Sample Preparation Technology Research Articles

Chromatography-Based Metabolomics as a Tool in Bioorganic Research of Honey

This review presents the latest research on chromatography-based metabolomics for bioorganic research of honey, considering targeted, suspect, and untargeted metabolomics involving metabolite profiling and metabolite fingerprinting. These approaches give an insight into the metabolic diversity of different honey varieties and reveal different classes of organic compounds in the metabolic profiles, among which, key metabolites such as biomarkers and bioactive compounds can be highlighted. Chromatography-based metabolomics strategies have significantly impacted different aspects of bioorganic research, including primary areas such as botanical origins, honey origin traceability, entomological origins, and honey maturity. Through the use of different tools for complex data analysis, these strategies contribute to the detection, assessment, and/or correlation of different honey parameters and attributes. Bioorganic research is mainly focused on phytochemicals and their transformation, but the chemical changes that can occur during the different stages of honey formation remain a challenge. Furthermore, the latest user- and environmentally friendly sample preparation methods and technologies as well as future perspectives and the role of chromatography-based metabolomic strategies in honey characterization are discussed. The objective of this review is to summarize the latest metabolomics strategies contributing to bioorganic research onf honey, with emphasis on the (i) metabolite analysis by gas and liquid chromatography techniques; (ii) key metabolites in the obtained metabolic profiles; (iii) formation and accumulation of biogenic volatile and non-volatile markers; (iv) sample preparation procedures; (v) data analysis, including software and databases; and (vi) conclusions and future perspectives. For the present review, the literature search strategy was based on the PRISMA guidelines and focused on studies published between 2019 and 2024. This review outlines the importance of metabolomics strategies for potential innovations in characterizing honey and unlocking its full bioorganic potential.

Spatial Proteomic Profiling of Colorectal Cancer Revealed Its Tumor Microenvironment Heterogeneity.

Colorectal cancer is a predominant malignancy with a second mortality worldwide. Despite its prevalence, therapeutic options remain constrained and surgical operation is still the most useful therapy. In this regard, a comprehensive spatially resolved quantitative proteome atlas was constructed to explore the functional proteomic landscape of colorectal cancer. This strategy integrates histopathological analysis, laser capture microdissection, and proteomics. Spatial proteome profiling of 200 tissue section samples facilitated by the fully integrated sample preparation technology SISPROT enabled the identification of more than 4000 proteins on the Orbitrap Exploris 240 from 2 mm2 × 10 μm tissue sections. Compared with normal adjacent tissues, we identified a spectrum of cancer-associated proteins and dysregulated pathways across various regions of colorectal cancer including ascending colon, transverse colon, descending colon, sigmoid colon, and rectum. Additionally, we conducted proteomic analysis on tumoral epithelial cells and paracancerous epithelium from early to advanced stages in hallmark rectum cancer and sigmoid colon cancer. Bioinformatics analysis revealed functional proteins and cell-type signatures associated with different regions of colorectal tumors, suggesting potential clinical implications. Overall, this study provides a comprehensive spatially resolved functional proteome landscape of colorectal cancer, serving as a valuable resource for exploring potential biomarkers and therapeutic targets.

Proteomics Meets Dose Response: A New Paradigm for Deciphering Drug Effects.

In a groundbreaking study, Eckert and colleagues introduce DecryptE, an innovative approach to dose-resolved proteomics that significantly advances our understanding of drug effects at the proteomic level. This method integrates cutting edge sample preparation and mass spectrometry technologies, establishing a robust platform for high-throughput proteome analysis. DecryptE enables the quantification of more than 7,000 proteins per hour and was employed to study 144 clinical drugs and research compounds, generating more than 1 million dose-response curves using Jurkat acute T-cell leukemia cells as a model system. The platform demonstrates outstanding reproducibility, ensuring reliable and consistent results across multiple experiments. By providing detailed information on drug potency and efficacy, DecryptE allows the identification of subtle changes in protein expression and facilitates the clustering of drugs based on their proteomic profiles. This study not only reveals novel drug mechanisms but also creates a comprehensive resource that can be utilized by the broader research community. Furthermore, it highlights the potential of integrating proteomics-and potentially other omics modalities in the future-with dose-response analysis to advance pharmacological research and improve therapeutic strategies.

Lectin-Based SP3 Technology Enables N-Glycoproteomic Analysis of Mouse Oocytes.

N-glycosylation is one of the most universal and complex protein post-translational modifications (PTMs), and it is involved in many physiological and pathological activities. Owing to the low abundance of N-glycoproteins, enrichment of N-glycopeptides for mass spectrometry analysis usually requires a large amount of peptides. Additionally, oocyte protein N-glycosylation has not been systemically characterized due to the limited sample amount. Here, we developed a glycosylation enrichment method based on lectin and a single-pot, solid-phase-enhanced sample preparation (SP3) technology, termed lectin-based SP3 technology (LectinSP3). LectinSP3 immobilized lectin on the SP3 beads for N-glycopeptide enrichment. It could identify over 1100 N-glycosylation sites and 600 N-glycoproteins from 10 μg of mouse testis peptides. Furthermore, using the LectinSP3 method, we characterized the N-glycoproteome of 1000 mouse oocytes in three replicates and identified a total of 363 N-glycosylation sites from 215 N-glycoproteins. Bioinformatics analysis revealed that these oocyte N-glycoproteins were mainly enriched in cell adhesion, fertilization, and sperm-egg recognition. Overall, the LectinSP3 method has all procedures performed in one tube, using magnetic beads. It is suitable for analysis of a low amount of samples and is expected to be easily adaptable for automation. In addition, our mouse oocyte protein N-glycosylation profiling could help further characterize the regulation of oocyte functions.

Plasma proteomic characterization of colorectal cancer patients with FOLFOX chemotherapy by integrated proteomics technology

BackgroundColorectal Cancer (CRC) is a prevalent form of cancer, and the effectiveness of the main postoperative chemotherapy treatment, FOLFOX, varies among patients. In this study, we aimed to identify potential biomarkers for predicting the prognosis of CRC patients treated with FOLFOX through plasma proteomic characterization.MethodsUsing a fully integrated sample preparation technology SISPROT-based proteomics workflow, we achieved deep proteome coverage and trained a machine learning model from a discovery cohort of 90 CRC patients to differentiate FOLFOX-sensitive and FOLFOX-resistant patients. The model was then validated by targeted proteomics on an independent test cohort of 26 patients.ResultsWe achieved deep proteome coverage of 831 protein groups in total and 536 protein groups in average for non-depleted plasma from CRC patients by using a Orbitrap Exploris 240 with moderate sensitivity. Our results revealed distinct molecular changes in FOLFOX-sensitive and FOLFOX-resistant patients. We confidently identified known prognostic biomarkers for colorectal cancer, such as S100A4, LGALS1, and FABP5. The classifier based on the biomarker panel demonstrated a promised AUC value of 0.908 with 93% accuracy. Additionally, we established a protein panel to predict FOLFOX effectiveness, and several proteins within the panel were validated using targeted proteomic methods.ConclusionsOur study sheds light on the pathways affected in CRC patients treated with FOLFOX chemotherapy and identifies potential biomarkers that could be valuable for prognosis prediction. Our findings showed the potential of mass spectrometry-based proteomics and machine learning as an unbiased and systematic approach for discovering biomarkers in CRC.

Pouring and Curing for Sample Preparation Technology for the Analysis of the Silicon-Aluminum Ratio of Molecular Sieves by Energy Dispersive X-Ray Fluorescence (EDXRF)

The silicon-aluminum ratio of the molecular sieves is a crucial control index for the quality of synthetic molecular sieves. Here a fast, simple and reliable sample preparation method, referred to as pouring and curing method, is described for determining the silicon-aluminum ratio of high silicon molecular sieves by energy dispersive x-ray fluorescence (EDXRF). Resin is used as the carrier with the addition of a curing agent, so that the sample powder is embedded in the resin matrix. By measuring sixteen random positions of the same cured sample, the homogeneity of the sample was evaluated to ensure the stability of the results. A double logarithmic calibration curve of the analysis line ratio and the corresponding concentration ratio of silicon and aluminum was established according to the binary ratio method. The results by EDXRF and inductively coupled plasma-optical emission spectrometer (ICP-OES) were compared with a relative standard error from 2.934 to 6.777%. The good precision shows that the pouring and curing method is a suitable for measuring the silicon-aluminum ratio of molecular sieves.

INNOVATIVE PROCESS LINE FOR MATERIAL SAMPLE PREPARATION

ABSTRACT. The successful establishment and sustainable development of mining enterprises depends on a number of factors, including geological research, management of mineral extraction and processing processes, and sample preparation technology. The sample preparation plants that are currently used have a multi-stage technological process, often with manual transfer of material between technological operations. Standard crushing and grinding equipment with a low degree of grinding, high metal consumption and overall dimensions is used. Separation of material by size also occurs on standard screens, which are poorly suited for sample preparation. The purpose of the work is to substantiate an innovative technological scheme for preparing mineral samples for laboratory analysis. The determining unit of the production line is a vibrating jaw crusher with an inclined crushing chamber, which has a degree of material grinding of more than 100 and allows one to obtain a final product with a particle size of -1 mm in one pass in an open crushing cycle, which reduces the number of overload devices that significantly affect sample clogging with material. Horizontal loading of the source material and unloading of the crushed material makes it possible to arrange all equipment in a horizontal plane, eliminate large-sized metal structures and ensure ease of maintenance. The vibrating screen of the improved design has a complex working profile, which provides a significant increase in the path of transportation of material particles along the sieving surface compared to its length, effective cleaning of the mesh and its visual inspection through the transparent lid, which eliminates clogging of the material of the previous sample. The working surface is a set of sieve cards that allow quick replacement of the worn part of the screening surface. The technical characteristics of the equipment and the general view of a small-sized modular crushing and grinding plant are presented.

Trends in Sample Preparation, Part I: Current State of the Field

This installment of “Sample Prep Perspectives” reports the results obtained from a new survey on sample preparation techniques. This is our first survey since 2016 and its findings are compared with the results of previous surveys dating from 1991. Results involve undercover trends in the field over the last generation and their impact. The survey investigated trends in areas such as which technologies are currently being used, sample loads, sample sizes, automation, the use of solid-phase extraction (SPE) devices (cartridges, disks, plates, tips), SPE chemistries, selection criteria, and problems encountered. Respondents were also asked about sample preparation technologies on the horizon.

Sample-to-answer sensing technologies for nucleic acid preparation and detection in the field.

Efficient sample preparation and accurate disease diagnosis under field conditions are of great importance for the early intervention of diseases in humans, animals, and plants. However, in-field preparation of high-quality nucleic acids from various specimens for downstream analyses, such as amplification and sequencing, is challenging. Thus, developing and adapting sample lysis and nucleic acid extraction protocols suitable for portable formats have drawn significant attention. Similarly, various nucleic acid amplification techniques and detection methods have also been explored. Combining these functions in an integrated platform has resulted in emergent sample-to-answer sensing systems that allow effective disease detection and analyses outside a laboratory. Such devices have a vast potential to improve healthcare in resource-limited settings, low-cost and distributed surveillance of diseases in food and agriculture industries, environmental monitoring, and defense against biological warfare and terrorism. This paper reviews recent advances in portable sample preparation technologies and facile detection methods that have been / or could be adopted into novel sample-to-answer devices. In addition, recent developments and challenges of commercial kits and devices targeting on-site diagnosis of various plant diseases are discussed.

Development of a multiplex assay to assess activated p300/CBP in circulating prostate tumor cells.

Reduced SIRT2 deacetylation and increased p300 acetylation activity leads to a concerted mechanism of hyperacetylation at specific histone lysine sites (H3K9, H3K14, and H3K18) in castration-resistant prostate cancer (CRPC). We examined whether circulating tumor cells (CTCs) identify patients with altered p300/CBP acetylation. CTCs were isolated from 13 advanced PC patients using Exclusion-based Sample Preparation (ESP) technology. Bound cells underwent immunofluorescent staining for histone modifying enzymes (HMEs) of interest and image capture with NIS-Elements software. Using the cBioPortal PCF/SU2C dataset, the response of CRPC to androgen receptor signaling inhibitors (ARSI) was analyzed in 50 subjects. Staining optimization and specificity revealed clear expression of acetyl-p300, acetyl-H3K18, and SIRT2 on CTCs (CK positive, CD45 negative cells). Exposure to A-485, a selective p300/CBP catalytic inhibitor, reduced p300 and H3K18 acetylation. In CRPC patients, a-p300 strongly correlated with its target acetylated H3k18 (Pearson's R = 0.61), and SIRT2 expression showed robust negative correlation with a-H3k18 (R = -0.60). A subgroup of CRPC patients (6/11; 55%) demonstrated consistent upregulation of acetylation based on these markers. To examine the clinical impact of upregulation of the CBP/p300 axis, CRPC patients with reduced deacetylase SIRT2 expression demonstrate shorter response times to ARSI therapy (5.9 vs. 12 mo; p = 0.03). A subset of CRPC patients demonstrate increased p300/CBP activity based on a novel CTC biomarker assay. With further development, this biomarker suite may be used to identify candidates for CBP/p300 acetylation inhibitors in clinical development.

Development and initial clinical testing of a multiplex assay evaluating histone modifier p300 acetylation in prostate cancer circulating tumor cells.

e17020 Background: Histone modifying enzymes (HMEs) hold promise as targets for drug discovery in cancer. Histone acetyltransferases and histone deacetylases dictate the presence of acetyl marks, which regulate chromatin and transcriptional activation. We previously developed a novel ‘histone array on a chip’ to measure HME dysregulation associated with the development of castration resistant prostate cancer (CRPC) (ACS Chem Biol 2017). This approach identified decreased SIRT2 expression and increased p300 activity leads to a concerted mechanism of hyperacetylation at specific histone lysine sites (H3K9, H3K14, and H3K18) in CRPC xenografts. These alterations were subsequently validated in castration-resistant tissues (BMC Cancer 2017). The goal of the current study was to determine if circulating tumor cells (CTCs) could be used as a therapeutic biomarker to identify patients with altered p300 acetylation activity. Methods: Blood was collected from 11 CRPC and 3 hormone-sensitive patients with advanced PC under an IRB-approved protocol. PBMCs were isolated with Ficoll-paque PLUS, and lymphocytes were depleted using anti-CD45 magnetic beads. CTCs were then captured with EpCAM labeled coated magnetic beads. Bound cells were isolated with Exclusion-based Sample Preparation (ESP) technology. Bound cells were moved through staining and washing wells prior to imaging at 20x on a fluorescent microscope. Images were analyzed with NIS Elements software. Results: We initially optimized the staining of acetyl-p300, total p300, H3K18ac, and SIRT2 in single cell prostate cancer cell cultures. Exposure to A-485, a selective p300/CBP catalytic inhibitor, reduced levels of p300 and H3K18 acetylation confirming assay specificity. CTCs from patient samples were then captured by EpCAM antibody binding and target staining revealed clear expression of acetyl-p300, acetyl-H3K18, SIRT2, and CK (cytokeratin). No expression of CD45+ cells (lymphocyte exclusion) is noted. Acetyl-p300 displayed a strong positive correlation with its target acetylated H3k18 (Pearson’s r = 0.61), and SIRT2 expression showed robust negative correlation with H3k18 (r = -0.60). A distinct group of 6/11 (55%) of CRPC patients demonstrated altered expression. In one subject with an untreated CSPC sample and subsequent CRPC sample ( > 12mo) a loss of SIRT2 expression, increased p300 activity, and histone H3 hyperacetylation are associated with the transition from hormone-sensitive to CRPC. Conclusions: Acetyl-p300, H3K18ac, and SIRT2 increase in a subset of CTC from patients with CRPC. These biomarkers are associated with increased CBP/p300 activity. This biomarker suite could potentially be used for noninvasive monitoring of outcomes and predicting tumor responses to CBP/p300 acetylation inhibitors in clinical development.

Neutron scattering studies on dynamics of lipid membranes.

Neutron scattering methods are powerful tools for the study of the structure and dynamics of lipid bilayers in length scales from sub Å to tens to hundreds nm and the time scales from sub ps to μs. These techniques also are nondestructive and, perhaps most importantly, require no additives to label samples. Because the neutron scattering intensities are very different for hydrogen- and deuterium-containing molecules, one can replace the hydrogen atoms in a molecule with deuterium to prepare on demand neutron scattering contrast without significantly altering the physical properties of the samples. Moreover, recent advances in neutron scattering techniques, membrane dynamics theories, analysis tools, and sample preparation technologies allow researchers to study various aspects of lipid bilayer dynamics. In this review, we focus on the dynamics of individual lipids and collective membrane dynamics as well as the dynamics of hydration water.

A59 GLOBAL PROTEOMIC PROFILING OF HUMAN COLONOID MONOLAYERS UNDERGOING IN VITRO CHRONIC DAMAGE

Abstract Background An in vitro damage model has been established in our lab using human colonoids grown as 2D monolayers. Upon being subjected to repeated rounds of air-liquid interface (ALI) growth and injury by submergence, these colonoid monolayers lost their barrier integrity and regrowth potential. Changes in mRNA expression and DNA methylation in genes from this human model of injury were similar to those that occur in Inflammatory Bowel Disease (IBD) and colon cancer. Significant morphological changes were observed in these monolayers after they were subjected to subsequent rounds of submergence injury, compared to when they were differentiated in ALI. Purpose Submergence injury is predicted to be involved in unfolded protein response (UPR) activation which can specifically alter translation. Hence proteomics studies will help undertand these changes. Method To determine if these changes are mirrored in the proteomes of damaged colonoids, we employed a Single-Plot, Solid-Phase-enhanced Sample Preparation (SP3) technology for Mass Spectrometry (MS) based proteomics analysis to characterize these monolayers at baseline, once they were differentiated in ALI, after one and five rounds of injury after differentiation in ALI, and after stimulation with the Toll-like receptor 5 (TLR5) agonist FliC. Hierarchical clustering, enrichment analysis, volcano plot analysis after pre-processing and normalization of the proteomics data set revealed differentially expressed proteins across various groups of monolayers. Result(s) Preliminary proteomic data analysis revealed changes in the profile of proteins involved in cellular differentiation, mitochondrial proteins, hypoxia upregulated proteins, those responsible for the maintenance and reorganization of the cytoskeletal structure and Golgi structure. These changes in protein profile may account for the significant morphological changes observed in these monolayers when subjected to submergence injury. Some outliers in monolayers subjected to microbial stimulation included proteins involved in regulation of extracellular matrix dependent motility and components of Adaptor Protein Complexes. Further studies are needed to ascertain if these account for the protective effect of FliC on these monolayers. Conclusion(s) This study suggests that the submergence injury to these healthy human derived colonoid monolayers leads to changes in their protein profile which mirror those seen in case of acute and chronic inflammation like IBD and colon cancer. It corroborates with the findings of gene expression and epigenetic analyses using the in vitro model established in our lab. Please acknowledge all funding agencies by checking the applicable boxes below CCC Disclosure of Interest None Declared

Advances on Hormones in Cosmetics: Illegal Addition Status, Sample Preparation, and Detection Technology.

Owing to the rapid development of the cosmetic industry, cosmetic safety has become the focus of consumers' attention. However, in order to achieve the desired effects in the short term, the illegal addition of hormones in cosmetics has emerged frequently, which could induce skin problems and even skin cancer after long-term use. Therefore, it is of great significance to master the illegal addition in cosmetics and effectively detect the hormones that may exist in cosmetics. In this review, we analyze the illegally added hormone types, detection values, and cosmetic types, as well as discuss the hormone risks in cosmetics for human beings, according to the data in unqualified cosmetics in China from 2017 to 2022. Results showed that although the frequency of adding hormones in cosmetics has declined, hormones are still the main prohibited substances in illegal cosmetics, especially facial masks. Because of the complex composition and the low concentration of hormones in cosmetics, it is necessary to combine efficient sample preparation technology with instrumental analysis. In order to give the readers a comprehensive overview of hormone analytical technologies in cosmetics, we summarize the advanced sample preparation techniques and commonly used detection techniques of hormones in cosmetics in the last decade (2012-2022). We found that ultrasound-assisted extraction, solid phase extraction, and microextraction coupled with chromatographic analysis are still the most widely used analytical technologies for hormones in cosmetics. Through the investigation of market status, the summary of sample pretreatment and detection technologies, as well as the discussion of their development trends in the future, our purpose is to provide a reference for the supervision of illegal hormone residues in cosmetics.

Evaluation of zero counts to better understand the discrepancies between bulk and single-cell RNA-Seq platforms

Recent advances in sample preparation and sequencing technology have made it possible to profile the transcriptomes of individual cells using single-cell RNA sequencing (scRNA-Seq). Compared to bulk RNA-Seq data, single-cell data often contain a higher percentage of zero reads, mainly due to lower sequencing depth per cell, which affects mostly measurements of low-expression genes. However, discrepancies between platforms are observed regardless of expression level. Using four paired datasets with multiple samples each, we investigated technical and biological factors that can contribute to this expression shift. Using two separate machine learning models we found that, in addition to expression level, RNA integrity, gene or UTR3 length, and the number of transcripts potentially also influence the occurrence of zeros. These findings could enable the development of novel analytical methods for cross-platform expression shift correction. We also identified genes and biological pathways in our diverse datasets that consistently showed differences when assessed at the single cell versus bulk level to assist in interpreting analysis across transcriptomic platforms. At the gene level, 25 genes (0.12%) were found in all datasets as discordant, but at the pathway level, 7 pathways (2.02%) showed shared enrichment in discordant genes.

Smartphone-controlled biosensor for viral respiratory infectious diseases: Screening and response

Outbreaks of emerging viral respiratory infectious diseases (VRIDs) including coronavirus disease 2019 (COVID-19) seriously endanger people's health. However, the traditional nucleic acid detection required professionals and larger instruments and antigen-antibody detection suffered a long window period of target generation. To facilitate the VRIDs detection in time for common populations, a smartphone-controlled biosensor, which integrated sample preparation (electromembrane extraction), biomarker detection (red-green-blue model) and remote response technology (a built-in APP), was developed in this work. With the intelligent biosensor, VRIDs could be recognized in the early stage by using endogenous hydrogen sulfide as the biomarker. Importantly, it only took 15 min to accomplish the whole process of screening and response to VRIDs. Moreover, the experimental data showed that this smartphone-controlled biosensor was suitable for ordinary residents and could successfully differentiate non-communicable respiratory diseases from VRIDs. To the best of our knowledge, this is the first time that a smartphone-controlled biosensor for screening and response to VRIDs was reported. We believe that the present biosensor will help ordinary residents jointly deal with the challenges brought by COVID-19 or other VRIDs in the future.

Magnet Integrated Fabric Phase Sorptive Extraction (MI-FPSE): A Powerful Green(er) Alternative for Sample Preparation

Green(er) sample preparation technologies still dominate as the anticipated improvement in all analytical protocols. Separation scientists all over the world continuously strive to comply with the Green Analytical Chemistry (GAC) demands. To follow this trend, microextraction techniques are constantly evolving to bridge the gap between Green Analytical Chemistry and sample pretreatment. A research group from Florida International University, Miami, Florida has introduced fabric phase sorptive extraction (FPSE) in 2014 that was considered as a new milestone in microextraction technologies at that time. Two years later, the same research group introduced an advantageous innovative configuration that combines the stirring and extraction mechanism into a single sample preparation device, keeping all the benefits originally offered by classical FPSE. Magnet integrated fabric phase sorptive extraction (MI-FPSE) was eventually introduced as a new, advantageous implementation of FPSE. This device exhibits the advantageous role of the increase in extraction kinetics through sample diffusion, resulting in improved extraction efficiency of the microextraction device and supports the need for combining processes for better promotion and implementation of the principles of Green Analytical Chemistry. The applications of MI-FPSE are presented herein, showing the essential role that this technique can play in analytical and bioanalytical sample preparation.

Research and Improvement of SBS Asphalt-Modifier Content Testing Technology Based on Infrared Spectrum

Infrared spectroscopy SBS asphalt-modifier content detection technology has been applied in practical projects, which can effectively ensure the quality of pavement engineering. If the composition ratio of modified asphalt is not clear, the accuracy of the test results will be affected, which is the disadvantage of conventional infrared spectroscopy detection methods. In this paper, the influence of different kinds of additives on the accuracy of the infrared spectrogram and SBS content test was analyzed, and the microscopic mechanism of the effect of additives on the accuracy of SBS modified asphalt content testing was uncovered. Meanwhile, the sample preparation technique was improved and the testing technique was optimized. Results demonstrate that the improved standard curve algorithm eliminates the effect of additives on test accuracy, and the effect of additives on test accuracy was eliminated by improving the standard curve algorithm. Without the addition of additives, the content of the modifier is accurately measured by simplifying the boundary conditions of the test technique. The preparation of samples by the dissolution method has more advantages than the tableting method and can be used as a new sample preparation technology. Improvements in sample preparation techniques and standard curve algorithms improve the safety, convenience, applicability, and accuracy of detection techniques.

Simultaneous single-cell genome and transcriptome sequencing in nanoliter droplet with digital microfluidics identifying essential driving genes

Single-cell genome and transcriptome sequencing investigates how genotype influences the phenotype of single cells, so as to comprehensively interpret biological inheritance and explain functional heterogeneity at the single-cell level. Current sample preparation technologies for simultaneous DNA and RNA sequencing of the same cell are cumbersome, expensive, and suffer from cross-contamination and limited sensitivity. Herein we describe DMF-DR-seq, a nanoliter-scale single-cell multi-omics sample preparation platform based on digital microfluidics. DMF-DR-seq integrates the major steps of single-cell isolation, DNA/RNA separation, and nucleic acid amplification in situ. The results confirm the enhanced ability of DMF-DR-seq relative to current state-of-the-art technology, with lower amplification bias, higher genome-wide coverage in DNA sequencing and better gene detection ability in RNA sequencing results. By using DMF-DR-seq, we identified the genome variation-induced abnormal transcriptome expression of single circulating tumor cells (CTCs) and cancer cells from multiple myeloma patients. The results identified potentially essential genes, known as transporters associated with antigen presentation (TAP1 and TAP2), that participate in the pathologic progress. The unique flexibility, sensitivity, and accuracy of DMF-DR-seq suggest its potential utility in deeper multi-omics analysis for inheritance mechanism study in single-cell biology.

Recent advances in microchip electrophoresis for analysis of pathogenic bacteria and viruses.

Life-threatening diseases, such as hepatitis B, pneumonia, tuberculosis, and COVID-19, are widespread due to pathogenic bacteria and viruses. Therefore, the development of highly sensitive, rapid, portable, cost-effective, and selective methods for the analysis of such microorganisms is a great challenge. Microchip electrophoresis (ME) has been widely used in recent years for the analysis of bacterial and viral pathogens in biological and environmental samples owing to its portability, simplicity, cost-effectiveness, and rapid analysis. However, microbial enrichment and purification are critical steps for accurate and sensitive analysis of pathogenic bacteria and viruses in complex matrices. Therefore, we first discussed the advances in the sample preparation technologies associated with the accurate analysis of such microorganisms, especially the on-chip microfluidic-based sample preparations such as dielectrophoresis and microfluidic membrane filtration. Thereafter, we focused on the recent advances in the lab-on-a-chip electrophoretic analysis of pathogenic bacteria and viruses in different complex matrices. As the microbial analysis is mainly based on the analysis of nucleic acid of the microorganism, the integration of nucleic acid-based amplification techniques such as polymerase chain reaction (PCR), quantitative PCR, and multiplex PCR with ME will result in an accurate and sensitive analysis of microbial pathogens. Such analyses are very important for the point-of-care diagnosis of various infectious diseases.