Broad-spectrum Screening Research Articles

Multi-signal sensor array based on a fluorescent nanozyme for broad-spectrum screening of pesticides

Accurate identification of broad-spectrum pesticides remains a challenge for existing rapid detection technologies based on natural cholinesterase. Sensor arrays show excellent promise, but typically rely on increasing receptors to improve sensing dimensionality, which is both cumbersome and imprecise. In this work, inspired by the multiple properties of a fluorescent nanozyme, we constructed a facile and simple sensor array based on the laccase-like activity, peroxidase-like activity, and fluorescent feature of one nanozyme (Cu-BDC-NH2). In the presence of pesticides, they produced differential positive responses and could be captured by linear discriminant analysis and hierarchical clustering analysis. Thus, different pesticides belonged to organophosphates, carbamates, organochlorines, pyrethroids, sulfonylureas, and diphenyloxazolines, respectively, have been successfully discriminated at 1 – 100 μg/mL. The differentiation of cross-contamination at different concentrations and compositions further validated the sensing performance. More importantly, they demonstrated strong immunity to interference as well as high feasibility for recognizing pesticides in fruits and vegetables, and performed precise identification for blind samples. Therefore, the sensor arrays provided a novel and reliable solution for broad-spectrum pesticide screening, which not only bridged the gap of existing rapid detection methods, but also opened up a new way for food safety regulation.

Development of a broad-spectrum one-step immunoassay for detection of glucocorticoids in milk using magnetosome-based immunomagnetic beads

Immunomagnetic beads provide novel tools for high-throughput immunoassay techniques. In this study, protein G (PG) was immobilized onto bacterial magentic particles (BMPs) using an additional cysteine residue at the C-terminus. A broad-spectrum monoclonal antibody against glucocorticoids (GCs) was attached to BMPs through PG-Fc interaction, generating BMP-PG-mIgG immunomagentic beads. A sensitive one-step immunoassay was developed for GCs based on combination of BMP-PG-mIgG and dexamethasone-horseradish peroxidase tracer (DMS-HRP). The developed assay exhibited half inhibitory concentrations (IC50) for dexamethasone (DMS), betamethasone (BMS), prednisolone (PNS), hydrocortisone (HCS), beclomethasone (BCMS), cortisone (CS), 6-α-methylprednisone (6-α-MPNS), fludrocortisone acetate (HFCS) of 0.98, 1.49, 2.42, 9.29, 1.63, 6.13, 7.3, and 4.89 ng/mL, respectively. The method showed recoveries ranging rates from 86.5 % to 117 % with a coefficient of variation less than 12.3 % in milk sample, which showed a good correlation with LC-MS/MS. Thus, the proposed assay offers a rapid and broad-spectrum screening tool for simultaneous detection of GCs in milk.

High-throughput screening of 54 alternative plasticizers in sesame oil using gas chromatography-quadrupole time-of-flight mass spectrometry

Restrictions on the use of phthalates have led to the wide use of alternative plasticizers (APs) such as organophosphate, adipate, citrate, and sebacate. However, because plasticizers combine with polymers in plastic products via unstable noncovalent bonds, they can easily migrate out of these products, causing environmental pollution. In particular, their migration out of food packaging, containers, and other food-contact materials and into food has raised great concerns. Toxicological studies have shown that APs contain potentially toxic substances that can affect endocrine functions and cause neurotoxicity, genotoxicity, and other adverse effects. Thus, their potential risks to food should not be underestimated. Sesame oil is a necessity in daily cooking. The results of risk monitoring in recent years have indicated that sesame oil often contains phthalates in excess of the standard limits. However, the potential risks of APs in sesame oil have not yet been reported. Some common detection methods for APs include gas chromatography-mass spectrometry, gas chromatography-triple quadrupole mass spectrometry, and liquid chromatography-triple quadrupole mass spectrometry. Unfortunately, these methods use low-resolution mass spectrometry and are limited by the resolution, scan rate, and analysis mode. Gas chromatography-quadrupole time-of-flight mass spectrometry (GC-Q-TOF/MS) has the advantages of high resolution, sensitivity, and analysis speed. In full-scan mode, GC-Q-TOF/MS can accurately collect the full-spectrum mass number of target compounds with low content levels in complex substrates, thereby realizing efficient screening and quantitative analysis. It shows outstanding advantages in the trace analysis of pesticide residues and pollutants. Furthermore, it features strong qualitative and high screening abilities. Establishment of a personal compound database and library (PCDL) addresses limitations in the number of compounds that can be measured and enables the rapid identification of targets without the use of standard products. In addition, increasing the number of targets for synchronous screening enables the retrospective analysis of new targets. In this study, a method based on GC-Q-TOF/MS was developed for the determination of 54 APs in sesame oil. The samples were extracted with acetonitrile and purified using a PSA/silica solid-phase extraction column. The mass-spectral information of the samples was then collected by GC-Q-TOF/MS in full-scan mode, and the 54 APs were searched using an established high-resolution mass-spectrum database to simultaneously achieve the broad-spectrum screening, qualitative identification, and quantitative analysis of multiple targets. The effects of different extraction solvents and purification methods on sample extraction and purification were compared. The accuracy of the screening results was improved by optimizing the GC-separation conditions, quality-extraction window, retention-time deviation, and other screening parameters. The screening detection limits (SDLs) of the 54 APs ranged from 0.01 to 0.02 mg/kg; specifically, the SDL of 41 compounds was 0.01 mg/kg and that of 13 compounds were 0.02 mg/kg. The limits of quantification were in the range of 0.02-0.04 mg/kg. A total of 80 sesame-oil samples were rapidly screened using this method under optimal conditions. Five APs were identified from the 80 sesame-oil samples and quantitatively analyzed using the matrix-matched external-standard method. The results of this quantitative methodology showed that the five APs had good linear relationships in the range of 0.01-0.2 mg/L, with all correlation coefficients greater than 0.99. The accuracy and precision of the method were verified using a standard recovery test with blank sesame-oil samples. Under the three standard levels of 0.04, 0.08, and 0.2 mg/kg, the recoveries of the five APs ranged from 71.3% to 97.8%, and the relative standard deviations (RSDs) ranged from 0.4% to 6.1%(n=6). The developed method is fast, accurate, sensitive, and has high throughput. Thus, it can realize the efficient screening, qualitative identification, and quantitative analysis of the 54 APs in sesame oil and provides a potential solution for the monitoring of other contaminants in food.

Bacillus velezensis and Paenibacillus peoriae Strains Effective as Biocontrol Agents against Xanthomonas Bacterial Spot

Gram-negative bacteria belonging to the Xanthomonas genus include plant pathogens representing a major challenge in the field of agriculture for a wide variety of economically important crops, such as tomato, pepper, and lettuce. Due to the massive usage of agrochemicals, Xanthomonas spp. are developing resistance to copper pesticides typically used to control microbial infections. An interesting alternative approach to control bacterial phytopathogens consists of using eco-friendly biocontrol agents, often beneficial microorganisms. Here, following the targeted, broad-spectrum screening of thousands of microorganisms isolated from different environmental locations, we isolated Bacillus velezensis strain 71 and Paenibacillus peoriae strain To99 displaying potent antagonistic activity against Xanthomonas spp. We found that oxydifficidin and polymyxin A secreted by B. velezensis 71 and P. peoriae To99, respectively, are mainly responsible for the anti-Xanthomonas activity. We further evaluated the performance of cell suspensions and cell-free supernatants of these isolates in controlling tomato bacterial spot disease in growth chamber and greenhouse conditions to validate the in vitro results. The overall results demonstrate the potential of treatments based on the secondary metabolites from both isolates and their cells as an alternative to copper-based chemicals to control leaf spot diseases caused by Xanthomonas spp. phytopathogens.

High-throughput untargeted screening of biotherapeutic macromolecules in equine plasma by UHPLC-HRMS/MS: Application to monoclonal antibodies and Fc-fusion proteins for doping control.

Many innovative biotherapeutics have been marketed in the last decade. Monoclonal antibodies (mAbs) and Fc-fusion proteins (Fc-proteins) have been developed for the treatment of diverse diseases (cancer, autoimmune diseases, and inflammatory disorders) and now represent an important part of targeted therapies. However, the ready availability of such biomolecules, sometimes characterized by their anabolic, anti-inflammatory, or erythropoiesis-stimulating properties, raises concerns about their potential misuse as performance enhancers for human and animal athletes. In equine doping control laboratories, a method has been reported to detect the administration of a specific human biotherapeutic in equine plasma; but no high-throughput method has been described for the screening without any a priori knowledge of human or murine biotherapeutic. In this context, a new broad-spectrum screening method involving UHPLC-HRMS/MS has been developed for the untargeted analysis of murine or human mAbs and related macromolecules in equine plasma. This approach, consisting of a "pellet digestion" strategy performed in a 96-well plate, demonstrates reliable performances at low concentrations (pmol/mL range) with high-throughput capability (≈100 samples/day). Targeting species-specific proteotypic peptides located within the constant parts of mAbs enables the "universal" detection of human biotherapeutics only by monitoring 10 peptides. As proof of principle, this strategy successfully detected different biotherapeutics in spiked plasma samples, and allowed, for the first time, the detection of a human mAb up to 10days after a 0.12 mg/kg administration to a horse. This development will expand the analytical capabilities of horse doping control laboratories towards protein-based biotherapeutics with adequate sensitivity, throughput, and cost-effectiveness.

Broad-Specificity Screening of Pyrethroids Enabled by the Catalytic Function of Human Serum Albumin on Coumarin Hydrolysis.

Sensing systems based on cholinesterase and carboxylesterase coupled with different transduction technologies have emerged for pesticide screening owing to their simple operation, fast response, and suitability for on-site analysis. However, the broad spectrum and specificity screening of pyrethroids over organophosphates and carbamates remains an unmet challenge for current enzymatic sensors. Human serum albumin (HSA), a multifunctional protein, can promote various chemical transformations and show a high affinity for pyrethroids, which offer a route for specific and broad-spectrum pyrethroid screening. Herein, for the first time, we evaluated the catalytic hydrolysis function of human serum albumin (HSA) on the coumarin lactone bond and revealed that HSA can act as an enzyme to catalyze the hydrolysis of the coumarin lactone bond. Molecular docking and chemical modifications indicate that lysine 199 and tyrosine 411 serve as the catalytic general base and contribute to most of the catalytic activity. Utilizing this enzymatic activity, a broad specific ratiometric fluorescence pyrethroids sensing system was developed. The binding energetics and binding constants of pesticides and HSA show that pyrethroids bind to HSA more easily than organophosphates and carbamates, which is responsible for the specificity of the sensing system. This study provides a general sensor platform and strategy for screening pesticides and reveals the catalytic activity of HSA on the hydrolysis of the coumarin lactone bond, which may open innovative horizons for the chemical sensing and biomedical applications of HSA.

Supramolecular solvent extraction and ambient mass spectrometry for the determination of organic contaminants in food packaging material

A rapid method based on a fast sample treatment with supramolecular solvents (SUPRASs) and ambient mass spectrometry (AMS) analysis was developed for the screening and quantification of organic contaminants in food packaging materials (FCMs). The suitability of SUPRASs made up of medium chain alcohols in ethanol:water mixtures was investigated, given their low toxicity, proven capacity for multi-residue analysis (since they provide a wide variety of interactions and multiple binding sites) and restricted access properties for simultaneous sample extraction and clean-up. Two families of emerging organic pollutants, bisphenols and organophosphate flame retardants, were targeted as representative compounds. The methodology was applied to 40 FCMs. Target compounds were quantitated using ASAP (atmospheric solids analysis probe)-low resolution MS and a broad-spectrum screening of contaminants was performed through spectral library search using direct injection probe (DIP) and high resolution MS (HRMS). The results showed the ubiquity of bisphenols and of some flame retardants, as well as the presence of other additives and unknown compounds in about half of the analyzed samples, which highlight the complex composition of FCMs and the possible associated health risks.

Detection of Matrix Metalloproteinase-1 in Human Saliva Based on a Pregnancy Test Strip Platform.

Matrix metalloproteinase (MMP) is closely correlated with tumorigenesis and progression. Establishing a low-cost, simple, rapid, and sensitive method for its detection is highly desired for the broad-spectrum screening of oral cancer. Herein, we combine the MMP-specific cleavage ability with magnetic separation technology and a commercial test strip to construct a sensitive biosensor to detect MMP-1 conveniently for the first time. The method involves two DNA probes, peptide-DNA1 and hCG-DNA2, where DNA1 and DNA2 are complementary sequences, and the peptide labeled with biotin can bind streptavidin-modified magnetic nanoparticles stably. The human chorionic gonadotropin (hCG) is the target of the pregnancy test strip. The cleavage reaction mediated by MMP-1 releases peptide-DNA1 and the hybridized hCG-DNA2 into the solution, and the hCG probe in the solution can develop color on the test strip for the determination of MMP-1 after magnetic separation. This method utilizes the high specificity of MMP-1's proteolytic cleavage and the high sensitivity of the test strip to the target probe, achieving a sensitive detection of MMP-1 with a visual detection limit of 65.5 pg/mL. The method shows better anti-interference and sensitivity than the enzyme-linked immunosorbent assay in the application of a biological sample matrix, suggesting its great potential for clinical diagnosis, especially for broad-spectrum oral cancer screening.

Screening of risky substances in sea cucumbers based on gas chromatography-orbitrap high-resolution mass spectrometry

建立了一种基于改良QuEChERs的样品前处理新方法,并通过气相色谱-静电场轨道阱高分辨质谱(GC-Orbitrap HRMS)对海参样品中的有机氯农药类(OCPs)、多环芳烃类(PAHs)、酞酸酯类(PAEs)、多氯联苯类(PCBs)及其他农药类(ACs)等10大类289种具有健康风险的有机污染物进行了高通量高精准的定性鉴别和定量分析。通过将传统的QuEChERs方法与柱净化方法结合,提出了一种基于改良QuEChERs的简便生物样品制备新方法,经弗洛里硅土(Florisil)柱净化后脂含量降低了99.9%,显著减少了基质效应对分析结果的干扰,可在高分辨质谱(6万分辨率)全扫描模式下对289种目标化合物同时进行高精准定性筛查和定量分析。该方法定量限约0.56~57.8 pg/g,线性范围约6个数量级,回收率范围为40%~120%。由于Q Exactive GC-Orbitrap HRMS具备较高的质量分辨率和灵敏度,因此对目标化合物的定量限显著优于常规的色谱和质谱分析方法,常规方法无法检出的超痕量有机污染物利用该研究开发的方法可进行精准定量。基于此高覆盖度多目标分析方法对养殖场采集的海参样品进行分析,共从海参体内检出100种有机污染物,其中PAHs、ACs、PAEs、OCPs类检出总含量相对高于其他类污染物,总含量均值分别为157.8、153.2、64.4和46.4 ng/g dw。9-氯芴、5-氯苊、3-甲基胆蒽等多种新污染物首次在海参样品中检出,但含量都非常低。该方法简单高效,定量限低,线性范围宽,结果准确。此高覆盖度多目标分析方法可广泛应用于各种水产品中风险物质的广谱筛查和精准定量,为食品安全和绿色养殖提供技术支撑。

PSMC2 is overexpressed in glioma and promotes proliferation and anti-apoptosis of glioma cells

BackgroundThis study aims to investigate the effect of PSMC2 expression on the clinical prognosis of glioma patients and its molecular mechanism.MethodsTCGA multi-tumor screening and survival analysis were combined to explore the differential expression of PSMC2 in multi-tumor. PSMC2 expression in glioma and normal tissues was detected by Western blot and RT-qPCR. Kaplan-Meier survival curve was used to visualize the effect of PSMC2 expression on the overall survival rate and disease-free survival rate of patients with glioma. The highly expressed cell line U343MG was selected to construct a PSMC2 knockdown model by siRNA transfection, and the effect of PSMC2 knockdown on cell proliferation ability was evaluated by CCK-8 assay. Gene-set enrichment analysis of PSMC2 co-expression genes was carried out to predict the molecular mechanism of their regulation of tumor cell phenotypes, and the analysis results were verified by flow cytometry and Western blot.ResultsThrough broad-spectrum screening of 31 kinds of tumors, we found that PSMC2 was upregulated in most tumors, but PSMC2 was most significantly overexpressed in gliomas and correlated with poor prognosis in glioma patients. The results of Western blot and qRT-PCR showed that PSMC2 was significantly overexpressed in glioma tissues. Further survival analysis revealed that the overall survival and disease-free survival of patients with low PSMC2 expression were significantly better than that of patients with high PSMC2 expression. The proliferation of U343MG cells was significantly inhibited after PSMC2 knockdown. Enrichment analysis of PSMC2 co-expression genes indicated that PSMC2 affected the apoptosis process. The expression of apoptosis-related proteins also significantly changed following PSMC2 knockdown.ConclusionsPSMC2 promotes the proliferation of glioma cells and inhibits the apoptosis, which is expected to be a potential therapeutic target for glioma.

Qualitative detection of pesticide residues using mass spectral data based on convolutional neural network

Excessive pesticide residues in crops directly threaten human life and health, so rapid screening and effective measurements of agricultural pesticides residues have important application significance in the field of food safety. It is imperative to detect different pesticide residue types in actual complex crop samples cause mixture analysis can provide more information than individual components. However, the accuracy of mixture analysis can be obviously affected by the impurities and noise disturbances. Purification and denoising will cost a lot of algorithm time. In this work, we used the problem transformation method to convert pesticide residues prediction into multi-label classification problem. In addition, a new convolutional neural network structure Pesticide Residues Neural Network (PRNet) was proposed to solve the problem of multi-label organophosphate pesticide residue prediction. The method of binary correlation and label energy set was used to adapt 35 pesticide residues labels. The Cross Entropy were used as loss functions for PRNet. The comprehensive comparison performances (e.g. 97% optimal accuracy rate) of PRNet is better than the other four models. By comparing the ROC curves of the five models, PRNet performs the best. The PRNet can separate the independent mass spectrometry data by different collision energy applied to phosphorus pesticide compounds through a three-channel structure. No complicated data preprocessing is required, the PRNet can extract the characteristics of different compounds more efficiently and presents high detecting accuracy and good model performance of multi-label mass spectrometry data classification. By inputting MS data of different instruments and adding more offset MS data, the model will be more transplantable and could lay the foundation for the wide application of PRNet model in rapid, on-site, accurate and broad-spectrum screening of pesticide residues in the future.

Gas-liquid microextraction coupled with magnetic-assisted dispersive solid-phase extraction clean-up for multi-residue pesticide analysis in fatty foods of animal origin

An effective sequential clean-up method by coupling gas-liquid microextraction (GLME) and magnetic-assisted dispersive solid phase extraction (d-SPE) termed as GLME-MA-d-SPE has been developed for multi-residue pesticide analysis in different fatty foods of animal origin. GLME is applied as a primary clean-up step to remove low-volatile interferences, followed by a secondary clean-up technique through adsorptive removal using d-SPE to eliminate other co-extracts like organic acids in fatty biological samples. As much as 99.3% of lipid substances were effectively eliminated by this powerful clean-up method, and the chromatographic analysis by GC-MS showed at least two orders of magnitude reduction for peaks of interference. Analytical results verified the accuracy and precision of this method with recoveries of 50 pesticides ranged from 60.5% to 119.7%, and RSDs of less than 20%. Permethrin was present in salmon, pork and egg samples, but the concentrations were within the maximum residue levels (MRLs) permitted by both national and international regulations. The GLME-MA-d-SPE technique minimizes matrix effects, and it exhibits significant potential as an analytical technique of food safety control systems for broad-spectrum screening trace-level environmental pollutants in complex biological matrices.

Development of a machine learning-based multimode diagnosis system for lung cancer.

As an emerging technology, artificial intelligence has been applied to identify various physical disorders. Here, we developed a three-layer diagnosis system for lung cancer, in which three machine learning approaches including decision tree C5.0, artificial neural network (ANN) and support vector machine (SVM) were involved. The area under the curve (AUC) was employed to evaluate their decision powers. In the first layer, the AUCs of C5.0, ANN and SVM were 0.676, 0.736 and 0.640, ANN was better than C5.0 and SVM. In the second layer, ANN was similar with SVM but superior to C5.0 supported by the AUCs of 0.804, 0.889 and 0.825. Much higher AUCs of 0.908, 0.910 and 0.849 were identified in the third layer, where the highest sensitivity of 94.12% was found in C5.0. These data proposed a three-layer diagnosis system for lung cancer: ANN was used as a broad-spectrum screening subsystem basing on 14 epidemiological data and clinical symptoms, which was firstly adopted to screen high-risk groups; then, combining with additional 5 tumor biomarkers, ANN was used as an auxiliary diagnosis subsystem to determine the suspected lung cancer patients; C5.0 was finally employed to confirm lung cancer patients basing on 22 CT nodule-based radiomic features.

Effective screening of antibiotic and coccidiostat residues in food of animal origin by reliable broad-spectrum residue screening tests

Foods of animal origin are controlled for antibiotic and coccidiostat residues. The rapid residue detection is possible using reliable broad-spectrum screening tests. This study’s objective using four microbial inhibition tests for the detection and identification of antibiotic and coccidiostat residues in different foods of animal origin: Premi®Test, EXP Ampulle test, Milchtest and Screening Test for Antibiotic Residues (STAR). Four hundred and thirty (430) food samples (165 animal tissues, 152 raw cow’s milk and 113 eggs) were randomly collected and screened. Using the Premi®Test, 18 samples were positive and 6 samples dubious. Using the EXP Ampulle test, 31 samples were positive and 2 samples dubious. Using the Milchtest, 15 samples were positive and 12 samples dubious. Using the STAR, 65 samples were positive with 62 samples positive on plates specific for beta-lactams and sulphonamides, 4 samples on plates specific for aminoglycosides, 8 samples on plates specific for macrolides and beta-lactams; and 7 samples on the plates specific for tetracyclines. Retesting using penicillinase and para-aminobenzoic acid (PABA) to confirm the presence of beta-lactams or sulphonamides all potentially positive tube test samples revealed 21 samples positive for beta-lactams and 27 samples positive for sulphonamides. Further testing of sulphonamide positive chicken samples revealed the positivity for coccidiostat salinomycin which was confirmed by testing with PABA, which counteracting salinomycin inhibition. Three hundred and sixty six (366) animal food samples were negative for antibiotic and coccidiostat residues. Microbial inhibition tests are preferred for initial antibiotic screening and have also proven useful for coccidiostat screening and post-screening. HIGHLIGHTS Foodstuffs of animal origin are subject to controls on antibiotic and coccidiostat residues. Microbial inhibition tests are still the preferred choice for the initial screening of antibiotic residues in food matrices and could become a useful tool for the screening of coccidiostat residues also. More specific post-screening analysis with PABA proved unexpectedly to be a reliable tool in the preliminary detection of coccidiostat residues in poultry meat and eggs.

A Survey of Antimicrobial Resistance Determinants in Category A Select Agents, Exempt Strains, and Near-Neighbor Species.

A dramatic increase in global antimicrobial resistance (AMR) has been well documented. Of particular concern is the dearth of information regarding the spectrum and prevalence of AMR within Category A Select Agents. Here, we performed a survey of horizontally and vertically transferred AMR determinants among Category A agents and their near neighbors. Microarrays provided broad spectrum screening of 127 Francisella spp., Yersinia spp., and Bacillus spp. strains for the presence/absence of 500+ AMR genes (or families of genes). Detecting a broad variety of AMR genes in each genus, microarray analysis also picked up the presence of an engineered plasmid in a Y. pestis strain. High resolution melt analysis (HRMA) was also used to assess the presence of quinolone resistance-associated mutations in 100 of these strains. Though HRMA was able to detect resistance-causing point mutations in B. anthracis strains, it was not capable of discriminating these point mutations from other nucleotide substitutions (e.g., arising from sequence differences in near neighbors). Though these technologies are well-established, to our knowledge, this is the largest survey of Category A agents and their near-neighbor species for genes covering multiple mechanisms of AMR.

Liquid chromatography-high resolution mass spectrometry for broad-spectrum drug screening of dried blood spot as microsampling procedure

BackgroundHyphenation of liquid chromatography (LC) with high-resolution mass spectrometry (HRMS) offers the potential to develop broad-spectrum screening procedures from low volumes of biological matrices. In parallel, dried blood spot (DBS) has become a valuable tool in the bioanalysis landscape to overcome conventional blood collection issues. Herein, we demonstrated the applicability of DBS as micro-sampling procedure for broad-spectrum toxicological screening. MethodsA method was developed on a HRMS system in data dependant acquisition (DDA) mode using an extensive inclusion list to promote collection of relevant data. 104 real toxicology cases were analysed, and the results were cross-validated with one published and one commercial screening procedures. Quantitative MRM analyses were also performed on identified substances on a triple quadrupole instrument as a complementary confirmation procedure. ResultsThe method showed limits of identification (LOIs) in appropriateness with therapeutic ranges for all the classes of interest. Applying the three screening approaches on 104 real cases, 271 identifications were performed including 14 and 6 classes of prescribed and illicit drugs, respectively. Among the detected substances, 23% were only detected by the proposed method. Based on confirmatory analyses, we demonstrated that the use of blood micro-samples did not impair the sensitivity allowing more identifications in the low concentration ranges. ConclusionA LC-HRMS assay was successfully developed for toxicological screening of blood microsamples demonstrating a high identification power at low concentration ranges. The validation procedure and the analysis of real cases demonstrated the potential of this assay by supplementing screening approaches of reference.

Megasonic Enhanced Photo Resist Removal Using Unique Solvent Free Phase-Fluids on an Advanced Single Wafer Resist Processing System

Removal of photoresist materials (resist stripping) is very straightforward in theory, but can be difficult and complicated in practice. Classical methods have included SPM (sulfuric peroxide module) /piranha, ozone and UV ozone, solvent based fluids and plasmas. All of the legacy methods include hazardous and toxic materials and/or high equipment expense as well as multi-step complexity. The stripping process becomes even more challenging by factors such as thick resist, cross-linked resist or sensitive metals or materials under the resist. This study was carried out In order to determine feasibility of the use of a unique phase-fluid based photoresist-stripper in commercial semiconductor resist removal process steps. Due to their highly dynamic inner structure, phase-fluids penetrate into the polymer network of photoresists and lift the material from the surface. As these water-based stripping fluids are non-aggressive, non-toxic, and require no special handling, the ability to apply them to industry standard resists will result in a reduction of toxic and dangerous chemistries and the environmental impact of their disposal. To investigate the impact of megasonic energy on the process time in both the active and rinse steps and therefore throughput, the study included the introduction of megasonics as a process variable in all test combinations. The study was broken down into two phases. Phase one consisted of a broad spectrum screening process incorporating nine standard photoresists Fig. 1 with various standard thermal treatments as well as a selection of five phase-fluid formulations. The tests were carried out in a single substrate stagnant immersion tank incorporating a megasonic transducer illustrated in Fig. 2. Process time was defined as the time from immersion of the substrate until the photoresist was completely removed. Process variables tested for each resist/phase-fluid combination included three process temperatures, ambient, 40c and 60c as well as the addition of megasonic energy at ambient with a frequency of 925KHz at a dosage of 1.5W/cm2 directly coupled. The results achieved in this phase one screening were used to select the best case phase-fluid, and process conditions for each of the screened photo resists. These conditions became the baseline parameters for phase two. Phase two of the study was carried out in actual single wafer production conditions on an EVG®100 Series Resist Processor equipped with a MegPie® large area megasonic transducer. Process recipes were established using phase one parameters and then refined for optimum total process time, including the actual phase-fluid process time and rinse time. Spin off and dry cycles were kept constant in all process recipes. Complete resist strip process times of <20 seconds were achieved with several photoresist phase fluid combinations while other combinations involving BCB based resist resulted in little to no removal. The results achieved in phase two testing demonstrate the feasibility of a phase-fluid based photo resist strip in a single wafer platform with megasonic enhancement for specific photo resists. Figure 1

RETRACTED ARTICLE: Peri-implantitis-associated methanogens: a preliminary report

Methanogens have already been described in periodontitis but not in peri-implantitis. Thirty peri-implantitis samples and 28 control samples were collected in 28 consenting peri-implantitis patients. PCR-sequencing of the 16S rRNA gene was used as a broad-spectrum screening method and results were further confirmed by real-time quantitative PCR targeting the mcrA genes. Results showed a methanogen community dominated by Methanobrevibacter oralis in 31/58 (51%) samples including 16/28 (57%) control samples and 15/30 (50%) peri-implantitis samples. Methanobrevibacter massiliense was detected in 5/58 (8.6%) samples including 3/28 (1%) control samples and 2/30 (6.7%) peri-implantitis samples. The prevalence of M. oralis or M. massiliense did not significantly differ in peri-implantitis and control samples (exact Fisher test, P = 0.61 and P = 0.67, respectively). Further ponderation of the methanogen load by the real-time quantitative PCR for actin human gene again indicated non-significant difference (Wilcoxon-Mann-Whitney test, P = 0.48 and P = 0.40, respectively). These data show that the prevalence of methanogens does not differ in peri-implantitis lesions and healthy sites, when individuals are their own control. These data do not allow assigning a specific pathogenic role to methanogens in peri-implantitis; methanogens rather are part of the commensal and normal flora of the oral cavity.

Ultrasensitive Single Fluorescence-Labeled Probe-Mediated Single Universal Primer-Multiplex-Droplet Digital Polymerase Chain Reaction for High-Throughput Genetically Modified Organism Screening.

As genetically modified (GM) technology develops and genetically modified organisms (GMOs) become more available, GMOs face increasing regulations and pressure to adhere to strict labeling guidelines. A singleplex detection method cannot perform the high-throughput analysis necessary for optimal GMO detection. Combining the advantages of multiplex detection and droplet digital polymerase chain reaction (ddPCR), a single universal primer-multiplex-ddPCR (SUP-M-ddPCR) strategy was proposed for accurate broad-spectrum screening and quantification. The SUP increases efficiency of the primers in PCR and plays an important role in establishing a high-throughput, multiplex detection method. Emerging ddPCR technology has been used for accurate quantification of nucleic acid molecules without a standard curve. Using maize as a reference point, four heterologous sequences ( 35S, NOS, NPTII, and PAT) were selected to evaluate the feasibility and applicability of this strategy. Surprisingly, these four genes cover more than 93% of the transgenic maize lines and serve as preliminary screening sequences. All screening probes were labeled with FAM fluorescence, which allows the signals from the samples with GMO content and those without to be easily differentiated. This fiveplex screening method is a new development in GMO screening. Utilizing an optimal amplification assay, the specificity, limit of detection (LOD), and limit of quantitation (LOQ) were validated. The LOD and LOQ of this GMO screening method were 0.1% and 0.01%, respectively, with a relative standard deviation (RSD) < 25%. This method could serve as an important tool for the detection of GM maize from different processed, commercially available products. Further, this screening method could be applied to other fields that require reliable and sensitive detection of DNA targets.

Biomonitoring application in the exposome assessment

Biomonitoring can be applied to assess internal exposure and environmental exposure by exposure markers with providing internal exposure to biological characterization and individual exposure information, which is a key tool to evaluate the risk exposure to disease by biological alternation information. With the development of high throughput, broad spectrum and high efficiency screening and detection technology, biomonitoring is defined as traditional biological monitoring (targeted monitoring) and non targeted monitoring analysis (exposomic approaches). An exposomic approach differs from traditional biomonitoring in that it can theoretically include all exposures of potential health significance, whether they are derived from exogenous sources. Both traditional and nontraditional biomonitoring methods should be used to understand the complexity of exposures faced throughout the lifespan. Through hybrid approaches, emerging techniques and the integration of bioinformatics, and developing the detection methods for low abundance chemicals, improving the differentiation ability between endogenous and exogenous chemical, the health outcomes and exposures can be widely recognized and characterized, which can finally contribute to improving the precise prevention and intervention for diseases under the new exposomic model.