Anti-Inflammatory Properties of the Citrus Flavonoid Diosmetin: An Updated Review of Experimental Models.

Inflammation is an essential contributor to various human diseases. Diosmetin (3',5,7-trihydroxy-4'-methoxyflavone), a citrus flavonoid, can be used as an anti-inflammatory agent. All the information in this article was collected from various research papers from online scientific databases such as PubMed and Web of Science. These studies have demonstrated that diosmetin can slow down the progression of inflammation by inhibiting the production of inflammatory mediators through modulating related pathways, predominantly the nuclear factor-κB (NF-κB) signaling pathway. In this review, we discuss the anti-inflammatory properties of diosmetin in cellular and animal models of various inflammatory diseases for the first time. We have identified some deficiencies in current research and offer suggestions for further advancement. In conclusion, accumulating evidence so far suggests a very important role for diosmetin in the treatment of various inflammatory disorders and suggests it is a candidate worthy of in-depth investigation.

Integrating CRISPR-Cas12a and rolling circle-amplified G-quadruplex for naked-eye fluorescent "off-on" detection of citrus Alternaria.

Alternaria is a plant pathogen that spreads globally and is prone to causing citrus brown spot disease and metabolizing mycotoxins, thus seriously hindering the development of this economic crop industry. Herein, a "label-free" and "turn on" visual fluorescent assay for citrus Alternaria based on CRISPR-Cas12a and rolling circle amplification (RCA) was described. Using ssDNA complementary to RCA primer as a trans-cleavage substrate for CRISPR-Cas12a, the two systems of CRISPR-Cas12a and RCA-amplified G-quadruplex were skillfully integrated. By using a portable light source for excitation, the positive sample produced obvious red fluorescence, while the negative sample remained almost colorless, making them easy to differentiate with the naked eye. In addition, the specificity was demonstrated by distinguishing Alternaria from other citrus disease related pathogens. Moreover, the practicality was verified by analyzing cultured Alternaria and Alternaria in actual citrus leaf and fruit samples. Therefore, this method may contribute to the on-site diagnosis of Alternaria.

Determination, temporal variation and potential health risk assessment of pesticide residues in grapes from South and Southwest China.

Pesticide residues in grapes from South and Southwest China were determined using the QuEChERS procedure and UHPLC-MS/MS and GC-MS/MS methods. The 4-year monitoring and survey showed 94.6% of the 1341 samples of grapes collected from eight main production areas contained one or multiple pesticide residues (above the respective LOQs). Overall, 40 pesticides were detected, including 24 fungicides, 12 insecticides, 2 acaricides and 2 plant growth modulators, of which one pesticide was unauthorised for use in treating grapes. Two or more pesticide residues were discovered in 87.4% of the samples (above the respective LOQs), and pesticide residues in 5.7% of the samples exceeded the MRLs, such as difenoconazole, cyhalothrin, propiconazole, etc. The main risk factors affecting the safety of grape before 2019 were difenoconazole, cyhalothrin and cyazofamid. After 2019, however, the frequency of occurrence of the above pesticides significantly declined, and the banned or restricted pesticides including omethoate were not found, which was credited to the stricter supervision and management policies by local governments. Despite the high detection rates and multi-residue occurrence of pesticides in grapes, about 84% of the samples were compliant with regulatory standards. Moreover, the accumulative chronic diet risk determined from ADI is very low. This study and timely monitoring can ensure that grape growers comply with GAP and minimise the occurrence of residues.

Determination, Quality, and Health Assessment of Pesticide Residues in Kumquat in China.

Pesticide residues in kumquat fruits from China, and the quality and chronic/acute intake risks in Chinese consumers, were assessed using the QuEChERS procedure and UHPLC-MS/MS and GC-MS/MS methods. Our 5-year monitoring and survey showed 90% of the 573 samples of kumquat fruits collected from two main production areas contained one or multiple residual pesticides. Overall, 30 pesticides were detected, including 16 insecticides, 7 fungicides, 5 acaricides, and 2 plant growth modulators, of which 2 pesticides were already banned. Two or more residual pesticides were discovered in 81% of the samples, and pesticide residues in 9.4% of the samples surpassed the MRLs, such as profenofos, bifenthrin, triazophos, avermectin, spirodiclofen, difenoconazole, and methidathion. The major risk factors on the safety of kumquat fruits before 2019 were profenofos, bifenthrin, and triazophos, but their over-standard frequencies significantly declined after 2019, which was credited to the stricter supervision and management policies by local governments. Despite the high detection rates and multi-residue occurrence of pesticides in kumquat fruits, about 81% of the samples were assessed as qualified. Moreover, the accumulative chronic diet risk determined from ADI is very low. To better protect the health of customers, we shall formulate stricter organic phosphorus pesticide control measures and stricter use guidelines, especially for methidathion, triazophos, chlorpyrifos, and profenofos. This study provides potential data for the design of kumquat fruit quality and safety control guidelines and for the reduction in health risks to humans.

Ascorbic acid-mediated in situ growth of gold nanostars for photothermal immunoassay of ochratoxin A.

Currently, the development of efficient mycotoxins detection methods, particularly using portable devices as readout devices, remains a great challenge. Herein, a photothermal enzyme-linked immunosorbent assay (ELISA) based on gold nanostars (AuNSs) for the detection of ochratoxin A (OTA) using a "thermometer" was proposed for the first time. AuNSs with photothermal conversion capacity were parepared using an ascorbic acid (AA)-mediated in situ growth methd. Quantification was based on the alkaline phosphatase catalyzing the dephosphorylation of ascorbic acid 2-phosphoate to AA, thereby converting OTA concentration to the amount of in situ synthesized AuNSs, thus achieving straightforward readout by temperature. Benefiting from the classical tyramine signal amplification strategy, a detection limit of 0.39 ng mL-1 was obtained. The recoveries of grape juice and maize samples spiked with 10 ng mL-1 and 30 ng mL-1 OTA ranged from 86.53% to 116.9%. Our method has great potential in on-site OTA detection for food safety.

RPA-CRISPR/Cas12a Combined with Rolling Circle Amplification-Enriched DNAzyme: A Homogeneous Photothermal Sensing Strategy for Plant Pathogens.

Alternaria is an endemic fungus associated with brown spot disease, which is one of the most serious citrus diseases. In addition, the mycotoxins metabolized by Alternaria threaten human health seriously. Herein, a novel homogeneous and portable qualitative photothermal method based on recombinase polymerase amplification (RPA), CRISPR/Cas12a, and rolling circle amplification (RCA) for the detection of Alternaria is described. Using RCA primers as substrates for CRISPR/Cas12a trans-cleavage, the two systems, RPA-CRISPR/Cas12a and RCA-enriched G-quadruplex/hemin DNAzyme, are intelligently combined. Target DNA at fg/µL levels can be detected with high specificity. Additionally, the practicability of the proposed method is demonstrated by analyzing cultured Alternaria from different fruit and vegetable samples, as well as citrus fruit samples collected in the field. Furthermore, the implementation of this method does not require any sophisticated equipment and complicated washing steps. Therefore, it has great potential to screen Alternaria in poor laboratories.

Construction of a portable immunosensor for the sensitive detection of carbendazim in agricultural products using a personal glucose meter.

Portable and sensitive detection of carbendazim (CBD) is highly desirable for food safety and environmental protection. Herein, a portable immunosensor for the sensitive detection of CBD is proposed based on alkaline phosphatase (ALP)-labeled and secondary antibody-modified gold nanoparticles (AuNPs). The quantification is based on ALP catalyzing the dephosphorylation of glucose-1-phosphate disodium salt to generate glucose, thus converting the concentration of CBD into glucose, thereby realizing the portable detection of CBD by personal glucose meter. Benefiting from signal amplification strategy that integrates the large specific surface area of AuNPs, the enzymatic reactions of terminal deoxynucleotidyl transferase and ALP, a low detection limit of 0.37 ng/mL for CBD is achieved. When this portable method is used to analyze citrus fruit, canned citrus, and cabbage, good-consistency results are obtained with the UPLC-MS/MS method. The good performance demonstrates the great potential of this portable method for CBD monitoring in resource-poor settings.

Seed-mediated in situ growth of photothermal reagent gold nanostars: Mechanism study and preliminary assay application.

Photothermal reagent-mediated portable detection platforms using thermometers as signal readers have received extensive attention due to their simplicity, low cost, and practicality. However, exploitation photothermal reagent with excellent photothermal conversion effect, convenient to synthesize, preferably without any modification for biosensing application, is still challenging. Herein, a simple and rapid seed-mediated in situ synthesis strategy has been developed for the preparation of gold nanostars (AuNSs) with remarkable photothermal conversion effect. By simply changing the seed size and component concentrations involved in the in situ synthesis process, AuNSs have adjustable geometries, allowing the photothermal conversion to be tuned to a high level optimal for biosensing. Meanwhile, an accurate understanding of the photothermal conversion mechanism is obtained by studying the relationship between the morphology of AuNSs and the photothermal effect. Subsequently, using ascorbic acid (AA) as a model target, the preliminary application of AuNSs in constructing a portable photothermal detection platform has been demonstrated. This in situ preparation strategy of AuNSs not only exhibits remarkable photothermal conversion effect, but also avoids complicated and time-consuming synthesis and modification. Therefore, it has great potential to be extended to portable detection of other targets by simply converting the concentration of the target to that of AA.

A CRISPR/Cas12a-based photothermal platform for the portable detection of citrus-associated Alternaria genes using a thermometer.

The outbreak of citrus brown spot because of Alternaria is one of the most destructive citrus diseases. Additionally, Alternaria species produce highly toxic mycotoxins. Mass screening is a valid method to control the spread of Alternaria. Morphological analysis and polymerase chain reaction combined with gene-sequencing technique are the most commonly used techniques for detecting Alternaria. However, they are limited by either low convenience and accuracy or low instrument accessibility and high cost. To balance the convenience, accuracy, test availability, and low cost, we develop a CRISPR/Cas12a-based photothermal platform for the portable detection of Alternaria genes using a thermometer. Using this platform, the Alternaria genes from the synthetic sequences and cultured fungus of citrus, tomato, and apple can be detected using a thermometer with a detection limit of 1.5 pM. With the aid of the CRISPR/Cas12a system, citrus-associated Alternaria can be specifically differentiated from other citrus disease-associated microorganisms. When the photothermal platform is applied to analyze the citrus fruit samples collected in the field, good-consistency results are obtained with the gene-sequencing technology. The excellent performance of this portable method shows that it can be applied to screen for Alternaria in resource-poor settings.

Simultaneous determination of pyflubumide and its metabolite in vegetables and fruits by ultrahigh performance liquid chromatography-tandem mass spectrometry.

A rapid and cost-effective analytical method based on ultrahigh-performance liquid chromatography-tandem mass spectrometry was designed and verified for simultaneously monitoring the novel acaricide pyflubumide and its metabolite (pyflubumide-des(2-methyl-1oxopropyl)) in vegetables and fruits. After the extraction with acetonitrile, the samples were purified by dispersive solid-phase extraction with multi-walled carbon nanotubes. Detection of the two target analytes was achieved within 3.0 min using a positive electrospray ionization mode. The average recovery, intra-day precision, and inter-day precision of the two analytes at three spiked levels (2, 20, and 100 µg/kg) were 75.0-101.0, 0.4-4.4, and 0.6-5.3%, respectively. The limit of quantification of two compounds was 2 µg/kg, which was far below the maximum residue limits of pyflubumide in foods established by Japan and South Korea. Finally, the concentrations of pyflubumide and its metabolite in the samples were 16.6 and 7.8 µg/kg respectively, which verified the practicability and reliability of the method. The method was used to efficiently detect pyflubumide and its metabolite in real samples and was confirmed to be robust and effective for routinely analyzing both pyflubumide and its metabolite in vegetable and fruit samples.

CRISPR-Cas12a based fluorescence assay for organophosphorus pesticides in agricultural products.

Herein, we propose a sensitive fluorescent assay for organophosphorus pesticides (OPs) detection based on a novel strategy of activating the CRISPR-Cas12a system. Specifically, acetylcholinesterase (AChE) hydrolyzes acetylthiocholine into thiocholine (TCh). Subsequently, TCh induces the degradation of MnO2 nanosheets and generates sufficient Mn2+ ions to activate the Mn2+-dependent DNAzyme. Then, as the catalytic product of activated DNAzyme, the short DNA strand activates the CRISPR-Cas12a system to cleave the fluorophore-quencher-labeled DNA reporter (FQ) probe effectively; thus, increasing the fluorescence intensity (FI) in the solution. However, in the presence of OPs, the activity of AChE is suppressed, resulting in a decrease in FI. Under optimized conditions, the limits of detection for paraoxon, dichlorvos, and demeton were 270, 406, and 218 pg/mL, respectively. Benefiting from the outstanding MnO2 nanosheets properties and three rounds of enzymatic signal amplification, the proposed fluorescence assay holds great potential for the detection of OPs in agricultural products.

The application of DNA-HRP functionalized AuNP probes in colorimetric detection of citrus-associated Alternaria genes.

The monitoring of the fungal genus Alternaria, which causes destructive brown spot disease in citruses worldwide and produces highly toxic mycotoxins, is extremely important to protect citrus and human health. In this work, we describe an ultrasensitive colorimetric method for the detection of genomic DNA of Alternaria from citrus fruit samples, using a system consisting of five groups of reporter probes. Each reporter probe is prepared by coupling recognition DNA and horseradish peroxidase (HRP) on the surface of gold nanoparticle (AuNP) through a convenient and low-cost freezing-assisted method. Meanwhile, the capture DNA is immobilized on magnetic bead (MB) via biotin-streptavidin reaction. Then, the capture DNA, target DNA, and five groups of AuNP-based reporter probes form a stable DNA-heptamer sandwich structure on the MB, and then HRP generates a blue signal for the subsequent colorimetric detection. It should be noted that AuNP with a large specific surface area drives abundant HRP anchoring, resulting in significant signal amplification. In addition, there are five groups of AuNP-based reporter probes, which further amplify the detection signal. As a result, the detection limit of the artificial target DNA is as low as 15.6 pM. Because the detection signal can be recorded visually without any special equipment, and its sensitivity is high, this method represents a suitable diagnostic tool for Alternaria genetic detection.

Enantioselective determination of phenthoate enantiomers in plant-origin matrices using reversed-phase high-performance liquid chromatography-tandem mass spectrometry.

Phenthoate is a chiral organophosphate pesticide with a pair of enantiomers which differ in toxicity, behavior and insecticidal activity, and its acute toxicity on human health owing to the inhibition of acetylcholinesterase highlights the need for enantioselective detection of enantiomers. Therefore, this study aimed to establish a simple rapid method for separation and detection of phenthoate enantiomers in fruits, vegetables and grains. The enantiomers were separated using reversed-phase high-performance liquid chromatography-tandem mass spectrometry for the first time. Rapid chiral separation (within 9 min) of the target compound was achieved on a chiral OJ-RH column with the mobile phase of methanol-water = 85:15(v/v), at a flow rate of 1 ml/min and a column temperature of 30°C. Acetonitrile and graphitized carbon black were used as the extractant and sorbent for pretreatment, respectively. This method provides excellent linearity (correlation coefficient ≥0.9986), high sensitivity (limit of quantification 5 µg/kg and limit of detection <0.25 µg/kg), satisfactory mean recoveries (76.2-91.0%) and relative standard deviation (intra-day RSDs ranged from 2.0 to 7.9% and inter-day RSDs ranged from 2.4 to 8.4%). In addition, a field trial to explore the stereoselective degradation of phenthoate enantiomers in citrus showed that (-)-phenthoate degraded faster than its antipode, resulting in the relative accumulation of (+)-phenthoate.

Determination of five pesticides in kumquat: Dissipation behaviors, residues and their health risk assessment under field conditions.

The present study was carried out to profile the dissipation patterns and residues of five pesticides (triazophos, profenofos, chlorpyrifos, etoxazole and bifenthrin) on kumquat using QuEChERS method coupled with HPLC-MS/MS. The corresponding dietary health risks were also estimated. In the method validation, satisfactory results of good linearity (r2 ≥ 0.9956), sensitivity (limits of quantification ≤0.01 mg/kg), recoveries (71.0-95.7%) with relative standard deviations (0.70-9.4%) were obtained. The half-lives of the five pesticides in kumquat were 13.6-38.5 d under field conditions according to first-order kinetics. Based on the final residue experiment, dietary exposure risks of profenofos, chlorpyrifos, etoxazole and bifenthrin were all acceptably low, with RQc and RQa values of 0.00199-0.122 and 0.00145-0.200, respectively. However, exposure intake of triazophos posed unacceptable acute and chronic health risks for Chinese residents, especially for children with RQa and RQc up to 4.25 and 2.19. Forbidden use suggestion of triazophos and recommended MRLs of profenofos and bifenthrin were put forward in kumquat for safe production and consumption. This work was significant in providing guidance on appropriate application and MRL establishment of pesticides in kumquat.

Dissipation of imidacloprid and its metabolites in Chinese prickly ash (Zanthoxylum) and their dietary risk assessment.

Dissipation of imidacloprid (IMI) and its metabolites (urea, olefin, 5-hydroxy, guanidine, 6-chloronicotinic acid) in Chinese prickly ash (CPA) was investigated using QuEChERS combined with UPLC-MS/MS. Good linearity (r2 ≥0.9963), accuracy (recoveries of 71.8-104.3%), precision (relative standard deviations of 0.9-9.4%), and sensitivity (limit of quantification ≤0.05 mg kg-1) were obtained. After application of IMI at dosage of 467 mg a.i. L-1 for three times with interval of 7 d, the dissipation dynamics of IMI in CPA followed first-order kinetics, with half-life of 6.48-7.29 d. IMI was the main compound in CPA, followed by urea and guanidine with small amounts of olefin, 5-hydroxy, and 6-chloronicotinic acid. The terminal residues of total IMI and its metabolites at PHI of 14-21 d were 0.16-7.80 mg kg-1 in fresh CPA and 0.41-10.44 mg kg-1 in dried CPA, with the median processing factor of 3.62. Risk assessment showed the acute (RQa) and chronic dietary risk quotients (RQc) of IMI in CPA were 0.020-0.083% and 0.052-0.334%, respectively. Based on the dietary structures of different genders and ages of Chinese people, the whole dietary risk assessment indicated that RQc was less than 100% for the general population except for 2- to 7-year-old children (RQc of 109.9%), implying the long-term risks of IMI were acceptable to common consumers except for children.

A multicolor enzyme-linked immunoassay method for visual readout of carbendazim.

Enzyme-linked immunosorbent assay (ELISA) with high specificity and sensitivity is one of the most popular techniques for detecting carbendazim (CBD), a commonly used benzimidazole fungicide in agriculture. However, the traditional ELISA based on the horseradish peroxidase (HRP)-3,3',5,5'-tetramethylbenzidine (TMB) system for CBD only displays the yellow color of TMB2+ from deep to light, making it difficult for the naked eye to judge whether CBD in fruits and vegetables exceeds the maximum residue limit. In this article, we intend to improve the traditional ELISA method to establish a multicolor signal output ELISA to achieve visual semiquantitative detection of CBD. This method is based on the optical properties of gold nanorods (AuNRs). After introducing AuNRs into TMB2+ solution, which was produced by the HRP-TMB system of traditional ELISA, AuNRs were quickly etched by TMB2+. Consequently, the longitudinal localized surface plasmon resonance peak of AuNRs shows a clear blue shift and a vivid color change. Different concentrations of CBD generate different amounts of TMB2+, which in turn leads to different etching degrees of AuNRs, and ultimately results in a rainbow-like color change. As a result, CBD from 0.08 to 100 ng mL-1 can be easily distinguished by the naked eye, which does not require any large instruments. Moreover, the colors displayed by 0.49 ng mL-1 (purple) and 0 ng mL-1 (pink) are significantly different from each other. It should be noted that 0.49 ng mL-1 is far below the most stringent maximum residue limit of CBD in the world. Additionally, the quantitative determination of CBD spiked in canned citrus, citrus fruits, chives, and cabbage samples showed satisfactory recoveries. The good performance of the AuNR-based ELISA makes it have a wide range of application prospects in food safety and international trade.

Ultrasound-assisted dispersive liquid-phase microextraction by solidifying L-menthol-decanoic acid hydrophobic deep eutectic solvents for detection of five fungicides in fruit juices and tea drinks.

An ecofriendly and efficient ultrasound-assisted deep eutectic solvents dispersive liquid-phase microextraction by solidifying the deep eutectic solvents-rich phase was developed to determine azoxystrobin, fludioxonil, epoxiconazole, cyprodinil, and prochloraz in fruit juices and tea drinks by high-performance liquid chromatography. A varieties of environmental hydrophobic deep eutectic solvents serving as extraction agents were prepared using L-menthol and decanoic acid as hydrogen-bond acceptor and hydrogen-bond donor, respectively. The deep eutectic solvents were ultrasonically dispersed in sample solutions, solidified in a freezer and easily harvested. The main variables were optimized by one-factor-at-a-time and response surface test. The new method performs well with relative recovery of 71.75-109.40%, linear range of 2.5-5000 µg/L (r ≥ 0.9968), detection limit of 0.75-8.45 µg/L, quantification limit of 2.5-25 µg/L,, and inter- and intraday relative standard deviations below 13.53 and 14.84%, respectively. As for the extraction mechanism, deep eutectic solvents were disposed into many fine particles in the solution and captured the analytes based on the changes of particle size and quantity in deep eutectic solvents droplets after extraction. The environmental method can successfully detect fungicide residues in real fruit juices and tea drinks.

Integrating multiple hybridization chain reactions on gold nanoparticle and alkaline phosphatase-mediated in situ growth of gold nanobipyramids: An ultrasensitive and high color resolution colorimetric method to detect the mecA gene of Staphylococcus aureus.

Colorimetry has been considered as a potential instrument-free platform for point-of-care genomic detection. However, it is limited by the poor sensitivity and low color resolution. Herein, we report a high-resolution colorimetric biosensor based on multiple hybridization chain reactions (HCRs) on gold nanoparticle (AuNP) and alkaline phosphatase (ALP)-mediated in situ growth of gold nanobipyramids (AuNBPs) for ultrasensitive detection of the Staphylococcus aureus (S. aureus) mecA gene. In our design, target DNA is hybridized with capture hairpin DNA on magnetic beads and then amplified by multiple HCRs on AuNP. Since biotin-labeled hairpin-structured nucleic acids are utilized to conduct HCRs, together with the large specific surface area of AuNP, the biotin- and streptavidin- based reaction results in a large amount of ALP on AuNP. With the aid of NADPH, ALP-mediated in situ growth of AuNBPs is observed, and a series of rainbow-like colors are associated with different target DNA concentrations. Through the multiple-amplification strategy produced by AuNP, HCRs, and enzymatic reactions, the target DNA as low as 2.71 pM can be detected with high specificity. Moreover, this method has been successfully applied to detect the mecA gene extracted from S. aureus. Therefore, the proposed method holds great potential in clinical diagnosis.

A sensitive and practical ELISA for analyzing naringenin in pummelo and herb samples.

Naringenin, a flavonoid compound found in pummelo, is a key biological active compound in some traditional Chinese medicines, including Citri reticulatae pericarpium, Citri reticulatae pericarpium viride, Aurantii fructus immaturus, and Aurantii fructus. These Chinese medicinal preparations are the peels or immature fruits of certain citrus species. Aiming at detecting naringenin in complex matrices such as pummelo and traditional Chinese medicines, we put forward a sensitive and practical indirect competitive enzyme-linked immunosorbent assay (icELISA) based on anti-naringenin monoclonal antibodies (anti-Nar-mAbs). The median inhibitory concentration (IC50) was 4.43 ng/mL, and the working range was 1.15-15.81 ng/mL. The findings of the icELISA for the analysis of naringenin in pummelo and herb samples had a good correlation with the ultra performance liquid chromatography (UPLC) methodology and showed good accuracy and reproducibility. These data demonstrated that the developed icELISA is reliable, accurate, and suitable for detecting naringenin in pummelo and traditional Chinese medicines.

Portable and quantitative detection of carbendazim based on the readout of a thermometer.

The detection of carbendazim (CBZ) is important for food safety and human health. However, most current analytical methods require large instruments and highly trained operators. In order to solve this problem, herein, an innovative portable and quantitative photothermal assay platform relying on a thermometer readout for the detection of CBZ has been developed. Gold nanoparticles (AuNPs), which exhibit a strong distance-dependent photothermal effect under specific laser irradiation, were utilized as indicators. The CBZ aptamer was introduced to protect AuNPs from salt-mediated aggregation. When CBZ is present, the binding event between CBZ and aptamer leads to the loss of the aptamer protective effect on AuNPs, and AuNP aggregation occurs. Under 650-nm laser irradiation, the increase in temperature associated with an AuNP-dependent photothermal effect is highly related to the CBZ concentration. Having the advantages of user-friendliness, low cost, quick response, and portability, this method has great potential for on-site applications.