Ethyl Carbamate Research Articles

Portable paper-based sheet for visual detection of ethyl carbamate in Chinese Baijiu based on aptamer recognition and laccase-like activity of cubic Ag2O nanoparticles

Ethyl carbamate (EC) is a small compound produced and stored during fermentation processes from ethanol, urea, citrulline, and carbamoyl phosphate. EC is commonly found in fermented foods and alcoholic beverages. Prolonged exposure to EC can cause nervous system diseases and liver damage in human beings. EC lacks stable physicochemical properties, and the presence of numerous interfering substances in alcoholic beverages poses challenges for its detection. Herein, it is found that EC1-34 aptamer bound to EC can enhance the laccase-like activity of cubic Ag2O nanoparticles (NPs). Subsequently, Ag2O NPs are wrapped with EC1-34 aptamer to prepare recognition probes, which are immobilized on a carrier membrane. Thus, a portable paper-based sheet for EC detection in Chinese Baijiu is designed. The visual detection limit of the developed paper-based sheet for EC is 10 μg⋅L-1, and shows an outstanding linear relationship within the range of 10-200 μg⋅L-1. Moreover, this colorimetric paper-based sheet has a good practicality in various Chinese Baijiu samples. It is believed that such paper-based sheet can facilitate portable inspection for EC in Baijiu, and may also become a beneficial tool for monitoring EC molecules in other fermented foods.

The "two-hump" evolution of ethyl carbamate (EC) and key precursor cyanide during Chinese Baijiu solid-state distillation

The "two-hump" evolution of ethyl carbamate (EC) and key precursor cyanide during Chinese Baijiu solid-state distillation

Ionic polymers as double-capture agents in an aptamer lateral flow assay strip for point-of-care detection of ethyl carbamate using peroxidase-like activity of bimetallic NiCo2O4 nanoparticles

A novel aptamer LFA (Apt-LFA) strip is first constructed for ethyl carbamate (EC) detection, in which cationic polyethyleneimine (PEI) and anionic polyacrylamide (APAM) are used as double-capture agents. The black NiCo2O4 nanoparticles (NCO NPs) encapsulated by EC1-34 aptamer are employed as recognition probes. The added EC will bind to EC1-34 aptamer on the probes, so that the test-line (T-line) immobilized with APAM can electrostatically capture the exposed NCO NPs. The control-line (C-line) sprayed with PEI can capture the excessive black recognition probes. The peroxidase-like activity of bimetallic NCO NPs is employed as signal amplification to improve the sensitivity of Apt-LFA strip. The naked eye discernible concentration of EC is 5 μg L−1 and the detection limit (LOD) is as low as 1.36 μg L−1. The Apt-LFA strip has the advantages of strong stability, simplicity and low cost, which provide a new method for EC detection and offer a new way for designing LFA.

A Chemometric Exploration of Potential Chemical Markers and an Assessment of Associated Risks in Relation to the Botanical Source of Fruit Spirits.

Chemometric evaluation of potentially harmful volatile compound and toxic metal(loid) distribution patterns in fruit spirits relating to distinct fruit classes most commonly used in spirit production highlighted the potential of several volatiles as candidates for differentiation markers while dismissing toxic metal(loid)s. Pome fruit and grape pomace spirits were mostly characterized by a lower abundance of n-propanol, methanol, ethyl acetate and acetaldehyde, while stone fruit spirits contained lower amounts of isoamyl alcohol and isobutanol. Chemometric analysis of the fruit spirit composition of aromatics identified additional potential markers characteristic for certain fruits-benzoic acid ethyl ester, benzyl alcohol, benzaldehyde, butanoic acid 3-methyl-ethyl ester, butanoic acid 2-methyl-ethyl ester and furfural. This study explored the variability in the risk potential of the investigated spirits, considering that some chemicals known to be detected in spirits are potent health hazards. Ethyl carbamate in combination with acetaldehyde showed a higher potential risk in stone fruit spirits, methanol in stone and pome fruit spirits and acetaldehyde in grape pomace spirits. It is of great interest to evaluate to what extent consumers' preference for spirits of distinct fruit types affects health risks. Consumers of stone fruit spirits are potentially at higher risk than those consuming pome fruit or grape pomace spirits.

Rapid measurement of ethyl carbamate in Chinese liquor by fast gas chromatography photoionization-induced chemical ionization mass spectrometry

As a common by-product during the production of alcoholic beverages, such as Chinese liquor, ethyl carbamate (EC) poses potential genotoxicity and is associated with the risk of various cancers. Hence, rapidly and accurately measuring the content of EC in liquor is critical to assess the product quality and risks of mass samples during the production process. In this study, a feasible method based on fast gas chromatography photoionization-induced chemical ionization mass spectrometry (FastGC-PICI-TOFMS) was developed for the analysis of EC in Chinese liquor. The rapid separation of EC in Chinese liquor was conducted using FastGC based on a thermostatic column set at 150 °C to eliminate the interferences of matrix effects. The PICI-TOFMS could realize accurate quantification of EC without any sample pre-treatment due to the efficient ionization of EC by the PICI source. As a result, the total analysis time for EC in Chinese liquor was less than 4 min. The limit of detection (LOD) for EC was 4.4 μg L−1. And the intra-day and inter-day precision were 3.2%–3.7 % and 1.6 %, respectively. Finally, the ability of the proposed method was preliminarily proved by high-throughput and accurate measurement of EC in four different flavors of Chinese liquors.

High Salt-Resistant Urethanase Degrades Ethyl Carbamate in Soy Sauce.

Urethanase is a promising biocatalyst for degrading carcinogen ethyl carbamate (EC) in fermented foods. However, their vulnerability to high ethanol and/or salt and acidic conditions severely limits their applications. In this study, a novel urethanase from Alicyclobacillus pomorum (ApUH) was successfully discovered using a database search. ApUH shares 49.4% sequence identity with the reported amino acid sequences. It belongs to the Amidase Signature family and has a conserved "K-S-S" catalytic triad and the characteristic "GGSS" motif. The purified enzyme overexpressed in Escherichia coli exhibits a high EC affinity (Km, 0.306 mM) and broad pH tolerance (pH 4.0-9.0), with an optimum pH 7.0. Enzyme activity remained at 58% in 12% (w/v) NaCl, and 80% in 10% (v/v) ethanol or after 1 h treatment with the same ethanol solution at 37 °C. ApUH has no hydrolytic activity toward urea. Under 30 °C, the purified enzyme (200 U/L) degraded about 15.4 and 43.1% of the EC in soy sauce samples (pH 5.0, 6.0), respectively, in 5 h. Furthermore, the enzyme also showed high activity toward the class 2A carcinogen acrylamide in foods. These attractive properties indicate their potential applications in the food industry.

Mechanism of Polygonum hydropiper reducing ethyl carbamate in Chinese rice wine (Huangjiu) brewing

Polygonum hydropiper (PH) is a rich source of active compounds and serves as a pivotal ingredient in Chinese rice wine (Huangjiu) production. This study investigates the impact of PH and Polygonum hydropiper extract (PHE) on ethyl carbamate (EC) production during Huangjiu fermentation. Our findings reveal that PH enhances the relative abundance of Bacillus subtilis in Huangjiu fermentation, thereby facilitating its interaction with Saccharomyces cerevisiae. Furthermore, PH modulates the urea metabolism of S. cerevisiae. In the PH-B. subtilis-S. cerevisiae fermentation system, the expression of DUR1,2 and DUR3 genes in S. cerevisiae is upregulated. This augmentation leads to increased urea uptake and metabolism by S. cerevisiae in the fermentation broth, subsequently reducing the urea concentration in the fermentation medium (The EC content in the CK group was approximately 355.55 % and 356.05 % higher than those in the PH and PHE groups, respectively). Consequently, PH demonstrates promise in reducing the EC concentration of Huangjiu, offering a novel approach to enhance the safety of Huangjiu consumption.

A Versatile Visual Molecular Imprinting-Driven Switchable Nanozyme Activity-Based Trimodal Assay and Logic Gate Circuits of Ethyl Carbamate.

Concerns regarding the hazard of the carcinogenic ethyl carbamate (EC) have driven attempts to exploit efficient, timely, straightforward, and economic assays for warning early food safety. Here, we proposed a novel molecularly imprinted polymer Co@MOF-MIP, with a high peroxidase (POD)-like activity and a bright blue fluorescence emission, to develop a versatile visual assay for colorimetric, fluorescent, and photothermal trimodal detection and logic gate outputting of EC. Briefly, the POD-like activity of Co@MOF-MIP made it to decompose H2O2 into ·OH for oxidizing colorless 3,3',5,5'-tetramethylbenzidine (TMB) into a blue oxTMB, resulting in a 660 nm irradiated photothermal effect and bursting the blue fluorescence of Co@MOF-MIP via inner filter effect, observing a decreased fluorescence signal together with an increased colorimetric and 660 nm irradiated photothermal signals. However, EC could specifically fill the imprinted cavities of Co@MOF-MIP to block the catalytic substrates TMB and H2O2 out of Co@MOF-MIP for further reacting with the inside catalytic center of Co2+, resulting in the transformation suppressing of TMB into oxTMB, yielding an EC concentration-dependent trimodal responses in fluorescence signal enhancement, colorimetric, and 660 nm irradiated photothermal signal decreases. Assisted by the portable devices such as smartphones and hand-held thermal imagers, a visual onsite portable trimodal analytical platform was proposed for EC fast and accurate detection with the low detection limits of 1.64, 1.24, and 1.78 μg/L in colorimetric, fluorescent, and photothermal modes, respectively. Interestingly, these reactive events could be programmed by the classical Boolean logic gate analysis to offer a novel promising avenue for the big data Internet of Things monitoring and warning early residual EC in a more intelligent, dynamical, fast, and accurate manner, safeguarding food safety.

Direct detection of ethyl carbamate in baijiu by molecularly imprinted electrochemical sensors based on perovskite and graphene oxide

Ethyl carbamate (EC), a carcinogen commonly found in Baijiu, requires an efficient detection method for quality control and monitoring. This study introduces a novel molecularly imprinted electrochemical sensor for sensitive and selective EC detection. We proposed a simple sol-gel method for the growth of perovskite-structured lanthanum manganate (LaMnO3) on graphene oxide (GO). A non-enzymatic electrochemical sensor was developed by coating a molecularly imprinted polymer synthesized via precipitation polymerization onto the surface of LaMnO3@GO. LaMnO3, with its superior three-dimensional nanocube structure, demonstrated excellent electrocatalytic activity, while the addition of GO provided a large specific surface area. The results indicate that the developed sensor exhibits exceptional recognition ability and electrochemical activity, which is attributed to the high affinity of LaMnO3@GO@MIP for EC. The sensor displays a broad linear range from 10 to 2000 μM, with a detection limit as low as 2.18 μM and long-term durability of 28 days. Notably, it demonstrates excellent selectivity, reproducibility, and stability even under different interference conditions. The sensor was successfully used to determine EC in real Baijiu samples. Overall, the sensor has broad application prospects for detecting trace contaminants in the field of food safety.

Crystal structure of urethanase from Candida parapsilosis and insights into the substrate-binding through in silico mutagenesis and improves the catalytic activity and stability

Ethyl carbamate (EC) is classified as a Class 2A carcinogen, and is present in various fermented foods, posing a threat to human health. Urethanase (EC 3.5.1.75) can catalyze EC to produce ethanol, CO2 and NH3. The urethanase (cpUH) from Candida parapsilosis can hydrolyze EC, but its low affinity and poor stability hinder its application. Here, the structure of cpUH from Candida parapsilosis was determined with a resolution of 2.66 Å. Through sequence alignment and site-directed mutagenesis, it was confirmed that cpUH contained the catalytic triad Ser-cisSer-Lys of the amidase family. Then, the structure-oriented engineering mutant N194V of urethanase was obtained. Its urethanase activity increased by 6.12 %, the catalytic efficiency (kcat/Km) increased by 21.04 %, and the enzyme stability was also enhanced. Modeling and molecular docking analysis showed that the variant N194V changed the number of hydrogen bonds between the substrate and the catalytic residue, resulting in enhanced catalytic ability. MD simulation also demonstrated that the introduction of hydrophobic amino acid Val reduced the RMSD value and increased protein stability. The findings of this study suggest that the N194V variant exhibits significant potential for industrial applications due to its enhanced affinity for substrate binding, improved catalytic efficiency, and increased enzyme stability.

Effective Ethyl Carbamate Prevention in Red Wines by Treatment with Immobilized Acid Urease.

Climate change poses several challenges in the wine industry, including increasing risks related to chemical food contaminants such as biogenic amines and ethyl carbamate (EC). In this work, we focused on urea removal in red wines by immobilized acid urease aiming at limiting EC formation during wine storage. By considering separable kinetics of catalyst deactivation and urea hydrolysis, it was possible to model the time course of urea removal in repeated uses in stirred batch reactors. Treatments based on immobilized urease of red wine enriched with 30 mg/L of urea allowed the reduction in the contaminant concentration to <5 mg/L. After 28.5 h of treatment, the observed urea level was reduced to about 0.5 mg/L, corresponding to a decrease in the potential ethyl carbamate (PEC) from 1662 μg/L to 93 μg/L, below the level of the non-enriched wine (187 μg/L). As a comparison, when treating the same wine with the free enzyme at maximum doses allowed by the EU law, urea and PEC levels decreased to only 12 mg/L and 415 μg/L respectively, after 600 h of treatment. These results show that, for red wines, urease immobilization is an effective strategy for urea removal and, thus, effective reduction in ethyl carbamate as a process contaminant. This study provides the scientific background for the future scaling-up of the process at an industrial level.

Mitigating ethyl carbamate in Chinese rice wine: Role of raspberry extract

This study investigated the use of raspberry extract (RBE) in mitigating ethyl carbamate (EC) accumulation in Chinese rice wine (Huangjiu), a traditional fermented beverage. It focused on the addition of RBE to the fermentation mash and its effects on EC levels. Results indicated a significant reduction in EC production, attributed to RBE's role in altering urea and citrulline catabolism and inhibiting arginine metabolism, thus preventing EC precursors from reacting with ethanol. Additionally, RBE enhanced the rice wine's flavor profile, as shown by volatile component and amino acid analysis. This study also explored RBE's impact on Saccharomyces cerevisiae (S. cerevisiae) arginine metabolism in a simulated fermentation environment, observing increased arginine, reduced urea and citrulline levels, altered enzyme activities, and gene expression changes in arginine metabolism and transport pathways. In conclusion, it clearly demonstrated RBE's efficacy in reducing EC content in Chinese rice wine, offering valuable insights for EC reduction strategies.

Optoelectronic performance of Jatropha oil-derived poly(ethyl carbamate) gel polymer electrolyte as quasi-solid-state solution for photoelectrochemical cells

Optoelectronic performance of Jatropha oil-derived poly(ethyl carbamate) gel polymer electrolyte as quasi-solid-state solution for photoelectrochemical cells

Investigating Safety and Technological Traits of a Leading Probiotic Species: Lacticaseibacillus paracasei.

Lacticaseibacillus spp. are genetically close lactic acid bacteria species widely used in fermented products for their technological properties as well as their proven beneficial effects on human and animal health. This study, the first to include such a large collection of heterogeneous isolates (121) obtained from international collections belonging to Lacticaseibacillus paracasei, aimed to characterize the safety traits and technological properties of this important probiotic species, also making comparisons with other genetically related species, such as Lacticaseibacillus casei and Lacticaseibacillus zeae. These strains were isolated from a variety of heterogeneous sources, including dairy products, sourdoughs, wine, must, and human body excreta. After a preliminary molecular characterization using repetitive element palindromic PCR (Rep-PCR), Random Amplification of Polymorphic DNA (RAPD), and Sau-PCR, particular attention was paid to safety traits, evaluating antibiotic resistance profiles, biogenic amine (BA) production, the presence of genes related to the production of ethyl carbamate and diaminobenzidine (DAB), and multicopper oxidase activity (MCO). The technological characteristics of the strains, such as the capability to grow at different NaCl and ethanol concentrations and different pH values, were also investigated, as well as the production of bacteriocins. From the obtained results, it was observed that strains isolated from the same type of matrix often shared similar genetic characteristics. However, phenotypic traits were strain-specific. This underscored the vast potential of the different strains to be used for various purposes, from probiotics to bioprotective and starter cultures for food and feed production, highlighting the importance of conducting comprehensive evaluations to identify the most suitable strain for each purpose with the final aim of promoting human health.

Highly branched thermoresponsive polysaccharide derivative in water. Partly substituted highly branched cyclic dextrin ethylcarbamate

A thermoresponsive highly branched polysaccharide derivative was revealed from commercially available highly branched cyclic dextrin (HBCD), originally synthesized from amylopectin. Eight samples of partially substituted ethyl carbamate derivatives of HBCD (HEC) were prepared with a degree of substitution DS ranging from 0.27 to 1.46. Three samples with DS = 0.88, 1.05, and 1.22 showed LCST type phase separation in water. The intrinsic viscosity and form factor in water were typical of the hyperbranched structure. The intermolecular interactions between HEC and iodine or 1-anilinonaphthalene-8-sulfonic acid (ANS) were appreciably different from those of the linear analog (AEC), suggesting that the locally bent helical conformation of highly branched HEC chains has a different interaction with small molecules. The phase diagram of HEC-water systems was accidentally similar to that of the linear chain with the same molar mass and DS, although the one phase region of the branched polymer chain-poor solvent system is usually wider than that of the corresponding linear chain. This is likely due to the lower hydration nature of the polymer segment of HEC chains than that of the corresponding linear chain.

Effect of cultures of synthetic starters on the flavour and ethyl carbamate content of rice wine

SummaryEthyl carbamate (EC) concentration is an important indicator of the safety of rice wine. In this study, the effects of replacing traditional starters with synthetic starters on the quality of rice wine were studied to provide a basis for the application of laboratory strains to explore the metabolic basis of EC formation in rice wine fermentation systems. The effects of synthetic starters on the physicochemical indices, free amino acids, flavour compounds, urea and EC of rice wine were studied. The results showed that the microorganism species and inoculation fermentation method strongly influenced the physicochemical and flavour properties of the rice wine, among which continuous co‐fermentation improved the mellowness and flavour of the rice wine. The concentrations of urea and EC in rice wine fermented with synthetic starters decreased significantly compared with those in the control group, in which the urea content decreased by up to 97.4% and the EC content decreased by up to 90.9%. In addition, the flavour compound data showed that the differences in the aromas of rice wine fermented with different starters were mainly due to the differences in the contents of alcohols and esters. These findings suggest that EC can be regulated by changes in starters and provide guidance for the optimal configuration of mixed fermentation systems.

Urea amidolyase displayed on the surface of Saccharomyces cerevisiae for ethyl carbamate reduction in Chinese Huangjiu

Chinese Huangjiu, as one of the most popular traditional alcoholic beverages, poses a series of potential food safety issues due to its relatively high content of the “2A” class carcinogen ethyl carbamate (EC). Urea in Huangjiu is the primary precursor of EC formation. To decrease the EC content in Huangjiu, the endogenous urea amidolyase (UA) of Saccharomyces cerevisiae, which can hydrolyze urea, was anchored on the cell wall by utilizing yeast surface display technology and the cells were applied to Huangjiu fermentation or added to commercial Huangjiu in this study. Four different anchoring proteins, namely Cwp2p, Sag1p, Flo1p, and Aga1p-Aga2p, were used to construct four corresponding surface display strains. Among them, ACCD strains with CAR1 knocked out and displaying UA in the cell wall by the anchoring protein Cwp2p based on AY14α, showed the best Urea and EC degradation efficiency during simulated Huangjiu fermentation. Huangjiu by achieving reductions of 89.9% and 56.4% in urea and EC contents, respectively, compared to the control strains. Furthermore, when the ACCD strain was used as an immobilized enzyme to treat commercial Huangjiu, reductions of 29.8% and 38.8% were observed in urea and EC contents, respectively. Moreover, after 10 cycles of reuse, the degradation of urea could still reach 74.53% of the first treatment. This study presents a novel strategy for the reduction of urea and EC content in Huangjiu, and provides implications for the reduction of EC and urea content in other alcoholic beverages.

Antiproliferative effect of indeno[1,2-d]thiazolo[3,2-a]pyrimidine analogues on IL-6 mediated STAT3 and role of the apoptotic pathway in albino Wistar rats of ethyl carbamate-induced lung carcinoma: In-silico, In-vitro, and In-vivo study

Lung cancer (LC) ranks second most prevalent cancer in females after breast cancer and second in males after prostate cancer. Based on the GLOBOCAN 2020 report, India represented 5.9% of LC cases and 8.1% of deaths caused by the disease. Several clinical studies have shown that LC occurs because of biological and morphological abnormalities and the involvement of altered level of antioxidants, cytokines, and apoptotic markers. In the present study, we explored the antiproliferative activity of indeno[1,2-d]thiazolo[3,2-a]pyrimidine analogues against LC using in-vitro, in-silico, and in-vivo models. In-vitro screening against A549 cells revealed compounds 9B (8-methoxy-5-(3,4,5-trimethoxyphenyl)-5,6-dihydroindeno[1,2-d]thiazolo[3,2-a]pyrimidine) and 12B (5-(4-chlorophenyl)-5,6-dihydroindeno[1,2-d]thiazolo[3,2-a]pyrimidine) as potential pyrimidine analogues against LC. Compounds 9B and 12B were docked with different molecular targets IL-6, Cyt-C, Caspase9, and Caspase3 using AutoDock Vina 4.1 to evaluate the binding affinity. Subsequently, in-vivo studies were conducted in albino Wistar rats through ethyl-carbamate (EC)- induced LC. 9B and 12B imparted significant effects on physiological (weight variation), and biochemical (anti-oxidant [TBAR’s, SOD, ProC, and GSH), lipid (TC, TG, LDL, VLDL, and HDL)], and cytokine (IL-2, IL-6, IL-10, and IL-1β) markers in EC-induced LC in albino Wistar rats. Morphological examination (SEM and H&E) and western blotting (IL-6, STAT3, Cyt-C, BAX, Bcl-2, Caspase3, and caspase9) showed that compounds 9B and 12B had antiproliferative effects. Accordingly, from the in-vitro, in-silico, and in-vivo experimental findings, we concluded that 9B and 12B have significant antiproliferative potential and are potential candidates for further evaluation to meet the requirements of investigation of new drug application.

A Straightforward, Sensitive, and Reliable Strategy for Ethyl Carbamate Detection in By-Products from Baijiu Production by Enzyme-Linked Immunosorbent Assay.

Baijiu is a renowned Chinese distilled liquor, notable for its distinctive flavor profile and intricate production process, which prominently involves fermentation and distillation. Ethyl carbamate (EC), a probable human carcinogen, can be potentially formed during these procedures, thus prompting significant health concerns. Consequently, the contamination of EC during Baijiu production has become an increasingly pressing issue. In this study, we developed a rapid and easily operable immunoassay for determining EC in the fermented materials used in Baijiu production. The development of a high-quality antibody specific to EC facilitated a streamlined analytical procedure and heightened method sensitivity. Furthermore, we systematically evaluated other essential parameters. Following optimization, the method achieved an IC50 value of 11.83 μg/kg, with negligible cross-reactivity against EC analogs. The recovery study demonstrated the method's good accuracy and precision, with mean recovery rates ranging from 86.0% to 105.5% and coefficients of variation all below 10%. To validate the feasibility of the technique, we collected and analyzed 39 samples simultaneously using both the proposed immunoassay and confirmatory gas chromatography-mass spectrometry (GC-MS). A robust correlation was observed between the results obtained from the two methods (R2 > 0.99). The detected EC levels ranged from 2.36 μg/kg to 7.08 μg/kg, indicating an increase during the fermentation process.

Solventless Synthesis of Zinc Sulphide Nanoparticles from Zinc Bis(diethyldithiocarbamate) as a Single Source Precursor.

This study explores the synthesis of nanoparticles through the thermal decomposition of single-source precursors, a method gaining popularity due to its low cost, minimal environmental toxicity, rapidity, scalability, and the ability to form nanoparticles with few defects. Zinc ethyl carbamate was synthesized and characterized using 1HNMR and infrared spectroscopy. Its purity was confirmed through microelemental analysis and melting point determination. The melting point of the complex was determined to be 165 °C. The thermogravimetric analyses indicated a one-step decomposition of zinc ethyl carbamate with a decomposition onset of of 200 °C, yielding a stable ZnS residue. Further thermal decomposition led to the formation of wurtzite phase ZnS nanoparticles, as evidenced by XRD. SEM micrographs displayed mixed spherical, and cubic unevenly sized, polydispersed nanoparticles, while EDX revealed approximately a 1 : 1 Zn to S ratio. Estimated band gap from the Tauc's plot gave 3.93 eV and 3.42 eV for the nanoparticles synthesized at 300 and 400 °C respectively. The wide difference in the band gaps may be as a result of the larger particles observed at 400 °C and the deformations in the sample as observed in the SEM.