Advancing HIC method development: Retention-time modeling and tuning selectivity with ternary mobile-phase systems.

The use of a ternary mobile-phase system comprising ammonium sulphate, sodium chloride, and phosphate buffer was explored to tune retention and enhance selectivity in hydrophobic interaction chromatography. The accuracy of the linear solvent-strength model to predict protein retention with the ternary mobile-phase system based on isocratic scouting runs is limited, as the extrapolated retention factor at aqueous buffer conditions (k0) cannot be reliably established. The Jandera retention model utilizing a salt concentration averaged retention factor (k¯0) in aqueous buffer for ternary systems overcomes this bottleneck. Gradient retention factors were derived based on isocratic scouting runs after numerical integration of the isocratic Jandera model, leading to retention-time prediction errors below 11 % for linear gradients. Furthermore, an analytical expression was formulated to predict HIC retention for both linear and segmented linear gradients, considering the linear solvent-strength (LSS) model within ternary salt systems, relying on a fixed k0. The approach involved conducting two gradient scouting runs for each of the two binary salt systems to determine model parameters. Retention-time prediction errors for linear gradients were below 12 % for lysozyme and 3 % for trypsinogen and α-chymotrypsinogen A. Finally, the analytical expression for a ternary mobile-phase system was used in combination with a genetic algorithm to tune the HIC selectivity. With an optimized segmented ternary gradient, a critical-pair separation for a mixture of 7 proteins was achieved within 15 min with retention-time prediction errors ranging between 0.7 and 15.7 %.

Lysozyme-sensitive plasmonic hydrogel nanocomposite for colorimetric dry-eye inflammation biosensing.

Detection of lysozyme levels in ocular fluids is considered crucial for diagnosing and monitoring various health and eye conditions, including dry-eye syndrome. Hydrogel-based nanocomposites have been demonstrated to be one of the most promising platforms for fast and accurate sensing of different biomolecules. In this work, hydrogel, electrospun nanofibers, and plasmonic nanoparticles are combined to fabricate a sensitive and easy-to-use biosensor for lysozyme. Poly(L-lactide-co-caprolactone) (PLCL) nanofibers were covered with silver nanoplates (AgNPls), providing a stable plasmonic platform, where a poly(N-isopropylacrylamide)-based (PNIPAAm) hydrogel layer allows mobility and good integration of the biomolecules. By integrating these components, the platform can also exhibit a colorimetric response to the concentration of lysozyme, allowing for easy and non-invasive monitoring. Quantitative biosensing operates on the principle of localized surface plasmon resonance (LSPR) induced by plasmonic nanoparticles. Chemical, structural, thermal, and optical characterizations were performed on each platform layer, and the platform's ability to detect lysozyme at concentrations relevant to those found in tears of patients with dry-eye syndrome and other related diseases was investigated by colorimetry and UV-Vis spectroscopy. This biosensor's sensitivity and rapid response time, alongside the easy detection by the naked eye, make it a promising tool for early diagnosis and treatment monitoring of eye diseases.

Developing portable and controllable fluorescence capillary imprinted sensor for visual detection Crohn's disease biomarkers.

Simultaneous detection of multiple biomarker levels is essential to improve the accuracy of early diagnosis. Introducing capillary will simplify procedure, less time, and reduce reagent consumption for point-of-care testing of biomarkers. Here, we developed a portable and controllable smartphone-integrated fluorescence capillary imprinted sensing platform for the accuracy visual detection of Crohn's disease biomarkers (lysozyme, Fe3+) using single-excitation/double-signal detection. A novel controllable capillary coating strategy was developed by static gas-driven coating method for synthesis uniform fluorescence capillary imprinted sensor (Si-CD/g-CdTe@MIP capillary sensor). When Fe3+ and lysozyme were added, the fluorescence intensity of Si-CD/g-CdTe@MIP capillary sensor was quenched at 426 nm and enhanced at 546 nm, respectively. This Si-CD/g-CdTe@MIP capillary sensor has high sensitivity and selectivity for quantification lysozyme and Fe3+ simultaneously with the detection limit of 0.098 nM and 0.20 nM, respectively. In addition, the smartphone-integrated Si-CD/g-CdTe@MIP capillary sensor was applied for the intelligent detection of lysozyme and Fe3+, in which the detection limit was calculated as 0.32 nM and 0.65 nM. The smartphone-integrated visual Si-CD/g-CdTe@MIP capillary sensor realized ultrasensitive microanalysis (18 µL/time) of biomarkers in health man and Crohn 's patients, providing a novel strategy for early diagnosis of Crohn 's disease.

Lysozyme functionalized silver nanoclusters as a dual channel optical sensor for the effective determination of glutathione.

This article describes the development of a facile and efficient fluorescence sensor for the determination of glutathione (GSH). Presence of the antioxidant glutathione in blood serum is considered as a biomarker for catastrophe like colorectal cancer. Silver nanoclusters with strong fluorescence and good water solubility synthesized from relatively cheaper precursors are one of the species very much explored in fluorescence sensors and bioimaging. Here, Chicken egg derived-lysozyme functionalized silver nanoclusters (Lyz AgNCs) with red fluorescence emission has been synthesized and developed to a turn-off fluorescence sensor for GSH through which colorimetric determination is also possible. Due to the ground state 'Ag-S' interaction between Lyz AgNCs and GSH, the determination of the analyte is possible from 1.00 × 10-5 M to 1.00 × 10-6 M via fluorimetric and from 9.00 × 10-6 to 8.00 × 10-7 M via spectrophotometric techniques with a limit of detection 2.86 × 10-7 M and 4.76 × 10-7 M, respectively. Selectivity of the sensor has been studied and applicability of the sensor in artificial blood serum samples has been demonstrated.

A sandwich FRET biosensor for lysozyme detection based on peptide-functionalized gold nanoparticles and FAM-labeled aptamer.

Lysozyme (LYZ) plays a crucial role in the body's immune defense system. Monitoring LYZ levels can provide valuable insights into the diagnosis and severity assessment of various diseases. Traditionally, antibody-based sandwich assays are employed for LYZ detection, but they are often time-consuming and operationally complicated. In this research, a novel sandwich FRET biosensor was developed, which enables rapid detection of LYZ based on peptide-functionalized gold nanoparticles (pAuNPs) and FAM-labeled aptamer (Apt-FAM). Initially, a mixture of Apt-FAM and pAuNPs resulted in partial quenching of the Apt-FAM fluorescence emission through an inner filter effect (IFE), with negligible energy transfer because of the electrostatic repulsion between the negatively charged pAuNPs and Apt-FAM. The introduction of LYZ into the mixture drove the specific binding of Apt-FAM and pAuNPs to LYZ, facilitating the formation of a pAuNPs-LYZ-aptamer sandwich structure. The formation of this complex drew the pAuNPs and Apt-FAM into close enough proximity to enable FRET to occur, which in turn effectively quenched the fluorescence emission of FAM. The decrease in FAM fluorescence intensity was correlated with the increasing concentration of LYZ. Thus, a sandwich FRET biosensor was successfully developed for LYZ detection with a linear detection range of 0-1.75 µM and a detection limit of 85 nM. Additionally, the biosensor allowed visual detection of LYZ in a 96-well microplate, with a rapid response time of just 15 s. This study introduces a innovative sandwich FRET biosensor that combines aptamer and peptide recognition elements, offering a fast and antibody-free method for protein detection.

Turn-off enzyme activity of histidine-rich peptides for the detection of lysozyme.

An assay that integrates histidine-rich peptides (HisRPs) with high-affinity aptamers was developed enabling the specific and sensitive determination of the target lysozyme. The enzyme-like activity of HisRP is inhibited by its interaction with a target recognized by an aptamer. In the presence of the target, lysozyme molecules progressively assemble on the surface of HisRP in a concentration-dependent manner, resulting in the gradual suppression of enzyme-like activity. This inhibition of HisRP's enzyme-like activity can be visually observed through color changes in the reaction product or quantified using UV-visible absorption spectroscopy. Under optimal conditions, the proposed colorimetric assay for lysozyme had a detection limit as low as 1 nM and exhibited excellent selectivity against other nonspecific interferents. Furthermore, subsequent research validated the practical applicability of the developed colorimetric approach to saliva samples, indicating that the assay holds significant potential for the detection of lysozymes in samples derived from humans.

The Effect of Hyperbaric Storage on the Nutritional Value and Retention of Certain Bioactive Proteins in Human Milk.

Human milk (HM) contains the essential macronutrients and bioactive compounds necessary for the normal growth and development of newborns. The milk collected by human milk banks is stored frozen and pasteurized, reducing its nutritional and biological value. The purpose of this study was to determine the effect of hyperbaric storage at subzero temperatures (HS-ST) on the macronutrients and bioactive proteins in HM. As control samples, HM was stored at the same temperatures under 0.1 MPa. A Miris HM analyzer was used to determine the macronutrients and the energy value. The lactoferrin (LF), lysozyme (LYZ) and α-lactalbumin (α-LAC) content was checked using high-performance liquid chromatography, and an ELISA test was used to quantify secretory immunoglobulin A (sIgA). The results showed that the macronutrient content did not change significantly after 90 days of storage at 60 MPa/-5 °C, 78 MPa/-7 °C, 111 MPa/-10 °C or 130 MPa/-12 °C. Retention higher than 90% of LYZ, α-LAC, LF and sIgA was observed in the HM stored at conditions of up to 111 MPa/-10 °C. However, at 130 MPa/-12 °C, there was a reduction in LYZ and LF, by 39 and 89%, respectively. The storage of HM at subzero temperatures at 0.1 MPa did not affect the content of carbohydrates or crude and true protein. For fat and the energy value, significant decreases were observed at -5 °C after 90 days of storage.

Fluorescence-Polarization-Based Assaying of Lysozyme with Chitooligosaccharide Tracers.

Lysozyme is a well-known enzyme found in many biological fluids which plays an important role in the antibacterial protection of humans and animals. Lysozyme assays are used for the diagnosis of a number of diseases and utilized in immunohistochemistry, genetic and cellular engineering studies. The assaying methods are divided into two categories measuring either the concentration of lysozyme as a protein or its activity as an enzyme. While the first category of methods traditionally uses an enzyme-linked immunosorbent assay (ELISA), the methods for the determination of the enzymatic activity of lysozyme use either live bacteria, which is rather inconvenient, or natural peptidoglycans of high heterogeneity and variability, which leads to the low reproducibility of the assay results. In this work, we propose the use of a chemically synthesized substrate of a strictly defined structure to measure in a single experiment both the concentration of lysozyme as a protein and its enzymatic activity by means of the fluorescence polarization (FP) method. Chito-oligosaccharides of different chain lengths were fluorescently labeled and tested leading to the selection of the pentasaccharide as the optimal size tracer and the further optimization of the assay conditions for the accurate (detection limit 0.3 µM) and rapid (<30 min) determination of human lysozyme. The proposed protocol was applied to assay human lysozyme in tear samples and resulted in good correlation with the reference assay. The use of synthetic fluorescently labeled tracer, in contrast to natural peptidoglycan, in FP analysis allows for the development of a reproducible method for the determination of lysozyme activity.

High hydrostatic pressure is similar to Holder pasteurization in preserving donor milk antimicrobial activity.

BACKGROUND: The microbiological safety of donor milk (DM) is commonly ensured by Holder pasteurization (HoP, 62.5 °C for 30 min) in human milk banks despite its detrimental effects on bioactive factors. We compared the antimicrobial properties of DM after Holder pasteurization treatment or High Hydrostatic Pressure processing (HHP, 350 MPa at 38 °C), a non-thermal substitute for DM sterilization. METHODS: We assessed lactoferrin and lysozyme concentrations in raw, HHP- and HoP-treated pools of DM (n = 8). The impact of both treatments was evaluated on the growth of Escherichia coli and Group B Streptococcus in comparison with control media (n = 4). We also addressed the effect of storage of HHP treated DM over a 6-month period (n = 15). RESULTS: HHP milk demonstrated similar concentrations of lactoferrin compared with raw milk, while it was significantly decreased by HoP. Lysozyme concentrations remained stable regardless of the condition. Although a bacteriostatic effect was observed against Escherichia coli at early timepoints, a sharp bactericidal effect was observed against Group B Streptococcus. Unlike HoP, these results were significant for HHP compared to controls. Stored DM was well and safely preserved by HHP. CONCLUSION: Our study demonstrates that this alternative sterilization method shows promise for use with DM in human milk banks. IMPACT: Antimicrobial activity of donor milk after High Hydrostatic Pressure treatment has not been clearly evaluated. Donor milk lactoferrin is better preserved by High Hydrostatic Pressure than conventional Holder pasteurization, while lysozyme concentration is not affected by either treatment. As with Holder pasteurization, High Hydrostatic Pressure preserves donor milk bacteriostatic activity against E. coli in addition to bactericidal activity against Group B Streptococcus. Donor milk treated by High Hydrostatic Pressure can be stored safely for 6 months.

Impact of COVID-19 vaccination on saliva immune barriers: IgA, lysozyme, and lactoferrin.

Understanding the role of salivary constituents, such as lactoferrin, lysozyme, and secretory immunoglobulin A (sIgA), in immune protection and defense mechanisms against microbial invasion and colonization of the airways is important in light of the ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. The salivary immune barrier in individuals affected by COVID-19 may contribute to disease prognosis. Thus, the aim of the present review is to evaluate the effect of COVID-19 vaccines on the immunological composition of saliva. IgA antibodies generated by vaccination can neutralize the virus at mucosal surfaces, whereas antimicrobial peptides, such as lysozyme and lactoferrin, have broad-spectrum antimicrobial activity. Collectively, these components contribute to the protective immune response of the oral cavity and may help minimize viral transmission as well as the severity of COVID-19. Measuring the levels of these components in the saliva of COVID-19-vaccinated individuals can help in evaluating the vaccine's ability to induce mucosal immunity, and it might also provide insights into whether saliva can be used in diagnostics or surveillance for monitoring immune responses following vaccination. This also has implications for viral transmission.

Electrochemical and thermal detection of allergenic substance lysozyme with molecularly imprinted nanoparticles.

Lysozyme (LYZ) is a small cationic protein which is widely used for medical treatment and in the food industry to act as an anti-bacterial agent; however, it can trigger allergic reactions. In this study, high-affinity molecularly imprinted nanoparticles (nanoMIPs) were synthesized for LYZ using a solid-phase approach. The produced nanoMIPs were electrografted to screen-printed electrodes (SPEs), disposable electrodes with high commercial potential, to enable electrochemical and thermal sensing. Electrochemical impedance spectroscopy (EIS) facilitated fast measurement (5-10 min) and is able to determine trace levels of LYZ (pM) and can discriminate between LYZ and structurally similar proteins (bovine serum albumin, troponin-I). In tandem, thermal analysis was conducted with the heat transfer method (HTM), which is based on monitoring the heat transfer resistance at the solid-liquid interface of the functionalized SPE. HTM as detection technique guaranteed trace-level (fM) detection of LYZ but needed longer analysis time compared to EIS measurement (30 min vs 5-10 min). Considering the versatility of the nanoMIPs which can be adapted to virtually any target of interest, these low-cost point-of-care sensors hold great potential to improve food safety.

[An integrated approach to reducing hyperesthesia of teeth in patients with underlying somatic pathology]. / Kompleksnyi podkhod v reabilitatsii patsientov s giperesteziei tverdykh tkanei zubov na fone somaticheskikh zabolevanii.

THE AIM OF THE STUDY: Was to analyze the effectiveness of therapeutic and preventive measures aimed at reducing hyperesthesia of hard dental tissues in patients with background somatic diseases. MATERIALS AND METHODS: The study involved 113 patients with increased tooth sensitivity and treated in the gastroenterological and endocrinological departments of the S.M. Kirov City Clinical Hospital No.3¼ in Astrakhan in the period from 2018 to 2021 at the age of 26-43 years. The main group included 52 patients with confirmed diagnoses of gastric and duodenal ulcer, pancreatitis and type II diabetes mellitus who were treated for dental hyperesthesia with an integrated approach. The control group included 61 patients with periodontal disease without background somatic pathologies in whom hyperesthesia was treated by remineralizing therapy. The effectiveness of the treatment was determined in dynamics on the 10th and 40th days of treatment using OHI-S, PMA indices, dental hypersensitivity prevalence (DHP), dental hypersensitivity intensity (DHI), Dental Sensitivity Index (DSI), Efficacy of Dental Sensitivity Index (EDSI). In addition, the pH of saliva, the activity of lysozyme and S-IgA, and the levels of cytokines IL-1ß, IL-4, IL-6, and IL-8 were determined. RESULTS: The average value of OHI-S in the main group on the 10th day of treatment decreased from 2.25±0.12 (poor level of hygiene) to 1.47±0.09 (satisfactory level). The PMA index in the main group also tended to decrease from 32.1±1.44% (moderate degree of gingivitis) to 20.5±2.08% (mild degree) on the 10th day of treatment. The average values of DPH, DPI, EDSI and DSI in the main group had a noticeable decrease already on the 10th day from the start of treatment (from 12.3±1.66% to 2.1±1.22%; from 2.5±0.48 to 1.2±0.16; from 48.3±1.14% to 40.8±1.71%; from 42.1±2.07% to 20.8±1, 65% respectively). In the main group on the 10th and 40th day of treatment the pH values of non-stimulated and stimulated saliva stabilized (from 4.61±0.12 to 6.94±0.07 and from 5.47±0.21 to 7.42±0.24, respectively), the activity of lysozyme increased (from 45.97±1.46% to 55.19±0.96%) alongside with secretory IgA (from 0.17±0.02 to 0.33±0.21 mg/ml). Also, indicators of cytokines IL-1ß, IL-4, IL-6, IL-8 tended to improve. The analysis of the control group revealed persistent mean values that did not yield to significant changes either in the course of treatment. CONCLUSION: Thus, in patients of the main group, the results obtained indicate an improvement in the dental status and activation of cytokine regulation, providing a combination of active components of the mineral complex. In controls the method of remineralizing therapy for tooth hyperesthesia alleviated dental hypersensitivity, but without significant improvement of the laboratory results.

Customizable bio-based coating of phase-transited lysozyme-COS for durable antibacterial and moisture management on wool fabric.

Textiles with efficient moisture management provide a comfortable microenvironment for human body. However, little attention has been paid to sweat-induced bacterial growth alongside. In this study, chitooligosaccharide (COS) was used to modify lysozyme (Lyz-COS) to obtain more excellent antibacterial activity. Lyz-COS could undergo an amyloid-like aggregation by reducing its disulfide bond and hydrogen bond triggered by thiourea dioxide (TD). The Phase-Transited Lyz-COS (PTL-COS) coating increases the hydrophilicity and antibacterial properties of wool fabrics, which can withstand 50 washing cycles and 100 rubbing cycles. In addition, two methods are proposed to customize Janus wool fabrics as desired. Method 1: The PTL-COS film was prepared first, and then the film was transferred to one side of the wool fabric. Method 2: Simply spray the PTL-COS solution on one side of the wool fabric. These two processes are simple to operate and can be customized on demand, enabling single transport of sweat and inhibiting sweat-induced bacterial growth. This work underlines the significance of chitooligosaccharide-modified PTL coatings for functionalization of textile surfaces and provides new insights into the development of more adaptable and smarter textiles and clothing.

Nanopore-Based Protein Identification.

The implementation of a reliable, rapid, inexpensive, and simple method for whole-proteome identification would greatly benefit cell biology research and clinical medicine. Proteins are currently identified by cleaving them with proteases, detecting the polypeptide fragments with mass spectrometry, and mapping the latter to sequences in genomic/proteomic databases. Here, we demonstrate that the polypeptide fragments can instead be detected and classified at the single-molecule limit using a nanometer-scale pore formed by the protein aerolysin. Specifically, three different water-soluble proteins treated with the same protease, trypsin, produce different polypeptide fragments defined by the degree by which the latter reduce the nanopore's ionic current. The fragments identified with the aerolysin nanopore are consistent with the predicted fragments that trypsin could produce.

Machine Learning Assisted Simultaneous Structural Profiling of Differently Charged Proteins in a Mycobacterium smegmatis Porin A (MspA) Electroosmotic Trap.

The nanopore is emerging as a means of single-molecule protein sensing. However, proteins demonstrate different charge properties, which complicates the design of a sensor that can achieve simultaneous sensing of differently charged proteins. In this work, we introduce an asymmetric electrolyte buffer combined with the Mycobacterium smegmatis porin A (MspA) nanopore to form an electroosmotic flow (EOF) trap. Apo- and holo-myoglobin, which differ in only a single heme, can be fully distinguished by this method. Direct discrimination of lysozyme, apo/holo-myoglobin, and the ACTR/NCBD protein complex, which are basic, neutral, and acidic proteins, respectively, was simultaneously achieved by the MspA EOF trap. To automate event classification, multiple event features were extracted to build a machine learning model, with which a 99.9% accuracy is achieved. The demonstrated method was also applied to identify single molecules of α-lactalbumin and ß-lactoglobulin directly from whey protein powder. This protein-sensing strategy is useful in direct recognition of a protein from a mixture, suggesting its prospective use in rapid and sensitive detection of biomarkers or real-time protein structural analysis.

Fructose and methylglyoxal-induced glycation alters structural and functional properties of salivary proteins, albumin and lysozyme.

Glycation process refers to reactions between reduction sugars and amino acids that can lead to formation of advanced glycation end products (AGEs) which are related to changes in chemical and functional properties of biological structures that accumulate during aging and diseases. The aim of this study was to perform and analyze in vitro glycation by fructose and methylglyoxal (MGO) using salivary fluid, albumin, lysozyme, and salivary α-amylase (sAA). Glycation effect was analyzed by biochemical and spectroscopic methods. The results were obtained by fluorescence analysis, infrared spectroscopy (total attenuated reflection-Fourier transform, ATR-FTIR) followed by multivariate analysis of principal components (PCA), protein profile, immunodetection, enzymatic activity and oxidative damage to proteins. Fluorescence increased in all glycated samples, except in saliva with fructose. The ATR-FTIR spectra and PCA analysis showed structural changes related to the vibrational mode of glycation of albumin, lysozyme, and salivary proteins. Glycation increased the relative molecular mass (Mr) in protein profile of albumin and lysozyme. Saliva showed a decrease in band intensity when glycated. The analysis of sAA immunoblotting indicated a relative reduction in intensity of its correspondent Mr after sAA glycation; and a decrease in its enzymatic activity was observed. Carbonylation levels increased in all glycated samples, except for saliva with fructose. Thiol content decreased only for glycated lysozyme and saliva with MGO. Therefore, glycation of salivary fluid and sAA may have the potential to identify products derived by glycation process. This opens perspectives for further studies on the use of saliva, an easy and non-invasive collection fluid, to monitor glycated proteins in the aging process and evolution of diseases.

Tracing Some Salivary Immune Elements in Iraqi SARS-2 Patients.

Saliva is one of the most significant components in maintaining oral homeostasis and symbiosis. It contains antimicrobial proteins and peptides, such as mucins, lactoferrin, lysozyme, lactoperoxidase, Catherine, statins, and antibodies (secretory immunoglobin A [sIgA]). Early defenses against respiratory infections rely heavily on mucosal immunity, especially secretory sIgA, which has several features and functions that make it suitable for mucosal defense. Salivary testing has been utilized to define mucosal immune responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Lysozyme has muramidase, with antimicrobial activity, and high concentrations in body fluids, such as saliva and tear. This research aimed to offer an update on how saliva components suppress viral infection and sustain health. A total of 50 individuals, including 30 SARS-2 patients and 20 non-infected subjects, in the age range of 32-54 years were enrolled in this study. Saliva specimens were obtained from polymerase chain reaction (PCR)-confirmed coronavirus disease 2019 (COVID-19) patients and non-infected participants. To collect saliva, the subjects were advised to swirl water over their lips three times, and 5.0 ml of saliva was collected. Samples were centrifuged at 800 x g for 10 min. Saliva was diluted at 1:2,000 with 1 × Diluent N. The immunoglobulin A (IgA) titer in saliva was detected. A spectrophotometer was used to measure the solution's change in absorbance at 550 nm. Measurements (salivary IgA and lysozyme) were made after 7, 30, and 60 days of confirmatory PCR COVID-19 test. The mean scores of salivary IgA levels were obtained at 17.85, 15.26, and 10.73 mg/dl in patients and 9.53, 10.33, and 9.21 mg/dl in healthy individuals after 7, 30, and 60 days, respectively. The salivary lysozyme activity levels in SARS-2 patients compared to controls were 9.7, 7.3, and 4.2 mg/dl versus 2.9, 3.4, and 3.77 mg/dl, respectively. The salivary IgA level was significantly higher in patients of a confirmatory test for COVID-19 compared to healthy individuals.

Development and Interlaboratory Evaluation of an LC-MS/MS Method for the Quantification of Lysozyme in Wine Across Independent Instrument Platforms.

BACKGROUND: Various processing aids and fining agents are used in winemaking to help improve sensory characteristics. Some of these materials may contain or be derived from allergenic foods, such as eggs. In order to ensure food safety and that products meet regulatory compliance, it is essential to have robust and effective analytical methods to verify the removal of allergenic proteins following their use. Current methods include ELISA and MS methods, which can target either whole foods or individual proteins, and provide either quantitative data or qualitative confirmation of proteins. MS methods offer the potential to test for multiple proteins within a single assay to improve cost and efficiency, whereas ELISA methods typically analyze for a single protein per assay. OBJECTIVE: This study focuses on the development of a LC-tandem MS (MS/MS) quantitative method for lysozyme in white wine and compares performance across two laboratories utilizing two different instrument platforms. METHODS: Lysozyme target peptides were selected by conducting bottom-up discovery proteomics. Candidate targets were evaluated using parallel reaction monitoring (PRM) or selected reaction monitoring (SRM) LC-MS/MS, depending on the instrument in each laboratory. Quantification of lysozyme was conducted using internal, stable isotope-labeled synthetic peptide standards. RESULTS: Three of eight candidate target peptides showed performance suitable for the final quantitative method. White wine spiked with 0.1 and 0.5 ppm lysozyme demonstrated quantitative recovery of 70-120%. While the PRM method delivered better repeatability, the SRM method gave higher quantitative recovery values. CONCLUSION: A targeted LC-MS/MS method for quantification of lysozyme in white wine has been developed and deployed on two different MS instrument platforms in two laboratories. HIGHLIGHTS: Both SRM and PRM targeted LC-MS/MS methodologies can be used for quantification of lysozyme in white wine. This study is among the first to evaluate an MS method for food allergen quantification in multiple laboratories.

Preparação e caracterização de nanocarreadores de beta-lactose à base de pectina e lisozima / Preparation and characterization of beta-lactose nanocarriers based on pectin and lysozyme

O objetivo deste trabalho foi preparar e caracterizar nanocarreadores via auto-organização a partir da pectina de citros e lisozima para o encapsulamento da ß-lactose. Foram estudadas três condições de interação entre os biopolímeros variando a razão molar pectina/lisozima (3:1, 2:1, 1:1, 1:2 e 1:3), o pH e o tempo de aquecimento. A confirmação da interação foi determinada por espectroscopia no infravermelho por transformada de Fourier (FTIR) e por calorimetria de varredura diferencial (DSC). Os espectros de infravermelho evidenciaram que ligações de hidrogênio foram as principais forças envolvidas na formação dos nanocarreadores e sugeriram a ausência de ß-lactose livre na superfície das nanopartículas. Os termogramas evidenciaram que as nanopartículas formadas na presença de ß-lactose têm maior estabilidade térmica do que as nanopartículas sem ß-lactose. Para ambas as formulações estudadas, na presença e na ausência de ß-lactose, a formação das nanopartículas ocorreu entre os valores de pKa e ponto isoelétrico (pI) da pectina e lisozima, respectivamente, sendo a melhor razão de interação pectina/lisozima 1:2, em pH 10, a 80 ºC por 30 min. As nanopartículas foram formadas via auto-organização e todos as partículas apresentaram distribuição de tamanho homogênea, formato esférico, diâmetro inferior a 100 nm e carga superficial negativa. A morfologia e o tamanho das partículas pouco alteraram com a incorporação da -lactose. A eficiência de encapsulação (EE) da ß-lactose foi superior a 96% para as concentrações estudadas. Ensaios preliminares in vitro, em células epiteliais de câncer de cólon (HCT-116), evidenciaram que as nanopartículas formadas são capazes de adentrar no meio intracelular, possivelmente, por via endocitose

This work aimed to prepare and characterize nanocarriers via self-assembly using citrus pectin and lysozyme for ß-lactose encapsulation. Three interaction conditions between the biopolymers were studied, varying the pectin/lysozyme molar ratio (3:1, 2:1, 1:1, 1:2 and 1:3), pH and heating time. Fourier transform infrared spectroscopy (FTIR) and differential scanning calorimetry (DSC) determined the interaction's confirmation. The infrared spectra showed that hydrogen bonds were the main forces involved in the formation of nanocarriers and suggested the absence of free ß-lactose on the surface of the nanoparticles. The thermograms showed that nanoparticles formed in the presence of ß-lactose have greater thermal stability than nanoparticles without ß-lactose. For both formulations studied, in the presence and absence of lactose, the formation of nanoparticles occurred between the pKa and isoelectric point (pI) values of pectin and lysozyme, respectively, with the best pectin/lysozyme interaction molar ratio 1:2, at pH 10, at 80 °C for 30 min. Nanoparticles were formed via self-assembly, and all particles presented homogeneous size distribution, spherical shape, diameter less than 100 nm, and negative surface charge. The morphology and size of the particles changed little with the incorporation of ß-lactose. The encapsulation efficiency (EE) of ß-lactose was higher than 96% for the concentrations studied. Preliminary in vitro assays in colon cancer epithelial cells (HCT-116) showed that the nanoparticles formed are capable of entering the intracellular medium, possibly via endocytosis

Recent applications of quantum dots in optical and electrochemical aptasensing detection of Lysozyme.

Lysozyme (Lyz) is a naturally occurring enzyme that operates against Gram-positive bacteria and leads to cell death. This antimicrobial enzyme forms the part of the innate defense system of nearly all animals and exists in their somatic discharges such as milk, tears, saliva and urine. Increased Lyz level in serum is an important indication of several severe diseases and so, precise diagnosis of Lyz is an urgent need in biosensing assays. Up to know, various traditional and modern techniques have been introduced for Lyz determination. Although the traditional methods suffer from some significant limitations such as time-consuming, arduous, biochemical screening, bacterial colony isolation, selective enrichment and requiring sophisticated instrumentation or isotope labeling, some new modern approaches like aptamer-based biosensors (aptasensors) and quantum dot (QD) nanomaterials are the main goal in Lyz detection. Electrochemical and optical sensors have been highlighted because of their adaptability and capability to decrease the drawbacks of common methods. Using an aptamer-based biosensor, sensor selectivity is enhanced due to the specific recognition of the analyte. Thereby, in this review article, the recent advances and achievements in electrochemical and optical aptasensing detection of Lyz based on different QD nanomaterials and detection methods have been discussed in detail.