to address this limitation, proposing a strategy of novel programmable DNA tweezers-SDA ultrasensitive signal amplification fluorescence sensing. We specifically investigate the effectiveness of this approach concerning signal amplification for two critical mycotoxins: aflatoxin B1 (AFB1) and zearalenone (ZEN). Results indicate that the detection ranges of AFB1 and ZEN via this strategy were 1-10,000 pg mL -1 and 10-100,000 pg mL -1, respectively, with corresponding detection limits of 0.933 pg mL -1 and 1.07 pg mL -1. Compared with the DNA tweezers direct detection method for mycotoxins, the newly constructed programmable DNA tweezers-SDA fluorescence sensing strategy achieved a remarkable 104-fold increase in the detection sensitivity for AFB1 and ZEN. SIGNIFICANCE: The constructed programmable DNA tweezers-SDA ultrasensitive signal-amplified fluorescence sensing strategy exhibits excellent detection performance for mycotoxins. The superb versatility of this strategy allows the developed method to be easily used for detecting other analytes by simply replacing the aptamer and cDNA, which has incredible potential in various fields such as food safety screening, clinical diagnostics, and environmental analysis.

Application of near-infrared-activated and ATP-responsive trifunctional upconversion nano-jelly for in vivo tumor imaging and synergistic therapy.

Upconversion nanoparticles (UCNPs)-mediated in-situ imaging and synergistic therapy may be an effective approach against tumors. However, it remains a challenge to improve therapeutic index and reduce toxicity. Here, we investigated the construction process of a three-layer (core-shell-shell) upconversion nano-jelly hydrogels (UCNJs) coated with stimulus-responsive deoxyribonucleic acid chains, aiming to achieve selective recognition of tumor cells and controlled release of drugs. The UCNJs have a NaYF4: Yb, Er core with an outer silica shell with embedded methylene blue (MB). Then the outer layer was coated with mesoporous silica and loaded with doxorubicin (DOX). Finally, polyacrylamide chains containing anti-adenosine triphosphate (ATP) aptamer sequences were assembled layer-by-layer on the surface of particles to form DNA hydrogels to lock DOX. Under near-infrared irradiation, green light (540 nm) emitted by UCNJs can be used for imaging, while red light (660 nm) is absorbed by MB. The latter generates singlet oxygen, resulting in photodynamic therapy (PDT) effect to inhibit tumor growth. UCNJs also can recognize ATP in tumor cells, leading to hydrogel degradation and DOX release. The hydrogel coating can increase drug-carrying capacity of mesoporous materials and improve biocompatibility. Therefore, the UCNJs has great potential advantages for application in the field of cancer diagnosis and treatment.

A facile dual-mode immunosensor based on speckle Ag-doped nanohybrids for ultrasensitive detection of Ochratoxin A.

Ochratoxin A (OTA) is a potent carcinogen, and is among the most dangerous mycotoxins in agricultural products. In this study, an ultrasensitive dual-mode immunosensor was developed for naked-eye and fluorescence detection of OTA based on Ag-doped core-shell nanohybrids (Ag@CSNH). Complete antigen-labeled Ag@CSNH (CA-Ag@CSNH) were used as a competitive bind and dual-mode probe. The diffused doping structure of CA-Ag@CSNH provided improved stability, color and fluorescence quencher performance. Antibodies modified magnetic beads were used as a capture probe. The competitive binding between OTA and CA-Ag@CSNH produced both color change and fluorescence quenching. Ultraviolet and fluorescence intensitie correlated linearly with OTA concentration ranges of 0.03-3 ng/mL and 10-10000 pg/mL, and limits of detection of 0.0235 ng/mL and 0.9921 pg/mL, respectively. The practical applicability of proposed strategy was demonstrated by analysis of OTA in spiked corn, soybean and flour samples. This study offers a new insight on multi-mode platforms for various applications.