Approaching Effective Differential Centrifugal Fractionation by Combining Image Analysis with Analytical Ultracentrifugation.

Centrifugation is one of the most commonly used methods for separation in biology and chemistry. However, effective fractionation is not always easy to obtain, as preparative centrifuge experiments are mostly conducted in an empirical way, even when it is guided by the quantitative results from analytical ultracentrifuge (AUC). Very few works have been performed to enhance the fractionation resolution of the differential centrifugation method in a swing-out rotor. This is primarily due to the absence of a characterization tool for sedimentation in the preparative centrifuge. In this study, we utilized image analysis to map the particle concentration distribution throughout the preparative centrifuge tube, revealing an unexpected and abnormal sedimentation process. By characterizing the sedimentation coefficient distributions of the fractionated product via AUC, we demonstrated that the overall sedimentation efficiency in a swing-out preparative centrifuge was significantly reduced. Furthermore, effective fractionation was confined to the intermediate phase of the entire sedimentation process. We propose that the mechanism here is a combination of the inverse Boycott effect and droplet sedimentation. The actual sedimentation process within a preparative centrifuge can be described by modifying the Lamm equation phenomenologically, which simply results in an effective sedimentation coefficient. Our work builds a foundation for determining the optimal preparative centrifugation conditions for various systems.

Oral immunization with recombinant L. lactis expressing GCRV-II VP4 produces protection against grass carp reovirus infection.

The hemorrhagic disease causing by grass carp reovirus (GCRV) infection, is associated with major economic losses and significant impact on aquaculture worldwide. VP4 of GCRV is one of the major outer capsid proteins which can induce an immune response in the host. In this study, pNZ8148-VP4/L. lactis was constructed to express recombinant VP4 protein of GCRV, which was confirmed by the Western-Blot and enzyme-linked immunosorbent assay. Then we performed the oral immunization for rare minnow model and the challenge with GCRV-II. After oral administration, pNZ8148-VP4/L. lactis can continuously reside in the intestinal tract to achieve antigen presentation. The intestinal and spleen samples were collected at different time intervals after immunization, and the expression of immune-related genes was detected by real-time fluorescence quantitative PCR. The results showed that VP4 recombinant L. lactis could induce complete cellular and humoral immune responses in the intestinal mucosal system, and effectively regulate the immunological effect of the spleen. The immunogenicity and the protective efficacy of the oral vaccine was evaluated by determining IgM levels and viral challenge to vaccinated fish, a significant level (P < 0.01) of antigen-specific IgM with GCRV-II neutralizing activity was able to be detected, which provided a effective protection in the challenge experiment. These results indicated that an oral probiotic vaccine with VP4 expression can provide effective protection for grass carp against GCRV-II challenge, suggesting a promising vaccine strategy for fish.

Evaluation on the effect of ice glazing with different compound additives on the quality of frozen stored (-23 °C) large yellow croaker (Pseudosciaena crocea).

BACKGROUND: Compounded ice glazing has been used in large yellow croaker to improve its quality during frozen storage. The ice glazing liquid is prepared by compound use of trehalose and tea polyphenols, and the moisture, protein-related properties and freshness of the fish have been evaluated during 300 days of frozen storage. RESULTS: The results showed that the addition of trehalose effectively reduced the loss of water. At the same time, it was difficult for ice crystals to grow under the action of trehalose, the average diameter could still be maintained at 111.25-119.85 µm. The combination with tea polyphenols could effectively maintain the protein structure and keep the total volatile base nitrogen (TVB-N) and K value within 11.84 mg/100 g and 13.18%, so that the freshness of the fish was always at the first level. CONCLUSION: In a word, the ice glazing with 5% trehalose and 8% tea polyphenols had the best preservation effect, which was recommended for the frozen storage. © 2022 Society of Chemical Industry.

Effects of Different Freezing Methods on Physicochemical Properties of Sweet Corn during Storage.

Fresh sweet corn has a series of physiological and biochemical reactions after picking due to the high moisture content, leading to damaged nutritional value. Rapid freezing of sweet corn after harvest can minimize tissue damage and quality deterioration. In this study, freshly harvested sweet corn was frozen by ultrasound-assisted freezing, brine freezing, strong wind freezing, and refrigerator freezing. The effects of different freezing methods on hardness, water loss, color, epidermal structure, soluble solids content, soluble sugars content, peroxidase (POD), catalase (CAT), and ascorbate peroxidase (APX) activities of frozen sweet corn during storage were investigated. The results showed that brine freezing and strong wind freezing could effectively reduce the quality loss of sweet corn, keep the color, soluble sugars, and soluble solids content of the sweet corn, delay the decrease in antioxidant enzyme activity, and maintain the quality of sweet corn during long term storage.

Improving the Accuracy of Pdot-Based Continuous Glucose Monitoring by Using External Ratiometric Calibration.

Continuous glucose monitoring (CGM) allows type I and II diabetes patients to track changes in their glucose levels, allowing detection of impending hypoglycemia or hyperglycemia. Polymer dots (Pdots) are candidates for use in implanted CGM systems due to their exceptional brightness, photostability, sensitivity, and biocompatibility. However, Pdot glucose transducers are oxygen-dependent, and changes in tissue oxygen levels affect their measurement accuracy. Here, we describe an external ratiometric calibration method that corrects for changes in tissue oxygen levels to improve measurement accuracy. This method uses the ratio of oxygen concentrations inside and outside the Pdot glucose transducer as an indicator of glucose concentration to correct for signal deviations caused by tissue oxygen fluctuations. A second oxygen-sensitive Pdot that is not conjugated with glucose oxidase is used to measure the oxygen concentration outside the Pdot glucose transducer. We describe the theoretical basis for this approach and demonstrate its effectiveness experimentally in a subcutaneous mouse implant model. This external ratiometric system achieves higher accuracy glucose measurements than previous Pdot-based CGM systems and comparable accuracy to current commercial CGM systems, demonstrating the utility of the external ratiometric calibration strategy.

Ratiometric pH sensing by fluorescence resonance energy transfer-based hybrid semiconducting polymer dots in living cells.

Intracellular pH plays a significant role in all cell activities. Due to their precise imaging capabilities, fluorescent probes have attracted much attention for the investigation of pH-regulated processes. Detecting intracellular pH values with high throughput is critical for cell research and applications. In this work, hybrid semiconducting polymer dots (Pdots) were developed and characterized and were applied for cell imaging and exclusive ratiometric sensing of intracellular pH values. The reported Pdots were prepared by blending a synthesized block polymer (POMF) and a semiconducting polymer poly[2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylenevinylene] (MEHPPV) to construct a fluorescence resonance energy transfer system for ratiometric sensing. Pdots showed many advantages, including high brightness, excellent photostability and biocompatibility, giving the pH probe high sensitivity and good stability. Our results proved the capability of POMF-MEHPPV Pdots for the detection of pH in living cells.

Hydration Kinetics for Alkaline Activation of Slag from Color Variation Data.

In this study, we explore a new method based on color variation data to derive the kinetics of the entire process of the hydration of alkali-activated slag (AAS). Using this image analysis technique, we can monitor the induction period that cannot be observed using conventional microcalorimetry techniques. Color variation was recorded across a sequence of 9999 images, which were processed via MATLAB software package. Further, an average pixel value (APV) was determined to represent the color in each image. Reaction parameters, such as color variation velocity v(t), reaction speed ε(t), and hydration degree α(t), that govern the entire hydration process were determined. On the basis of the reaction parameters and a Krstulovic-Dabic kinetic model, integral and differential equations were derived to simulate the three basic processes of AAS hydration. Equations describing the reaction kinetics of AAS with solutions of three different concentrations of NaOH were extracted using this method.

Reversible Ratiometric NADH Sensing Using Semiconducting Polymer Dots.

Reduced nicotinamide adenine dinucleotide (NADH) is a key coenzyme in living cells due to its role as an electron carrier in redox reactions, and its concentration is an important indicator of cell metabolic state. Abnormal NADH levels are associated with age-related metabolic diseases and neurodegenerative disorders, creating a demand for a simple, rapid analytical method for point-of-care NADH sensing. Here we develop a series of NADH-sensitive semiconducting polymer dots (Pdots) as nanoprobes for NADH measurement, and test their performance in vitro and in vivo. NADH sensing is based on electron transfer from semiconducting polymer chains in the Pdot to NADH upon UV excitation, quenching Pdot fluorescence emission. In polyfluorene-based Pdots, this mechanism resulted in an on-off NADH sensor; in DPA-CNPPV Pdots, UV excitation resulted in NADH-sensitive emission at two wavelengths, enabling ratiometric detection. Ratiometric NADH detection using DPA-CNPPV Pdots exhibits high sensitivity (3.1 µM limit of detection), excellent selectivity versus other analytes, reversibility, and a fast response (less than 5 s). We demonstrate applications of the ratiometric NADH-sensing Pdots including smartphone-based NADH imaging for point-of-care use.

Monitoring Metabolites Using an NAD(P)H-sensitive Polymer Dot and a Metabolite-Specific Enzyme.

We introduce an NAD(P)H-sensitive polymer dot (Pdot) biosensor for point-of-care monitoring of metabolites. The Pdot is combined with a metabolite-specific NAD(P)H-dependent enzyme that catalyzes the oxidation of the metabolite, generating NAD(P)H. Upon UV illumination, the NAD(P)H quenches the fluorescence emission of Pdot at 627 nm via electron transfer, and also fluoresces at 458 nm, resulting in a shift from red to blue emission at higher NAD(P)H concentrations. Metabolite concentration is quantified ratiometrically-based on the ratio of blue-to-red channel emission intensities, with a digital camera-with high sensitivity and specificity. We demonstrate phenylalanine biosensing in human plasma for a phenylketonuria screening test, quantifying several other disease-related metabolites (lactate, glucose, glutamate, and ß-hydroxybutyrate), and a paper-based assay with smartphore imaging for point-of-care use.

Artificial Metalloprotein Nanoanalogues: In Situ Catalytic Production of Oxygen to Enhance Photoimmunotherapeutic Inhibition of Primary and Abscopal Tumor Growth.

Photoimmunotherapy (PIT) has shown enormous potential in not only eliminating primary tumors, but also inhibiting abscopal tumor growth. However, the efficacy of PIT is greatly limited by tumor hypoxia, which causes the attenuation of phototherapeutic efficacy and is a feature of the immunosuppressive tumor microenvironment (TME). In this study, one type of brand-new artificial metalloprotein nanoanalogues is developed via reasonable integration of a "phototherapy-enzymatic" RuO2 and a model antigen, ovalbumin (OVA) for enhanced PIT of cancers, namely, RuO2 -hybridized OVA nanoanalogues (RuO2 @OVA NAs). The RuO2 @OVA NAs exhibit remarkable photothermal/photodynamic capabilities under the near-infrared light irradiation. More importantly, the photoacoustic imaging and immunofluorescence staining confirm that RuO2 @OVA NAs can remarkably alleviate hypoxia via in situ catalysis of hydrogen peroxide overexpressed in the TME to produce oxygen (O2 ). This ushers a prospect of concurrently enhancing photodynamic therapy and reversing the immunosuppressive TME. Also, OVA, as a supplement to the immune stimulation induced by phototherapy, can activate immune responses. Finally, further combination with the cytotoxic T-lymphocyte-associated protein 4 checkpoint blockade is reported to effectively eliminate the primary tumor and inhibit distant tumor growth via the abscopal effect of antitumor immune responses, prolonging the survival.

Light-harvesting metal-organic framework nanoprobes for ratiometric fluorescence energy transfer-based determination of pH values and temperature.

Light-harvesting nanoprobes were developed by self-assembly of nanoscale metal-organic frameworks (NMOFs) and stimuli-responsive polymers for fluorometric sensing of pH values and temperature. Two kinds of fluorescent NMOFs (acting as the energy donor) and stimuli-responsive polymers conjugated to fluorophores (acting as energy acceptors) were prepared and characterized. The NMOFs include zirconium(IV) and π-conjugated dicarboxylate ligands. The fluorophores inclued cyaine dyes and a Bodipy dye. The energy donor and energy acceptor form a Förster resonance energy transfer (FRET) nanosystem. In the light-harvesting system, the chain lengths of the stimuli-responsive polymers vary when the local pH value or temperature change. Ratiometric sensing of pH and temperature was accomplished by monitoring fluorescence. pH values were can be sensed between 3.0 and 8.0 under 420 nm excitation and by ratioing the emission peaks at 645 and 530 nm. Temperature can be sensed in the range from 25 to 50 °C under 550 nm excitation and by ratioing the emission peaks at 810 and 695 nm. The nanoprobes display excellent water dispersibility and cell membrane permeability. They were applied to image pH values and temperature in HeLa cells. Graphical abstract Schematic presentation of an effective strategy to fabricate light-harvesting nanoprobes by self-assembly of MOFs and stimuli-responsive polymers for ratiometric pH and temperature sensing. The distance as the polymer length between energy donor and acceptor is crucial for energy transfer efficiency.

Construction of Thermo-Responsive Elastin-Like Polypeptides (ELPs)-Aggregation-Induced-Emission (AIE) Conjugates for Temperature Sensing.

In this work, an aggregation-induced emission (AIE) molecule (tetraphenylethene derivative, TPE-COOH) was conjugated to elastin-like polypeptides (ELPs40) via an amide bond to form ELPs40-TPE. The successful synthesis of ELPs40-TPE was confirmed by Circular Dichroism spectroscopy, gel electrophoresis, UV-vis absorption, and fluorescence emission spectroscopy. ELPs40-TPE possessed both amphiphilicity and the features of an AIE, and the fluorescence intensity was dependent on the local temperature. The Hela cells imaging indicated that ELPs40-TPE has great potential for bio-imaging applications because of its advantages of high fluorescence intensity, good water-solubility, and remarkable biocompatibility.

The application of ice glazing containing D-sodium erythorbate combined with vacuum packaging to maintain the physicochemical quality and sweet/umami non-volatile flavor compounds of frozen stored large yellow croaker (Pseudosciaena crocea).

Ice glazing containing 0.3 % D-sodium erythorbate (DSE), combined with vacuum packaging, was used as a method to maintain the quality of large yellow croaker during frozen storage. This study aimed to assess various aspects, including water properties (water holding capacity and moisture distribution), protein-related characteristics (secondary and tertiary structure of myofibrillar protein), freshness indicators (K value and total volatile base nitrogen (TVB-N)), and non-volatile flavor compounds (free amino acids (FAAs) and nucleotides) in samples stored for 300 days at -23 °C. The results showed that vacuum packaging had a significant inhibitory effect on the growth of ice crystal. Notably, it successfully maintained the cross-sectional area of nearly all ice crystals below 20,000 µm2, effectively curtailing water loss. Simultaneously, the combination of vacuum packaging with the complex ice glaze effectively mitigated the degradation of IMP and free amino acids, maintaining low levels of K value (12.85 %) and TVB-N (11.28 mg N/100 g) throughout the 300-day frozen storage, retaining first-class freshness. Among the various treatment modalities assessed, the combined application of vacuum packaging and 0.3 % DSE-infused ice glazing emerged as the most effective in terms of preservation outcomes. This efficacious combination shows promising potential for the frozen storage of aquatic products and is therefore recommended for practical implementation.

Amino acids inhibit the photodynamic inactivation effect by hindering cellular oxidative stress.

Photodynamic inactivation (PDI) could utilize light to activate reactive oxygen species (ROS) produced by photosensitizers to kill bacteria for preservation. To delve into the complex effects arising during the post-harvest PDI processing, we conducted experiments using Pseudomonas reinekei, a food spoilage bacteria extracted from rotten Pakchoi. Through analyzing the metabolomics results, we discovered that methionine (Met) and glutamate (Glu) exhibited significant inhibitory effects during the PDI process. The oxidative stress generated by light treatment resulted in a reduction of 30.31% and 36.37% in the levels of Met and Glu, respectively. The data also showed that exogenous Met and Glu reduced intracellular oxidative stress levels, increased peroxidase activity, and prevented the damage of intracellular material and cell membrane deformation. That amino acids could inhibit the effect of PDI by hindering oxidative stress. Therefore, the amino acid content should be considered when applying PDI to treat Met- or Glu-rich foods.

Prolong the shelf-life of the Pakchoi seedlings through the ammonium glycyrrhizinate.

Pakchoi seedlings (Brassica chinensis L.) is susceptible to damage and spoilage during harvest and transport, leading to significant quality deterioration and financial losses. This study explored the use of ammonium glycyrrhizinate (AG) to address these issues. AG self-assembles into macromolecules at room temperature, blocking stomata and regulating respiration rates in Pakchoi seedlings. Additionally, it disrupts bacterial cell biofilm and inhibits its synthesis. While AG has been used in medicine, its application in the food industry remains limited. The study found that incorporating AG in Pakchoi seedlings preserves water content and total soluble solids (TSS), while preventing declines in catalase (CAT), Vitamin C (VC), and chlorophyll during storage. AG also reduced malondialdehyde (MDA) levels and maintained peroxidase (POD) and superoxide dismutase (SOD) activities. At a concentration of 4.25 g L-1, AG enhanced radical scavenging ability and extended the shelf life of Pakchoi seedlings by inhibiting bacteria and postponing senescence.

Dual-Mode Ce-MOF Nanozymes for Rapid and Selective Detection of Hydrogen Sulfide in Aquatic Products.

Increasing concern over the safety of consumable products, particularly aquatic products, due to freshness issues, has become a pressing issue. Therefore, ensuring the quality and safety of aquatic products is paramount. To address this, a dual-mode colorimetric-fluorescence sensor utilizing Ce-MOF as a mimic peroxidase to detect H2S was developed. Ce-MOF was prepared by a conventional solvothermal synthesis method. Ce-MOF catalyzed the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) by hydrogen peroxide (H2O2) to produce blue oxidized TMB (oxTMB). When dissolved, hydrogen sulfide (H2S) was present in the solution, and it inhibited the catalytic effect of Ce-MOF and caused the color of the solution to fade from blue to colorless. This change provided an intuitive indication for the detection of H2S. Through steady-state dynamic analysis, the working mechanism of this sensor was elucidated. The sensor exhibited pronounced color changes from blue to colorless, accompanied by a shift in fluorescence from none to light blue. Additionally, UV-vis absorption demonstrated a linear correlation with the H2S concentration, ranging from 200 to 2300 µM, with high sensitivity (limit of detection, LOD = 0.262 µM). Fluorescence intensity also showed a linear correlation, ranging from 16 to 320 µM, with high selectivity and sensitivity (LOD = 0.156 µM). These results underscore the sensor's effectiveness in detecting H2S. Furthermore, the sensor enhanced the accuracy of H2S detection and fulfilled the requirements for assessing food freshness and safety.

Recognition and detection of histamine in foods using aptamer modified fluorescence polymer dots sensors.

Histamine has been known as a momentous cause of biogenic amine poisoning. Therefore, the content of histamine in foods is strictly required to be controlled within a certain range. Here, an aptamer fluorescent sensor was developed for detection of histamine. Poly [(9, 9-di-n-octylfluorenyl-2, 7-diyl)-alt-(benzo [2,1,3] thiadia-zol-4, 8-diyl)] (PF8BT) and the styrene maleic anhydride copolymer (PSMA) were used for the preparation of PF8BT-Polymer dots (PF8BT-Pdots). PF8BT-Pdots and the cyanine3-phosphoramidite (Cy3) were linked through aptamer to achieve the ratiometric detection for histamine. PF8BT-Pdots were partly quenched by Cy3 due to the fluorescence resonance energy transfer (FRET), when the histamine molecule was recognized by aptamer on the surface of PF8BT-Pdots. A linear range (3-21 µmol/L) was obtained for histamine detection with a low limit of detection (LOD = 0.38 µmol/L). PF8BT aptamer Pdots (PF8BT-A) were used to detect histamine in simply treated aquaculture water and tuna. The cell imaging of HeLa cells presented a good biosecurity and outstanding fluorescent imaging capability of PF8BT-A. The aptamer fluorescent sensors provided a new platform for rapid and accurate detection of histamine in aquatic products and had great potential for the application in food safety and quality control.

Development of packaging films based on UiO-66 MOF loaded melatonin with antioxidation functions for spinach preservation.

Spinach tends to deteriorate after harvest due to physiological metabolic activities. As a natural, pollution-free, and environmentally friendly preservative, melatonin (MT) can effectively maintain the quality of fruits and vegetables after harvest and delay senescence. To enhance the preservation effect of MT, this study developed antioxidant films using MT-loaded UiO-66 metal-organic framework (MOF) nanoparticles. This approach effectively extends the shelf life of spinach while preserving its quality. The underlying mechanism involves leveraging the microporous structure and stability of UiO-66 MOF. Experimental results obtained from the packaging films demonstrated significant improvements in both mechanical strength and antioxidant properties when UiO-66 was loaded with MT at a concentration of 0.20 mg/mL and combined with sodium alginate. Freshness preservation experiments also indicated the effective preservation effect of these films on spinach. In conclusion, the results of this study suggest that MT-loaded UiO-66 MOF is a promising active packaging material for spinach preservation.

Alizarin complexone modified UiO-66-NH2 as dual-mode colorimetric and fluorescence pH sensor for monitoring perishable food freshness.

Food prone to spoilage has a huge food safety hazard, threatening people's health, so early detection of food spoilage is a continuous and urgent need. Herein, we developed a dual-mode response sensor, alizarin complexone@UiO-66-NH2, which can accurately detect pH. The sensor demonstrated significant changes in color from pale yellow to deep pink, while the fluorescence shifted from light blue to blue violet. Moreover, both UV absorption and fluorescence intensity showed a linear correlation with pH raging from 4.5 to 7.5. These results indicate that the sensor effectively responds to pH, making it suitable for detecting the freshness of perishable food. To put this into practice, we integrated the sensor with cellulose-based filter paper to determine the freshness of shrimp and beef, which was proved to be effective in assessing freshness. In the future, it can be combined with intelligent colorimetric and fluorescence instruments to achieve visual detection.

Affinity precipitation of human serum albumin using a thermo-response polymer with an L-thyroxin ligand.

BACKGROUND: Affinity precipitation has been reported as a potential technology for the purification of proteins at the early stage of downstream processing. The technology could be achieved using reversible soluble-insoluble polymers coupled with an affinity ligand to purify proteins from large volumes of dilute solution material such as fermentation broths or plasma. In this study, a thermo-response polymer was synthesized using N-methylol acrylamide, N-isopropyl acrylamide and butyl acrylate as monomers. The molecular weight of the polymer measured by the viscosity method was 3.06 × 104 Da and the lower critical solution temperature (LCST) was 28.0°C.The recovery of the polymer above the LCST was over 95.0%. Human serum albumin (HSA) is the most abundant protein in the human serum system, and it has important functions in the human body. High purity HSA is required in pharmaceuticals. Safe and efficient purification is a crucial process during HSA production. RESULTS: A thermo-response polymer was synthesized and L-thyroxin immobilized on the polymer as an affinity ligand to enable affinity precipitation of HSA. The LCST of the affinity polymer was 31.0°C and the recovery was 99.6% of its original amount after recycling three times. The optimal adsorption condition was 0.02 M Tris-HCl buffer (pH 7.0) and the HSA adsorption capacity was 14.9 mg/g polymer during affinity precipitation. Circular dichroism spectra and a ForteBio Octet system were used to analyze the interactions between the affinity polymer and HSA during adsorption and desorption. The recovery of total HSA by elution with 1.0 mol/L NaSCN was 93.6%. When the affinity polymer was applied to purification of HSA from human serum, HSA could be purified to single-band purity according to SDS-PAGE. CONCLUSION: A thermo-response polymer was synthesized and L-thyroxin was attached to the polymer. Affinity precipitation was used to purify HSA from human serum.