Ultra-low Quantities Research Articles

Structural Characterization of a Pathogenic Antibody Underlying Vaccine-Induced Immune Thrombotic Thrombocytopenia (VITT).

Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a rare but dangerous side effect of adenoviral-vectored COVID-19 vaccines. VITT had been linked to production of autoantibodies recognizing platelet factor 4 (PF4). Here, we characterize anti-PF4 antibodies obtained from a VITT patient's blood. Intact mass measurements indicate that a significant fraction of these antibodies represent a limited number of clones. MS analysis of large antibody fragments (the light chain and the Fc/2 and Fd fragments of the heavy chain) confirms the monoclonal nature of this component of the anti-PF4 antibodies repertoire and reveals the presence of a mature complex biantennary N-glycan within the Fd segment. Peptide mapping using two complementary proteases and LC-MS/MS was used to determine the amino acid sequence of the entire light chain and over 98% of the heavy chain (excluding a short N-terminal segment). The sequence analysis allows the monoclonal antibody to be assigned to the IgG2 subclass and verifies that the light chain belongs to the λ-type. Incorporation of enzymatic de-N-glycosylation into the peptide mapping routine allows the N-glycan in the Fab region of the antibody to be localized to the framework 3 region of the VH domain. This novel N-glycosylation site is the result of a single mutation within the germline sequence. Peptide mapping also provides information on lower-abundance (polyclonal) components of the anti-PF4 antibody ensemble, revealing the presence of all four subclasses (IgG1-IgG4) and both types of the light chain (λ and κ). This case study demonstrates the power of combining the intact, middle-down, and bottom-up MS approaches for meaningful characterization of ultralow quantities of pathogenic antibodies extracted directly from patients' blood.

Abstract 5026: Microfluidic capillary electrophoresis optimization of high-throughput cfDNA and pDNA measurements for enabling cancer diagnostics and gene therapy development

Abstract Rapid and accurate electrophoresis characterization can be an important tool for cancer researchers and therapy manufacturers. Advances in NGS of low molecular weight cfDNA for diagnostics may benefit from high-throughput checks of cfDNA sample quality with ultra-low quantities of sample. On the other hand, high molecular weight pDNA characterization and separation of supercoiled, linear and open-linear conformations aids in quality control of these payloads for personalized medicine and in particular cancer vaccines and gene therapies. This work shows optimization of established microfluidic capillary electrophoresis to enable the measurement of cfDNA and pDNA. cfDNA study was completed on plasma reformatted from whole blood tubes using Janus G3 Blood iQ workstation. The cfDNA samples were extracted using commercial cfDNA extraction kits. Using a LabChip GX Touch Biologics Characterization system: gel optimization, marker solution dilution, and inclusion of a low molecular weight DNA standard improved the sensitivity of cfDNA detection. This afforded a new electrophoresis assay with linear dynamic range of 50-1000 pg/uL and sensitivity of <25 ng/uL. pDNA, supercoiled DNA and linear DNA standards were prepared according to published procedures and measured on the LabChip GX Touch Biologics Characterization system. A new ladder system was developed along with gel optimization to develop a new High Sensitivity Assay platform capable of baseline resolving each conformation and providing fragment DNA sizing and purity information. Taken together, this work provides researchers new tools to leverage automated microfluidic capillary electrophoresis for DNA analysis in both upstream and manufacturing processes. Citation Format: Dipti Mehta, Gayatri Gautam, Adriana Coll de Pena, Menel Ben Frej, Anubhav Tripathi, James White. Microfluidic capillary electrophoresis optimization of high-throughput cfDNA and pDNA measurements for enabling cancer diagnostics and gene therapy development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5026.

Nanomaterials promote the fast development of electrochemical MiRNA biosensors.

Cancer has become the leading cause of death worldwide. In recent years, molecular diagnosis has demonstrated great potential in the prediction and diagnosis of cancer. MicroRNAs (miRNAs) are short oligonucleotides that regulate gene expression and cell function and are considered ideal biomarkers for cancer detection, diagnosis, and patient prognosis. Therefore, the specific and sensitive detection of ultra-low quantities of miRNA is of great significance. MiRNA biosensors based on electrochemical technology have advantages of high sensitivity, low cost and fast response. Nanomaterials show great potential in miRNA electrochemical detection and promote the rapid development of electrochemical miRNA biosensors. Some methods and signal amplification strategies for miRNA detection in recent years are reviewed herein, followed by a discussion of the latest progress in electrochemical miRNA detection based on different types of nanomaterial. Future perspectives and challenges are also proposed for further exploration of nanomaterials to bring breakthroughs in electrochemical miRNA detection.

Trace amounts of fluorinated surfactant additives enable high performance zinc-ion batteries

Aqueous zinc-ion batteries are promising alternatives to lithium-ion batteries for grid-scale energy storage. However, the practical application of AZIBs is challenged by side reactions and unsatisfactory performance. A new electrolyte additive is reported that can inhibit side reactions on the Zn anode and enlarge the working potential window of aqueous electrolytes. Trace amounts of perfluorooctanoic acid (PFOA) is capable of facilitating long-term reversible Zn deposition in AZIBs due to C-F chains ordered orientation absorbing on the electrode surface. The molecular dynamics results show that the as-formed electronegative absorbing layer act as a zincophilic layer to regulate the ions in the electrolyte. The electrolyte with PFOA additive enables a long lifespan of the Zn anode for >2000 hours. Also, the PFOA additive is used at one of the lowest amounts of reported additives in AZIBs. This strategy provides a simple and low-cost method to tackle common issues in AZIBs. Owing to C-F chains ordered orientation absorbing on the electrode surface, ultralow quantities of perfluorooctanoic acid (PFOA) additive (0.25 mM) in AZIBs is capable of enlarging working window to 2.1 V and facilitating long-term reversible Zn deposition (2200 hours), a high rate performance (153 mAh g −1 at 5 A −1 ) is achieved in Zn||Na 0.65 Mn 2 O 4 cells.

Probing the Role of Atomic Defects in Photocatalytic Systems through Photoinduced Enhanced Raman Scattering

Even in ultralow quantities, oxygen vacancies (VO) drastically impact key properties of metal oxide semiconductors, such as charge transport, surface adsorption, and reactivity, playing central roles in functional materials performance. Current methods used to investigate VO often rely on specialized instrumentation under far from ideal reaction conditions. Hence, the influence of VO generated in situ during catalytic processes has yet to be probed. In this work, we assess in situ extrinsic surface VO formation and lifetime under photocatalytic conditions which we compare to photocatalytic performance. We show for the first time that lifetimes of in situ generated atomic VO play more significant roles in catalysis than their concentration, with strong correlations between longer-lived VO and higher photocatalytic activity. Our results indicate that enhanced photocatalytic efficiency correlates with goldilocks VO concentrations, where VO densities must be just right to encourage carrier transport while avoiding charge carrier trapping.

Colorimetric optical nanosensors for trace explosive detection using metal nanoparticles: advances, pitfalls, and future perspective

Warfare threats and acts of terror are challenging situations encountered by defense agencies across the globe and are of growing concern to the general public, and security-minded policy makers. Detecting ultra-low quantities of explosive compounds in remote locations or under harsh conditions for anti-terror purposes as well as the environmental monitoring of residual or discarded explosives in soil, remains a major challenge. The use of metal nanoparticles (NPs) for trace explosive detection has drawn considerable interest in recent years. For nano-based explosive sensor devices to meet real-life operational demands, analytical parameters such as, long-shelf life, stability under harsh conditions, ease-of-use, high sensitivity, excellent selectivity, and rapid signal response must be met. Generally, the analytical performance of colorimetric-based nanosensor systems is strongly dependent on the surface properties of the nanomaterial used in the colorimetric assay. The size and shape properties of metal NPs, surface functionalisation efficiency, and assay fabrication methods, are factors that influence the efficacy of colorimetric explosive nanosensor systems. This review reports on the design and analytical performances of colorimetric explosive sensor systems using metal NPs as optical signal transducers. The challenges of trace explosive detection, advances in metal NP colorimetric explosive design, limitations of each methods, and possible strategies to mitigate the problems are discussed.

Highly active Ag/C nanoparticles containing ultra-low quantities of sub-surface Pt for the electrooxidation of glycerol in alkaline media

The practical application of Pt-based technologies depends on the lowering in the costs and one way to face this challenge is by reducing the amount of Pt. Herein, we decorate Ag nanoparticles with ultra-small (0.8 %) quantities of Pt and test them for the electrooxidation of glycerol. We showed that the Pt atoms of our catalyst are one order of magnitude more active (in terms of mass activity) than Pt/C commercial catalyst. By performing in situ FTIR measurements, electrolysis experiments and HPLC analysis, we showed that both catalysts form mainly glycerate and lactate, but Pt/C is more prone to poisoning and to form carbonate. We suggest that this different behavior is attributed to the high dispersion of the Pt atoms on Ag nanoparticles. The lower probability of finding neighboring Pt atoms diminishes the formation of multiple bonded intermediates, which are precursors for the formation of carbonate and some poisoning intermediates.

Future requirements for non-decimal unit prefixes in chemical measurement

The 2019 revision of the International System of Units (SI), including the redefinition of the mole, and the progress of analytical chemistry technologies allowing single-molecule resolution has brought into sharp focus the dilemma between expressing measurements of ultra-low quantities of elementary entities in terms of amount of substance or in terms of counting quantities. We should prefer the use of amount of substance for these measurements for a number of reasons including to allow intensive and extensive quantities in chemistry to be distinguished and to separate these measurements from those involving the counting of non-elementary entities. The discussion article highlights how a new, non-decimal approach to unit prefixes might in future enable amount of substance to be used usefully down to its natural limit—a single elementary entity. A new prefix symbol ‘σ’ is proposed to represent the multiplier 1/{NA}.

Сравнение режимов возбуждения флуоресценции полупроводниковых квантовых точек на основе селенида кадмия для биомедицинских приложений

Fluorescence spectroscopy is a powerful tool used in applied biological and medical research. Colloid semi-conductor quantum dots are promising fluorescent tags for simultaneous detection of different biopathogens. The techniques employing these tags can be improved by selecting the optimal modes for signal excitation and detection. The aim of the present work was to derive a mathematical expression to describe the signal-to-noise ratios in the pulsed and modulated excitation modes. Below, we compare these two modes of fluorescence excitation in ultralow quantities of quantum dots. We demonstrate that modulated excitation should be preferred for CdSe/ZnS quantum dots given that signal accumulation time is over 100 mc and the photosensor is exposed to background light of > 1 μW.

Development of a Method to Implement Whole-Genome Bisulfite Sequencing of cfDNA from Cancer Patients and a Mouse Tumor Model.

The goal of this study was to develop a method for whole genome cell-free DNA (cfDNA) methylation analysis in humans and mice with the ultimate goal to facilitate the identification of tumor derived DNA methylation changes in the blood. Plasma or serum from patients with pancreatic neuroendocrine tumors or lung cancer, and plasma from a murine model of pancreatic adenocarcinoma was used to develop a protocol for cfDNA isolation, library preparation and whole-genome bisulfite sequencing of ultra low quantities of cfDNA, including tumor-specific DNA. The protocol developed produced high quality libraries consistently generating a conversion rate >98% that will be applicable for the analysis of human and mouse plasma or serum to detect tumor-derived changes in DNA methylation.

Laser ion mobility spectrometry in the detection of ultra-low quantities of explosives.

The paper presents the results of research and development of ion mobility spectrometer with laser ion sources for detecting ultra-low quantities of explosives in air. Particular attention is paid to the mechanisms of the formation of negative ions. It is shown that laser ionization with respect to ion mobility spectrometry provides an increase of sensitivity and selectivity of detection. The ion mobility spectra of the most common nitro explosives are investigated. The detection threshold for laser ion mobility spectrometer was reached at the level of 10-14 g/cm3 (for trinitrotoluene).

Electrooxidation of Hexacyanoferrate(II) Anions in the Microfabricated Electrochemical Sensor-Array System

The field of application of micro-electrodes is very wide: from electroanalytical applications (detection of ultra-low quantities of analyte, biosensors, etc.), kinetic studies of very fast and complex reactions, to measurements in solutions of very low conductivity, etc.1-3 Often several micro-electrodes are combined into sensor-arrays for simultaneous analysis of various parameters. In this work the microfabrication technique was used to prepare a six-channel sensor-array chip.4 The sensor-array has been fabricated of borosilicate wafer containing Pt electrodes and connection pads and the silicon wafer with through holes, which were bonded using adhesion bonding method with SU-8. Although the fabrication process of two wafers is simple, the extensively perforated silicon wafer is brittle and can break down during bonding step. However, with the customized bonding process, a good bonding was achieved and all the chips were applicable after dicing. The cyclic voltammetry and electrochemical impedance experiments were carried out in a three-electrode electrochemical system to characterize the prepared sensor-array chips. Sputtered platinum was used for micro-electrodes, and Ag/AgCl/sat. KCl as a reference electrode. The high surface area Pt counter electrode has been used for studies. It was shown that the measured current density depends on the electrode potential sweep rate, however both linear and radial diffusion mass transfer components are important at high electrode potential scanning rate. When using slow potential sweep rates (v = 0.005 V s‒1) the pseudo steady-state has been achieved and the electrodes behaved as the micro-electrodes. It was shown that the pseudo steady-state current density depends linearly on the concentration of potassium hexacyanoferrate(II) in the 1.0 M KCl aqueous solution. Based on the electrochemical characterization data it can be concluded that the produced sensor array is suitable for future research including BOD biosensor-array construction. The sensor-array developed can be modified with microorganisms by immobilizing the bacteria into the cavities fabricated onto/into the chip. Acknowledgements This research was supported by the EU through the European Regional Development Fund (Centre of Excellence, 2014-2020.4.01.15-0011), Institutional Research Grant IUT20-13, and Estonian Science Foundation (Grant ETF 9136).

Declining malaria, rising of dengue and Zika virus: insights for mosquito vector control.

The fight against mosquito-borne diseases is a challenge of huge public health importance. To our mind, 2015 was an extraordinary year for malaria control, due to three hot news: the Nobel Prize to Youyou Tu for the discovery of artemisinin, the development of the first vaccine against Plasmodium falciparum malaria [i.e. RTS,S/AS01 (RTS,S)], and the fall of malaria infection rates worldwide, with special reference to sub-Saharan Africa. However, there are major challenges that still deserve attention, in order to boost malaria prevention and control. Indeed, parasite strains resistant to artemisinin have been detected, and RTS,S vaccine does not offer protection against Plasmodium vivax malaria, which predominates in many countries outside of Africa. Furthermore, the recent outbreaks of Zika virus infections, occurring in South America, Central America and the Caribbean, represent the most recent of four arrivals of important arboviruses in the Western Hemisphere, over the last 20years. Zika virus follows dengue (which slyly arrived in the hemisphere over decades and became more aggressive in the 1990s), West Nile virus (emerged in 1999) and chikungunya (emerged in 2013). Notably, there are no specific treatments for these arboviruses. The emerging scenario highlights that the effective and eco-friendly control of mosquito vectors, with special reference to highly invasive species such as Aedes aegypti and Aedes albopictus, is crucial. The concrete potential of screening plant species as sources of metabolites for parasitological purposes is worthy of attention, as elucidated by the Y. Tu's example. Notably, plant-borne molecules are often effective at few parts per million against Aedes, Ochlerotatus, Anopheles and Culex young instars, can be used for the rapid synthesis of mosquitocidal nanoformulations and even employed to prepare cheap repellents with low human toxicity. In addition, behaviour-based control tools relying to the employ of sound traps and the manipulation of swarming behaviour (i.e. "lure and kill" approach) are discussed. The importance of further research on the chemical cues routing mosquito swarming and mating dynamics is highlighted. Besides radiation, transgenic and symbiont-based mosquito control approaches, an effective option may be the employ of biological control agents of mosquito young instars, in the presence of ultra-low quantities of nanoformulated botanicals, which boost their predation rates.

The influence of PTFE used as basic covered electrode binder on weld metal acicular ferrite formation

Weld metals obtained from covered electrodes agglomerated with polytetrafluorethylene presented ultra-low diffusible hydrogen content and appreciable quantities of acicular ferrite. This article investigates the polymer influence on its constituent formation. The microstructure produced by consumables containing this component presented acicular ferrite quantities higher than expected in the literature for weld metals with the same carbon, silicon and manganese content. This difference could not be related to welding parameters employed. The residual chemical element evaluation indicated nitrogen as being mainly responsible for the microstructural variances observed. The operational characteristics of classic E7018 and polymer-agglomerated electrodes showed the lesser weld metal nitrogen absorption must be associated with two main factors. The dissolved nitrogen of the metallic drops collected during the polymer-agglomerated electrode welding showed most effective shielding atmosphere. The carbon content of the polymer-agglomerated electrode indicated an atmosphere rich in this element. This is in accordance with a best shielding process hypothesis. The metallic drops’ average size, dissolved nitrogen content as a function of size and voltage oscillograms indicated metallic transfer mode changes.

Phase-Based Electrochemical Biosensors for Detection of Pathogenic Bacteria

Bacterial pathogens are universally present in the environment and easily contaminate food and water to cause serious food-borne illness to humans. Detecting these pathogens in ultra low quantities is critical to prevent major outbreaks of food-borne diseases. Several approaches have been explored for detecting pathogenic bacteria in food and water of which electrochemical biosensors offer unique advantages including rapid detection, high sensitivity and easy-to-use testing procedure. However the specificity of detection and the cost of the biosensor heavily depend on the type and nature of the bio-recognition element used. Enzymes and antibodies are highly susceptible to environment and have low stability when used on electrodes. In this work, bacteriophage (virus) has been explored as an alternative bio-recognition element for the detection of pathogenic bacteria. More specifically, a wild type T2 phage was utilized as recognition element for direct detection of Escherichia coli, ATCC 11303. The T2 phage was immobilized on carbon nanotube modified screen-printed carbon electrodes (SPE) using a molecular tethering approach developed by us.1 The binding of phage to the carbon nanotubes was verified by X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM). The phage-modified electrodes were used to specifically detect E. coli. Electrochemical impedance spectroscopy was used to detect the impedance variations resulting from the binding of E. coli to T2 phage. The phage-based biosensor showed high sensitivity towards the detection of E. coli and the controls in the absence of phage did not show any signal for the E. coli detection.

The influence of carbonization elimination on the environmental safety and efficiency of vehicle operation

Poor quality of motor fuels affects the environmental safety and effi ciency of vehicle operation. Solving the problem by modernizing the refi nery to raise the quality of petroleum products to the level of modern European standards requires substantial capital investment and cannot be implemented in the short term in Russia and other countries that have a transition economy. The aim of this work is to develop a low-cost method for improving the environmental safety and effi ciency of vehicle operation by eliminating carbon formation in engines. It was shown in the theoretical analysis of the infl uence of carbon on temperature conditions inside the engine, which determines the characteristics performance of engines and the composition of exhaust gases, that the removal of carbon formation leads to a reduction in fuel consumption, toxic emissions, and the requirements of vehicles with gasoline engines for octane number of gasoline used. The method of carbonization elimination has been developed. The effective removal of carbon and signifi cant improvement of the environmental and operational properties of fuels and vehicle characteristics is provided by introducing into the engine fuel in ultra-low quantities the multifunctional additive. The results of extensive research and testing of the additive are presented.

Marine sediment pore‐water profiles of phosphate d18O using a refined micro‐extraction

Phosphorus cycling in the ocean is influenced by biological and geochemical processes that are reflected in the oxygen isotope signature of dissolved inorganic phosphate (Pi). Extending the Pi oxygen isotope record from the water column into the seabed is difficult due to low Pi concentrations and small amounts of marine porewaters available for analysis. We obtained porewater profiles of Pi oxygen isotopes using a refined protocol based on the original micro‐extraction designed by Colman (2002). This refined and customized method allows the conversion of ultra‐low quantities (0.5 – 1 µmol) of porewater Pi to silver phosphate (Ag3PO4) for routine analysis by mass spectrometry. A combination of magnesium hydroxide co‐precipitation with ion exchange resin treatment steps is used to remove dissolved organic matter, anions, and cations from the sample before precipitating Ag3PO4. Samples as low as 200 µg were analyzed in a continuous flow isotope ratio mass spectrometer setup. Tests with external and laboratory internal standards validated the preservation of the original phosphate oxygen isotope signature (δ18OP) during micro extraction. Porewater data on δ18OP has been obtained from two sediment cores of the Moroccan margin. The δ18OP values are in a range of +19.49 to +27.30‰. We apply a simple isotope mass balance model to disentangle processes contributing to benthic P cycling and find evidence for Pi regeneration outbalancing microbial demand in the upper sediment layers. This highlights the great potential of using δ18OP to study microbial processes in the subseafloor and at the sediment water interface.

Nanoparticle-based optical biosensors for the direct detection of organophosphate chemical warfare agents and pesticides

Neurotoxic organophosphates (OP) have found widespread use in the environment for insect control. In addition, there is the increasing threat of use of OP based chemical warfare agents in both ground based warfare and terrorist attacks. Together, these trends necessitate the development of simple and specific methods for discriminative detection of ultra low quantities of OP neurotoxins. In our previous investigations a new biosensor for the direct detection of organophosphorus neurotoxins was pioneered. In this system, the enzymatic hydrolysis of OP neurotoxins by organophosphate hydrolase (OPH) generated two protons in each hydrolytic turnover through reactions in which P–X bonds are cleaved. The sensitivity of this biosensor was limited due to the potentiometric method of detection. Recently, it was reported that a change in fluorescence properties of a fluorophore in the vicinity of gold nanoparticles might be used for detection of nanomolar concentrations of DNA oligonucleotides. The detection strategy was based on the fact that an enhancement or quenching of fluorescence intensity is a function of the distances between the gold nanoparticle and fluorophore. While these reports have demonstrated the use of nanoparticle-based sensors for the detection of target DNA, we observed that the specificity of enzyme–substrate interactions could be exploited in similar systems. To test the feasibility of this approach, OPH-gold nanoparticle conjugates were prepared, then incubated with a fluorescent enzyme inhibitor or decoy. The fluorescence intensity of the decoy was sensitive to the proximity of the gold nanoparticle, and thus could be used to indicate that the decoy was bound to the OPH. Then different paraoxon concentrations were introduced to the OPH–nanoparticle–conjugate–decoy mixtures, and normalized ratio of fluorescence intensities were measured. The greatest sensitivity to paraoxon was obtained when decoys and OPH–gold nanoparticle conjugates were present at near equimolar levels. The change in fluorescence intensity was correlated with concentration of paraoxon presented in the solution.