Responses of gut microbiota in crocodile lizards (Shinisaurus crocodilurus) to changes in temperature.

The gut microbiota plays an essential role in maintaining the health and fitness of the host organism. As a critical environmental variable, temperature exerts significant effects on animal survival and reproduction. Elevated temperatures can influence the composition and function of the animal gut microbiota, which may have potentially detrimental effects on the host. The crocodile lizard (Shinisaurus crocodilurus) is an ancient and currently endangered reptile species due to human hunting and habitat destruction. Given the predicted shifts in global temperatures in the next century, it is important to understand how warming affects the gut microbiota of these vulnerable lizards, which remains unclear. To determine how the microbial communities change in crocodile lizards in response to warming, we analyzed the gut microbiota under five temperature conditions (22°C, 24°C, 26°C, 28°C, and 30°C) using 16S rRNA high-throughput sequencing. Results showed that the dominant phyla, Proteobacteria and Bacteroidetes, in gut microbiota were not significantly affected by temperature variations, but increasing temperature altered the structure and increased the community richness of the gut microbiota. In addition, warming changed the abundance of Pseudomonas aeruginosa and Actinobacteria, which may have negative effects on the physiological health of the crocodile lizards. Functional prediction analysis demonstrated that the functional pathways enriched in crocodile lizards were mainly related to metabolism, with no significant differences observed in these pathways at KEGG pathway level 1 after warming. These results provide valuable insights into the ecological adaptations and regulatory mechanisms employed by crocodile lizards in response to warming, which may be of benefit for their conservation.

Fluorimetric identification of sulfonamides by carbon dots embedded photonic crystal molecularly imprinted sensor array.

Identification of sulfonamides (SAs) residues in food is vital for human health. A set of 4-channel sensor array was constructed by carbon dots (CDs) embedded in photonic crystal molecularly imprinted (PCMIP@CDs) film which included 3 PCMIP@CDs units and 1 PCNIP@CDs unit to determine typical SAs: sulfadimethoxine, sulfathiazole, sulfaguanidine, sulfamethazine, sulfadiazine. Under the optimal conditions, the response time of the sensor array was only 200 s. Moreover, 300 fluorescence response signals (4 sensor units × 5 sulfonamides × 3 concentrations × 5 repeats) were processed by pattern recognition technique to analyze the ability of the sensor array to recognize 5 kinds of SAs. Subsequently, the linear discrimination analysis (LDA) method was used to identify the five SAs simultaneously with 100 % classification accuracy and the limit of detection was 0.01-0.26 nmol/L. Moreover, the proposed method can effectively identify-five SAs in water and fish samples.

Histamine detection in fish samples based on indirect competitive ELISA method using iron-cobalt co-doped carbon dots labeled histamine antibody.

Due to complex matrixes and specific reagent deficiency, the rapid detection of histamine is still a challenge to date. Based on the high peroxidase-like activity of iron-cobalt co-doped carbon dots, an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) was established for histamine detection using the mimic enzyme labeled with histamine antibody (His-Ab). Through the competitive binding of the labeled His-Ab to solid-phase and sample antigens, histamine content was detected with a linear range of 2.5-150 µg mL-1. The detection limit based on 3σ/K was 0.50 mg kg-1, which was much lower than those of commercial His-kit and HPLC methods. The ic-ELISA method was applied to histamine detection in fish samples with the recovery of (103.4 ± 0.5)%, which was in accord with those of commercial His-kit and HPLC methods. The results indicated that the established ic-ELISA method was suitable for rapid detection of histamine in fish samples with high accuracy, sensitivity and stability.

Rapid extraction of domoic acid by a magnetic molecularly imprinted silica before HPLC measurement.

A magnetic molecularly imprinted silica solid was obtained by sol-gel polymerization for the separation of domoic acid. The solid showed rapid adsorption kinetics with an adsorption equilibrium time of 5 min. The solid showed affinity to domoic acid under the interference of tryptophan and could be repeatedly used for 5 times at least. The solid was used as a solid-phase-extraction sorbent for the extraction of domoic acid from clam samples before measurement with liquid chromatography. The detection limit of 0.20 mg kg-1 was lower than the allowable limits in several countries or areas. The recoveries in the spiked samples were 88% approximately.

Aptamer-based fluorometric determination of chloramphenicol by controlling the activity of hemin as a peroxidase mimetic.

A method for the aptamer-based determination of chloramphenicol (CAP) was developed by exploiting the peroxidase mimicking activity of hemin. The method includes two hemin-modified DNA probes termed P1 and P2. P1, which was modified at its 5' end with one hemin monomer, contains the CAP-binding sequence. The hybridization between P1 and P2 brings the two hemin monomers in close proximity, resulting in the formation of a hemin dimer with low peroxidase mimicking activity. The duplex structure was dehybridized in the presence of CAP. The formed hemin monomer featured a strong peroxidase mimicking activity and catalyzed the conversion of non-fluorescent tyramine into fluorescent dityramine by hydrogen peroxide. Fluorescence (with an excitation/emission maxima at 320 and 410 nm, respectively) increased linearly in the 0.1 ng mL-1 to 10 ng mL-1 CAP concentration range. The detection limit based on the 3σ/k criterion reached 0.07 ng mL-1. The proposed assay was successfully employed for CAP detection in (spiked) honey samples with recoveries of 94.3-117.2%. Given its high sensitivity and good stability, this method shows potential in providing a platform for antibiotic detection.

Spectrophotometric detection of fenthion in foods after extraction by magnetic zirconia.

A magnetic material, Fe3O4@ZrO2, was used to enrich an organophosphorus pesticide, fenthion. After enrichment the Fe3O4@ZrO2 was treated with NaOH to elute and hydrolyze fenthion. The hydrolysis products, orthophosphate ions, combine to molybdate ions to yield molybdophosphoric acid, which was further reduced with SnCl2 to obtain a blue phosphatemolybdate. The content of orthophosphate ions as well as fenthion can be determined through the spectrophotometric method. Adsorption and elution conditions were optimized to obtain an enrichment factor of 12.5. The proposed composite method was successfully used to analyze the fenthion residues on the skin of cucumber and apple samples. The limit of detection was as low as 0.037 mg/Kg, which was close to that of the gas chromatography-mass spectrometer method. The method was simple, rapid, and economic, suitable for the rapid screen of fenthion and the other organophosphorus pesticide in mass samples.

Molecularly imprinted polymer-based photonic crystal sensor array for the discrimination of sulfonamides.

In this paper, molecular imprinting and photonic crystal techniques were combined to construct a four-channel sensor array for the simultaneous identification of various sulfonamides. The assay was composed of four units. Three of these units were prepared using sulfaguanidine, sulfamethazine, or sulfathiazole as template molecules. The fourth unit was prepared without a template molecule. The preparation was optimized to obtain maximum identification with a molar ratio of template, monomer, and cross-linker of 1:50:10. The response time was as short as 10 min. For demonstration, six sulfonamides were selected as analytes. The Bragg diffraction patterns of analytes at different concentrations were measured using the sensor array. Data obtained were analyzed using linear discrimination analysis (LDA) and principal component analysis (PCA). LDA can be applied for SAs discrimination. The message ratios of 87.6%, 94.4%, and 95.8% for six SAs at 10-4 mol L-1, 10-6 mol L-1, and 10-8 mol L-1 were achieved using LDA. The sensor array identified the mixture containing various SAs with an LDA coefficient of 86.1%, thereby indicating that the sensor array had a strong anti-interference ability. The sensor array was used to identify six SAs in fish samples. The measured data in spiked samples were consistent with the fingerprint collected from standard solutions. The accuracy rate reached 90.9%, indicating that the array can be used to identify SAs from food samples.

Rapid detection of trace malachite green using a fluorescence probe based on signal amplification through electrostatic self-assembly of CdTe QDs and polystyrene microsphere.

A fluorescence probe was delicately designed for the detection of malachite green (MG) in water and fish samples. Through the electrostatic self-assembly of CdTe QDs on the surface of polystyrene (PS) microspheres, the fluorescence signal was amplified. After grafting molecularly imprinted film, the fluorescence probe of MIP@PS@CdTe was fabricated and applied to the detection of MG based on fluorescence quenching. The linear range of MG detection was 0.01-20 µmol L-1, and the detection limit was 4.7 nmol L-1 (3σ, n = 9) which was much lower than those of the previous reports. The recoveries of MG in aquaculture water and fish samples ranging from 87.6% to 105.4% illustrated that the detection by MIP@PS@CdTe probe was accurate and reliable.

A dichromatic label-free aptasensor for sulfadimethoxine detection in fish and water based on AuNPs color and fluorescent dyeing of double-stranded DNA with SYBR Green I.

A dichromatic label-free aptasensor was described for sulfadimethoxine (SDM) detection. Compared with the binding of SDM-aptamer to SDM, the higher affinity of aptamer to cDNA may result in the hybridization of dsDNA. In the presence of SDM, the aptamer specifically binds to SDM, leading to a blue color of AuNPs in deposit and fluorescence at 530 nm in supernatant after adding cDNA and SGI. With no target of SDM, AuNPs protected with the aptamer re-disperse in PBS with a red color, and no fluorescence occurs in supernatant. Based on the principle, SDM can be quantitatively detected through both fluorescent emission and AuNPs color changes with recoveries ranging from 99.2% to 102.0% for fish and from 99.5% to 100.5% for water samples. An analytical linear range of 2-300 ng mL-1 was achieved with the detection limits of 3.41 ng mL-1 for water and 4.41 ng g-1 for fish samples (3σ, n = 9).

Detection of Malachite Green using a colorimetric aptasensor based on the inhibition of the peroxidase-like activity of gold nanoparticles by cetyltrimethylammonium ions.

A specific and sensitive colorimetric aptasensor is described for the determination of Malachite Green (MG). It is exploiting the inhibition of the peroxidase-like activity of gold nanoparticles (AuNPs). The AuNPs act as enzyme mimics that catalyze the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) by H2O2 to yield a dark blue solution. The catalytic activity is inhibited by hexadecyl trimethyl ammonium ion, specifically by cetyltrimethylammonium bromide (CTAB), which causes the aggregation of AuNPs. If a (negatively charged) RNA-aptamer against MG is added, it binds to the positively charged CTAB and prevents aggregation. This enhances the enzyme mimicking activity of the AuNPs and leads to the formation of a dark blue solution. However, in the presence of MG, the aptamer binds to MG, and leads to the aggregation of AuNPs again. The aggregated AuNPs possess a light blue color. A colorimetric method (best performed at 650 nm) was work out that can detect MG in a concentration range from 10 to 500 nmol L-1. The detection limit based on 3σ/k criterion is 1.8 nmol L-1. The assay is highly specific and accurate. Recoveries from spiked real samples (aquaculture water) ranged from 80% to 120%. Graphical abstract Based on the inhibition of cetyltrimethyal ammonium ion and the enhancement of RNA-aptamer, the differences of the peroxidase-like activities of AuNPs can be greatly enlarged with and without MG, by which a colorimetric aptasensor can be constructed for the detection of Malachite Green (MG).

Label-Free Fluorescence-Based Aptasensor for the Detection of Sulfadimethoxine in Water and Fish.

Fluorescence-based aptasensors possess high sensitivity but are complicated and usually require multistep labeling and modification in method design, which severely limit the practical applications. Here, a label-free fluorescence-based aptasensor, consisting of aptamer, gold nanoparticles (AuNPs), and cadmium telluride (CdTe) quantum dots (QDs), was developed for the detection of sulfadimethoxine (SDM) in water and fish based on the specific recognition of SDM-aptamer and the inner filter effect of QDs and AuNPs. In the absence of a target, AuNPs dispersed in salt solution because of the aptamer protection, which could effectively quench the fluorescence emission of QDs, while in the presence of SDM, AuNPs aggregated due to the specific recognition of SDM-aptamer to SDM, which resulted in fluorescence recovery. A linear response of SDM concentrations in the range of 10-250 ng mL-1 ( R2 = 0.99) was obtained, and the detection limit was 1.54 ng mL-1 (3σ, n = 9), far below the maximum residue limit (100 ng mL-1) of SDM in edible animal tissues regulated by China and the European Commission. The fluorescence-based aptasensor was applied to the detection of SDM in aquaculture water and fish samples with high accuracy, excellent precision, and ideal selectivity. The results indicated that the developed aptasensor was simple in design, easy to operate, and could be used to detect rapidly and accurately SDM in water and fish samples.

Rapid detection of sulfaguanidine in fish by using a photonic crystal molecularly imprinted polymer.

A photonic crystal (PC) sensor was prepared through molecular imprinting by using dispersed SiO2 microspheres as PC, sulfaguanidine (SG) as template, methacrylic acid as monomer, and ethylene glycol dimethacrylate as crosslinker. The preparation conditions were optimized as follows: molar ratio of template, monomer, and crosslinker was 1:10:4; pH was 6.0 in phosphate buffer; and volume fraction of methanol was 15%. The relationship between wavelength shift of reflection peak and SG concentration was found as: Δλ = 7.8887 lg(C) + 79.9664. The response time was only 5 min. The limit of detection was 2.8 × 10-10 mol L-1. The sensor exhibited higher specificity for SG than its sulfonamide analogs. The sensor maintained original sensing performance after five cycles of usage. The recoveries of SG ranged from 93.8% to 111.2% in lake water and from 88.5% to 115.2% in fish samples. This finding suggested that the sensor can be used in food samples with complicated matrix.

Ratiometric fluorescence probe of MIPs@CdTe QDs for trace malachite green detection in fish.

A facile and practical ratiometric fluorescence probe based on two CdTe quantum dots (QDs) coated with molecularly imprinted polymers (MIPs) was prepared for the detection of trace malachite green (MG) in fish. Two CdTe QDs coated with MIPs were fabricated by a one-pot method using MG, (3-aminopropyl) triethoxysilane (APTES) and tetraethyl orthosilicate (TEOS) as template, functional monomer, and cross-linker, respectively. CdTe QDs with λem 530 nm (gQDs) and 630 nm (rQDs) were used as the referential fluorophore and target sensitive fluorophore, respectively. The fluorescence intensity of gQDs remained unchanged in the presence of MG, while the fluorescence of rQDs could be quantitatively quenched by MG based on the strategy of fluorescence resonance energy transfer. The ratiometric fluorescence probe (MIPs@gQDs&rQDs) was characterized by transmission electron microscopy and Fourier transform infrared spectroscopy. The linear range of MG detection was 0.1-32 µmol L-1 with a detection limit of 8.8 µg kg-1. The constructed probe has been successfully applied to the detection of MG in fish with the recoveries of 92.3-109.1%, which were validated by the method of HPLC. The result indicated that the probe possessed rapid response, wide linear range, high sensitivity, and relatively high selectivity, and was low-cost and easy in operation in the detection of MG in fish samples.

Detection of malachite green in fish based on magnetic fluorescent probe of CdTe QDs/nano-Fe3O4@MIPs.

A magnetic fluorescent probe of CdTe QDs/nano-Fe3O4@MIPs was prepared using CdTe QDs and Fe3O4 nanoparticles as co-nucleus and molecularly imprinted polymers (MIPs) as specific recognition sites based on a reverse microemulsion method. With the specific enrichment and magnetic separation properties, the probe of CdTe QDs/nano-Fe3O4@MIPs was used to detect malachite green (MG) in fish samples. The TEM analysis showed that the particles of CdTe QDs/nano-Fe3O4@MIPs were spherical with average diameter around 53nm, and a core-shell structure was well-shaped with several Fe3O4 nanoparticles and CdTe QDs embedded in each of the microsphere. Quick separation of the probes from solutions could be realized with a magnet, indicating the excellent magnetic property of CdTe QDs/nano-Fe3O4@MIPs. The probe exhibited high specific adsorption towards MG and excellent fluorescence emission at λem 598nm. The fluorescence of CdTe QDs/nano-Fe3O4@MIPs could be linearly quenched by MG at the concentrations from 0.025 to 1.5µmolL-1. The detection limit was 0.014µmolL-1. The average recovery of spiked MG in fish samples was 105.2%. The result demonstrated that the as-prepared CdTe QDs/nano-Fe3O4@MIPs could be used as a probe to the detection of trace MG in fish samples.

Detection of trace tetracycline in fish via synchronous fluorescence quenching with carbon quantum dots coated with molecularly imprinted silica.

A novel fluorescence-based sensor combining synchronous fluorescence spectroscopy (SFS) with molecularly imprinted polymers (MIPs) was fabricated with reverse microemulsion method. Tetracycline (TC), (3-aminopropyl) triethoxysilane (APTES), tetraethyl orthosilicate (TEOS) and carbon quantum dots (CDs) were used as template, functional monomer, cross-linker and signal sources respectively in the probe preparation. A synchronous fluorescence emission (λem) at 355nm was observed for the prepared MIP-coated CDs (MIP@CDs) particles when the wavelength interval (Δλ) was set as 70nm, and the synchronous fluorescence intensity could be rapidly and efficiently quenched by TC based on inner filter effect (IFE). The quenching efficiencies of synchronous fluorescence intensity was linearly fitted with tetracycline (TC) concentrations ranging from 0.1 to 50µmolL-1 with a detection limit (DL) of 9nmolL-1 (3σ, n=9). The MIP@CDs was used as a probe to detect TC in fish samples with the recoveries ranging from 98.4% to 103.1% and the relative standard deviation less than 6.0%. The results illustrated that the as-prepared MIP@CDs could be applied to the detection of trace TC in fish samples with rapidity, high sensitivity and accuracy.

Biomimetic ELISA detection of malachite green based on molecularly imprinted polymer film.

A highly selective and sensitive enzyme-linked immunosorbent assay (ELISA) was developed for the detection of malachite green (MG) using a molecularly imprinted polymer (MIP) film as bionic antibody. The MIP film, based on the self-polymerization of dopamine, was fabricated on the surfaces of a 96-well microplate. It showed specific recognition for MG in aqueous solution. A direct competitive ELISA method was established with the sensitivity reaching 10.31µgL-1 and the detection limit being 0.3µgL-1. The cross-reactivity of two structural analogues to MG was less than 10%. The average recovery tested by MG standard spiking was 88.8% for bass and 90.4% for water, and the relative standard deviations were less than 3.6%. All the above results indicated that the developed method could be used to detect MG in fish and water samples rapidly, specifically and accurately.

Rapid detection of malachite green in fish based on CdTe quantum dots coated with molecularly imprinted silica.

A sensitive fluorescence sensor for the detection of malachite green (MG) was fabricated by grafting molecularly imprinted polymers (MIPs) onto the surface of CdTe quantum dots (QDs). The MIP-coated QDs were synthesized via a reverse microemulsion method using (3-aminopropyl)triethoxysilane (APTES) and tetraethyl orthosilicate (TEOS) as functional monomer and cross-linker, respectively. The optimum molar ratio of MG, functional monomer and cross-linker was 1:3:10. The MIP-coated QDs exhibited uniform spheres with diameter around 49nm and excellent fluorescence emission at λex 370nm. A linear relationship with two segments between the relative fluorescence intensities and the MG concentrations ranging from 0.08 to 20µmol·L-1 could be obtained with a detection limit of 12µg·kg-1. The fluorescent probe was successfully applied to the determination of MG in fish samples with the spiked recoveries ranging from 94.3% to 109.5% which were in accordance with those of the measurement by HPLC-UV.

Biomimetic ELISA detection of malachite green based on magnetic molecularly imprinted polymers.

A direct competitive enzyme-linked immunosorbent assay (ELISA) method was used for the detection of malachite green (MG) with a high sensitivity and selectivity using magnetic molecularly imprinted polymers (MMIPs) as a bionic antibody. MMIPs were prepared through emulsion polymerization using Fe3O4 nanoparticles as magnetic nuclei, MG as a template, methacrylic acid (MAA) as a functional monomer, ethylene glycol dimethacrylate (EGDMA) as a crosslinking agent and span-80/tween-80 as mixed emulsifiers. The MMIPs were characterized by scanning electron micrographs (SEM), thermal-gravimetric analyzer (TGA), Fourier transform infrared spectrometer (FT-IR) and vibrating sample magnetometer (VSM), respectively. A high magnetic saturation value of 54.1emug-1 was obtained, resulting in rapid magnetic separation of MMIPs with an external magnet. The IC50 of the established ELISA method was 20.1µgL-1 and the detection limit (based on IC85) was 0.1µgL-1. The MMIPs exhibited high selective binding capacity for MG with cross-reactivities less than 3.9% for MG structural analogues. The MG spiking recoveries were 85.0%-106% with the relative standard deviations less than 4.7%. The results showed that the biomimetic ELISA method by using MMIPs as bionic antibody could be used to detect MG rapidly in fish samples with a high sensitivity and accuracy.

Determination of domoic acid in shellfish extracted by molecularly imprinted polymers.

A selective sample cleanup method using molecularly imprinted polymers was developed for the separation of domoic acid (a shellfish toxin) from shellfish samples. The molecularly imprinted polymers for domoic acid was prepared by emulsion polymerization using 1,3,5-pentanetricarboxylic acid as the template molecule, 4-vinyl pyridine as the functional monomer, ethylene glycol dimethacrylate as the crosslinker, and Span80/Tween-80 (1:1 v/v) as the composite emulsifiers. The molecularly imprinted polymer showed high affinity to domoic acid with a dissociation constant of 13.5 µg/mL and apparent maximum adsorption capacity of 1249 µg/g. They were used as a selective sorbent for the detection of domoic acid from seafood samples coupled with high-performance liquid chromatography. The detection limit of 0.17 µg/g was lower than the maximum level permitted by several authorities. The mean recoveries of domoic acid from clam samples were 93.0-98.7%. It was demonstrated that the proposed method could be applied to the determination of domoic acid from shellfish samples.