查看更多>>摘要：The tetanus neurotoxin (TeNT) is one of the most toxic proteins known to man, which prior to the use of the vaccine against the TeNT producing bacteria Clostridium tetani, resulted in a 20% mortality rate upon infection. The clinical detrimental effects of tetanus have decreased immensely since the introduction of global vaccination programs, which depend on sustainable vaccine production. One of the major critical points in the manufacturing of these vaccines is the stable and reproducible production of high levels of toxin by the bacterial seed strains. In order to minimize time loss, the amount of TeNT is often monitored during and at the end of the bacterial culturing. The different methods that are currently available to assess the amount of TeNT in the bacterial medium suffer from variability, lack of sensitivity, and/or require specific antibodies. In accordance with the consistency approach and the three Rs (3Rs), both aiming to reduce the use of animals for testing, in-process monitoring of TeNT production could benefit from animal and antibody-free analytical tools. In this paper, we describe the development and validation of a new and reliable antibody free targeted LC-MS/ MS method that is able to identify and quantify the amount of TeNT present in the bacterial medium during the different production time points up to the harvesting of the TeNT just prior to further upstream purification and detoxification. The quantitation method, validated according to ICH guidelines and by the application of the total error approach, was utilized to assess the amount of TeNT present in the cell culture medium of two TeNT production batches during different steps in the vaccine production process prior to the generation of the toxoid. The amount of TeNT generated under different physical stress conditions applied during bacterial culture was also monitored.
Nwokwu, Chukwumaobim D.Bari, Saif Mohammad IshraqHutson, K. HopeBrausell, Clay...
11页
查看更多>>摘要：Exosomes encapsulate genomic and proteomic biomarkers for non-invasive diagnosis and disease monitoring. However, exosome surface-markers heterogeneity is a major drawback of current isolation methods. Here, we report a direct, one-step exosome sampling technology, ExoPRIME, for selective capture of CD63(+) exosome subpopulations using an immune-affinity protocol. Microneedles (300 mu m x 30 mm), functionalized with anti-CD63 antibodies, were incubated under various experimental conditions in conditioned astrocyte medium and astrocyte-derived exosome suspension. The probe's capture efficiency and specificity were validated using FluoroCet assay, immunofluorescent imaging, and OMICS analyses. Significantly higher exosomes were captured by probes incubated for 16 hat 4 C-0 in enriched exosomal suspension (23 x 10(6) exosomes per probe) vis-a-vis 2 hat 4 C-0 (12 x 10(6)) and 16 hat 22 C-0 (3 x 10(6)) in conditioned cell media. Our results demonstrate the application of ExoPRIME over a broad dynamic range of temperature and incubation parameters, offering flexibility for any desired application. ExoPRIME permits the use and re-use of minimal sample volumes (<= 200 mu L) can be multiplexed in arrays, and integrated into a lab-on-a-chip platform to achieve parallel, high-throughput isolation of different exosome classes in a semi-automated workstation. This platform could provide direct exosomal analysis of biological fluids since it can elegantly interface with existing room-temperature, picomolar-range nucleic acid assays to provide a clinical diagnostic tool at the point of care.
查看更多>>摘要：Surface-enhanced Raman scattering (SERS) based on plasmonic metal nanoparticles and semiconductors has been used as performance-enhancing structures for sensing trace chemicals. We have selected a case of oxide functional oxide organic nanostructure between ZnFe2O4 and ZnO, denoted as ZZF. By decorating such nanostructure with AuNPs, to identify R6G in varying concentrations (10-6 M - 10-12 M), an enhancement factor of 1.6 x 108 was observed. The material was used for the identification of melamine in the concentration range of 0.39 mu M-7.92 mu M. This high-performance nanocomposite provides improved melamine sensitivity towards SERS and the limit of detection as low as 0.39 mu M. The Au-ZZF SERS substrate can yield a SERS enhancement factor of 1.37 x 107. The experimental performance demonstrates that excellent SERS enhancement is due to electrons movement within ZZF and Au nanoparticles. Owing to its easy and effective synthesis methodology, this sensitive and specific SERS substrate is a promising technique to detect trace chemicals. We further study the best energetically favorable orientation of melamine molecules over the substrate leading to the SERS activity using density functional theoretical study.
查看更多>>摘要：Non-targeted metabolomics is increasingly applied in various applications for understanding biological processes and finding novel biomarkers in living organisms. However, high-confidence identity confirmation of metabolites in complex biological samples is still a significant bottleneck, especially when using single-stage mass analysers. In the current study, a complete workflow for alternating in-source fragmentation on a time-of-flight mass spectrometry (TOFMS) instrument for non-targeted metabolomics is presented. Hydrophilic interaction liquid chromatography (HILIC) was employed to assess polar metabolites in yeast following ESI parameter optimization using experimental design principles, which revealed the key influence of fragmentor voltage for this application. Datasets from alternating in-source fragmentation high resolution mass spectrometry (HRMS) were evaluated using open-source data processing tools combined with public reference mass spectral databases. The significant influence of the selected fragmentor voltages on the abundance of the primary analyte ion of interest and the extent of in-source fragmentation allowed an optimum selection of qualifier fragments for the different metabolites. The new acquisition and evaluation workflow was implemented for the non-targeted analysis of yeast extract samples whereby more than 130 metabolites were putatively annotated with more than 40% considered to be of high confidence. The presented workflow contains a fully elaborated acquisition and evaluation methodology using alternating in-source fragmentor voltages suitable for peak annotation and metabolite identity confirmation for non-targeted metabolomics applications performed on a single-stage HRMS platform.
查看更多>>摘要：Single-cell analysis of proteins is critical to gain precise information regarding the mechanisms that dictate the heterogeneity in cellular phenotypes and their differential response to internal and external stimuli. However, tools that allow sensitive and easy measurement of proteins in individual cells are still limited. The emerging semiconductor-based bioelectronics may provide a new approach to overcome the challenges in this field, however its utility in single-cell protein analysis has not been explored. In this study, we investigated multiple protein detection in single cells by MoS2 field effect transistors (MoS2-FETs) modified with specific biological probes. First, beta-actin antibody was connected to the surface of MoS2-FETs by covalent bonds, and the fabricated device was tested using beta-actin solution with concentrations from 10(-9) to 10(-3) mu g/mu L. Next, we examined the application of MoS2-FET for protein analysis in complex biological samples, and the device showed electrical signal response to human embryonic kidney cell line HEK293T in a dose-dependent manner. Furthermore, we applied this method to analyze individual liver cancer MHCC-97L cells, targeting four cellular proteins, including beta-actin, epidermal growth factor receptor, sirtuin-2, and glyceraldehyde-3-phosphate dehydrogenase. The devices modified with corresponding probes could identify the target proteins and showed cell number-dependent responses. As a proof of principle, we demonstrated sensitive and multiplexed detection of proteins in single cells using MoS2-FETs. The biosensor and this detection method are cost-efficient and user-friendly with broad application prospects in biological studies and clinical diagnosis.
查看更多>>摘要：Early diagnosis and timely management of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) are the keys to preventing the spread of the epidemic and controlling new infection clues. Therefore, strengthening the surveillance of the epidemic and timely screening and confirming SARS-CoV-2 infection is the primary task. In this work, we first proposed the idea of activating CRISPR-Cas12a activity using double-stranded DNA amplified by a three-dimensional (3D) DNA walker. We applied it to the design of an electrochemiluminescent (ECL) biosensor to detect the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) gene. We first activated the cleavage activity of CRISPR-Cas12a by amplifying the target DNA into a segment of double-stranded DNA through the amplification effect of a 3D DNA walker. At the same time, we designed an MXene based ECL material: PEI-Ru@Ti3C2@AuNPs, and constructed an ECL biosensor to detect the RdRp gene based on this ECL material as a framework. Activated CRISPR-Cas12a cleaves the single-stranded DNA on the surface of this sensor and causes the ferrocene modified at one end of the DNA to move away from the electrode surface, increasing the ECL signal. The extent of the change in electrochemiluminescence reflects the concentration of the gene to be measured. Using this system, we detected the SARS-CoV-2 RdRp gene with a detection limit of 12.8 aM. This strategy contributes to the rapid and convenient detection of SARS-CoV-2-associated nucleic acids and promotes the clinical application of ECL biosensors based on CRISPR-Cas12a and novel composite materials.
Alnajrani, Mohammed N.Aljohani, Maher M.Chinnappan, RajaAlsager, Omar A....
8页
查看更多>>摘要：Dabigatran etexilate (DBG) is a new anticoagulant drug (commercially sold under the names Pradaxa (R) and PradaxTM) that replaces Warfarin, the landmark agent for anticoagulation therapy. Inadequate administration of DBG or in the cases of massive bleeding that occurs after renal impairment, DBG therapy can carry a substantial life-threatening risks. One of the major limitations of DBG treatment is the lack of a simple and quick tool for measuring its level in blood in the case of massive bleedings or emergency operations. In this work, we have incorporated a previously isolated aptamer for DBG to develop a simple competitive lateral flow aptasensor (LFA) for the determination of DBG in buffer and blood samples. A full-length 60-mer aptamer as well as a truncated 38-mer aptamer were conjugated to gold nanoparticles (AuNPs) via thiol-Au coupling chemistry. After appropriate AuNP surface passivation steps, the aptamer's core region was hybridized with 8-mer biotinylated sequences. The conjugated particles could be capture on the test line by the interaction of the biotin molecules with a previously deposited streptavidin. Incubation of the conjugated particles with DBG causes the aptamer to undergo a conformational change that releases the 8-mer biotinylated sequences and result in the disappearance of the test line. Lysozyme protein was used to construct the control line that non-specifically interacts with the conjugated particles whether or not the target compound is present. The developed LFA achieves 20 nM detection level in buffer and blood samples, operates within the nanomolar range, and shows excellent selectivity against potential interfering molecules. The developed sensor could help assessing the levels of DBG in medical conditions that require rapid interventions.
NSTL
Elsevier