Correction: Immunoassay-aptasensor for the determination of tumor-derived exosomes based on the combination of magnetic nanoparticles and hybridization chain reaction.

[This corrects the article DOI: 10.1039/D0RA10159A.].

Diatoms pass through the gastrointestinal barrier and lead to false-positive: an animal experiment.

The diatom test has been used by forensic pathologist as standard for drowning, but the occurrence of false-positive results (presence of diatoms found in the tissues of subjects who died from causes other than drowning) draws criticism regarding the specificity of the test. Diatoms within food or water can be ingested through the gastrointestinal tract. However, the mechanisms of how the diatoms reach distant organs such as the lung, liver, and kidney have not been studied. In this article, we simulated the process of diatoms entering the gastrointestinal tract using gastric lavage on experimental rabbits. Diatoms are detected in lymph from a lymphatic vessel at the root of the mesentery, portal vein blood, aortic blood, lung, liver, and kidney samples in the gavage group. Of diatoms, 76.24% were the centric diatom, 99.86% of diatoms have a maximum size of less than 50 µm, and most of diatoms concentrate in the lung. Our study provided the evidence supporting the theory that the diatoms could pass through the gastrointestinal barrier and reach the rabbits' other internal organs. The diatoms could reach internal organs through the portal vein and lymphatic vessel at the root of the mesentery. This provides us new insight into our understanding of false-positive diatom test in forensic pathology.

Microbial community analyses provide a differential diagnosis for the antemortem and postmortem injury of decayed cadaver: An animal model.

Differentiating antemortem injury from postmortem injury of decayed cadavers is one of the difficult issues in forensic science. Forensic pathologists identify antemortem injury according to the macroscopic and microscopic vital reactions taken place after being injured. However, the decomposition would render those vital reactions ineffective. Microbiomes have been widely used in forensic science due to their succession with time and sensitivity to vary of environment. In this study, microbiomes were introduced to determine whether the bacterial communities can be used to distinguish between the ante- and postmortem injuries through an animal experiment. Our findings showed that the differences of bacterial community were increasingly apparent from the 6th to 9th day after the wound created when the types of wounds were unidentified by morphological examination due to decomposition. The biomarkers at the genus level could effectively distinguish between injury types, Among them, Enterococcus and Enterobacter were only observed in the antemortem injured group, while Staphylococcus and Acinetobacter were only in the postmortem injured group. It is possible to tell whether cadaveric injuries developed before or after death by detecting differences in the bacterial communities of putrefying wounds. This study provides a new perspective for the differences between ante- and postmortem injuries and provides a promising method for us to identify the ante- and postmortem wounds, especially in decomposed cadavers.

CRISPR/Cas12a Coupling with Magnetic Nanoparticles and Cascaded Strand Displacement Reaction for Ultrasensitive Fluorescence Determination of Exosomal miR-21.

Exosomal MicroRNA-21 (miRNA-21, miR-21) is significantly up-regulated in blood samples of patients with lung cancer. Exosomal-derived miR-21 can be used as a promising biomarker for the early diagnosis of lung cancer. This paper develops a fluorescent biosensor based on the combination of magnetic nanoparticles (MNPs), cascade strand displacement reaction (CSDR) and CRISPR/Cas12a to detect the exosomal miR-21 from lung cancer. The powerful separation performance of MNPs can eliminate the potential interference of matrix and reduce the background signal, which is very beneficial for the improvement of specificity and sensitivity. The CSDR can specifically transform one miR-21 into plenty of DNA which can specifically trigger the trans-cleavage nuclease activity of Cas12a, resulting in the cleavage of ssDNA bi-labeled with fluorescent and a quencher. Under the optimized experimental conditions, the developed fluorescence biosensor exhibited high sensitivity and specificity towards the determination of exosomal-derived miR-21 with a linear range from 10 to 1 × 105 fM and a low detection limit of about 0.89 fM. Most importantly, this method can be successfully applied to distinguish the exosomal miR-21 from the lung cancer patients and the healthy people.

The fluorescent aptasensor based on CRISPR-Cas12a combined with TdT for highly sensitive detection of cocaine.

Ultrasensitive and specific detection of cocaine is of great significance for monitoring cocaine abuse. Herein, a fluorescent aptasensor via coupling CRISPR-Cas12a, with magnetic nanoparticles (MNPs), split-aptamer, and terminal deoxynucleotidyl transferase (TdT), was developed for the detection of cocaine. In short, the complete cocaine aptamer is split into two parts, one is modified on magnetic nanoparticles (MNPs) and the other is free. The presence of cocaine will mediate the binding of these two segments. Then TdT will mediate the extension to form an ultra-long sequence that can bind with multiple CRISPR-Cas12a resulting in the trans-cleavage activity of CRISPR-Cas12a being triggered. Thence, the DNA reporter which is bi-labeled with fluorophore and quencher is cleaved resulting in the generation of a fluorescence signal. The developed fluorescent aptasensor realizes the detection of cocaine with excellent sensitivity and specificity. The detection limit is low down to 33 pM, and the linear range is from 330 to 1.65 × 105 pM. Most importantly, this fluorescent aptasensor can be successfully applied to the determination of cocaine in human plasma samples.

Highly sensitive electrochemical determination of rutin based on the synergistic effect of 3D porous carbon and cobalt tungstate nanosheets.

Rutin, a flavonoid found in fruits and vegetables, is a potential anticancer compound with strong anticancer activity. Therefore, electrochemical sensor was developed for the detection of rutin. In this study, CoWO4 nanosheets were synthesized via a hydrothermal method, and porous carbon (PC) was prepared via high-temperature pyrolysis. Successful preparation of the materials was confirmed, and characterization was performed by transmission electron microscopy, scanning electron microscopy, and X-ray photoelectron spectroscopy. A mixture of PC and CoWO4 nanosheets was used as an electrode modifier to fabricate the electrochemical sensor for the electrochemical determination of rutin. The 3D CoWO4 nanosheets exhibited high electrocatalytic activity and good stability. PC has a high surface-to-volume ratio and superior conductivity. Moreover, the hydrophobicity of PC allows large amounts of rutin to be adsorbed, thereby increasing the concentration of rutin at the electrode surface. Owing to the synergistic effect of the 3D CoWO4 nanosheets and PC, the developed electrochemical sensor was employed to quantitively determine rutin with high stability and sensitivity. The sensor showed a good linear range (5-5000 ng/mL) with a detection limit of 0.45 ng/mL. The developed sensor was successfully applied to the determination of rutin in crushed tablets and human serum samples.

Evaluation of Inspection Efficiency of Diatom Artificial Intelligence Search System Based on Scanning Electron Microscope.

OBJECTIVES: To explore the application values of diatom artificial intelligence (AI) search system in the diagnosis of drowning. METHODS: The liver and kidney tissues of 12 drowned corpses were taken and were performed with the diatom test, the view images were obtained by scanning electron microscopy (SEM). Diatom detection and forensic expert manual identification were carried out under the thresholds of 0.5, 0.7 and 0.9 of the diatom AI search system, respectively. Diatom recall rate, precision rate and image exclusion rate were used to detect and compare the efficiency of diatom AI search system. RESULTS: There was no statistical difference between the number of diatoms detected in the target marked by the diatom AI search system and the number of diatoms identified manually (P>0.05); the recall rates of the diatom AI search system were statistically different under different thresholds (P<0.05); the precision rates of the diatom AI system were statistically different under different thresholds(P<0.05), and the highest precision rate was 53.15%; the image exclusion rates of the diatom AI search system were statistically different under different thresholds (P<0.05), and the highest image exclusion rate was 99.72%. For the same sample, the time taken by the diatom AI search system to identify diatoms was only 1/7 of that of manual identification. CONCLUSIONS: Diatom AI search system has a good application prospect in drowning cases. Its automatic diatom search ability is equal to that of experienced forensic experts, and it can greatly reduce the workload of manual observation of images.

Pathway of Diatoms Enter Experimental Rabbits through the Lymphatic System of the Digestive Tract.

OBJECTIVES: To study whether diatoms can enter the body through the lymphatic system of the digestive tract. METHODS: Twenty experimental rabbits were divided into the test group and the control group randomly, and intragastric administration was performed with 20 mL water sample from the Pearl River and 20 mL ultrapure water, respectively. After 30 min, lymph, lungs, livers and kidneys were extracted for the diatom test. The concentration, size and type of diatoms were recorded. RESULTS: The concentration of diatoms of the test group was higher than that of the control group (P<0.05). In the test group, Stephanodiscus, Coscinodiscus, Cyclotella, Melosira, Nitzschia, Synedra, Cymbella, and Navicula were detected; in the control group, Stephanodiscus, Coscinodiscus and Cyclotella were detected. The long diameter and the short diameter of diatoms of the test group were higher than those of the control group (P<0.05). In the test group, 1-2 diatoms were detected in 3 lung samples and 2 liver samples, which were Stephanodiscus or Cyclotella, and no diatoms were detected in the kidney samples; in the control group, 1-2 diatoms were detected in 2 lung samples and 3 liver samples, which were Stephanodiscus or Coscinodiscus, and no diatoms were detected in the kidney samples. CONCLUSIONS: Diatoms can enter the body through the lymphatic fluid, which is one of the reasons for the presence of diatoms in tissues and organs of non-drowning cadavers.

Effects of Digestive Temperature and Time on Diatom Test.

OBJECTIVES: To study the effects of temperature and time for diatoms digestion and find out suitable digestive temperature and time. METHODS: Eighty pieces of liver tissues were collected, each piece of tissue was 2 g, and 2 mL Pearl River water was added to each piece of tissue. The digestion temperature was set at 100 ℃, 120 ℃, 140 ℃, 160 ℃, 180 ℃ and the digestion time was set at 40, 50, 60, 70, 80 min. The liver tissue and water mixture were divided into 8 portions in each group. All the samples were tested by microwave digestive - vacuum filtration - automated scanning electron microscopy method. The quantity of diatom recovered and the quality of residue on the membrane were recorded. RESULTS: When the digestion time was set to 60 min, there were statistically significant differences in the number of diatoms recovered at different temperatures (P<0.05). The maximum number of diatoms recovered was (28 797.50±6 009.67) at 140 ℃, and the minimum residue was (0.60±0.28) mg at 180 ℃. When the digestion temperature was set at 140 ℃, there were statistically significant differences in the number of diatoms recovered at different digestion times (P<0.05). The number of diatoms recovered was the highest at 40 min, it was up to (20 650.88±1 950.29), and the residue quality of each group had no statistical significance among different digestion time groups(P>0.05). CONCLUSIONS: The effect of diatom digestion is related to temperature and time. When the digestion temperature was 140 ℃ and the digestion time was 40, 50 and 60 min, it is favorable for diatom test.

Comparison of Application of MD-VF-Auto SEM Method and Plankton Gene Multiplex PCR System in the Diagnosis of Drowning.

OBJECTIVES: To compare the application effect of microwave digestion - vacuum filtration - automated scanning electron microscopy (MD-VF-Auto SEM) method and plankton gene multiplex PCR system in the diagnosis of drowning. METHODS: Lung, liver and kidney tissue of 10 non-drowning cases and 50 drowning cases were prepared for further MD-VF-Auto SEM method analysis and plankton gene multiplex PCR system analysis. The positive detection rate of the two methods in each tissue was calculated. RESULTS: The positive rate of the MD-VF-Auto SEM method detecting diatoms in drowning cases was 100%, and few diatoms were detected in the liver and kidney tissues of 6 non-drowning cases. By using the plankton gene multiplex PCR system, the diatom positive rate of drowning cases was 84%, and all the non-drowning cases were negative. There were significant differences in the positive rate of the liver, kidney tissues between MD-VF-Auto SEM method and plankton gene multiplex PCR system (P<0.05), as well as the total positive rate of cases. However, no significant differences were found in the positive rates of lung tissues (P>0.05). CONCLUSIONS: MD-VF-Auto SEM method is more sensitive than plankton gene multiplex PCR system in diatom test. But the plankton gene multiplex PCR system can also detect plankton other than diatoms. Combination of the two methods can provide a more reliable basis for the diagnosis of drowning.

Application of Diatoms Quantitative Analysis in the Diagnosis of Drowning.

OBJECTIVES: To retrospectively analyze diatom test cases of corpses in water and discuss the value of quantitative analysis of diatoms in the diagnosis of drowning. METHODS: A total of 490 cases of water-related death were collected. They were divided into drowning group and postmortem immersion group according to the cause of death. Diatoms in lung, liver, kidney tissue and water sample were analyzed quantitatively by microwave digestion-vacuum filtration-automated scanning electron microscopy (MD-VF-Auto SEM) method. The ratios of content of diatoms in lung tissue and water sample (CL/CD) were calculated. RESULTS: The results of diatom test for three organs (lung, liver and kidney) were all positive in 400 cases (85.5%); the content of diatom in lung, liver, kidney tissues, and water samples of drowning group were (113 235.9±317 868.1), (26.7±75.6), (23.3±52.2) and (12 113.3±21 760.0) cells/10 g, respectively; the species of diatom were (7.5±2.8), (2.6±1.9), (2.9±2.1) and (8.9±3.0) types, respectively; the CL/CD of drowning group and postmortem immersion group were (100.6±830.7) and (0.3±0.4), respectively. CONCLUSIONS: Quantitative analysis of diatoms can provide supportive evidence for the diagnosis of drowning, and the parameter CL/CD can be introduced into the analysis to make a more accurate diagnosis of drowning.

Concordance analysis of diatom types and patterns in lung tissue and drowning medium in laboratory animal model.

Diatom test has been widely used in the diagnosis of drowning and inferring the drowning site. One of the issues is whether the concordance of the diatom types and patterns between the drowning victim's organs and media should be considered an essential requirement for the diagnosis of drowning. In this study, lung tissues from 20 rabbits and drowning media were studied by the Microwave Digestion-Vacuum Filtration-Automated Scanning Electron Microscopy method, and four methods, type consistency, coefficient of similarity, squared-chord distance, and cluster analysis, were introduced to analyze the diatom types and patterns for evaluating the value of diatom consistency in drowning cases. The results showed that diatom types and patterns in lung tissues do not perfectly match the drowning medium, and they are sometimes concordant with the drowning medium sampled from other than drowning site. We should be cautious when using diatom detection to infer drowning sites.

Highly sensitive electrochemical determination of rutin based on the synergistic effect of 3D porous carbon and cobalt tungstate nanosheets / 药物分析学报

Rutin,a flavonoid found in fruits and vegetables,is a potential anticancer compound with strong anti-cancer activity.Therefore,electrochemical sensor was developed for the detection of rutin.In this study,CoWO4 nanosheets were synthesized via a hydrothermal method,and porous carbon(PC)was prepared via high-temperature pyrolysis.Successful preparation of the materials was confirmed,and character-ization was performed by transmission electron microscopy,scanning electron microscopy,and X-ray photoelectron spectroscopy.A mixture of PC and CoWO4 nanosheets was used as an electrode modifier to fabricate the electrochemical sensor for the electrochemical determination of rutin.The 3D CoWO4 nanosheets exhibited high electrocatalytic activity and good stability.PC has a high surface-to-volume ratio and superior conductivity.Moreover,the hydrophobicity of PC allows large amounts of rutin to be adsorbed,thereby increasing the concentration of rutin at the electrode surface.Owing to the syn-ergistic effect of the 3D CoWO4 nanosheets and PC,the developed electrochemical sensor was employed to quantitively determine rutin with high stability and sensitivity.The sensor showed a good linear range(5-5000 ng/mL)with a detection limit of O.45 ng/mL.The developed sensor was successfully applied to the determination of rutin in crushed tablets and human serum samples.

Determination of miRNA derived from exosomes of prostate cancer via toehold-aided cyclic amplification combined with HRP enzyme catalysis and magnetic nanoparticles.

MicroRNAs (miRNAs) play a significant role in tumorigenesis and tumor development. Exosomal microRNA-141 (miRNA-141, miR-141) has been reported to be overexpressed in prostate cancer (PCa) and has become a potential biomarker for the diagnosis of PCa. Herein, a novel fluorescent biosensor based on toehold-aided cyclic amplification combined with horseradish peroxidase (HRP) enzyme catalysis and magnetic nanoparticles (MNPs) was designed for determination of the exosomes-derived microRNA-141 (miRNA-141, miR-141). The synergy of HRP enzyme catalysis and toehold mediated strand display reaction (TSDR) increase the sensitivity of the method, and the good separation ability of MNPs ensures the specificity of the method. Therefore, under the optimized experimental conditions, the highly sensitive and specific detection of miRNA-141 can be realized, and the detection limit is as low as 10 fM. More importantly, the biosensor successfully determinates the exosomal miR-141 in the plasma of patients with PCa.

Identification of diatom taxonomy by a combination of region-based full convolutional network, online hard example mining, and shape priors of diatoms.

Diatom test is one of the commonly used diagnostic methods for drowning in forensic pathology, which provides supportive evidence for drowning. However, in forensic practice, it is time-consuming and laborious for forensic experts to classify and count diatoms, whereas artificial intelligence (AI) is superior to human experts in processing data and carrying out classification tasks. Some AI techniques have focused on searching diatoms and classifying diatoms. But, they either could not classify diatoms correctly or were time-consuming. Conventional detection deep network has been used to overcome these problems but failed to detect the occluded diatoms and the diatoms similar to the background heavily, which could lead to false positives or false negatives. In order to figure out the problems above, an improved region-based full convolutional network (R-FCN) with online hard example mining and the shape prior of diatoms was proposed. The online hard example mining (OHEM) was coupled with the R-FCN to boost the capacity of detecting the occluded diatoms and the diatoms similar to the background heavily and the priors of the shape of the common diatoms were explored and introduced to the anchor generation strategy of the region proposal network in the R-FCN to locate the diatoms precisely. The results showed that the proposed approach significantly outperforms several state-of-the-art methods and could detect the diatom precisely without missing the occluded diatoms and the diatoms similar to the background heavily. From the study, we could conclude that (1) the proposed model can locate the position and identify the genera of common diatoms more accurately; (2) this method can reduce the false positives or false negatives in forensic practice; and (3) it is a time-saving method and can be introduced.

[Extraction of exosome by gel electrophoresis microfluidic chip and determination of miRNA-21 in exosome of human plasma].

We developed a high-efficiency microfluidic chip for extracting exosomes from human plasma. We collected peripheral blood from normal human, designed and fabricated a microfluidic chip based on nanoporous membrane and agarose gel electrophoresis to isolate exosomes. The extracted exosomes were characterized by transmission electron microscopy, nanosight and Western blotting, the morphology, concentration and particle size of exosomes were identified and analyzed. Meanwhile, we used ultracentrifugation and microfluidic chip to isolate exosomes separately. The particle size and concentration of the exosomes extracted by two methods were compared and analyzed, and their respective extraction efficiency was discussed. Finally, the expression level of miRNA-21 in exosomes was analyzed by RT-PCR. The microfluidic chip isolated (in 1 hour) high-purity exosomes with size ranging from 30-200 nm directly from human plasma, allowing downstream exosomal miRNA analysis. By comparing with ultracentrifugation, the isolation yield of microfluidic chip was 3.80 times higher than ultracentrifugation when the volume of plasma sample less than 100 µL. The optimized parameters for exosome isolation by gel electrophoresis microfluidic chip were: voltage: 100 V; concentration of agarose gel: 1.0%; flow rate of injection pump: 0.1 mL/h. The gel electrophoresis microfluidic chips could rapidly and efficiently isolate the exosomes, showing great potential in the research of exosomes and cancer biomarkers.

Immunoassay-aptasensor for the determination of tumor-derived exosomes based on the combination of magnetic nanoparticles and hybridization chain reaction.

The detection of tumor-related exosomes is of great significance. In this work, a fluorescence aptasensor was designed for the determination of tumor-related exosomes based on the capture of magnetic nanoparticles (MNPs) and specific recognition of an aptamer. MNPs were used as substrates to capture the exosomes by modifying the CD63 antibody on the MNP surface. Probe 1 consists of PDL-1 aptamer sequence and a section of other sequences. PDL-1 expression was observed on the surface of exosomes; the aptamer of PDL-1 could combine with PDL-1 with high affinity. Thus, the immunoassay-type compounds of "MNPs-exosomes-probe 1" were formed. The other section of probe 1 triggered the HCR with probe 2 and probe 3 and formed the super-long dsDNA. The addition of GelRed resulted in the generation of an amplified fluorescence signal. The proposed design demonstrated a good linearity with the exosome concentration ranging from 300 to 107 particles per mL and with a low detection limit of 100 particles per mL. This aptasensor also exhibited high specificity for tumor-related exosomes, and was successfully applied in biological samples.

Scale-Up Synthesis and Identification of GLYX-13, a NMDAR Glycine-Site Partial Agonist for the Treatment of Major Depressive Disorder.

GLYX-13, a NMDAR glycine-site partial agonist, was discovered as a promising antidepressant with rapidly acting effects but no ketamine-like side effects. However, the reported synthetic process route had deficiencies of low yield and the use of unfriendly reagents. Here, we report a scaled-up synthesis of GLYX-13 with an overall yield of 30% on the hectogram scale with a column chromatography-free strategy, where the coupling and deprotection reaction conditions were systematically optimized. Meanwhile, the absolute configuration of precursor compound of GLYX-13 was identified by X-ray single crystal diffraction. Finally, the activity of GLYX-13 was verified in the cortical neurons of mice through whole-cell voltage-clamp technique.