Immunomagnetic Separation Research Articles

Shiga toxin-producing Escherichia coli (STEC) in meat and leafy greens available in the Swedish retail market – Occurrence and diversity of stx subtypes and serotypes

Shiga toxin-producing Escherichia coli (STEC) is a major cause of foodborne illness, ranging from mild diarrhea to permanent kidney failure. This study summarizes the results of four surveys performed at different time periods, which investigated the occurrence and characteristics of STEC in beef, lamb and leafy greens available in the Swedish retail market. Such data is required when assessing the public health risk of varying types of STEC in different foods, and for establishing risk management measures. Samples from domestic and imported products were collected based on their availability in the retail market. The occurrence of STEC was investigated in 477 samples of beef, 330 samples of lamb and 630 samples of leafy greens. The detection of virulence genes (stx1, stx2, eae) was performed using real-time PCR followed by the isolation of bacteria from stx-positive enriched samples using immunomagnetic separation or an immunoblotting method. All STEC isolated from the food samples was further characterised in terms of stx subtyping and serotyping through whole genome sequencing. STEC was isolated from 2 to 14 % of beef samples and 20 to 61 % of lamb samples, depending on the region of origin. STEC was not isolated from samples of leafy greens, although stx genes were detected in 11 (2 %) of the samples tested. In total, 5 of the 151 sequenced STEC isolates from meat contained stx2 and eae, of which 4 such combinations had the stx2a subtype. The stx2 gene, stx2a in particular, is strongly associated with serious disease in humans, especially in combination with the eae gene. The isolates belonged to 20 different serotypes. Two isolates from beef and one from lamb belonged to the serotype O157:H7 and contained genes for stx2 and eae. Overall, several combinations of stx subtypes were found in isolates from beef, whereas stx1c, either alone or together with stx2b, was the dominant combination found in STEC from lamb. In conclusion, STEC was rare in whole meat samples of domestic beef in the Swedish retail market, whereas such bacteria were frequently found in minced meat and whole meat samples of imported beef and domestic and imported lamb. Although the number of isolates containing genes linked to an increased risk of severe disease was low, beef and lamb in the Swedish retail market is a common source of human exposure to potentially pathogenic STEC.

Nanobody-based immunomagnetic separation platform for rapid isolation and detection of Salmonella enteritidis in food samples.

Rapid separation and identification of Salmonella enteritidis (S. enteritidis) in food is of great importance to prevent outbreaks of foodborne diseases. Herein, by using O and H antigens as targets, an epitope-based bio-panning strategy was applied to isolate specific nanobodies towards S. enteritidis. This method constitutes an efficient way to obtain specific antibody fragments and test pairwise nanobodies. On this basis, a double nanobody-based sandwich enzyme-linked immunosorbent assay (ELISA) coupled with immunomagnetic separation (IMS) was developed to rapid enrich and detect S. enteritidis in food. The detection limit of the IMS-ELISA was 3.2×103 CFU/mL. In addition, 1CFU of S. enteritidis in food samples can be detected after 4-h cultivation, which was shortened by 2h after IMS. The IMS-ELISA strategy could avoid matrix interference and shorten the enrichment culture time, which has great potential for application in monitoring bacterial contamination in food.

Research advances of porous organic framework materials on enrichment and detection of mycotoxins

Mycotoxins are a class of toxic secondary metabolites produced by fungi. These substances are carcinogenic, teratogenic, and mutagenic, and cause serious harm to the human body; thus, they have attracted wide attention worldwide. Establishing accurate, rapid, and sensitive methods for the detection of mycotoxins is of great significance. Chromatography is a commonly used technology for mycotoxin detection. However, it is challenging to use in the direct analysis of these metabolites because of the wide variety and distribution of mycotoxins, their complex sample matrix, and their very low content in actual samples. Therefore, the development of suitable sample pretreatment methods for the efficient separation and enrichment of mycotoxins is necessary. In recent years, porous organic framework materials, which are represented by metal-organic frameworks (MOFs) and covalent-organic frameworks (COFs), have been widely applied in the sample pretreatment of mycotoxins owing to their many advantages, which include a large specific surface area, high porosity, adjustable pore size, diverse frame structures, uniform active site distribution, and modifiable structures. In addition, MOF/COF materials feature excellent fluorescence and electrochemical properties, rendering them highly suitable for mycotoxin analysis and sensing. In this article, the recent applications of MOF/COF materials in conventional sample pretreatment methods (e. g., solid-phase extraction, dispersive solid-phase extraction, magnetic solid-phase extraction, and immunomagnetic bead separation) for mycotoxin separation and enrichment are reviewed. Research on the use of MOF/COF materials for the fluorescence and electrochemical sensing of mycotoxins is also summarized. Finally, the existing challenges and future development trends of these materials are discussed and prospected to provide a reference for future research on the applications of MOF/COF materials in mycotoxin detection and analysis.

Comparison of four commercial immunomagnetic separation kits for the detection of Cryptosporidium.

Cryptosporidium spp. are protozoan parasites of significant health importance found in environmental waters globally. Four commercially available Cryptosporidium-specific immunomagnetic separation (IMS) kits used in various water sample matrices were analysed and compared. Beads were characterised by flow cytometry and tested for the recovery efficiencies for oocysts spiked into different matrices: river water sediment, clay sample, and filter backwash sample. Results showed that Dynabeads™ Cryptosporidium and Waterborne Crypto-Grab™ kits contained immunoglobulin IgM antibody-coated beads. In contrast, the BioPoint CryptoBead and the TCS Isolate kits contained immunoglobulin IgG antibody-coated beads. BioPoint CryptoBead was significantly coated with more antibodies and were able to capture oocysts more rapidly compared to the other beads. Recovery efficiencies of Dynabeads™, TCS Isolate® beads, and BioPoint CryptoBead ranged from 55 to 93% when tested against different sample matrices, with BioPoint CryptoBead resulting in the highest at 93% in reagent-grade water and Dynabeads™ at 55%, the lowest against clay samples. The Waterborne beads did not perform well on any samples, with recovery efficiencies ranging from 0 to 8%. Fluorescence microscopy analyses showed that both the IMS method and the sample matrix processed affect the quality of the membranes, with the cleanest samples for microscopy examination observed from BioPoint CryptoBead.

Rapid Isolation of Low-Level Carbapenem-Resistant E. coli from Water and Foods Using Glycan-Coated Magnetic Nanoparticles.

Carbapenem-resistant Enterobacterales (CRE) are one of the major global issues needing attention. Among them, carbapenemase-producing (CP) E. coli strains are commonly found in clinical and biological samples. Rapid and cost-effective detection of such strains is critical in minimizing their deleterious impact. While promising progress is being made in rapid detection platforms, separation and enrichment of bacteria are required to ensure the detection of low bacterial counts. The current separation methods, such as centrifugation, filtration, electrophoresis, and immunomagnetic separation, are often tedious, expensive, or ineffective for clinical and biological samples. Further, the extraction and concentration of antimicrobial-resistant bacteria (ARB) are not well documented. Thus, this study assessed the applicability of cost-effective glycan-coated magnetic nanoparticles (gMNPs) for simple and rapid extraction of CP E. coli. The study included two resistant (R)strains: Klebsiella pneumoniae carbapenemase (KPC)-producing E. coli (R: KPC) and New Delhi metallo-β-lactamase (NDM)-producing E. coli (R: NDM). A susceptible E. coli (S) strain was used as a control, a reference bacterium. The gMNPs successfully extracted and concentrated E. coli (R) and E. coli (S) at low concentrations from large volumes of buffer solution, water, and food samples. The gMNPs concentrated up to two and five times their initial concentration for E. coli (R) and E. coli (S) in the buffer solution, respectively. In water and food samples, the concentration of E. coli (S) and E. coli (R) were similar and ranged 1-3 times their initial inoculation. A variation in the concentration from different food samples was seen, displaying the impact of food microstructure and natural microflora. The cost-effective and rapid bacterial cell capture by gMNPs was achieved in 15 min, and its successful binding to the bacterial cells in the buffer solution and food matrices was also confirmed using Transmission Electron Microscopy (TEM). These results show promising applications of gMNPs to extract pathogens and ARB from biological samples.

Improved analysis of derivatized steroid hormone isomers using ion mobility-mass spectrometry (IM-MS).

Over the last decade, applications of ion mobility-mass spectrometry (IM-MS) have exploded due primarily to the widespread commercialization of robust instrumentation from several vendors. Unfortunately, the modest resolving power of many of these platforms (~40-60) has precluded routine separation of constitutional and stereochemical isomers. While instrumentation advances have pushed resolving power to >150 in some cases, chemical approaches offer an alternative for increasing resolution with existing IM-MS instrumentation. Herein we explore the utility of two reactions, derivatization by Girard's reagents and 1,1-carbonyldiimidazole (CDI), for improving IM separation of steroid hormone isomers. These reactions are fast (≤30min), simple (requiring only basic lab equipment/expertise), and low-cost. Notably, these reactions are structurally selective in that they target carbonyl and hydroxyl groups, respectively, which are found in all naturally occurring steroids. Many steroid hormone isomers differ only in the number, location, and/or stereochemistry of these functional groups, allowing these reactions to "amplify" subtle structural differences and improve IM resolution. Our results show that resolution was significantly improved amongst CDI-derivatized isomer groups of hydroxyprogesterone (two-peak resolution of Rpp = 1.10 between 21-OHP and 11B-OHP), deoxycortisone (Rpp = 1.47 between 11-DHC and 21-DOC), and desoximetasone (Rpp = 1.98 between desoximetasone and fluocortolone). Moreover, characteristic collision cross section (DTCCSN2) measurements can be used to increase confidence in the identification of these compounds in complex biological mixtures. To demonstrate the feasibility of analyzing the derivatized steroids in complex biological matrixes, the reactions were performed following steroid extraction from urine and yielded similar results. Additionally, we applied a software-based approach (high-resolution demultiplexing) that further improved theresolving power (>150). Overall, our results suggest that targeted derivatization reactions coupled with IM-MS can significantly improve the resolution of challenging isomer groups, allowing for more accurate and efficient analysis of complex mixtures.

<i>In vitro</i> production of myeloid-derived suppressor cells from peripheral blood monocytes

Myeloid-derived suppressor cells (MDSCs) are of interest as key regulators of the immune response for the development and improvement of cellular technologies in biomedicine. Enhancing the suppressive activity of these cells is important for developing therapies for autoimmune diseases and miscarriages, and their suppression may be useful in the treatment of cancer, since MDSCs are known to suppress antitumor immunity. However, there is a problem that prevents the active study of MDSCs, i.e., the difficulty in obtaining sufficient numbers of this cell population. Isolation of MDSCs in cancer patients poses an ethical challenge. Moreover, these MDSC may differ in subpopulation composition and suppressive activity due to individual factors. Researchers who generate human MDSC from bone marrow cells may also face similar problems. Therefore, finding a reliable and affordable source of these cells to facilitate the study of their functions is extremely important. Attempts to obtain human MDSCs in vitro have been ongoing for a long time. GM- CSF, IL-6, IL- 1β, IL-4, PGE2, LPS, M-CSF, IFNγ are described as factors that induce the ex vivo MDSC differentiation. However, despite multiple factors used, not all protocols are clearly reproducible, leading to generation of a sufficient number of cells in the target population. Previously, we had also developed a scheme for MDSC differentiation from CD11b+ cells derived from human peripheral blood, which made it possible to obtain a tangible but still insufficient percentage of cells to study functional activity.
 To increase the number of MDSCs in cultures, we developed a protocol aimed for differentiation of these cells from peripheral blood monocytes (CD14+ cells) previously transformed into PCMO (programmed cells of monocytic origin). The monocytes isolated by immunomagnetic separation were cultured in a de-differentiating medium (complete culture medium supplemented with M-CSF, IL-3 and β-mercaptoethanol) for one week. Later on, the medium was replaced by the addition of GM-CSF, being cultured for three days, followed by addition of LPS and IL-1β in order to induce suppressive activity. We have found that culturing CD14+ cells on a two-week schedule with prior creation of dedifferentiation conditions resulted in a slightly decreased percentage of viable cells in culture. However, there was a trend towards an increased ratio of MDSCs in culture (from an average of 34 to 40%) and an increase in their suppressive activity (arginase and IDO expression). The percentage of Arg+ cells increased by average of 10%, and IDO+ cells, by 16%. Moreover, the percentage of mature M-MDSCs was significantly (several-fold) higher when compared with differentiation protocol using CD11b+ cells. Hence, this method of MDSCs production enables us to increase the number of cells belonging to the conditionally “mature” monocyte subpopulation of MDSCs, as well as the percentage of functional suppressor cells in the population. The described scheme may be used to improve the quality of studies aimed at modulating MDSC functions in order to develop new therapeutic approaches.

Inertia-magnetic microfluidics for rapid and high-purity separation of malignant tumor cells

The existence of many background cells and the size overlap between white blood cells (WBCs) and malignant tumor cells (MTCs) pose a challenge for the efficient separation of MTCs from effusions or bloods. Herein, we proposed an inertia-magnetic cell separation (IMCS) device integrating a trapezoidal spiral chip with an immunomagnetic chip for high-throughput, high-recovery, and high-purity separation of MTCs from malignant pleural and peritoneal effusions (MPPEs). First, the effects of flow rate and particle size on separation performances in two separate chips were investigated. Then, the performances of the integrated IMCS device were characterized using blood samples spiked with three different tumor cells (A549, MCF-7, and MDA-MB-231 cells). The results showed that all the three tumor cells were separated from the WBCs with a high-throughput of ∼ 3200 μL/min, a high-recovery ratio of > 90%, and a high-purity of > 70%. Finally, we successfully separated MTCs from four clinical MPPE samples with an average purity of > 70%. As compared with conventional microfluidic devices using single technologies, our IMCS device combines the advantages of both passive size-dependent separation technology and immunomagnetic separation technology and can accurately separate tumor cells from WBCs of comparable sizes at a high throughput. Therefore, we envision that the high-purity MTC separation ability of our IMCS device can improve the sensitivity and efficiency of cytological examination.

Elucidating Structures of Protein Complexes by Collision-Induced Dissociation at Elevated Gas Pressures.

Ion activation methods carried out at gas pressures compatible with ion mobility separations are not yet widely established. This limits the analytical utility of emerging tandem-ion mobility spectrometers that conduct multiple ion mobility separations in series. The present work investigates the applicability of collision-induced dissociation (CID) at 1 to 3 mbar in a tandem-trapped ion mobility spectrometer (tandem-TIMS) to study the architecture of protein complexes. We show that CID of the homotetrameric protein complexes streptavidin (53 kDa), neutravidin (60 kDa), and concanavalin A (110 kDa) provides access to all subunits of the investigated protein complexes, including structurally informative dimers. We report on an "atypical" dissociation pathway, which for concanavalin A proceeds via symmetric partitioning of the precursor charges and produces dimers with the same charge states that were previously reported from surface induced dissociation. Our data suggest a correlation between the formation of subunits by CID in tandem-TIMS/MS, their binding strengths in the native tetramer structures, and the applied activation voltage. Ion mobility spectra of in situ-generated subunits reveal a marked structural heterogeneity inconsistent with annealing into their most stable gas phase structures. Structural transitions are observed for in situ-generated subunits that resemble the transitions reported from collision-induced unfolding of natively folded proteins. These observations indicate that some aspects of the native precursor structure is preserved in the subunits generated from disassembly of the precursor complex. We rationalize our observations by an approximately 100-fold shorter activation time scale in comparison to traditional CID in a collision cell. Finally, the approach discussed here to conduct CID at elevated pressures appears generally applicable also for other types of tandem-ion mobility spectrometers.

Applications of magnetic nanomaterials in the fabrication of lateral flow assays toward increasing performance of food safety analysis: Recent advances

Up to now, the life of humans is complex, and it has been under attack by numerous factors. In other words, human beings are relationship with living in a healthy and clean environment. Remarkably, life on the planet Earth has been threatened by food contamination in all areas of the world. Over the last few years, the portability of analytical methods has received considerable attention in biosensors. Among these, lateral flow assays (LFAs) can consider one of the critical types of portable sensing platforms. The vast majority of conventional LFAs operate according to the gold nanoparticles (AuNPs) signaling. However, the presence of NaCl in real samples can decrease the performance of the AuNPs-LFA. Thereby, the substitute of AuNPs with other types of nanomaterials (NMs) or using hybrid NMs can boost the application of these sensing biodevices in different real samples. Elaborately, magnetic nanomaterials (MNMs) can play a significant role in developing sensing approaches, especially later flow assays LFAs. These NMs not only do quantitative measurements by coupling with an external reader, but they can also act as an immunomagnetic separation agent. In addition, ease of use, less coat, and size uniformity can highlight their application in food safety. Therefore, the application of MNMs in LFA with different formats can significantly reduction in the limit of detection (LOD) of the analyte without methodically complicating the analytical procedure. In this review, we attempted to highlight the recent progress and technical breakthroughs of MNMs in LFA for food safety.

Immunomagnetic separation for isolation and enrichment of Lacticaseibacillus paracasei strain Shirota from human feces

Probiotics are live microorganisms that, when consumed in adequate amounts, confer health benefits on the host. The beneficial effects of probiotics are believed to be exerted when the microbes reach the intestine alive and engage in metabolic activity. The number of ingested probiotics that are able to reach the intestine is much fewer than that of the commensal intestinal bacteria. This makes it difficult to characterize the physiological state of probiotics in vivo owing to interference by other bacteria. Here, we established and evaluated an immunomagnetic separation (IMS)-based approach using a strain-specific monoclonal antibody to isolate Lacticaseibacillus paracasei strain Shirota (LcS) from human feces. Six healthy adults ingested a bottle of LcS-containing commercially available fermented milk each day for 4 consecutive days, and feces were collected after the final ingestion. Our approach successfully yielded a collection of more than 70 % of LcS from fecal samples in all subjects. Furthermore, the proportion of LcS among total bacteria, which initially stood at approximately 1 % before IMS, surged to over 70 % after IMS. This substantial increase signifies a concentration of LcS several-fold higher than the original fecal ratio. We conclude that LcS can be separated and enriched from human feces with acceptable level of efficiency and specificity by using our IMS approach.

Use of adipose derived stem cells accelerates the healing process in third-degree burns

IntroductionBurns are defined as a traumatic injury, usually of thermal origin, that affects the epithelial and adjacent tissue and is classified according to the depth reached. Tissue repair involved in this type of injury is often a challenge both due to its severity and the multiplicity of complications. Regenerative medicine has focused on the use of low-level laser photobiomodulation therapy (LLLT) and adipose-derived stem cells (ADSC), especially in the early stages of the process, to promote better healing and shorten repair time. Therefore, aim of this study was to evaluate the action of LLLT (660 nm) and ADSC in the repair process of burned skin tissue and investigate the association of the techniques (LLLT and ADSC). Materials and methodsAn in vivo study was carried out using 96 rats (Wister) with a scald burn model at a temperature of 95ºC, exposing the animal's back for 14 s. Animals were randomized into seven groups and three periods, five, 14 and 21 days. The groups included GC: Control group, ADSC-: Group treated with CD49d negative cells, ADSC+ : Group treated with positive CD49d cells, CULT: Group treated with conventional isolation cells, LLLT: Group treated only with LLLT Low Power Laser, ADSC-LLLT: Group treated with CD49d negative cells and LLLT. ADSC+LLLT: Group treated with positive CD49d cells and LLLT. The groups treated with LLLT (660 nm; 5 J/cm2) received irradiation three times a week, on alternate days for five, 14 and 21 days, according to the time of biopsy. ADSC-treated groups received one to three applications of the cells in a total volume of 1000 μL starting soon after the surgical debridement of the burn. Photographic monitoring was carried out at 5, 14 and 21 days after the beginning of the experiment to assess the degree of lesion contraction. Macroscopic, morphometric and histopathological analyzes were performed. ResultsWe showed significant re-epithelialization as well as an improvement in the healing process in the ADSC+, LLLT and ADSC+LLLT groups. We observed effects in the reduction of the inflammatory phase, increase in angiogenesis, decrease in oedema, greater collagen deposition, and better organization of the extracellular matrix compared to the other treatments. Moreover, the immunomagnetic separation of ADSC cells through the expression of the CD49d protein proved to be a useful means to obtain a more homogeneous population of cells with a role in tissue regeneration compared to the ADSC- and CULT groups. ConclusionIn conclusion, the association of ADSC+ with LLLT was effective in accelerating the burn repair process, stimulating cell proliferation and formation of more normal skin tissue.

Coupling Isotopic Shifts with Collision Cross-Section Measurements for Carbohydrate Characterization in High-Resolution Ion Mobility Separations.

Herein, we introduce a two-dimensional strategy to better characterize carbohydrate isomers. In a single experiment, we can derive cyclic ion mobility-mass spectrometry (cIMS-MS)-based collision cross-section (CCS) values in conjunction with measuring isotopic shifts through the relative arrival times of light and heavy isotopologues. These isotopic shifts were introduced by permethylating carbohydrates with either light, CH3, or heavy, CD3, labels at every available hydroxyl group to generate a light/heavy pair of isotopologues for every individual species analyzed. We observed that our calculated CCS values, which were exclusively measured for the light isotopologues, were orthogonal to our measured isotopic shifts (i.e., relative arrival time values between heavy and light permethylated isotopologues). Our permethylation-induced isotopic shifts scaled well with increasing molecular weight, up to ∼m/z 1300, expanding the analysis of isotopic shifts to molecules 3-4 times as large as those previously studied. Our presented use of coupling CCS values with the measurement of isotopic shifts in a single cIMS-MS experiment is a proof-of-concept demonstration that our two-dimensional approach can improve the characterization of challenging isomeric carbohydrates. We envision that our presented 2D approach will have broad utility for varying molecular classes as well as being amenable to many forms of derivatization.

Immunomagnetic Separation Combined with RCA-CRISPR/Cas12a for the Detection of Salmonella typhimurium on a Figure-Actuated Microfluidic Biosensor.

A figure-actuated microfluidic biosensor was developed for the rapid and sensitive detection of Salmonella typhimurium using immunomagnetic separation to separate target bacteria and rolling circle amplification (RCA) combined with CRISPR/Cas12a to amplify the detection signal. The magnetic nanoparticles (MNPs) modified with the capture antibodies (MNPs@Ab1) and RCA primer linked with recognized antibodies (primer@Ab2) were first used to react with S. typhimurium, resulting in the formation of MNPs@Ab1-S. typhimurium-primer@Ab2 complexes. Then, the RCA and CRISPR/Cas12a reagents were successively pumped into the chamber and incubated at the appropriate conditions. With the help of a 3D-printed signal detector, the fluorescence signal was collected and analyzed using the smartphone APP for the determination of bacterial concentration. This biosensor exhibited a wide linear range for the detection of S. typhimurium with a low limit of detection of 1.93 × 102 CFU/mL and a mean recovery of about 106% in the spiked milk sample.

Isolation of Mouse Neutrophils.

Neutrophils represent the first line of defense against bacterial and fungal pathogens. Indeed, patients with inherited or acquired qualitative and quantitative neutrophil defects are at high risk for developing bacterial and fungal infections and suffering adverse outcomes from these infections. Therefore, research aiming at defining the molecular factors that modulate neutrophil effector function under homeostatic conditions and during infection is essential for devising strategies to augment neutrophil function and improve the outcomes of infected individuals. This article describes reproducible density-gradient-centrifugation-based as well as positive and negative immunomagnetic selection protocols that can be applied in any laboratory to harvest large numbers of highly enriched and highly viable neutrophils from the bone marrow of mice. In another protocol, we also present a method that combines gentle enzymatic tissue digestion with a positive immunomagnetic selection technique or fluorescence-activated cell sorting (FACS) to harvest highly pure and highly viable preparations of neutrophils directly from mouse tissues such as the kidney, the liver, or the spleen. Mouse neutrophils isolated by these protocols can be used to examine several aspects of cellular function ex vivo, including pathogen binding, phagocytosis, and killing, neutrophil chemotaxis, oxidative burst, degranulation, and cytokine production, and for performing neutrophil adoptive transfer experiments. © 2023 Wiley Periodicals LLC. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA. Basic Protocol 1: Isolation of Neutrophils from Mouse Bone Marrow Using Positive Immunomagnetic Separation Alternate Protocol 1: Purification of Neutrophils from Bone Marrow Using Negative Immunomagnetic Separation Alternate Protocol 2: Purification of Neutrophils from Bone Marrow Using Histopaque-Based Density Gradient Centrifugation Basic Protocol 2: Isolation of Neutrophils from Mouse Tissues Using Positive Immunomagnetic Separation Alternate Protocol 3: Isolation of Neutrophils from Mouse Tissues Using FACS.

Permethylation and Metal Adduction: A Toolbox for the Improved Characterization of Glycolipids with Cyclic Ion Mobility Separations Coupled to Mass Spectrometry.

Lipids are an important class of molecules involved in various biological functions but remain difficult to characterize through mass-spectrometry-based methods because of their many possible isomers. Glycolipids, specifically, play important roles in cell signaling but display an even greater level of isomeric heterogeneity as compared to other lipid classes stemming from the introduction of a carbohydrate and its corresponding linkage position and α/β anomericity at the headgroup. While liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) remains the gold standard technique in lipidomics, it is still unable to characterize all isomeric species, thus presenting the need for new, orthogonal, methodologies. Ion mobility spectrometry-mass spectrometry (IMS-MS) can provide an additional dimension of information that supplements LC-MS/MS workflows, but has seen little use for glycolipid analyses. Herein, we present an analytical toolbox that enables the characterization of various glycolipid isomer sets using high-resolution cyclic ion mobility separations coupled with mass spectrometry (cIMS-MS). Specifically, we utilized a combination of both permethylation and metal adduction to fully resolve isomeric sphingolipids and ceramides with our cIMS-MS platform. We also introduce a new metric that can enable comparing peak-to-peak resolution across varying cIMS-MS pathlengths. Overall, we envision that our presented methodologies are highly amenable to existing LC-MS/MS-based workflows and can also have broad utility toward other omics-based analyses.

Sensitive Molecular Detection for Salmonella Enterica Serovars Typhimurium

Introduction: Salmonella infections contribute significantly to gastroenteritis cases, with the National Salmonella Reference Laboratory reporting 500 isolates in 2022. However, traditional culture-based methods for detecting Salmonella in samples can take 4 to 7 days to confirm a positive result, which poses health risks due to delayed detection. Given these health risks, swift and accurate detection methods are essential to minimize both false-positive and false-negative outcomes. Salmonella, a gram-negative bacterium within the Enterobacteriaceae family, demonstrates remarkable hardiness, surviving for several weeks in dry environments and months in water. Although most serotypes of Salmonella cause relatively mild gastroenteritis, some, particularly those transmitted from animals to humans, can lead to severe, life-threatening conditions
 Methods: The qRT-PCR procedure involved the design of primers and probes targeting the same genes as the mPCR assay. These primer sets were reconfigured to generate smaller amplicons suitable for qRT-PCR systems
 Results: qRT-PCR process, TaqMan probes were meticulously designed for specific target genes: FAM dye was employed to detect STM2745, Cy5 dye was used for STM4492, and Rox dye was utilized to detect. A standard curve was constructed using Typhimurium LT2 genomic DNA. Each sample underwent duplicate analysis, and Rotor-Gene software was employed to assign threshold values for each channel.
 Conclusion: The effectiveness of our qPCR assay for the detection of Salmonella across a diverse array of matrices. Notably, our results unveiled distinct limits of detection for Salmonella in various samples. Specifically, a parallel vein, the deployment of a PCR assay, leveraging an immunomagnetic separation technique for DNA extraction, was studied by another group. While subsequent analysis of Salmonella detected via our assay may necessitate the full ISO SMT method for live culture isolation, this supplementary step can be seamlessly conducted alongside qRT-PCR.

Competitive ratiometric fluorescent lateral flow immunoassay based on dual emission signal for sensitive detection of chlorothalonil

In this study, we develop a competitive ratiometric fluorescent lateral flow immunoassay (CRF-LFIA) based on dual emission fluorescence signal, which has great advantage in visual and quantitative detection of Chlorothalonil (CTN). Red-emitted fluorescent magnetic nanobeads (FMNBs) and green-emitted aggregation-induced emission fluorescent microsphere (AIEFM) are synthesized and conjugated to antibodies and antigens respectively, resulting in competitive binding with the analyte. The ratiometric fluorescence signal which comes from the overlap of these two fluorescence emissions. FMNBs probes also provide immunomagnetic separation (IMS) to enrich the analysts and resist complex matrix effects. This strip generates a visually discernible yellow-to-green fluorescence color change in the presence of CTN (2 ng/mL), which could be incisively observed by naked eye. Moreover, the limit of detection (LOD) reached 0.152 ng/mL by measurement of color (Red-Green-Blue, RGB) signals. Method validation shows a good correlation between CRF-LFIA and LC-MS/MS.

Single-cell lipidomics enabled by dual-polarity ionization and ion mobility-mass spectrometry imaging

Single-cell (SC) analysis provides unique insight into individual cell dynamics and cell-to-cell heterogeneity. Here, we utilize trapped ion mobility separation coupled with dual-polarity ionization mass spectrometry imaging (MSI) to enable high-throughput in situ profiling of the SC lipidome. Multimodal SC imaging, in which dual-polarity-mode MSI is used to perform serial data acquisition runs on individual cells, significantly enhanced SC lipidome coverage. High-spatial resolution SC-MSI identifies both inter- and intracellular lipid heterogeneity; this heterogeneity is further explicated by Uniform Manifold Approximation and Projection and machine learning-driven classifications. We characterize SC lipidome alteration in response to stearoyl-CoA desaturase 1 inhibition and, additionally, identify cell-layer specific lipid distribution patterns in mouse cerebellar cortex. This integrated multimodal SC-MSI technology enables high-resolution spatial mapping of intercellular and cell-to-cell lipidome heterogeneity, SC lipidome remodeling induced by pharmacological intervention, and region-specific lipid diversity within tissue.

Colorimetric immunosensing using liposome encapsulated MnO2 nanozymes for SARS-CoV-2 antigen detection

Development of specific signal reporters with signal amplification effect are highly needed for sensitive and accurate detection of pathogen. Herein, we design a colorimetric immunosensing nanosystem based on liposome encapsulated quantum dots-sized MnO2 nanozyme (MnO2QDs@Lip) as a signal reporter for ultrasensitive and fast detection of SARS-CoV-2 antigen. The pathogenic antigens captured and separated by antibody-conjugated magnetic beads (MBs) are further connected with antibody-modified MnO2QDs@Lip to form a sandwich-like immunocomplex structure. After triggered release, MnO2 QDs efficiently catalyze colorless 3,3′,5,5′-tetramethylbenzidine (TMB) to blue oxidized TMB, which can be qualitatively observed by naked eyes and quantitatively analyzed by UV–Vis spectra or smartphone platforms. By taking advantages of immuno-magnetic separation, excellent peroxidase-like catalytic activity of MnO2 QDs, and high encapsulation efficiency of MnO2QDs@Lip, ultrasensitive detection of SARS-CoV-2 antigen ranging from 0.1 pg/mL to 100 ng/mL is achieved within 20 min. The limit of detection (LOD) is calculated to be 65 fg/mL in PBS buffer. Furthermore, real clinical samples of SARS-CoV-2 antigens can be effectively identified by this immunosensing nanosystem with excellent accuracy. This proposed detection nanosystem provides a strategy for simple, rapid and ultrasensitive detection of pathogens and may shed light on the development of new POCT detection platforms for early diagnosis of pathogens and surveillance in public health.