Detection of sensitivity and vestigiality of presumptive tests for swabbed blood stains.

It is a common practice in forensic casework to use presumptive tests for blood stains before DNA extraction and testing. Stains are usually swabbed and then the swabs are sent for analysis. The Kastle-Meyer (KM) and Leucomalachite green (LMG) presumptive tests for blood are widely used, and their sensitivities have been thoroughly tested in the literature in solution and directly on stains, but not on swabbed stains to mimic casework. In this study, the sensitivity of the KM and LMG tests was tested on eight blood dilutions on cotton fabric and ceramic tile that were stained and subsequently swabbed. Both tests showed sensitivity up to 1:5000, which is slightly lower than reported values in solution or directly on stain but still highly effective in most cases. Stains were also cleaned with common agents, then swabbed and re-tested. Stained ceramic tiles cleaned with soap/water or bleach gave mixed positive and negative results for the 1:10 dilution, presumably due to variance in how thoroughly each investigator cleaned the stain, and other dilutions were undetectable after cleaning. The LMG test gave false positives for bleach cleaned stains, due to reagent reactivity with bleach. Surprisingly, blood was detectible up to the 1:100 dilution with both tests on stained cotton fabric that was cleaned in a washing machine with detergent and dried. Ultimately the KM and LMG presumptive tests remain effective tools for swabbed blood stains, and their practicality for cleaned stains is dependent on material containing the stain, cleaning agent and processing.

Selection and characterization of bispecific aptamers against malachite green and leucomalachite green.

In order to develop multi-residues rapid detection, the bispecific aptamers against malachite green (MG) and leucomalachite green (LMG) were isolated by the capture systematic evolution of ligands by exponential enrichment (Capture-SELEX). After thirteen rounds of selection, the enriched ssDNA pools were sent for high-throughput sequencing. Nine aptamer candidates (A1-A9) were picked out to test their specificity by gold nanoparticles (AuNPs) colorimetric assay. Three aptamers (A2, A3, A5) with good selectivity were truncated to verify their affinity by fluorescence assay. Finally, three truncated aptamers (A2-a, A3-a, A5-a) with bispecificity and high affinity were identified. For LMG, the dissociation constant (Kd) of them were 8.4 ± 0.8 nM, 8.2 ± 1.2 nM, and 13.7 ± 1.4 nM, respectively. For MG, Kd of them were 3.4 ± 0.3 µM, 2.3 ± 0.2 µM, 3.0 ± 0.2 µM. Among them, A3-a is the best. Our work will provide novel probes for the development of multi-residues rapid detection as well as opportunities for multiple target aptamer discovery.

Development of a PRiME cartridge purification method for rapid determination of malachite green and leucomalachite green in Chinese softshell turtle.

A high-throughput PRiME (process, robustness, improvements, matrix effects, ease of use) sample purification procedure was developed to simplify the multiple steps of traditional SPE in extracting the malachite green and leucomalachite green in Chinese softshell turtle (Pelodiscus sinensis). The sample loading volume, extracting solvent type, and pH value of the employed PRiME hydrophilic-lipophilic balance cartridge for sample purification were optimized to be 3 mL, acetonitrile, and pH 5, respectively. In comparison with traditional SPE, the PRiME process is cost-effective, solvent-saving, and simple to operate, which only consists of a passing through step without traditional sorbent conditioning and impurity washing. Afterward, eluate was analyzed by ultra-performance liquid chromatography-tandem mass spectrometry, and the proposed method was validated for linearity (R2 > 0.9992), intraday precision (2.44-3.22%), interday precision (3.28-6.58%), sensitivity (LOD ≤ 0.18 µg/kg and, LOQ ≤ 0.60 µg/kg), and recovery (88.7-94.1%, RSD < 6.79%). The results indicated that the PRiME technique can simplify the sample preparation procedure by avoiding the tedious steps, such as conditioning, washing, etc. It would be of significant interest for environmental and food safety applications in the market of Chinese softshell turtle and related products.

Arsenic Monitoring in Water by Colorimetry Using an Optimized Leucomalachite Green Method.

Arsenic contamination of drinking water is a global concern. Standard laboratory methods that are commonly used for arsenic detection in water, such as atomic absorption spectroscopy and mass spectroscopy, are not suitable for mass monitoring purposes. Autonomous microfluidic detection systems combined with a suitable colorimetric reagent could provide an alternative to standard methods. Moreover, microfluidic detection systems would enable rapid and cost efficient in situ monitoring of water sources without the requirement of laborious sampling. The aim of this study is to optimize a colorimetric method based on leucomalachite green dye for integration into a microfluidic detection system. The colorimetric method is based on the reaction of arsenic (III) with potassium iodate in acid medium to liberate iodine, which oxidizes leucomalachite green to malachite green. A rapid colour development was observed after the addition of the dye. Beer's law was obeyed in the range between 0.07⁻3 µg mL-1. The detection limit and quantitation limit were found to be 0.19 and 0.64 µg mL-1, respectively.

A sensitive electrochemical impedance immunosensor for determination of malachite green and leucomalachite green in the aqueous environment.

Application of malachite green (MG) and leucomalachite green (LMG) in fish farm water causes an environmental problem. This study proposes for the first time a sensitive and convenient electrochemical impedance spectroscopy (EIS) method for determining MG and LMG by a bovine serum albumin-decorated gold nanocluster (BSA-AuNC)/antibody composite film-based immunosensor. In order to improve the analytical performance, the glassy carbon electrode (GCE) was modified with 1, 4-phenylenediamine to form a stable layer, and then, BSA-AuNCs were covalently bound to the GCE. An adequate quantity of the polyclonal antibody of LMG was immobilized onto the surface of the BSA-AuNCs by the chemical reaction of EDC/NHS. The sensors can respond to the specific target based on specific covalent bonding. The experimental parameters, such as the pH, incubating concentration, and time, have been investigated and optimized. The calibration curve for LMG was linear in the range of 0.1~10.0 ng/mL with the limit of detection (LOD) 0.03 ng/mL. Furthermore, the sum of MG and LMG was detected in fish farm water by MG reduction. The recovery was between 89.7 % and 99.2 % in spiked samples. The EC sensor method was also compared with the ELISA method and validated by the LC-MS/MS method, which proves its great promise as a field instrument for the rapid monitoring of MG and LMG pollution. Graphical abstract 1, 4-Phenylenediamine and BSA-AuNC/antibody-decorated glassy carbon electrodes have been used for the impedimetric detection of the sum of malachite green and leucomalachite green via specific immuno-binding.

Magnetic solid-phase extraction for determination of the total malachite green, gentian violet and leucomalachite green, leucogentian violet in aquaculture water by high-performance liquid chromatography with fluorescence detection.

In this study, magnetic multi-walled carbon nanotube nanoparticles were synthesized and used as the adsorbent for the sums of malachite green, gentian violet and leucomalachite green, leucogentian violet in aquaculture water samples followed by high performance liquid chromatography with fluorescence detection. This method was based on in situ reduction of chromic malachite green, gentian violet to colorless leucomalachite green, leucogentian violet with potassium borohydride, respectively. The obtained adsorbent combines the advantages of carbon nanotubes and Fe3 O4 nanoparticles in one material for separation and preconcentration of the reductive dyes in aqueous media. The structure and properties of the prepared nanoparticles were characterized by transmission and scanning electron microscopy, X-ray diffraction, and Fourier-transform infrared spectroscopy. The main parameters affecting the adsorption recoveries were investigated and optimized, including reducing agent concentration, type and amount of sorbent, sample pH, and eluting conditions. Under the optimum conditions, the limits of detection in this method were 0.22 and 0.09 ng/mL for malachite green and gentian violet, respectively. Product recoveries ranged from 87.0 to 92.8% with relative standard deviations from 4.6 to 5.9%. The results indicate that the sorbent is a suitable material for the removal and concentration of triphenylmethane dyes from polluted environmental samples.

Evaluating the persistence of malachite green residues in tilapia and pacu fish.

Although banned in food-producing animals, residues of malachite green (MG) and its primary metabolite, leucomalachite green (LMG), have been found in fish due to illegal use in aquaculture and the release of industrial wastewater, which represent a serious risk to food and environmental securities. This study aimed to investigate the residue depletion profile of MG and LMG in edible tissues of Nile tilapia (Oreochromis niloticus) and pacu (Piaractus mesopotamicus) cultured simultaneously under the same environmental conditions to support control measures in case of abuse. An analytical method involving QuEChERS sample preparation and liquid chromatography coupled to tandem mass spectrometry was developed, validated, and applied to quantify MG and LMG residues in fish fillets from two depletion experiments after treatment by immersion bath (MG at 0.10 mg L-1 for 60 min). During the experiment, the average water temperature was 30 ºC, while the pH was 6.9. The method is selective, precise (CV = 0.4 - 22%) and accurate (recovery 92 - 114%). The limits of detection and quantification are 0.15 and 0.5 ng g-1, respectively. In both species, the sum of MG and LMG residues were quantified up to the 32nd day post-exposure, and the concentrations were significantly higher in the pacu fillets (up to 3284 ng g-1) than in Nile tilapia (up to 432 ng g-1). The sums of MG and LMG residues were below 2 ng g-1 at 44 days and 342 days for Nile tilapia and pacu, respectively - the Minimum Required Performance Limit (MRPL) for analytical methods intended to monitor forbidden substances in food according to old European Commission guidelines. The persistence of MG residues in pacu may be attributed to its higher lipid content, which favors the accumulation of the non-polar metabolite LMG. These results provide insights into the concern about human, animal, and environmental health risks resulting from unauthorized use or aquatic contamination by industrial wastewater containing MG residues.

Distribution and Probabilistic Risk Assessment of Antibiotics, Illegal Drugs, and Toxic Elements in Gastropods from Southeast China.

We investigated fourteen antibiotics, three illegal drugs, and two toxic elements in commercially available gastropods from southeast China. The data revealed high detection frequencies (DFs) for florfenicol (61.32%), florfenicol amine (47.33%), and thiamphenicol (39.88%), with maximum concentrations of 1110, 2222, and 136 µg/kg wet weight (ww), respectively. The DFs of illegal drugs were 3.54% for leucomalachite green and 0.3% for chloramphenicol. The average levels of Cd and As were 1.17 and 6.12 mg/kg ww, respectively. All chemicals presented diverse DFs in different sampling months. The highest DFs of florfenicol, florfenicol amine, and thiamphenicol were in July. The health risk assessment showed that targeted hazard quotients (THQs) of antibiotics, Cd, and As for children, teens, and adults were all less than one. Notably, the toxic elements (Cd and As) were identified as the primary health risk in gastropods, contributing to over 90% of the total THQs.

Dual-color immunochromatographic test strip for simultaneous sensitive detection of malachite green and leucomalachite green residues in fish and shrimp meat samples.

A sensitive dual immunochromatographic test strip (dual-ICTS) was developed to detect malachite green (MG) and its metabolite, leucomalachite green (LMG), using two types of gold nanoparticles: round-shaped (red) and star-shaped (blue). The detection limits were determined to be 0.221 µg L-1 for MG and 0.214 µg L-1 for LMG, respectively. The dual-ICTS provided a cut-off value of 1.8 µg L-1 for MG and LMG detection. The dual-ICTS successfully detected MG and LMG in food samples, with recovery rates ranging from 86 % to 116 %. The dual-ICTS was evaluated by correlation analysis between the proposed assay and the well-established enzyme-linked immunosorbent assay in the MG and LMG detection. This is the first report on the development of the ICTS that can detect both MG and LMG at the same time within only 5 min, making it a sensitive and rapid tool for on-site detection.

Optimization of leucomalachite green degradation through electron beam and persulfate oxidation using box-behnken design and life cycle assessment.

This study explored the treatment of Leucomalachite Green (LMG) solutions using an electron beam and sodium persulfate (Na2S2O8), employing Box-Behnken design (BBD) to optimize operational variables such as absorbed dose, initial pH and Na2S2O8 concentration. The findings highlighted an optimal absorbed dose of 4.5 kGy, a Na2S2O8 concentration of 1.0 mM, and an initial pH of 6, leading to a remarkable 97.77% removal of LMG. The adjusted R2 for the model indicated a close match of 1.4% between predicted and actual outcomes under these optimized conditions, affirming the quadratic model's suitability for predicting the LMG removal process using combined EB and Na2S2O8. To assess the environmental impact of the LMG treatment, the study applied SimaPro 9.4 with the TRACI tool, examining ten distinct environmental impact categories. The results unveiled that deionized water and Na2S2O8 exhibited a notable impact on global warming (GW) and ecotoxicity (ET) in controlled laboratory settings. Furthermore, a comparative analysis of four scenarios shed light on the environmental implications of different energy sources. Notably, electricity generated from waste incineration demonstrated a substantial influence on all environmental indicators. In contrast, natural gas emerged as the cleanest source for electricity generation, offering a promising avenue for reducing environmental impacts. This study presents a practical method for addressing dye contaminants through the employment of EB in conjunction with Na2S2O8, with potential implications for broader applications.

A highly sensitive hyperbranched Au plasmonic blackbody immunochromatographic assay for detection of leucomalachite green in fish and shrimp.

A novel immunochromatographic assay (ICA) based on hyperbranched Au plasmonic blackbodies (AuPBs) with a smartphone readout was fabricated for the detection of leucomalachite green (LMG) in fish and shrimp products. The ICA was carried out in a competitive immunoassay format with AuPBs as labels. The developed AuPBs-ICA allowed for the LMG detection with a low detection limit (0.15 µg L-1) within 5 min by the smartphone reader. With the label-AuPBs ICA, the color intensity response was linearly related to the concentrations of the LMG (0.2 -1.7 µg L-1). The test line signal could be clearly distinguished at a 1.7 µg L-1 LMG as a cut-off level by the naked eye, which is lower than the conventional colloidal gold nanoparticle (2 µg L-1) and star-shaped nanoparticles (1.9 µg L-1) labeling. LMG contamination in samples was determined with the proposed AuPBs-ICA and the enzyme-linked immunosorbent (ELISA). The AuPBs-ICA results showed good agreement with those from the ELISA. The proposed AuPBs-ICA has the potential to be used as a rapid, sensitive, and simple device for the analysis of LMG residues in fish and shrimp samples.

SERS detection of Hg2+ and aflatoxin B1 through on-off strategy of oxidase-like Au@HgNPs/carbon dots.

In this work, cerium-doped carbon dots (Ce-CDs) both as a reducing agent and template hybrid gold nanoparticles (AuNPs) with weak oxidase-like (OXD) activity was synthesized for the detection of Hg2+ and aflatoxin B1 (AFB1). The AuNPs can catalyze efficiently mercury ion (Hg2+) reduction to the metallic (Hg0) to form Au-Hg amalgam (Au@HgNPs). The obtained Au@HgNPs with strong OXD-like activity oxidize without Raman-active leucomalachite green (LMG) into the Raman-active malachite green (MG) and simultaneously as the SERS substrates by the formed Raman "hot spot" through MG-induced Au@HgNPs aggregation. While AFB1 was introduced resulting in the SERS intensity decreasing due to Hg2+ with AFB1 via carbonyl group to inhibit the aggregation of Au@HgNPs. The work paves a new path for the design of a nanozyme-based SERS protocol for tracing Hg2+ and AFB1 residues in foodstuff analysis.

[Sulfonated magnetic graphite carbon nitride solid-phase extraction-ultra performance liquid chromatography-tandem mass spectrometry for screening malachite green and leucomalachite green in freshwater fish].

Malachite green (MG) and its metabolite, leucomalachite green (LMG), exert toxic effects on the human body. The use of these dyes is illegal, but they are still detected in aquatic products. Freshwater fish are aquatic products with the high non-qualified rates. Therefore, the sensitive screening of MG and LMG in freshwater fish is of great importance to ensure the safety of aquatic products. Owing to the low contents of MG and LMG in fish and the complex matrix of actual samples, sample preparation is required before detection to purify impurities and enrich the target compounds. Graphite carbon nitride (GCN), a polymer material composed of C, N, and H, has good chemical and thermal stability, a large specific surface area, and a large number of active sites. It has a wide range of application prospects in adsorption and can be used in food safety testing when compounded with Fe3O4 to form magnetic graphite carbon nitride (MGCN). In this study, sulfonated magnetic graphite carbon nitride (S-MGCN) was prepared by further functionalizing MGCN with sulfonic acid. After characterization by scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FT-IR), and vibrating sample magnetometry (VSM), a magnetic solid-phase extraction (MSPE) method based on S-MGCN was established to extract MG and LMG from freshwater fish. The targets were screened using ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Following sulfonic acid functionalization, S-MGCN showed increased electrostatic interactions based on the MGCN adsorption mechanism, which includes hydrogen bonds and π-π interactions; thus, its adsorption efficiency was significantly improved. The matrix effects were -42.21% and -33.77% before functionalization, -11.40% and -7.84% after functionalization, thus confirming that S-MGCN has significant matrix removal ability. Given that S-MGCN demonstrated excellent efficiency as an MSPE adsorbent, the adsorption conditions for S-MGCN were optimized. The optimal conditions were as follows: adsorbent dosage, 15 mg; adsorption time, 2 min; solution pH, 5; and ionic strength, not adjusted. Under these conditions, the adsorption efficiency of S-MGCN could reach 94.2%. Different organic solvents were used to elute adsorbed MG and LMG, and the desorption efficiency peaked when 1%(v/v) ammonia acetonitrile was used as the elution solvent. The elution volume was also optimized, and a maximum desorption efficiency of 93.2% was obtained when 1 mL of 1%(v/v) ammonia acetonitrile was added to S-MGCN. The limits of detection (LODs) and quantification (LOQs) of the two targets were determined at signal-to-noise ratios (S/N) of 3 and 10, respectively. The LODs and LOQs were 0.075 µg/kg and 0.25 µg/kg, respectively. The linear ranges of the two target compounds were 0.25-20.0 µg/kg with correlation coefficients (r) greater than 0.998. To assess accuracy and precision, we prepared spiked samples at three levels (low, medium, and high) with six parallel samples per level (n=6). The recoveries ranged from 88.8% to 105.9%. The intra- and inter-day relative standard deviations were 5.4%-13.7% (n=6) and 3.3%-11.1% (n=3), respectively. Compared with the national standard method, the proposed method features simpler sample pretreatment procedures, less use of organic reagents (5 mL), and a shorter extraction time (2 min); moreover, the method does not require complicated elution steps, and the eluent can be directly analyzed by UPLC-MS/MS. The test results of actual samples were consistent with those obtained via the national standard method, thus confirming the practical feasibility of the developed method. The proposed MSPE method based on S-MGCN is an efficient and environmentally friendly method that could provide a new methodological reference for the sensitive screening of MG and LMG in actual samples.

Enzyme-Free Tandem Reaction Strategy for Surface-Enhanced Raman Scattering Detection of Glucose by Using the Composite of Au Nanoparticles and Porphyrin-Based Metal-Organic Framework.

In this work, an S hybrid nanosheet with multiple functions is synthesized by in situ modification of gold nanoparticles (AuNPs) onto two-dimensional (2D) metalloporphyrinic metal-organic framework (MOF) (Cu-tetra(4-carboxyphenyl)porphyrin chloride(Fe(III)), designated as AuNPs/Cu-TCPP(Fe). Cu-TCPP(Fe) nanosheets contribute peroxidase-like activity, and AuNPs have glucose oxidase (GOx) mimicking performance, which induce the cascade catalysis reactions to convert glucose into hydrogen peroxide (H2O2), and then, by using AuNP catalysis, H2O2 oxidizes the no Raman-active leucomalachite green (LMG) into the Raman-active malachite green (MG). Simultaneously, in the presence of AuNPs, sensitive and selective surface-enhanced Raman scattering (SERS) determination of glucose can be achieved. The bioenzyme-free SERS assay based on such AuNPs/Cu-TCPP(Fe) nanosheets is used for detection of glucose in saliva, showing good recovery from 96.9 to 100.8%. The work paves a new way to design a nanozyme-based SERS protocol for biomolecule analysis.

Development of qualitative and quantitative immunochromatographic strip test assay for rapid and simple detection of leucomalachite green residual in aquatic animals.

A rapid and simple immunochromatographic strip test assay based on competitive format was developed for leucomalachite green (LMG) detection. LMG-bovine serum albumin and rabbit anti-sheep IgG were immobilized on nitrocellulose membrane for the test line and control line, respectively. Anti-LMG-colloidal gold conjugate was immobilized onto the conjugate pad. For qualitative detection, the cut-off limit of the strip test was determined at 2 µg/L by the naked eye. For quantitative analysis, the working range of the LMG detection was 0.7-2 µg/L with LOD at 0.28 µg/L. A one-step immunochromatographic strip test for LMG detection can be completed within 5 min without any incubation, washing and blocking steps. Analysis results of LMG in aquatic animals obtained from the immunochromatographic strip test were in good agreement with those realized from enzyme-link immunosorbent assay. The developed the immunochromatographic strip test offered rapid detection as a simple (one-step), cost-effective, instrument-free assay and no need for handling reagents.

[Simultaneous and rapid determination of malachite green and leucomalachite green by a label-free colorimetric aptasensor].

A label-free colorimetric aptasensor, using a bispecific aptamer (A3) as a sensing probe, gold nanoparticles (AuNPs) as an indicator, and NaCl solution as an aggregation inducer, was successfully developed for the simultaneous, rapid and visual detection of malachite green (MG) and leucomalachite green (LMG) in aquatic products. This method is based on the aptamer A3 having bispecific binding ability with MG and LMG, making it an ideal recognition receptor for MG and LMG. It can adsorb on the AuNPs and protect AuNPs against salt-induced aggregation, maintaining the red color of the solution. When MG or LMG was added to a solution, aggregation of AuNPs was specifically induced by desorption of aptamer from the AuNPs surface upon formation of the aptamer-target complex. Therefore, the salt could trigger aggregation of AuNPs and the solution color was changed from red to blue. This color change allowed the qualitative determination of MG and LMG visually, and quantitative determination by measuring the ratio of the absorbances at 520 nm and 650 nm. In this study, 50 µL of the nucleic acid aptamer A3 (final concentrations 150 nmol/L) and 150 µL of AuNPs (final concentrations 1.25 nmol/L) were incubated at room temperature (RT) for 6 min, then 50 µL of the sample was added and incubated at RT for 30 min, and finally 50 µL NaCl solution (final concentrations 150 mmol/L) was added. After 4 min, the solution color change was observed, and the absorbances at 520 nm and 650 nm were measured. Under the optimal conditions, MG and LMG could be detected specifically without any cross-reactivity with sulfadiazine (SDZ) and nitrofurantoin (NFT). The absorbance were related to the concentrations of MG and LMG, and a good linear relationship was obtained in the range of 0-17.5 µmol/L. The correlation coefficients (R2) were 0.9938 and 0.9715, respectively. The limits of detection of MG and LMG were 6.93 nmol/L and 6.38 nmol/L, respectively. The spiked recoveries of MG and LMG ranged from 88.60% to 93.30% and 101.80% to 107.00%, respectively. The relative standard deviations (RSDs) of MG and LMG ranged from 2.27% to 3.55% and 2.62% to 3.75%, respectively. This colorimetric method is simple, rapid, sensitive, and allows visual, and it can provide a new method for the simultaneous and rapid determination of the MG and LMG in aquatic products.

Accurate SERS detection of malachite green in aquatic products on basis of graphene wrapped flexible sensor.

Malachite Green (MG) is a banned pesticide for aquaculture products. As a required inspection item, its fast and accurate determination before the products' accessing market is very important. Surface enhanced Raman scattering (SERS) is a promising tool for MG sensing, but it requires the overcoming of several problems such as fairly poor sensitivity and reproducibility, especially laser induced chemical conversion and photo-bleaching during SERS observation. By using a graphene wrapped Ag array based flexible membrane sensor, a modified SERS strategy was proposed for the sensitive and accurate detection of MG. The graphene layer functioned as an inert protector for impeding chemical transferring of the bioproduct Leucomalachite Green (LMG) to MG during the SERS detection, and as a heat transmitter for preventing laser induced photo-bleaching, which enables the separate detection of MG and LMG in fish extracts. The combination of the Ag array and the graphene cover also produced plentiful densely and uniformly distributed hot spots, leading to analytical enhancement factor up to 3.9 × 108 and excellent reproducibility (relative standard deviation low to 5.8% for 70 runs). The proposed method was easily used for MG detection with limit of detection (LOD) as low as 2.7 × 10-11 mol L-1. The flexibility of the sensor enable it have a merit for in-field fast detection of MG residues on the scale of a living fish through a surface extraction and paste transferring manner. The developed strategy was successfully applied in the analysis of real samples, showing good prospects for both the fast inspection and quantitative detection of MG.

In vitro interactions of malachite green and leucomalachite green with hepatic drug-metabolizing enzyme systems in the rainbow trout (Onchorhyncus mykiss).

Malachite green (MG) has been widely used in aquaculture to treat a number of microbial and parasitic diseases. It is currently banned in the EU because of the high cytotoxicity and carcinogenic activity, which is also shared by leucomalachite green (LMG), a reduced MG metabolite that can persist in fish tissues for months. There is scant information about the ability of either compound to interact with drug metabolizing enzymes in fish. Therefore we evaluated the in vitro effects of MG and LMG (25, 50 and 100µM) on some DMEs and glutathione (GSH) content in rainbow trout liver subfractions. LMG did not affect any of the examined parameters. In contrast, MG proved to deplete GSH and to depress to a various extent the activities of NAD(P)H cytochrome c reductase, 7-ethoxycoumarin O-deethylase, 1-naphthol uridindiphosphoglucuronyl-transferase and maximally those of 7-ethoxyresorufin O-deethylase (EROD) and glutathione S-transferase (GST) accepting 1-chloro2,4-dinitrobenzene (CDNB) as substrate. The inhibition mechanisms of EROD and GST were investigated by means of non-linear Michaelis-Menten kinetics and Lineweaver-Burk plots using 0.175-8µM MG. The calculated IC50 for EROD was 7.1µM, and the inhibition appeared to be competitive (Ki 2.78±0.24µM). In the case of GST, the calculated IC50 was 0.53µM. The inhibition was best described as competitive toward GSH (Ki 0.39±0.02µM) and of mixed-type toward CDNB (Ki 0.64±0.06µM). Our findings indicate that, contrary to LMG, MG behaves as a relatively strong inhibitor of certain liver DMEs and can reversibly bind GSH.

Bispecific Monoclonal Antibody-Based Multianalyte ELISA for Furaltadone Metabolite, Malachite Green, and Leucomalachite Green in Aquatic Products.

A new multianalyte immunoassay was designed to screen furaltadone metabolite 5-morpholinomethyl-3-amino-2-oxazolidone (AMOZ), malachite green (MG), and leucomalachite green (LMG) in aquatic products using a bispecific monoclonal antibody (BsMAb). Gradient drug mutagenesis methods were separately used to prepare an anti-3-nitrobenzaldehyde-derivatized AMOZ (3-NPAMOZ) hybridoma cell line that was hypoxanthine-guanine-phosphoribosyltransferase (HGRPT) deficient and an anti-LMG hybridoma cell line that was thymidine kinase (TK) deficient. BsMAb recognizing 3-NPAMOZ and LMG was generated using hybrid-hybridomas of HGRPT and TK deficient cell lines. For AMOZ and LMG, respectively, the BsMAb-based indirect competitive ELSIA (ic-ELISA) values of 1.7 ng/mL and 45.3 ng/mL and detection limits of 0.2 ng/mL and 4.8 ng/mL. To establish the ic-ELISA, 3-NPAMOZ derivatized from AMOZ with 3-nitrobenzaldehyde and LMG reduced from MG by potassium borohydride was recognized by BsMAb. Recoveries of AMOZ, MG, and LMG in aquatic products were satisfactory and correlated with HPLC analysis. Thus, the multianalyte ic-ELISA is suitable for rapid quantification of AMOZ, MG, and LMG in aquatic products.

Rapid analysis of malachite green and leucomalachite green in fish muscles with surface-enhanced resonance Raman scattering.

Surface-enhanced resonance Raman scattering (SERRS) coupled with gold nanospheres was applied for rapid analysis of the hazardous substances malachite green (MG) and leucomalachite green (LMG) in fish muscle tissues. The lowest concentration of MG that could be detected was 0.5ngmL(-1) with high linear correlation (R(2)=0.970-0.998) between MG concentration and intensities of characteristic Raman peaks. A simplified sample preparation method taking less than 1h for recovering MG and LMG in fish fillets was developed for SERRS analysis, and 4-8 samples could be handled in parallel. MG and LMG could be detected in extracts of tilapia fish fillets at as low as 2ngg(-1) with SERRS and a simple principle component analysis method. For six other fish species, the lowest detectable concentration of MG ranged from 1ngg(-1) to 10ngg(-1). This study provides a new sensitive approach for the detection of trace amounts of the prohibited drugs MG and LMG in muscle food, which has the potential for rapidly screening a large number of samples.