Limulus Amebocyte Lysate Research Articles

Physicochemical characterization of biological and synthetic forms of two lipid A-based TLR4 agonists

Toll-like receptor (TLR) agonists are recognized as potential immune-enhancing adjuvants and are included in several licensed vaccines. Monophosphoryl lipid A (MPL®, GlaxoSmithKline) is one such TLR4 agonist that has been approved for use in human vaccines, such as Cervarix and Shingrix. Due to the heterogeneous nature of biologically derived MPL and the need for safer and more potent adjuvants, our groups have developed the novel TLR4 agonist candidates, BECC438 and BECC470 using the Bacterial Enzymatic Combinatorial Chemistry (BECC) platform. BECC438 and BECC470 have been included in studies to test their adjuvant potential and found to be effective in vaccines against both viral and bacterial disease agents. Here, we report detailed biophysical characterization of BECC438 and BECC470 purified from a biological source (BECC438b and BECC470b, respectively) and synthesized chemically (BECC438s and BECC470s, respectively). Both BECC438s and BECC470s have identical acyl chain configurations, BECC438s is bis-phosphorylated and BECC470s is mono-phosphorylated with the removal of the 4′ phosphate moiety. We determined the phase transition temperatures for the acyl chains of BECC438b and BECC470b and found them to be different from those exhibited by their synthetic counterparts. Furthermore, the phosphate groups of BECC438b and BECC470b are more highly hydrated than are those of BECC438s and BECC470s. In addition to exploring the BECC molecules’ biophysical features in aqueous solution, we explored potential formulation of BECC438 and BECC470 with the aluminum-based adjuvant Alhydrogel and as part of an oil-in-water emulsion (Medimmune Emulsion or ME). All of the lipid A analogues could be fully absorbed to Alhydrogel or incorporated onto ME. Surprisingly, the BECC470s molecule, unlike the others, displayed a nearly baseline signal when monitored using a Limulus amebocyte lysate (LAL) endotoxin detection system. Despite this, it was shown to behave as an agonist for human and mouse TLR4 when tested using multiple cell-based systems. This work paves the way for further formulation optimization of two chemically defined TLR4 agonists that are showing great promise as vaccine adjuvants.

Plasma lipopolysaccharide elevations in cattle associated with early-stage infection by Fasciola hepatica.

Fasciolosis is an endemic zoonotic parasitic disease with significant impacts on human health and both animal health and production. Early post-infection impacts on the host remain unclear. The objective of this study was to determine the changes, if any, to levels of endotoxin in cattle plasma in response to early-stage infection with Fasciola hepatica. Thirty-six (36) commercial bred cattle were experimentally infected with approximately 400 viable metacercariae. Plasma lipopolysaccharide (endotoxin) levels were examined on 24 occasions from 0 h before infection to 336 h after infection using the Limulus Amoebocyte Lysate chromogenic end point assay and compared with that of six (6) uninfected control animals. Peak lipopolysaccharide levels in infected animals were reached at 52 h after infection and returned to pre-infection levels at time 144 h after infection. Infected animals had significantly elevated lipopolysaccharide levels between 24 and 120 h after infection when compared to uninfected animals. The mean change in endotoxin units (EU)/mL over time after infection was statistically significant in infected animals. Elevations of lipopolysaccharide occurred in all infected animals suggesting a possible repeatable and titratable endotoxemia conducive to therapeutic agent model development.

Using Limulus Amebocyte Lysate (LAL) to discover the presence of endotoxins in the tanks at the Pritzker Marine Biology Research Center

Endotoxins are harmful for many organisms, including those that live in the water. Therefore, it is important to identify effective ways in identifying if they are present in water environments. Research has been conducted to show that Limulus amebocyte lysate (LAL) is an effective way to determine if endotoxins are present. In this study, LAL was used as the main reagent to detect if endotoxins were present in the tanks at the Pritzker Marine Biology Research Center. It is believed that LAL will be able to detect the presence of any endotoxins within the tanks at Pritzker. The first part of the procedure to do this was to collect 5mL of water from each tank at Pritzker. After the LAL reagent was dissolved in LAL Reagent Water, the gel-clot assay was then produced. 0.1mL of the reagent was added to each assay tube. 0.1mL of each product sample or control was transferred into each assay tube, beginning with the negative control and ending with the highest endotoxin concentration. The contents of the tubes were mixed and incubated undisturbed in a 37 C heating block for 60 minutes. After incubation, each tube was examined for gelation. A positive test is defined as the formation of a firm gel capable of maintaining its integrity when the assay tube is inverted 180 degrees. A negative test is characterized by the absence of gel or by the formation of a viscous mass which does not hold when the assay tube is inverted. Endotoxins were detected in an average of 13 tanks at the Pritzker. To expand upon these findings, quantifying how many endotoxins are present in the tanks and what types of endotoxins are present in the tanks will be tested. Biology department funds at the New College of Florida This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Endotoxin contamination of nanoparticle formulations: A concern in vaccine adjuvant mechanistic studies.

The increasing awareness of endotoxin contamination has raised important questions during the study of the mechanism of action of the vaccine adjuvants. The endotoxins or lipopolysaccharides (LPS) can contaminate vaccine formulations contributing to result misinterpretations of the in vitro and in vivo studies. In this short communication, we considered the suitability of the Limulus amebocyte lysate (LAL) assay to quantify chitosan (Chit) nanoparticle (NP) endotoxin contamination to use them in a comparative in vitro immunotoxicology study using both LPS-free (LF) and non-LF Chit NPs. It was shown that chit NPs had a masking effect on endotoxin levels, hampering a reliable conclusion about the effect of their contamination. Neither non-LF nor LF Chit NPs induced the production of ROS in RAW 264.7 cells or IL-6 and TNF-α in PBMCs. The lack of effect of non-LF NPs was not expected and likely due to the NPs masking effect, more evident for higher deacetylation degree Chit. Overall, to prevent questionable results, nanomaterials should be produced under endotoxin-free conditions.

Occupational Bioaerosol Exposures Associated with Poultry Farming

ABSTRACT This study aimed to investigate occupational exposure to particulate dust, endotoxin, and (1–3)-β-D-glucan among workers involved in various poultry farming activities. A total of 298 personal samples were collected from randomly selected individuals from exposure groups based on distinct poultry farming activities comprising broiler farms, rearing, laying, hatchery, and catching activities. Aside from the inhalable particulate dust concentration that was determined, filter extracts were also analyzed for (1–3)-β-D-glucan and endotoxin using the endpoint Glucatell® and Limulus amoebocyte lysate (LAL) assays, respectively. Data were analyzed using STATA 12 and linear regression models developed. The mean (GM) dust particulate concentration was 11.04 mg/m3 (GSD = 3.87); 2298 endotoxin units (EU/m3) (GSD = 10.56) and 149 ng/m3 for (1–3)-β-D-glucan (GSD = 4.62). A modest positive correlation was observed between log-transformed endotoxin and (1–3)-β-D-glucan concentrations (Pearson r = 0.44, p < .001), whilst a moderate negative correlation was observed for inhalable dust particulate and (1–3)-β-D-glucan (Pearson r = −0.33, p < .001). However, there was a very poor correlation between inhalable dust and endotoxin (Pearson r = −0.02, p < .001). In the regression models, exposure group based on the nature of farming activity explained 50% of the variability in dust particulate and glucan levels. For dust particulate, rearing activities were significant predictors of higher dust levels compared to hatchery work, while rearing, laying, broiler and catching activities were significant predictors of higher endotoxin or glucan levels. Furthermore, working in a small broiler was a significant determinant of elevated glucan exposures. Farms using automated laying activities had significantly higher particulate levels compared to those using manual laying activities. This study revealed that workers engaged in poultry farming activities were exposed to significantly high levels of inhalable particulate dust, endotoxin, and (1–3)-β-D-glucan concentrations, posing an increased risk for adverse respiratory health effects in these farm workers.

HDL and LDL have distinct, opposing effects on LPS-induced brain inflammation

Endotoxemia and sepsis induce neuroinflammation and increase the risk of neurodegenerative disorders although the mechanism by which peripheral infection leads to brain inflammation is not well understood. While circulating serum lipoproteins are known immunometabolites with the potential to modulate the acute phase response and cross the blood brain barrier, their contribution to neuroinflammation during systemic infection is unknown. The objective of this study was to elucidate the mechanisms by which lipoprotein subclasses modulate lipopolysaccharide (LPS)-induced neuroinflammation. Adult C57BL/6 mice were divided into 6 treatment groups, including a sterile saline vehicle control group (n = 9), an LPS group (n = 11), a premixed LPS + HDL group (n = 6), a premixed LPS + LDL group (n = 5), a HDL only group (n = 6) and an LDL only group (n = 3). In all cases injections were administered intraperitoneally. LPS was administered at 0.5 mg/kg, and lipoproteins were administered at 20 mg/kg. Behavioural testing and tissue collection was performed 6 h post-injection. The magnitude of peripheral and central inflammation was determined by qPCR of pro-inflammatory genes in fresh liver and brain. Metabolite profiles of liver, plasma and brain were determined by 1H NMR. Endotoxin concentration in the brain was measured by the Limulus Amoebocyte Lysate (LAL) assay. Co-administration of LPS + HDL exacerbated both peripheral and central inflammation, whilst LPS + LDL attenuated this inflammation. Metabolomic analysis identified several metabolites significantly associated with LPS-induced inflammation, which were partially rescued by LDL, but not HDL. Endotoxin was detected at significantly greater concentrations in the brains of animals that received LPS + HDL compared to LPS + saline, but not those that received LPS + LDL. These results suggest that HDL may promote neuroinflammation through direct shuttling of endotoxin to the brain. In contrast, LDL was shown to have anti-neuroinflammatory properties in this study. Our results indicate that lipoproteins may be useful targets in neuroinflammation and neurodegeneration associated with endotoxemia and sepsis.

High-throughput determination of lipopolysaccharide by flow injection analysis using dipicolylamine-type fluorescent probe

Endotoxin (lipopolysaccharide: LPS), a bacterial toxin, is currently quantified by batch analysis using a reagent made from Limulus amebocyte lysate (LAL). Although the LAL method is sensitive, a long measurement time and gelation have hindered its application to online water quality monitoring. In this paper, we report a novel high-throughput analytical technique that combines flow injection analysis (FIA) and a dipicolylamine-type fluorescent probe, which was previously reported to exhibit strong fluorescence emission in response to LPS. A comparison of normal and reverse FIA systems revealed that the normal FIA offers more rapid detection with throughput of 30 samples per hour, which is the highest among known LAL methods and alternative LPS detection methods that have been recently reported. On the other hand, the reverse FIA requires much less reagent (480 μL per sample). Continuous online monitoring of water quality and analysis of real samples spiked with LPS were realized by this technique.

Assessment of endotoxin levels of water in hemodialysis centers in Iraq

The significance of endotoxin-free, sterile dialysis fluid for long-term, high-quality hemodialysis treatment is obvious and highly desired[MF1] . The current study aimed to determine endotoxin for the water quality used in twenty hemodialysis treatment centers. Eighty samples (40 x dialysates and 40 x dialysis water) were tested for endotoxin using the LAL gel clot method. The results revealed a variation in the quality of the produced water that makes it unaccepted compared to the international standard (ANSI/AAMI/ISO-23500). Endotoxin levels were higher than (0.25 EU/ml) in 40% of dialysis water and 30% of dialysate water, higher than the recommended international standards. The results conclude that systematic water quality monitoring in hemodialysis services is essential for patient safety and health problems. Keywords: Endotoxin; Water quality; Dialysis fluid; LAL test; Hemodialysis patient

Effect of antimicrobial photodynamic therapy on the reduction of bacteria and virulence factors in teeth with primary endodontic infection.

To evaluate the effectiveness of supplemental photodynamic therapy for improving the bacterial removal and the levels of lipopolysaccharide (LPS) and lipoteichoic acid (LTA) by conducting a clinical trial. Twenty-four root canals with pulp necrosis and periapical lesion were selected and randomly divided into conventional group using endodontic treatment with chemo-mechanical preparation (CMP) alone (n=12) and a group using antimicrobial photodynamic therapy (aPDT) after CMP (n=12). The samples were collected before and after CMP (conventional group) and after photodynamic therapy (aPDT group). A photosensitizer (0.005% methylene blue) was applied to the root canal for 3 minutes after CMP, whereas aPDT was performed by using a red laser with a power of 30Mw and energy density of 9J/cm2 for 90 s per root canal. Culture technique was performed to determine the bacterial colony forming units. LPS and LTA levels were quantified by using limulus amoebocyte lysate (LAL) assay and enzyme-linked immunosorbent assay (ELISA), respectively. All samples showed growth of viable bacteria on Fastidious Anaerobe Agar (FAA), with an average of 5.19×105 CFU/ mL. CMP was effective in decreasing viable bacteria (p<0.05), whereas there was a significant decrease (p<0.05) in the samples treated with aPDT compared to those submitted to CMP. LPS and LTA were detected in all initial samples, with mean values of 20.561 EU/mL and 430.91 pg/mL, respectively. Both CMP and aPDT groups significantly decreased the levels of LPS and LTA (p<0.05), with a statistical difference between the groups regarding aPDT (p<0.05). Photodynamic therapy as an adjunct to CMP proved to be effective in improving root canal disinfection and reducing the LPS and LTA levels in teeth with primary endodontic infection.

Ophthalmic viscoelastics commonly used in cataract surgery: A microbiota investigation.

To survey commonly used, sterile ophthalmic viscoelastic materials used during routine cataract surgery for the presence of bacterial DNA and/or viable bacteria and endotoxin quantification. Samples from three different ophthalmic viscoelastic manufacturers and three different production lots per manufacturer were collected for 16 S ribosomal ribonucleic acid (rRNA) sequencing and conventional aerobic and capnophilic bacterial culture. Other samples of viscoelastic material from the same three manufacturers were collected for endotoxin quantification using a commercially available Limulus amebocyte lysate (LAL) assay. Statistical analysis was performed using Sigma Plot 14.0, and R v4.0.2.0. Differences (p ≤ .05) between sample collection sites in total DNA concentration, microbial richness, mean intra-group distances, and endotoxin quantification alongside reagent controls were evaluated. Culture yielded two isolates, identified as Staphylococcus epidermidis and Bacillus megaterium. 16 S rRNA sequencing revealed no differences between brands in richness or overall composition. The most common bacterial DNA detected across all brands was Staphylococcus sp., Cutibacterium sp., Flavobacterium sp., and Lactobacillus sp. A significant difference was found between the median endotoxin concentration between Anvision and Hyvisc® viscoelastic (Anvision: 0.171 EU/mL, Hyvisc®: 0.03 EU/mL; p < .001). No brand-specific differences in bacterial DNA were detected in the viscoelastic materials. Staphylococcus, Cutibacterium, Flavobacterium, and Lactobacillus were the dominant contributors to the bacterial DNA detected. Although Anvision viscoelastic samples contained significantly more endotoxin than Hyvisc® viscoelastic samples, endotoxin concentrations were below the FDA limit of 0.2 EU/mL for both manufacturers. These data further the understanding of inflammatory outcomes following cataract surgery.

Intralaboratory Validation of a Kinetic Turbidimetric Assay Based on Limulus Amebocyte Lysate (LAL) for Assessing Endotoxin Activity in Cow Milk.

Mastitis, one of the most common diseases in dairy cattle, causes severe losses in the dairy sector worldwide and affects animal welfare. The disease is characterized by an inflammatory reaction of the mammary gland and is mainly caused by bacterial infections, including both Gram-negative and Gram-positive bacteria. The release of endotoxins associated to bacterial lysis is a weighty factor in the clinical course of Gram-negative associated mastitis and should be taken into consideration when using antibiotics in the treatment of these infections. Therefore, endotoxin detection in milk samples would be of help in the management of bovine mastitis. With this aim, we have validated a kinetic turbidimetric assay based on Limulus amebocyte lysate (LAL) for the quantification of endotoxins in milk samples. The assay was adapted to this particular matrix by incorporating filtration and dilution of the milk samples in the procedure. Our results demonstrate the robustness and usefulness of the assay, which allows the identification of coliform mastitis in milk samples from affected cows and the quantification of endotoxin activity in bulk and commercial milk samples. Further studies are required to evaluate the performance of the assay in mastitis milk samples associated to Gram-negative bacteria other than Escherichia coli as well as during the clinical course of these Gram-negative mastitis or after their treatment with antibiotics.

Highly Sensitive Electrochemical Endotoxin Sensor Based on Redox Cycling Using an Interdigitated Array Electrode Device

The Limulus amebocyte lysate (LAL) reaction-based assay, the most commonly used endotoxin detection method, requires a skilled technician. In this study, to develop an easy-to-use and highly sensitive endotoxin sensor, we created an electrochemical endotoxin sensor by using an interdigitated array electrode (IDAE) device with advantages of amplifiable signals via redox cycling and portability. We added Boc-Leu-Gly-Arg-p-aminophenol (LGR-pAP) as an electrochemical substrate for an LAL reaction and detected p-aminophenol (pAP) released from LGR-pAP as a product of an endotoxin-induced LAL reaction via an IDAE device. The IDAE device showed a great redox cycling efficiency of 79.8%, and a 4.79-fold signal amplification rate. Then, we confirmed that pAP was detectable in the presence of LGR-pAP through chronoamperometry with the potential of the anode stepped from -0.3 to 0.5 V vs. Ag/AgCl while the cathode was biased at -0.3 V vs. Ag/AgCl. Then, we performed an endotoxin assay by using the IDAE device. Our endotoxin sensor detected as low as 0.7 and 1.0 endotoxin unit/L after the LAL reaction for 1 h and 45 min, respectively, and these data were within the cut-off value for ultrapure dialysis fluid. Therefore, our highly sensitive endotoxin sensor is useful for ensuring medical safety.

Endotoxin content in neonatal formulas, fortification, and lactoferrin products: association with outcomes and guidance on acceptable limits

While endotoxin (lipopolysaccharide) can be harmful and contribute to morbidity and mortality with Gram-negative sepsis or necrotizing enterocolitis in preterm infants, non-toxic amounts are produced as part of the neonatal microbiome and may be present in enteral nutrition and medications administered. The United States Food and Drug Administration has given guidance for endotoxin concentration limits for intravenous medications and fluids of 5 endotoxin units/kg/hour (120 endotoxin units/kg/day), but no guidance for amounts of endotoxin in enteral products. To determine baseline exposure to infants in the neonatal intensive care unit, we examined endotoxin content of enteral formulas and fortification used for preterm infants, as well as bovine lactoferrin products. We also examined endotoxin exposure and outcomes in very low birth weight infants. Endotoxin content was measured using kinetic chromogenic limulus amebocyte lysate analysis. Daily endotoxin exposure from enteral formulas ranged between < 75 to 7110 endotoxin units/kg and from lactoferrin products from 7 to 3720 endotoxin units/kg. In examining neonatal outcomes from a bovine lactoferrin product studied at three different escalating doses (100, 200, and 300 mg/kg/day), we measured endotoxin in the lactoferrin product and daily exposure was 1089 (N = 10), 2178 (N = 10) and 3287 (N = 11) endotoxin units/kg, respectively. There were no cases of necrotizing enterocolitis or mortality and no lactoferrin-related adverse effects in these patients. Enteral endotoxin daily exposures from lactoferrin products are similar to amounts in preterm enteral nutrition and appear safe and not associated with patient harm. Testing enteral products and establishing safety limits may improve care of high risk patients.

Risk of false conformity decisions in LAL gel-clot test due to false-negative results

Endotoxins are impurities capable of causing multiple deleterious effects to health. Therefore, the results of Limulus horseshoe crab amebocyte lysate (LAL) coagulation test in injectable drugs should be reliable. However, the reliability of qualitative results is still a challenge for analytical chemists and microbiologists. Thus, the objective of this work is to estimate the risk of false compliance/non-compliance decisions in LAL test based on the probability of true-negative and false-negative results through likelihood ratio value (LHR). For this, 11 injectable drugs were analyzed in two dilution schemes. A total of 1560 tests were performed using 41 different lots of LAL. The results from the confirmation of lysate sensitivity were used to obtain a logistic regression model, thus enabling to estimate the probability of negative results and consequently the LHR of the drug tests. LHR values were found to be between about 102 (114) and 105 (117,206), which corresponds to evidence of moderately strong to extremely strong negative results. Higher LHR values were obtained using lower dilution schemes. The use of logistic regression and calculation of LHR values can be useful to assess the reliability of qualitative results obtained by other chemical or microbiological methods.

Yeast Expressed Hybrid Peptide CLP Abridged Pro-Inflammatory Cytokine Levels by Endotoxin Neutralization.

The aim of this study was to apply a strategy to express a recombinant CLP peptide and explore its application as a product derived from natural compounds. The amphiphilic CLP peptide was hybridized from three parent peptides (CM4, LL37, and TP5) and was considered to have potent endotoxin-neutralizing activity with minimal cytotoxic and hemolytic activity. To achieve high secretion expression, an expression vector of pPICZαA-HSA-CLP was constructed by the golden gate cloning strategy before being transformed into Pichia pastoris and integrated into the genome. The recombinant CLP was purified through the Ni-NTA affinity chromatography and analyzed by SDS-PAGE and mass spectrometry. The Limulus amebocyte lysate (LAL) test exhibited that the hybrid peptide CLP inhibited lipopolysaccharides (LPS) in a dose-dependent manner and was significantly (p < 0.05) more efficient compared to the parent peptides. In addition, it essentially diminished (p < 0.05) the levels of nitric oxide and pro-inflammatory cytokines (including TNF-α, IL6, and IL-1β) in LPS-induced mouse RAW264.7 macrophages. As an attendant to the control and the parental peptide LL37, the number of LPS-induced apoptotic cells was diminished compared to the control parental peptide LL37 (p < 0.05) with the treatment of CLP. Consequently, we concluded that the hybrid peptide CLP might be used as a therapeutic agent.

Quality control of endotoxin measurement in dust samples/Qualitätskontrolle der Endotoxinmessung in Staubproben

Endotoxin activity determination is a common tool and a surrogate marker for bioaerosol assessment and is used at workplaces especially for this purpose. The reliability of the methods used for quantification in airborne dust samples is important for exposure estimation and thus for assessing the workplace situation. Within the scope of this study, the reproducibility of the endotoxin activity values determined with the two common methods, the LAL test and the rFC test, should be determined and compared. 21 water-based dust extracts from electrostatic dust collectors (EDC) were used for repeated endotoxin quantification using LAL and rFC test. As a result, twelve independent standard curves (0-50 EU/ml) were applied showing a median coefficient of variation (CV) of 9% for rFC and 12% for LAL test. The measured endotoxin activities of the samples could be reproduced within one test as well on three consecutive days. The CV of the 7- and 8-fold measured samples was below 20%. in 92% (LAL) and 93% (rFC) of the samples. Multiple measurements of the same sample showed that the results of LAL and rFC tests are highly reproducible. To eliminate interfering factors detected by recovery of spiked samples, dilutions of the samples are valuable methodological tools. This study demonstrates that quality-assured quantification of endotoxin activity is possible with both methods and is suitable for workplace measurements using the respective Standard Operation Procedure (SOP).

Abstract 1587: Using Limulus Amebocyte Lysate (LAL) to Discover the Presence of Endotoxins in the Tanks at the Pritzker Marine Biology Research Center

Abstract 1587: Using Limulus Amebocyte Lysate (LAL) to Discover the Presence of Endotoxins in the Tanks at the Pritzker Marine Biology Research Center

Evaluation of Recombinant Cascade Reagent PyroSmart NextGen&lt;sup&gt;®&lt;/sup&gt; and Limulus Amebocyte Lysate Equivalency in a Plate and Tube Reader for Bacterial Endotoxins Testing

PyroSmart NextGen® is a recombinant cascade reagent (rCR) for the detection and quantification of bacterial endotoxins developed using cloned genes derived from the Limulus polyphemus horseshoe crab genome. Requirements for use of this alternative reagent include analysis of analytical performance, method suitability, and test result equivalency to Limulus amoebocyte lysate (LAL) reagents used in the compendial Bacterial Endotoxins Test (BET). The plate reader evaluation has been expanded to address two long-standing user preferences, with the inclusion of the tube reader method increasing the sensitivity of endotoxin detection from 0.005 EU/mL to 0.001 EU/mL. The utilization of PyroSmart NextGen® with the two different instrument types also allows for a more comprehensive equivalency analysis. Furthermore, the comparison results demonstrate that PyroSmart NextGen® detects equivalent levels of autochthonous endotoxin in water samples when compared to LAL reagents. Overall, this study provides the first large-scale example of equivalency analysis utilizing a robust rCR and has verified that PyroSmart NextGen® meets the expectations for alternative reagents.

Ultramicro and ultrasensitive detection of lipopolysaccharides based on triple-signal amplification via ultrafast ATRP and an ultramicroelectrode.

Highly sensitive testing of trace lipopolysaccharides (LPS) is very important due to their high toxicity to the human body. Here, an ultrasensitive electrochemical sensor requiring only 5 μL solution was developed for LPS detection via triple-signal amplification based on ultrafast atom transfer radical polymerization (UATRP) and a Au ultramicroelectrode (UME). Firstly, the Au UME was modified with gold nanoparticles (nAu) and an LPS aptamer (Apt) in turn. When the Apt recognized LPS, the ATRP initiator of 4-(bromomethyl)phenylboronic acid (BPA) could be tethered to the electrode by covalent cross-linking between the phenylboronic acid moiety and the cis-diol site of LPS. Then UATRP was conducted for 2.5 min with nitrogen-doped carbon quantum dots (N-CQDs) as the photocatalyst and methylacrolein (MLA) as the monomer. After the electroactive probes of Ag nanoparticles (AgNPs) were formed on the surface of poly(MLA) by the silver mirror reaction, the electrochemical sensor was successfully prepared. Under the optimal conditions, the sensor exhibited a lower detection limit and a wider linear range when it was compared with a similar assay for LPS. In particular, the LOD of 7.99 × 10-2 pg mL-1 was better than that of the limulus amoebocyte lysate (LAL)-based technique, which is the gold standard for LPS detection. In the end, the sensor reported in this paper showed good selectivity and satisfactory feasibility for LPS detection in real biological samples and food products. The results obtained from the drug, blood and potable water samples laid a strong foundation for its clinical applications and application in other fields.

Endotoxins Affecting Human Health during Agricultural Practices: An Overview

Agricultural operations and the processing sector generate dust laden with endotoxin in the workplace. Endotoxin, a pro-inflammatory agent, has adverse effects on health, especially in the lungs, as exposure to endotoxin reduces lung function capacity. Endotoxin exposure to workers and its harmful impact on the health of agricultural workers needs to be studied in detail for future interventions to reduce exposure to endotoxin. The review can help to identify the analytical methods used to determine endotoxin exposure in agriculture. A detailed study of the research articles published in the last two decades related to agriculture and allied fields was carried out. In the agricultural sector, Pantoea agglomerans, a Gram-negative bacterium, was predominantly present. The filters were stored at a temperature of −20 °C, and E. coli 055: B5 was the predominately used standard to analyze the endotoxin. The quantitative kinetic Limulus Amebocyte Lysate test was the most common detection method for quantifying endotoxin. Control strategies to reduce endotoxin exposure are also emphasized in this review.