A sensitive CRISPR/Cas12a-assisted fluorescent aptasensor for rapid detection of food allergens.

Food allergens elicit abnormal immune system responses among allergic individuals and sensitive detection for allergenic ingredient is greatly significant. To address this need, a novel fluorescent aptasensor, assisted by Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), have been developed for food allergens. In this study, aptamer offers distinctive recognition capabilities in binding specific targets, while CRISPR-associated-12a protein (Cas12a) holds precise cis-cleavage for cutting fluorescent signal probes. Notably, the utilization of Cas12a cis-cleavage activity, rather than trans-cleavage, eliminates the necessity for additional fluorescent probes, thus reducing interference between substances and enhancing sensitivity. Throughout the process, complementary DNA (cDNA) plays a crucial dual role in target recognition conversion and signal presentation, representing a key challenge and innovative aspect of this study. To evaluate the performance of the aptasensor, lysozyme (LYS) is employed as a representative model target of food allergens. Under optimal conditions, the developed aptasensor could achieve an exceptional low limit of detection (LOD) of 6.10 pM with a dynamic detection range of 10 pM-320 pM. The aptasensor demonstrates high selectivity and great recovery rates. This strategy yields promising outcomes, holding the potential to serve as a valuable reference for various food allergens detection.

The Development of Aptamer-Based Gold Nanoparticle Lateral Flow Test Strips for the Detection of SARS-CoV-2 S Proteins on the Surface of Cold-Chain Food Packaging.

The COVID-19 pandemic over recent years has shown a great need for the rapid, low-cost, and on-site detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In this study, an aptamer-based colloidal gold nanoparticle lateral flow test strip was well developed to realize the visual detection of wild-type SARS-CoV-2 spike proteins (SPs) and multiple variants. Under the optimal reaction conditions, a low detection limit of SARS-CoV-2 S proteins of 0.68 nM was acquired, and the actual detection recovery was 83.3% to 108.8% for real-world samples. This suggests a potential tool for the prompt detection of SARS-CoV-2 with good sensitivity and accuracy, and a new method for the development of alternative antibody test strips for the detection of other viral targets.

Effect of Vacuum Roasting on Total Selenium Content of Selenium-Enriched Rapeseed, Maillard Reaction Products, Oxidative Stability and Physicochemical Properties of Selenium-Enriched Rapeseed Oil.

Selenium-enriched rapeseed (SER) is an emerging oil seed. Roasting is beneficial in improving oil yield and promoting the release of micronutrients into SER oil, but high temperatures and dry air lead to selenium loss and fatty acid degradation in SER. To minimize the selenium loss and improve the SER oil quality, this study investigated the effects of vacuum (VC) roasting (90-170 °C for 30 min) on the SER selenium content, Maillard reaction products, oxidative stability, and physicochemical properties of SER oil, with conventional dry air (DA) roasting as the control. The results showed that the selenium loss in VC-roasted SER meals increased from 7.17 to 19.76% (90-170 °C for 30 min), which was 47.13 to 80.48% of that in DA-roasted SER meals, while no selenium was detected in the SER oils. Compared to DA roasting, VC roasting (90-170 °C for 30 min) reduced lipid oxidation products (LOPs), Maillard reaction products (MRPs), and benzo[a]pyrene contents, and increased carotenoids, unsaturated fatty acid contents, reaching a maximum oil yield of 35.58% at a lower temperature (130 °C for 30 min). Selenium contents exhibited a highly significant negative correlation with MRPs and LOPs (p ≤ 0.005). The VC roasting retarded selenium loss and improved SER oil quality compared to conventional DA roasting.

Establishment of the multi-component bone-on-a-chip: to explore therapeutic potential of DNA aptamers on endothelial cells.

Background: Despite great efforts to develop microvascular bone chips in previous studies, current bone chips still lacked multi-component of human-derived cells close to human bone tissue. Bone microvascular endothelial cells (BMECs) were demonstrated to be closely related to the glucocorticoid (GC)-induced osteonecrosis of the femoral head (ONFH). Tumor necrosis factor-alpha (TNF-α) aptamer has been proved to bind to its receptor and block cascade activities. Objective: There are two main objectives in this study: 1) to establish a multi-component bone-on-a-chip within the microfluidic system in vitro, 2) to explore the therapeutic potential of TNF-α aptamer on BMECs in the GC-induced ONFH model. Methods: Histological features of clinical samples were analyzed before BMECs isolation. The functional bone-on-a-chip consists of the vascular channel, stromal channel and structure channel. GC-induced ONFH model was established based on the multi-component of human-derived cells. Truncation and dimerization were performed on a previously reported DNA aptamer (VR11). BMECs apoptosis, cytoskeleton and angiogenesis status in the ONFH model were observed by the TUNEL staining and confocal microscope. Results: The multi-component of BMECs, human embryonic lung fibroblasts and hydroxyapatite were cultured within the microfluidic bone-on-a-chip. TNF-α was found up-regulated in the necrotic regions of femoral heads in clinical samples and similar results were re-confirmed in the ONFH model established in the microfluidic platform by detecting cell metabolites. Molecular docking simulations indicated that the truncated TNF-α aptamer could improve the aptamer-protein interactions. Further results from the TUNEL staining and confocal microscopy showed that the truncated aptamer could protect BMECs from apoptosis and alleviate GC-induced damages to cytoskeleton and vascularization. Conclusion: In summary, a microfluidic multi-component bone-on-a-chip was established with 'off-chip' analysis of cell metabolism. GC-induced ONFH model was achieved based on the platform. Our findings provided initial evidence on the possible potentials of TNF-α aptamer as a new type of TNF-α inhibitor for patients with ONFH.

Quantitative Detection of Natural Rubber Content in Eucommia ulmoides by Portable Pyrolysis-Membrane Inlet Mass Spectrometry.

Eucommia ulmoides gum (EUG) is a natural polymer predominantly consisting of trans-1,4-polyisoprene. Due to its excellent crystallization efficiency and rubber-plastic duality, EUG finds applications in various fields, including medical equipment, national defense, and civil industry. Here, we devised a portable pyrolysis-membrane inlet mass spectrometry (PY-MIMS) approach to rapidly, accurately, and quantitatively identify rubber content in Eucommia ulmoides (EU). EUG is first introduced into the pyrolyzer and pyrolyzed into tiny molecules, which are then dissolved and diffusively transported via the polydimethylsiloxane (PDMS) membrane before entering the quadrupole mass spectrometer for quantitative analysis. The results indicate that the limit of detection (LOD) for EUG is 1.36 µg/mg, and the recovery rate ranges from 95.04% to 104.96%. Compared to the result of pyrolysis-gas chromatography (PY-GC), the average relative error is 1.153%, and the detection time was reduced to less than 5 min, demonstrating that the procedure was reliable, accurate, and efficient. The method has the potential to be employed to precisely identify the rubber content of natural rubber-producing plants such as Eucommia ulmoides, Taraxacum kok-saghyz (TKS), Guayule, and Thorn lettuce.

Study on in vitro NR biosynthesis by rapid quantitative determination of substrate depletion.

A convenient and nonradioactive method for quantifying in vitro NR biosynthesis is presented that is based upon the quantitation of substrate depletion by ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS). NR oligomers could be in vitro biosynthesized with the enzyme source from Hevea brasiliensis (Hevea) or Taraxacum kok-saghyz (TKS) by exogenous monomers (IPP) and initiators (FPP). The IPP incorporation rate and FPP consumption rate were 62.24% and 51.14% respectively when the washed rubber particles (WRP) of Hevea was the enzyme source. The IPP incorporation rate and the FPP consumption rate were 74.49% and 95.90% respectively when the sediment bottom fraction (BF) of Hevea was the enzyme source. The in vitro NR biosynthesis can be divided into two stages:(1) the initiation reaction of FPP, which occurs more in BF, and (2) the growth reaction of IPP, which occurs more in WRP. In addition, the IPP incorporation and FPP consumption rates were 59.39% and 34.15% respectively when the BF of TKS was selected as an enzyme source.

Research on the Impact Initiation Behavior of PTFE/Al/Ni2O3 Reactive Materials.

PTFE/Al reactive material is an energetic material that releases energy under impact conditions, resulting in a wide range of application prospects. In order to improve its damage ability-considering the higher heat of the reaction per unit mass when Ni2O3 is involved in the aluminothermic reaction-we designed and studied PTFE/Al/Ni2O3, a reaction material based on polytetrafluoroethylene (PTFE). We also designed two other kinds (PTFE/Al, PTFE/Al/CuO) for comparative study, with the mass fraction of the metal oxides added at 10%, 20%, and 30%, respectively. The quasi-static compression properties and impact initiation behavior of the material were investigated by a universal material testing machine and a drop hammer test. The reaction process of different materials under a high strain rate was recorded using a high-speed camera. The results show that with the increase in Ni2O3 content, the strength of the PTFE/Al/Ni2O3 reactive material shows an increasing trend followed by a decreasing trend. Among the three reactive materials, when the content of Al/Ni2O3 reaches 30 wt.%, the reaction duration is the longest (at 4 ms) and the reaction fireball is the largest. The addition of Ni2O3 is helpful to improve the reactivity and reaction duration of the PTEF/Al reactive material.

Evolution of Interferon-Gamma Aptamer with Good Affinity and Analytical Utility by a Rational In Silico Base Mutagenesis Post-SELEX Strategy.

The Systematic Evolution of Ligands by EXponential enrichment (SELEX) is conventionally an effective method to identify aptamers, which are oligonucleotide sequences with desired properties to recognize targets specifically and sensitively. However, there are some inherent limitations, e.g., the loss of potential high-affinity sequences during biased iterative PCR enrichment processes and the limited structural diversity of the initial library, which seriously restrict their real-world applications. To overcome these limitations, the in silico base mutagenesis post-SELEX strategy based on the low Gibbs free energy (ΔG) and genetic algorithm was developed for the optimization of the interferon-gamma aptamer (B1-4). In the process of evolution, new sequences were created and the aptamer candidates with low ΔG values and advanced structures were produced. After five rounds of selection, systematic studies revealed that the affinity of the newly developed evolutionary aptamer (M5-5) was roughly 10-fold higher than that of the parent aptamer (B1-4), and an aptasensor detection system with a limit-of-detection (LOD) value of 3.17 nM was established based on the evolutionary aptamer. The proposed approach provided an efficient strategy to improve the aptamer with low energy and a high binding ability, and the good analytical utility thereof.

High-Throughput Aptamer Microarrays for Fluorescent Detection of Multiple Organophosphorus Pesticides in Food.

A novel high-throughput aptamer microarray fluorescent method based on thioflavin T (ThT) was established for the sensitive detection of phoxim, parathion, fensulfothion, and isocarbophos. In this work, the aptamers in binding buffer tended to have the antiparallel G-quadruplex structure, which can bind ThT and release its potential fluorescence signal. However, when the organophosphorus pesticides (OPs) were present, partial aptamers preferred to bind them, forcing the displacement of ThT from the G-quadruplex and resulting in the significant decrease in fluorescence signal. Under optimal experimental conditions (12T spacer, 300 nM aptamer, and 80 µM ThT), the OP aptamer microarray has low limits of detection of 25.4 ng/mL for phoxim, 12.0 ng/mL for parathion, 7.7 ng/mL for fensulfothion, and 9.9 ng/mL for isocarbophos. The accuracy and reliability of the method is further verified by testing the recovery rate of OPs spiked in two different complicated sample matrices (pears and radishes). It is worth mentioning that not only the developed aptamer microarray technology has low sensitivity and a broad spectrum, but it also allows for high-throughput and rapid analysis of a variety OPs, which overcomes some of the shortcomings of other OP detection methods.

Spatial-temporal variations of reference evapotranspiration and its driving factors in cold regions, northeast China.

Reference evapotranspiration ([Formula: see text]) is an important indicator for hydrometeorological change, which integrates atmospheric and surface conditions, and its downward trends have been reported in many regions over the past several decades. Cold regions constitute an important ecological barrier in China; however, few studies focus on change in [Formula: see text] in cold regions. Especially in the cold region of northeast China (CRNEC), as one of the national strategic grain bases, understanding spatial-temporal variations of [Formula: see text] is important for agriculture water management and ecological protection. This study selected the observations at 113 national meteorological stations located in CRNEC and evaluated the trends of [Formula: see text] and their driving factors from 1961 to 2017. Results indicate that annual [Formula: see text] increases from the northeast to the southwest of CRNEC and has an insignificant decreasing trend in the whole study period, in which 33 stations (29.2%) show significant decreasing trends and only 19 stations (16.8%) show significant increasing trends at the 95% confidence level. An abrupt change in [Formula: see text] data is detected from 1994. Reasons for this abrupt change in [Formula: see text] vary largely over the study areas. Analysis shows that wind speed and minimum air temperature are the two major factors that control the change of [Formula: see text] before 1994. It also shows that wind speed and actual vapor pressure are the two major controlling factors after 1994. We also found that [Formula: see text] shows a certain correlation with Pacific Decadal Oscillation and Western Pacific Index, but there is a significant correlation between meteorological factors and teleconnection factors related to [Formula: see text]. These findings will promote agricultural water management and improve water ecological protection in the CRNEC. We investigated changes in reference evapotranspiration relationships with atmospheric circulation and its attributions over the cold regions in northeast China during 1961 ~ 2017. The results indicate that the wind speed and minimum air temperature are the two major factors that control the change of ET0 before 1994, and wind speed and actual vapor pressure are the two major controlling factors after 1994. We also found that ET0 shows a certain correlation with Western Pacific Index in the whole period.

[Effect of graphene oxide on the function of erythrocytes].

The biocompatibility of nanomaterials has attracted much attention. Graphene oxide (GO) is a nanomaterial widely used in biomedicine, but its toxicity can not be ignored. In this study, the effect of GO on the blood system (the hemolysis rate, the fragility of erythrocyte, and acetylcholinesterase activity) was systematically investigated. The results showed that the hemolysis rate of erythrocytes was lower than 8% when the GO concentration was below 100 µg/mL (P<0.01). GO at low concentration levels (<5 µg/mL) had no significant effect on the fragility of erythrocytes, but GO at high concentration (10 µg/mL) increased the fragility of erythrocytes (P=0.01). Moreover, GO increased the activity of acetylcholinesterase on erythrocytes. The concentration of 20 µg/mL graphene oxide with the size >5 µm (LGO) increased the activity of acetylcholinesterase by 42.67% (P<0.05). Then molecular dynamics simulation was used to study how GO interacted with acetylcholinesterase and increased its activity. The results showed that GO was attached to the cell membrane, thus may provide an electronegative environment that helps the hydrolysate to detach from the active sites more quickly so as to enhance the activity of acetylcholinesterase.

A steroid-induced osteonecrosis model established using an organ-on-a-chip platform.

Bone microvascular endothelial cells (BMECs) constitute the central part of the femoral head's intramural microenvironment network and have an essential role in the development of steroid-induced osteonecrosis of the femoral head. Recently, the rapid development of microfluidic technology has led to innovations in the fields of chemistry, medicine and life sciences. It is now possible to use microfluidics organ-on-a-chip techniques to assess osteonecrosis. In the present study, BMECs were cultured on a microfluidic organ-on-a-chip platform to explore the pathogenesis of femoral-head necrosis. The aim of the present study was to explore the effects of different interventions on BMECs and study the pathogenesis of steroid-induced osteonecrosis through a microfluidic organ-on-a-chip platform. Methods including SU-8 lithography were used to produce a microfluidic organ-on-a-chip and human umbilical vein endothelial cells (HUVECs) were used to test whether it was possible to culture cells on the chip. Subsequently, a set of methods were applied for the isolation, purification, culture and identification of BMECs. Hydroxyapatite (HA) was used for co-culture, dexamethasone was used at different concentrations as an intervention in the cells and icariin was used for protection. BMECs were isolated and cultured from the femoral head obtained following total hip arthroplasty and were then inoculated into the microfluidic organ-on-a-chip for further treatment. In part I of the experiment, HUVECs and BMECs both successfully survived on the chip and a comparison of the growth and morphology was performed. HA and BMECs were then co-cultured for comparison with the control group. The cell growth was observed by confocal microscopy after 24 h. In part II, the effects of different concentrations of glucocorticoid (0.4 or 0.6 mg/ml dexamethasone) and the protection of icariin were evaluated. The morphology of BMECs and the cleaved caspase-3/7 content were observed by immunofluorescence staining and confocal microscopy after 24 h. In the microfluidic organ-on-a-chip, the response of the cells was able to be accurately observed. In part I, at the same concentration of injected cells, BMECs exhibited improved viability compared with HUVECs (P<0.05). In addition, it was indicated that HA was not only able to promote the germination and growth of BMECs but also improve the survival of the cells (P<0.05). In part II, it was identified that dexamethasone was able to induce BMECs to produce cleaved caspase 3/7; the caspase 3/7 content was significantly higher than that in the blank control group (P<0.05) and a dose correlation was observed. Icariin was able to inhibit this process and protect the microvascular structure of BMECs. The content of cleaved caspase 3/7 in the icariin-protected group was significantly lower than that in the group without icariin (P<0.05). It was concluded that BMECs are more likely to survive than HUVECs and HA promoted the growth of BMECs on the microfluidic organ-on-a-chip platform. Glucocorticoid caused damage to BMECs through the production of cleaved caspase 3/7, which was observed through the microfluidic organ-on-a-chip platform, and icariin protected BMECs from damage.

[Execution, assessment and improvement methods of motor imagery for brain-computer interface].

Motor imagery (MI) is an important paradigm of driving brain computer interface (BCI). However, MI is not easy to control or acquire, and the performance of MI-BCI depends heavily on the performance of the subjects' MI. Therefore, the correct execution of MI mental activities, ability evaluation and improvement methods play important and even critical roles in the improvement and application of MI-BCI system's performance. However, in the research and development of MI-BCI, the existing researches mainly focus on the decoding algorithm of MI, but do not pay enough attention to the above three aspects of MI mental activities. In this paper, these problems of MI-BCI are discussed in detail, and it is pointed out that the subjects tend to use visual motor imagery as kinesthetic motor imagery. In the future, we need to develop some objective, quantitatively visualized MI ability evaluation methods, and develop some effective and less time-consumption training methods to improve MI ability. It is also necessary to solve the differences and commonness of MI problems between and within individuals and MI-BCI illiteracy to a certain extent.

Advances and perspectives of aptasensors for the detection of tetracyclines: A class of model compounds of food analysis.

The residual tetracyclines in food are frequently applied as the model compounds to develop aptasensors. Until now, more than 100 advanced aptasensors towards tetracyclines have been developed and published in English. This review summarizes and discusses comprehensively these advanced aptasensors, in terms of the principle designs, applied frontier transducers/materials, working performance, and advantages/disadvantages. The aptasensors are classified according to the inherent transduction techniques, i.e., optics, optics-electricity, optics-mass, and electricity-mass. Moreover, the present challenges such as the limited specificity and limited affinity of the aptamers, the future prospects and trends such as further combination with other advanced materials and technologies, and the urgent need of expanding the practical application were discussed and prospected. We hope this review can serve as a powerful tool for both tracing the development progresses of aptasensors and providing adequate references for further development of aptasensing methods for food-related analytes.

Simultaneous determination of zearalenone and ochratoxin A based on microscale thermophoresis assay with a bifunctional aptamer.

Nowadays, contamination of various mycotoxins in crops and their products exposes increasing risks to human health. Efficient determination methods are urgently needed. Herein, a bifunctional aptamer and a simple aptasensor based on microscale thermophoresis assay (MST) were constructed for the first time for simultaneous determination of two mycotoxins, i.e. zearalenone (ZEN) and ochratoxin A (OTA). The bifunctional aptamer was engineered by splicing a ZEN aptamer and an OTA aptamer with a linker according to the structure analysis of aptamers. The binding mechanism of the bifunctional aptamer to ZEN and OTA were revealed basing on the molecular docking studies. The MST assay proved that the bifunctional aptamer showed high affinity and specificity towards ZEN and OTA. Furthermore, a bifunctional aptamer-based MST-aptasensor was developed for simultaneous detection of ZEN and OTA in corn oil sample. The MST-aptasensor provided a limit of detection (LOD) of 0.12 nM, with satisfactory recoveries of 93.31-104.19% and excellent selectivity, indicating that the bifunctional aptamer and MST-aptasensor had great potential in practical applications.

Structural identification of sour compounds in wine and tea by ambient ionization mass spectrometry according to characteristic product ion and neutral loss.

Sourness is an important food taste for human. A rapid, accurate method was used to generalize the structure similarity and diversity of sour compounds. Based on the product ion and neutral loss of sour compounds, ambient ionization techniques combined with quadrupole-Orbitrap mass spectrometry (AI-Q-Orbitrap) was employed. According to the behavior of sour compounds in the process of high collision dissociation (HCD) of MS/MS, three fragmentation pathway schemes were proposed: (1) charge-driven fragmentation and CO2 loss, (2) six-membered ring rearrangement and Cα-Cß cleavage, and (3) elimination rearrangement and H2O, CO2 and CO loss in succession. Besides, structure information about characteristic product ions and characteristic neutral losses was summarized. Finally, multi-class sour compounds including monoacids, diacids, polyacids and phenolic acids in wine and tea were identified and compared. Therefore, sour compounds and their structure information can be determined by AI-MS based on characteristic product ion and neutral loss.

Development of a lateral flow aptamer assay strip for facile identification of theranostic exosomes isolated from human lung carcinoma cells.

Exosomes are Extracellular Vesicles (EV) that own unique structural features and functions and have gradually become the hot research spot in recent years. The tumor-derived exosomes contain various types of useful biological information, and medical identification of exosomes relied on the specific characterization of membrane surface proteins. In this study, in order to rapidly identify non-small cell lung cancer (NSCLC)-derived exosomes, based on an aptamer against CD63 protein on exosome membrane, a low cost lateral flow aptamer assay (LFAA) test strip using nanogold particles as visualization probes was successfully developed for facile identification of A549 exosomes isolated from human lung carcinoma cells diluted from 6.4 × 109 particles/mL herein.

Proteomic Landscape Has Revealed Small Rubber Particles Are Crucial Rubber Biosynthetic Machines for Ethylene-Stimulation in Natural Rubber Production.

Rubber particles are a specific organelle for natural rubber biosynthesis (NRB) and storage. Ethylene can significantly improve rubber latex production by increasing the generation of small rubber particles (SRPs), regulating protein accumulation, and activating many enzyme activities. We conducted a quantitative proteomics study of different SRPs upon ethylene stimulation by differential in-gel electrophoresis (DIGE) and using isobaric tags for relative and absolute quantification (iTRAQ) methods. In DIGE, 79 differentially accumulated proteins (DAPs) were determined as ethylene responsive proteins. Our results show that the abundance of many NRB-related proteins has been sharply induced upon ethylene stimulation. Among them, 23 proteins were identified as rubber elongation factor (REF) and small rubber particle protein (SRPP) family members, including 16 REF and 7 SRPP isoforms. Then, 138 unique phosphorylated peptides, containing 129 phosphorylated amino acids from the 64 REF/SRPP family members, were identified, and most serine and threonine were phosphorylated. Furthermore, we identified 226 DAPs from more than 2000 SRP proteins by iTRAQ. Integrative analysis revealed that almost all NRB-related proteins can be detected in SRPs, and many proteins are positively responsive to ethylene stimulation. These results indicate that ethylene may stimulate latex production by regulating the accumulation of some key proteins. The phosphorylation modification of REF and SRPP isoforms might be crucial for NRB, and SRP may act as a complex natural rubber biosynthetic machine.

Characteristic synergistic cytotoxic effects toward cells in graphene oxide dressing with cadmium and copper ions.

The increasing applications of graphene oxide (GO) in bio-medicine, environment and other fields enhance the exposure possibility of human beings to GO. Studies have been performed to address the in vitro toxicity of GO; however, little information on the in vivo biological consequence of GO with other common disasters is available, especially when cells are co-exposed to GO and common metal ions. To explore the influence and possible mechanisms of such co-exposure scenarios, a series of tests of cell viability, membrane integrity, reactive oxygen species (ROS), cell morphology, and Cd2+ distribution, were conducted. The results showed that the synergistic toxic mechanisms of GO and Cd2+, initiated from the adhesion of GO on HeLa cells, and followed by the recruitment of Cd2+ ions around the cell membrane, impaired the membrane integrity, morphology and adhesion capability, and triggered cell toxicity. The synergistic toxic mechanism of GO and Cu2+ mainly correlated to ROS, while no obvious relationship with membrane integrity was observed. The findings are envisaged to facilitate the application of GO in biology and related fields.