Novel electrochemiluminescence sensing platform for ultrasensitive detection of malathion residue in tea based on SiO2NSs doped Luminol/AgNPs as a signal amplification strategy.

To address the potential hazards of organophosphorus pesticides (OPs) residues in tea, an electrochemiluminescence (ECL) aptasensor based on functionalized nanomaterials was constructed in this work. Firstly, gold nanoparticles (AuNPs) were attached on the surface of multi-walled carbon nanotubes (MWCNTs) by the constant potential electrodeposition to form a compound, and it was utilized to provide excellent immobilization sites for complementary DNA (cDNA). Subsequently, composite nanomaterials were synthesized by a one-pot method with aminated Luminol/silver nanoparticles@silica nanospheres (NH2-Luminol/Ag@SiO2NSs). Finally, NH2-Luminol/Ag@SiO2NSs was combined with a malathion aptamer (Apt) to obtain signal probes (SPs) for the construction of an aptasensor. The aptasensor had a wide linear range (1×10-3-1×103 ng/mL) and a low limit of detection (LOD) (0.3×10-3 ng/mL). It had the virtues of high sensitivity, wonderful stability and excellent specificity, which could be used for the detection of malathion residue in tea. The work provides a proven way for the construction of a rapid and ultrasensitive aptasensor with low-cost.

Combination of Capture-SELEX and Post-SELEX for procymidone-specific aptamer selection and broad-specificity aptamer discovery, and development of aptamer-based lateral flow assay.

BACKGROUND: Due to its wide application, procymidone has become one of the pesticides with high detection rates in supervision and sampling. Therefore, it is necessary to establish a rapid and efficient method for the detection of procymidone. However, an important bottleneck restricting the development of rapid detection methods of procymidone is that its specific recognition elements are rarely reported. In this work, Capture-SELEX and post-SELEX were used in aptamer screening, and the obtained aptamers were used to construct an aptamer-based lateral flow assay (LFA). RESULTS: Firstly, a specific aptamer Seq15 was obtained for procymidone by Capture-SELEX, and its dissociation constant (Kd) was 24.22 nM. Secondly, post-SELEX was used to analyze and modify Seq15 to improve its performance, and the Kd of the truncated sequence Seq15-2 was 21.28 nM. In addition to this, the broad-specificity aptamer Seq17-1 was obtained via post-SELEX. Seq17-1 could broadly recognize dicarboximide fungicides (procymidone, iprodione, chlozolinate, dimethachlon and vinclozolin) and their metabolic derivative (3,5-dichloroaniline). Finally, the specific aptamer-based LFA of procymidone was constructed, and the limit of detection (LOD) was 0.79 ng/mL. Meanwhile, the LODs of dicarboximide fungicides and their metabolic derivative were 0.62, 0.64, 0.71, 0.69, 0.64 and 0.66 ng/mL, respectively. The above LFAs were highly specific and stable, and had been successfully used for the detection of vegetable samples. SIGNIFICANCE: Under the combination of Capture-SELEX and Post-SELEX, this study not only provides specific recognition elements for rapid detection of procymidone, but also provides new ideas for the discovery of broad-specificity aptamers. Combining broad-specificity primary detection and single-specificity quantification, a composite aptamer-based LFA detection platform has been developed, which significantly improves detection efficiency.

Multifunctional nanoenzyme lateral flow immunoassay strip for rapid and ultrasensitive detection of carbofuran in vegetables.

A lateral flow immunoassay strip (LFIAS) is one of the most frequently rapid test technologies for carbofuran (CAR). Nevertheless, the LFIAS has a poor quantitative capability and low sensitivity. And, it also requires often complex sample handling steps, making testing time longer. In this study, Fe3O4 nanoparticles were successively modified with MIL-100(Fe)-based metal-organic framework (MOF) and chloroplatinic acid hexahydrate to obtain a core-shell complex of Fe3O4-MOF-Pt. The complex had a peroxidase-mimicking activity catalytic function that enabled signal amplification and sensitivity enhancement. Upon coupling with carbofuran monoclonal antibody (CAR-mAb), the magnetic separation properties of the probe enabled target-specific enrichment. The LFIAS based on Fe3O4-MOF-Pt nanocomposites could detect CAR in the range of 0.25-50 ng mL-1 with a limit of detection (LOD) of 0.15 ng mL-1, enabling colorimetric and catalytic analysis. In addition, the method showed high specificity and stability for detecting CAR in various vegetables, and recovery rates of the spiked samples were 91.40%-102.40%. In conclusion, this study provided one-stop detection of "target enrichment-visual inspection". While lowering the LOD, it reduced the detection time and improved the detection efficiency. The multifunctional Fe3O4-MOF-Pt nanocomposite provides an idea for the construction of novel multifunctional probes to improve the detection performance of conventional LFIAS.

Preparation of dual recognition adsorbents based on molecularly imprinted polymers and aptamer for highly sensitive recognition and enrichment of ochratoxin A.

In light of the significant risks that mycotoxins posed to public health and environmental safety, this research developed an adsorbent MIPs/Apt/AuNPs@ZIF-67 (MA-AZ) utilizing a dual-recognition approach combining molecularly imprinted polymers (MIPs) and aptamer (Apt). This innovative method enabled the effective and highly selective recognition and enrichment of ochratoxin A (OTA). ZIF-67 was utilized as a carrier with a substantial specific surface area, and gold nanoparticles (AuNPs) were loaded on its surface to fix the thiol-modified Apt on the surface of the carrier. Then, an initiator was used to initiate a polymerization reaction, and the generated MIPs coated Apt/AuNPs@ZIF-67, thereby synthesizing the MA-AZ with a "synergistic recognition" effect. The Apt significantly increased the number of recognition sites within the imprinted cavities, and MIPs played roles in identifying targets, fixing and protecting Apt. The combination of the both produced the effect of "1+1>2". The study on the adsorption performance of MA-AZ found that the adsorption capacity of MA-AZ could reach 65.1 mg/g, and the imprinted factor was 5.48. In addition, MA-AZ exhibited excellent stability, specificity, reusability and recovery rate. Thus, this study offers valuable insights for the recognition and enrichment of hazardous substances, and helps to promote the rapid development of safety detection.

Enhanced detection of Aspergillus flavus in peanut kernels using a multi-scale attention transformer (MSAT): Advancements in food safety and contamination analysis.

In this study, a multi-scale attention transformer (MSAT) was coupled with hyperspectral imaging for classifying peanut kernels contaminated with diverse Aspergillus flavus fungi. The results underscored that the MSAT significantly outperformed classic deep learning models, due to its sophisticated multi-scale attention mechanism which enhanced its classification capabilities. The multi-scale attention mechanism was utilized by employing several multi-head attention layers to focus on both fine-scale and broad-scale features. It also integrated a series of scale processing layers to capture features at different resolutions and incorporated a self-attention mechanism to integrate information across different levels. The MSAT model achieved outstanding performance in different classification tasks, particularly in distinguishing healthy peanut kernels from those contaminated with aflatoxigenic fungi, with test accuracy achieving 98.42±0.22%. However, it faced challenges in differentiating peanut kernels contaminated with aflatoxigenic fungi from those with non-aflatoxigenic contamination. Visualization of attention weights explicitly revealed that the MSAT model's multi-scale attention mechanism progressively refined its focus from broad spatial-spectral features to more specialized signatures. Overall, the MSAT model's advanced processing capabilities marked a notable advancement in the field of food quality safety, offering a robust and reliable tool for the rapid and accurate detection of Aspergillus flavus contaminations in food.

Novel cross-linkable fluorescent probe with oriented antibody to enhance lateral immunoassay strip for the detection of acetamiprid.

Time-resolved fluorescent lateral immunoassay strip (TRFLIS) is a reliable and rapid method for detecting acetamiprid. However, its sensitivity is often affected by the structural patterns and stability of the fluorescent probe. Researchers have shown significant interests in using goat anti-mouse IgG (GaMIgG) which is indirectly bound to time-resolved fluorescent microsphere (TRFM) and antibody. This allowed for oriented modification of the antibody. However, the stability of fluorescent probe in this binding mode remained unexplored. Herein, 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride was innovatively used as a cross-linking agent to enhance the binding of antibody to GaMIgG, which improved the stability of the fluorescent probe. Under optimal working conditions, this strategy exhibited a wide linear response range of 5-700 ng/mL. Its limit of detection (LOD) was 0.62 ng/mL, the visual LOD was 5 ng/mL, and the limit of quantification (LOQ) of 2.06 ng/mL. Additionally, under tomato matrix, leek matrix and Chinese cabbage matrix, the linear response ranges were 5-400, 5-300, and 5-700 ng/mL, with LODs of 0.16, 0.60, and 0.41 ng/mL, with LOQs of 0.53, 2.01 and 1.37 ng/mL, respectively. In conclusion, this strategy effectively reduced the dosage of acetamiprid antibody compared with TRFM directly linking acetamiprid antibody, and greatly increased the sensitivity of TRFLIS. Meanwhile, it demonstrated outstanding specificity and accuracy in acetamiprid detection and had been successfully applied to vegetable samples. This method enables rapid and accurate detection of large-volume samples by combining qualitative and quantitative methods. As such, it has great potential in the development of low-cost and high-performance immunochromatographic platforms.

Metal-organic framework-based aptasensor utilizing a novel electrochemiluminescence system for detecting acetamiprid residues in vegetables.

The work was based on N-(4-Aminobutyl)-N-ethylisoluminol (ABEI)-functionalized Fe-MIL-101 and gold nanoparticles (AuNPs) as sensing materials, and an electrochemiluminescence (ECL) aptasensor was constructed for detecting acetamiprid. As a metal-organic framework (MOF) material, Fe-MIL-101, was renowned for its unique three-dimensional network structure and efficient catalytic capability. ABEI, a common ECL reagent, was widely applied. ABEI was introduced into the Fe-MIL-101 structure as a luminescence functionalization reagent to form Fe-MIL-101@ABEI. This approach avoided limitations on the loading capacity of luminescent reagents imposed by modification and encapsulation methods. With character of excellent catalytic activity and ease of bioconjugation, AuNPs offered significant advantages in biosensing. Leveraging the reductive properties of ABEI, AuNPs were reduced around Fe-MIL-101@ABEI, resulting in the modified luminescent functionalized material denoted as Fe-MIL-101@ABEI@AuNPs. An aptamer was employed as a recognition element and was modified accordingly. The aptamer was immobilized on Fe-MIL-101@ABEI@AuNPs through gold-sulfur (Au-S) bonds. After capturing acetamiprid, the aptamer induced a decrease in the ECL signal intensity within the ABEI-hydrogen peroxide (H2O2) system, enabling the quantitative detection of acetamiprid. The aptasensor displayed remarkable stability and repeatability, featured a detection range of 1×10-3-1×102 nM, and had a limit of detection (LOD) of 0.3 pM (S/N=3), which underscored its substantial practical application potential.

Novel electrochemiluminescence aptasensor based on AuNPs-ABEI encapsulated TiO2 nanorod for the detection of acetamiprid residues in vegetables.

Gold nanoparticles (AuNPs)@N-(4-aminobutyl)-N-ethylisoluminol (ABEI)@Titanium dioxide nanorods (TiO2NRs) were used as sensing materials to produce a unique encapsulated nanostructure aptasensor for the detection of acetamiprid residues in this work. ABEI, an analog of luminol, was extensively used as an electrochemiluminescence (ECL) reagent. The ECL mechanism of ABEI- hydrogen peroxide (H2O2) system had connections to a number of oxygen-centered free radicals. TiO2NRs improved ECL response with high electron transfer and a specific surface area. AuNPs were easy to biolabel and could catalyze H2O2 to enhance ECL signal. AuNPs were wrapped around TiO2NRs by utilizing the reduction property of ABEI to form wrapped modified nanomaterials. The sulfhydryl-modified aptamer bound to the nanomaterial by forming gold-sulfur (Au-S) bonds. The aptamer selectively bound to its target with the addition of acetamiprid, which caused a considerable decrease in ECL intensity and enabled quantitative detection of acetamiprid. The aptasensor showed good stability, repeatability and specificity with a broad detection range (1×10-2-1×103 nM) and a lower limit of detection (3 pM) for acetamiprid residues in vegetables. Overall, this aptasensor presents a simple and highly sensitive method for ECL detecting acetamiprid, with potential applications in vegetable safety monitoring.

Portable multichannel detection instrument based on time-resolved fluorescence immunochromatographic test strip for on-site detecting pesticide residues in vegetables.

In this work, a portable multichannel detection instrument based on time-resolved fluorescence immunochromatographic test strip (TRFIS) was proposed for on-site detecting pesticide residues in vegetables. Its hardware consisted of a silicon photodiode and excitation light source array, a mainboard of the lower machine with STMicroelectronics 32 (STM32) and a linear stepping motor. While detecting, cardboard with 6-channel TRFIS was pulled into the cassette by the stepping motor. The peak area of the test (T) line and control (C) line of each TRFIS was sampled and calculated by software, then the concentration of the detected pesticide was obtained according to the ratio of the T to C value. This instrument could sample 6-channel TRFIS within 30 s simultaneously, and it exhibited excellent accuracy with a 2.5% average coefficient of variation for each channel (n = 12). In addition, the TRFIS was constructed by using europium oxide time-resolved fluorescent microspheres to label the monoclonal antibody against acetamiprid and form a fluorescent probe, which was fixed on the binding pad. The TRFIS was used for the detection of acetamiprid in celery cabbage, cauliflower and baby cabbage. This instrument was used to complete the qualitative and quantitative analysis of the TRFIS, so as to enhance the practical application of the detection method. This TRFIS possessed excellent linearity ranging from 0.25 mg kg-1 to 1.75 mg kg-1 for the detection of acetamiprid, and the limit of detection were 0.056-0.074 mg kg-1 in the different vegetable matrix. The platform combines the accuracy and portability of traditional test strips with the highly sensitive and efficient fluorescence intensity recognition function of detection equipment, which shows a great application prospect of multi-channel rapid detection of small molecule pollutants in the field.

Paper-based biosensors relying on core biological immune scaffolds for the detection of procymidone in vegetables.

In order to achieve a highly sensitive detection of procymidone in vegetables, three paper-based biosensors based on a core biological immune scaffold (CBIS) were developed, which were time-resolved fluorescence immunochromatography strips with Europium (III) oxide (Eu-TRFICS). Goat anti-mouse IgG and europium oxide time-resolved fluorescent microspheres formed secondary fluorescent probes. CBIS was formed by secondary fluorescent probes and procymidone monoclonal antibody (PCM-Ab). The first type of Eu-TRFICS (Eu-TRFICS-(1)) fixed secondary fluorescent probes on a conjugate pad, and PCM-Ab was mixed with a sample solution. The second type of Eu-TRFICS (Eu-TRFICS-(2)) fixed CBIS on the conjugate pad. The third type of Eu-TRFICS (Eu-TRFICS-(3)) was directly mixed CBIS with the sample solution. They solved the problems of steric hindrance of antibody labeling, insufficient exposure of antigen recognition region and easy loss of activity in traditional methods. They realized multi-dimensional labeling and directional coupling. They replaced the loss of antibody activity. And the three types of Eu-TRFICS were compared, among which Eu-TRFICS-(1) was the best detection choice. Antibody usage was reduced by 25% and sensitivity was increased by 3 times. Its detection range was 1-800 ng/mL, the limit of detection (LOD) was 0.12 ng/mL with the visible LOD (vLOD) of 5 ng/mL.

Phylogenetic Analysis of Some Species of the Anopheles hyrcanus Group (Diptera: Culicidae) in China Based on Complete Mitochondrial Genomes.

Some species of the Hyrcanus group are vectors of malaria in China. However, the member species are difficult to identify accurately by morphology. The development of sequencing technologies offers the possibility of further studies based on the complete mitochondrial genome. In this study, samples of mosquitoes of the Hyrcanus group were collected in China between 1997 and 2015. The mitochondrial genomes of ten species of the Hyrcanus group were analyzed, including the structure and base composition, codon usage, secondary structure of tRNA, and base difference sites in protein coding regions. Phylogenetic analyses using maximum-likelihood and Bayesian inference were performed based on mitochondrial genes and complete mitochondrial genomes The mitochondrial genome of 10 Hyrcanus group members ranged from 15,403 bp to 15,475 bp, with an average 78.23% (A + T) content, comprising of 13 PCGs (protein coding genes), 22 tRNAs, and 2 rRNAs. Site differences between some closely related species in the PCGs were small. There were only 36 variable sites between Anopheles sinensis and Anopheles belenrae for a variation ratio of 0.32% in all PCGs. The pairwise interspecies distance based on 13 PCGs was low, with an average of 0.04. A phylogenetic tree constructed with the 13 PCGs was consistent with the known evolutionary relationships. Some phylogenetic trees constructed by single coding regions (such as COI or ND4) or combined coding regions (COI + ND2 + ND4 + ND5 or ND2 + ND4) were consistent with the phylogenetic tree constructed using the 13 PCGs. The phylogenetic trees constructed using some coding genes (COII, ND5, tRNAs, 12S rRNA, and 16S rRNA) differed from the phylogenetic tree constructed using PCGs. The difference in mitochondrial genome sequences between An. sinensis and An. belenrae was very small, corresponding to intraspecies difference, suggesting that the species was in the process of differentiation. The combination of all 13 PCG sequences was demonstrated to be optimal for phylogenetic analysis in closely related species.

High frequency of Voltage-gated sodium channel (VGSC) gene mutations in Aedes albopictus (Diptera: Culicidae) suggest rapid insecticide resistance evolution in Shanghai, China.

BACKGROUND: Dengue fever is an infectious disease that is imported into Shanghai, China and requires prevention and control measures. Controlling the vector Aedes albopictus through insecticide use is a key approach to dengue control. However, the rapid evolution of insecticide resistance in Ae. albopictus has raised concerns about the failure of dengue control efforts. Knockdown resistance (kdr) caused by point mutations in the voltage-gated sodium channel (VGSC) gene is a primary mechanism of pyrethroid resistance. In this study, we investigated the kdr mutations of Ae. albopictus in Shanghai and evaluated the trend in its evolution. METHODOLOGY/PRINCIPAL FINDINGS: We collected 17 populations of Ae. albopictus from 15 districts in Shanghai in 2020, extracted genomic DNA from individual mosquitoes, and amplified Domain II, III, and IV in VGSC using PCR. Following sequencing, we obtained 658 VGSC sequences. We detected the nonsynonymous mutations V1016G, I1532T, and F1534S/C/I, among which V1016G and F1534C/I were reported in Shanghai for the first time and F1534I was a novel mutant allele in Ae. albopictus. The overall mutation frequency was 84.65%, with individual mutation frequencies ranging from 46.81% to 100%, excluding the Fengxian District population, which had a frequency of 0%. The V1016G and I1532T mutation types accounted for 7.14% and 3.42%, respectively. The mutant allele at codon 1534 accounted for 63.98% of all mutations, including TCC/S (62.77%), TGC/C (1.06%), and ATC/I (0.15%). We identified and classified five intron types in Domain III by length, including A (83 bp, 12.07%), B (68 bp, 87.30%), C (80 bp, 0.16%), D (72 bp, 0.16%), and E (70 bp, 0.31%). Individuals with intron B had a significant mutation tendency at codon 1534 relative to intron A (chi-square test, p < 0.0001). We found no correlation between mutation frequency and the amount of pyrethroid used (Pearson correlation, p = 0.4755). CONCLUSIONS/SIGNIFICANCE: In recent years, kdr mutations in the Ae. albopictus population in Shanghai have rapidly evolved, as evidenced by an increase in mutation types and significantly increased mutation frequency. The F1534I/ATC mutant allele was found to be a novel mutation, F1534C/TGC was reported for the first time in Shanghai, and intron B in Domain III was significantly associated with mutation frequency at codon 1534. Continuous monitoring of resistance changes and strict regulation of insecticide use are required.

Genetic structure of Aedes albopictus (Diptera: Culicidae) populations in China and relationship with the knockdown resistance mutations.

BACKGROUND: Mosquito control is needed to prevent dengue fever, which is mainly spread by Aedes albopictus in China. Application of insecticides is one of the main mosquito control methods; however, this approach can fail due to the knockdown resistance (kdr) gene mutation that causes decreased sensitivity to insecticides in Ae. albopictus. The kdr mutation patterns among different regions in China differ significantly. However, the underlying mechanism and factors that influence kdr mutation remain unclear. To explore the potential influence of genetic background on the development of insecticide resistance in Ae. albopictus, we analyzed the genetic structure of Ae. albopictus populations in China and its correlation with major kdr mutations. METHODS: We collected Ae. albopictus from 17 sites in 11 provinces (municipalities) across China from 2016 to 2021 and extracted the genomic DNA from individual adult mosquitoes. We selected eight microsatellite loci for genotyping, and based on microsatellite scores, we estimated intraspecific genetic diversity, population structure, and effective population size. The association between the intrapopulation genetic variation and F1534 mutation rate was evaluated by the Pearson correlation coefficient. RESULTS: Based on variation analysis of the microsatellite loci of 453 mosquitoes representing 17 populations throughout China, more than 90% of the variation occurred within individuals, whereas only about 9% of the variation occurred among populations, indicating that field populations of Ae. albopictus are highly polymorphic. The northern populations tended to belong to gene pool I (BJFT 60.4%, SXXA 58.4%, SDJN 56.1%, SXYC 46.8%), the eastern populations tended to belong to pool III (SH 49.5%, JZHZ 48.1%), and the southern populations tended to belong to three different gene pools. Moreover, we observed that the greater the fixation index (FST), the lower the wild-type frequency of F1534 of VSGC. CONCLUSIONS: The degree of genetic differentiation among Ae. albopictus populations in China was low. These populations were divided into three gene pools, in which the northern and eastern pools are relatively homogeneous, while the southern gene pool is heterogeneous. The possible correlation between its genetic variations and kdr mutations is also noteworthy.

Spatio-temporal distribution patterns and quantitative detection of aflatoxin B1 and total aflatoxin in peanut kernels explored by short-wave infrared hyperspectral imaging.

Aflatoxin contamination in peanut kernels seriously harms the health of humans and causes significant economic losses. Rapid and accurate detection of aflatoxin is necessary to minimize its contamination. However, current detection methods are time-consuming, expensive and destructive to samples. Therefore, short-wave infrared (SWIR) hyperspectral imaging coupled with multivariate statistical analysis was used to investigate the spatio-temporal distribution patterns of aflatoxin, and quantitatively detect the aflatoxin B1 (AFB1) and total aflatoxin in peanut kernels. In addition, Aspergillus flavus contamination was identified to prevent the production of aflatoxin. The result of validation set demonstrated that SWIR hyperspectral imaging could predict the contents of the AFB1 and total aflatoxin accurately, with residual prediction deviation values of 2.7959 and 2.7274, and limits of detection of 29.3722 and 45.7429 µg/kg, respectively. This study presents a novel method for the quantitative detection of aflatoxin and offers an early warning system for its potential application.

Multiplex strategy electrochemical platform based on self-assembly dual-site DNA tetrahedral scaffold for one-step detection of diazinon and profenofos.

In the work, based on self-assembly dual-site DNA tetrahedral scaffold (DTS), thionine (Thi), and 6-(Ferrocenyl)hexanethiol (Fc6S), a multiplex strategy electrochemical platform was fabricated for the simultaneous detection of profenofos (PFF) and diazinon (DZN). Thi and Fc6S were used to label aptamers for the synthesis of probes respectively. Notably, Thi and Fc6S engendered recognizable DPV peaks at different potentials to achieve simultaneous detection of PFF and DZN. In addition to increasing the conductivity of the electrode, the combination of carboxylic acid functionalized multi-walled carbon nanotubes and ferroferric oxide nanoparticles could also increase its higher specific surface area of the electrode interface to adsorb more DTS. Because of the mechanical rigidity of the DTS, the DTS could keep a complementary chain upright and provide more binding sites for aptamers, the binding efficiency between the complementary chain and 2 binding aptamers could be improved. Comparing the aptasensors performance of single-strand DNA with that of the DTS with complementary strands, the benefits of the DTS were highlighted in this system. Under optimal conditions, the detection limits of PFF and DZN were both 3.33 pg/mL and the detection ranges were both 1.00 × 101-1.00 × 107 pg/mL. Meanwhile, the recoveries of PFF and DZN were 87.15%-117.34% and 91.20%-114.19%, respectively. The aptasensor could realize the simultaneous detection of PFF and DZN in vegetables. Furthermore, the aptasensor also had good stability and selectivity. This strategy could provide a good reference for developing effective aptasensors for the simultaneous detection of other small molecules and toxins.

Mitochondrial COI and Cytb gene as valid molecular identification marker of sandfly species (Diptera: Psychodidae) in China.

The accurate identification of sandfly species is crucial because some species transmit medically significant diseases, including leishmaniasis, bartonellosis and sandfly fever. However, due to the high similarity of the external morphology in sandfly species, identification can only be performed using internal morphological characteristics after dissection, which is time consuming and requires highly experienced staff. Thus, the introduction of suitable molecular markers may solve these identification problems. This study screened suitable DNA barcodes to identify common sandfly species in China. The phlebotomine sandflies were collected from Sichuan, Henan and Hainan Provinces from 2014 to 2016. The species were identified by the morphological characteristics of the pharyngeal armature and spermatheca. The genomic DNA of sandfly was extracted individually, and mitochondrial DNA (mtDNA) cytochrome C oxidase subunit I (COI) and cytochrome B (Cytb) as well as the 18S subunit of ribosomal DNA (rDNA) were amplified using polymerase chain reaction (PCR). Additionally, intraspecific and interspecific differences (p-distance) were calculated to evaluate the feasibility of the three gene fragments as a DNA barcode. The phylogeny trees of all sandfly species in this study were constructed using neighbor joining (NJ) method. Six species were identified by the morphological features, belonging to Phlebotomus and Sergentomyia, as Ph. chinensis s. l., Ph. stantoni, Se. bailyi, Se. iyengari, Se. squamirostris, and Se. squamipleuris. Analysis based on three gene fragments revealed some degree of intraspecific polymorphism among these sandfly species in China. The largest intraspecific variation occurred in Ph. chinensis s. l. (mtDNA COI, p-distance = 0.042; mtDNA Cytb, p-distance = 0.071), but the 18S rDNA fragment showed a small variation (p-distance = 0.005). The ranges of interspecific p-distances for mtDNA COI and mtDNA Cytb were 0.138 - 0.231 and 0.128 - 0.274, respectively. However, the interspecific p-distances of 18S rDNA are relatively low ranging from 0.003 to 0.055. Both mitochondrial COI and Cytb gene fragments are valid molecular identification markers in theses sandfly species. The topological structure of phylogeny trees based on mtDNA COI, mtDNA Cytb and 18S rDNA genes were all consistent with morphological classification. And we also found there were significant intraspecies differences within Ph. chinensis s. l. (0.006-0.071) and Se. bailyi (0.002-0.032) based on mtDNA Cytb gene fragment. Sequence alignment data suggested that Ph. chinensis s. l. from Sichuan should be Ph. sichuanensis, and the sandfly specimen collected from Henan was Ph. chinensis s. s.. There could be cryptic species in Se. bailyi from China.

Interference-resistant aptasensor with tetrahedral DNA nanostructure for profenofos detection based on the composites of graphene oxide and polyaniline.

In this work, an interference-resistant electrochemical aptasensor that could detect profenofos in vegetables was constructed based on complexes of graphene oxide and polyaniline (GO@PANI) and gold nanoparticles-tetrahedral DNA nanostructure (Au-TDN). Compared with a single chain aptamer, the tetrahedral DNA nanostructure is highly stable and allows the aptamer on this structure to stand in a highly ordered position on an electrode surface. Moreover, the AuNPs are biocompatible and can protect the activity of the aptamer, which can improve the assembly success rate of Au-TDN. Besides, the conductivity of PANI had been tremendously enhanced thanks to the existence of GO, which improved the dispersion of PANI. The GO@PANI was prepared by a chemical synthesis method, which had a large surface area and was able to adsorb many Au-TDN. Under optimal working parameters, the constructed aptasensor exhibited good electrochemical sensing performance with a detection limit of 10.50 pg/mL and a linear range of 1.0 × 102-1.0 × 107 pg/mL. In addition, it was employed in detecting profenofos in vegetables with a good recovery rate of 90.41-116.37 %. More importantly, the aptasensor also has excellent stability and high selectivity. This study provides a promising method to avoid interference in the detection of profenofos by sensors.

Broad-specificity time-resolved fluorescent immunochromatographic strip for simultaneous detection of various organophosphorus pesticides based on indirect probe strategy.

The massive use of organophosphorus pesticides (OPs) poses a great threat to food safety, human health and environmental protection. As there are many kinds of pesticides, their detection is facing a severe challenge. The simultaneous detection of multiple organophosphorus pesticides in one test is a problem to be solved at present. In this paper, a time-resolved fluorescent immunochromatographic (TRFIA) strip is prepared by using broad-specificity antibodies (Abs) of OPs as the recognition element. Abs were connected to europium oxide latex microspheres using sheep anti-mouse antibodies (SaMIgG) to form an indirect probe. This strategy could effectively realize signal amplification, and could save the amount and protect the activity of Abs. After the detection, the color change of the test line (T-line) was observed to make qualitative judgment under UV-light (365 nm). Then, the images of the positive sample were analyzed by using ImageJ to complete the quantitative detection. Under optimal construction and operating conditions, the limit of detection of the strip could reach 0.53 ng g-1. And the TRFIA strip performed well in the additive test of vegetable samples. It is inexpensive to prepare, convenient to carry, and easy to operate. More importantly, it improves the detection efficiency and meets the needs of rapid field testing of a large number of samples.

A novel label-free electrochemiluminescence aptasensor using a tetrahedral DNA nanostructure as a scaffold for ultrasensitive detection of organophosphorus pesticides in a luminol-H2O2 system.

In this work, a new type of Au-tetrahedral DNA nanostructure (Au-TDN) was originally proposed and successfully applied in an electrochemiluminescence aptasensor to detect organophosphorus pesticides (Ops). The aptamers modified with -SH could be covalently bonded with gold nanoparticles (AuNPs) to form a tetrahedron structure, and there were independent probes at each vertex of the tetrahedron, which could increase the probability of specific binding with Ops. The originally designed structure could not only maintain a stable tetrahedral configuration, but also combined with the target to improve the sensitivity of the sensor. Meanwhile, silver nanoparticles (AgNPs) could catalyze the chemical reaction between luminol and H2O2 to generate a variety of intermediates called reactive oxygen species (ROS) for signal enhancement. Factors that had important influences on the aptasensor, such as the concentration of Au-TDN, the incubation time, and the pH value of the buffer, were optimized in this trial. According to the final results, the limit of detection (LOD) of 3 pg mL-1 (S/N = 3) for methyl parathion, the LOD of 0.3 pg mL-1 (S/N = 3) for parathion and the LOD of 0.03 pg mL-1 (S/N = 3) for phoxim were obtained, respectively. Moreover, the novel tetrahedral structure could be replaced by different types of aptamers to expand its application range and lay a foundation for the development of portable rapid detection devices for pesticide residues.

Human-like performance umami electrochemical biosensor by utilizing co-electrodeposition of ligand binding domain T1R1-VFT and Prussian blue.

Over the past decades, due to the desire for artificial umami flavors, apparatuses for detecting the umami taste have constantly been developed. Nevertheless, most information on umami is still acquired through human sensory assessment, which makes it difficult to establish an umami standard or quantify the umami flavor. In this study, the ligand binding domain called venus flytrap (VFT) domain of the umami taste receptor protein T1R1 was used as a recognition element, and an electrochemical biosensor based on a double-signal amplification strategy was constructed using single-walled carbon nanotubes (SWCNTs) and Prussian blue (PB). Moreover, the umami taste of four representative umami substances, inosine-5'-monophosphate (IMP), monosodium L-glutamate (MSG), beefy meaty peptide (BMP), and sodium succinate (WSA), were successfully quantitatively measured using differential pulse voltammetry (DPV) at an electrochemical workstation. Based on an equation (S/N = 3), the low detection limits (LODs) of IMP, MSG, BMP, and WSA were 0.1, 0.1, 0.1, and 0.01 pM, respectively. Meanwhile, a normalized signal intensity of more than 90% was kept for 4 days. The results showed that the biosensor could be used to detect umami substances with high sensitivity and selectivity, and was shown to have human-like performance. To develop the T1R1-VFT biosensor using the above-mentioned method, we utilized the ligand binding domain of the human umami receptor, rather than the entire umami receptor protein, which had a complex structure, having the following advantages: volume reduction, simplicity, and stability. This method has great potential for the detection of umami tastes, instead of using sensory evaluation, and for the development of new artificial flavorings.