Demand-Driven Multi-Target Sample Preparation on Resource-Constrained Digital Microfluidic Biochips

Microfluidic lab-on-chips offer promising technology for the automation of various biochemical laboratory protocols on a minuscule chip. Sample preparation (SP) is an essential part of any biochemical experiments, which aims to produce dilution of a sample or a mixture of multiple reagents in a certain ratio. One major objective in this area is to prepare dilutions of a given fluid with different concentration factors, each with certain volume, which is referred to as the demand-driven multiple-target (DDMT) generation problem. SP with microfluidic biochips requires proper sequencing of mix-split steps on fluid volumes and needs storage units to save intermediate fluids while producing the desired target ratio. The performance of SP depends on the underlying mixing algorithm and the availability of on-chip storage, and the latter is often limited by the constraints imposed during physical design. Since DDMT involves several target ratios, solving it under storage constraints becomes even harder. Furthermore, reduction of mix-split steps is desirable from the viewpoint of accuracy of SP, as every such step is a potential source of volumetric split error. In this article, we propose a storage-aware DDMT algorithm that reduces the number of mix-split operations on a digital microfluidic lab-on-chip. We also present the layout of the biochip with -storage cells and their allocation technique for . Simulation results reveal the superiority of the proposed method compared to the state-of-the-art multi-target SP algorithms.

Novel Applications of Microextraction Techniques Focused on Biological and Forensic Analyses

In recent years, major attention has been focused on microextraction procedures that allow high recovery of target analytes, regardless of the complexity of the sample matrices. The most used techniques included liquid-liquid extraction (LLE), solid-phase extraction (SPE), solid-phase microextraction (SPME), dispersive liquid-liquid microextraction (DLLME), microextraction by packed sorbent (MEPS), and fabric-phase sorptive extraction (FPSE). These techniques manifest a rapid development of sample preparation techniques in different fields, such as biological, environmental, food sciences, natural products, forensic medicine, and toxicology. In the biological and forensic fields, where a wide variety of drugs with different chemical properties are analyzed, the sample preparation is required to make the sample suitable for the instrumental analysis, which often includes gas chromatography (GC) and liquid chromatography (LC) coupled with mass detectors or tandem mass detectors (MS/MS). In this review, we have focused our attention on the biological and forensic application of these innovative procedures, highlighting the major advantages and results that have been accomplished in laboratory and clinical practice.

Development of a sample preparation procedure for Sr isotope analysis of Portland cements

The 87Sr/86Sr isotope ratio can, in principle, be used for provenancing of cement. However, while commercial cements consist of multiple components, no detailed investigation into their individual 87Sr/86Sr isotope ratios or their influence on the integral 87Sr/86Sr isotope ratio of the resulting cement was conducted previously. Therefore, the present study aimed at determining and comparing the conventional 87Sr/86Sr isotope ratios of a diverse set of Portland cements and their corresponding Portland clinkers, the major component of these cements. Two approaches to remove the additives from the cements, i.e. to measure the conventional 87Sr/86Sr isotopic fingerprint of the clinker only, were tested, namely, treatment with a potassium hydroxide/sucrose solution and sieving on a 11-µm sieve. Dissolution in concentrated hydrochloric acid/nitric acid and in diluted nitric acid was employed to determine the 87Sr/86Sr isotope ratios of the cements and the individual clinkers. The aim was to find the most appropriate sample preparation procedure for cement provenancing, and the selection was realised by comparing the 87Sr/86Sr isotope ratios of differently treated cements with those of the corresponding clinkers. None of the methods to separate the clinkers from the cements proved to be satisfactory. However, it was found that the 87Sr/86Sr isotope ratios of clinker and cement generally corresponded, meaning that the latter can be used as a proxy for the clinker 87Sr/86Sr isotope ratio. Finally, the concentrated hydrochloric acid/nitric acid dissolution method was found to be the most suitable sample preparation method for the cements; it is thus recommended for 87Sr/86Sr isotope analyses for cement provenancing. Graphical abstract

Mechanical Degradation Behavior of Single Crystal LiNixMnyCozO2 Cathode in Li-ion Battery by Indentation Analysis

LiNixMnyCozO2 (NMC) is among the most promising cathode materials for commercial Li-ion batteries due to its high electrochemical performance. However, NMC composite cathode is still plagued with limited cyclic performance, which is influenced by its structural stability during the cycling process. The cathode, which comprises of the active material, polymeric binder, and porous conductive matrix, often exhibits large structural variation during the electrochemical cycling process. This inevitably increases the challenge of measuring the mechanical properties of the material. Even though single crystal NMC possesses better stability as compared to the polycrystalline NMC, the electrochemical performance degradation of single crystal NMC cathode remains relatively unexplored. Different sample preparation methods are compared systematically in accordance to the previous report, and a new method of sample preparation is proposed. Nanoindentation instrument is used to measure the elastic modulus and hardness of the single crystal NMC particles. The measured elastic modulus and hardness of NMC particles, under different electrochemical environments, are dependent on a large number of nanoindentation experiments and statistical analysis of the result obtained from the carefully prepared samples. The sample preparation method is the key factor that can significantly influence the nanoindentation experiment results of the NMC particles. This work shows that the mechanical properties of the single crystal NMC particles degrade significantly with number of electrochemical cycles. The decreasing elastic modulus with the number of electrochemical cycles can be fitted using a two-parameter logarithm model.

One for All and All in One: Modified Silica Kit-based Protocol for simultaneous sample-specific Extraction of DNA from a Variety of Source Materials

Protocols utilized for the extraction of DNA vary significantly with regards to steps involved and duration of the overall procedure due to material-specific requirements for ensuring the highest possible yield in recovery of DNA. This variation mostly affects aspects of sample preparation and digestion steps required to release the DNA from the sample material.In contexts such as the development of new PCR-based assays - which always includes a test of species-specificity - reference samples from a number of species are utilized, requiring extraction of DNA from a variety of source materials, each with their specific conditions for effective isolation of DNA.The method presented here follows the strategy of synchronizing sample material-specific aspects such as sample preparation and digestion in such a way that one common protocol can be utilized for the actual extraction and purification of the DNA, allowing for an overall more efficient extraction process, while maintaining optimized conditions for DNA recovery.

Automated Sample Preparation and Data Collection Workflow for High-Throughput In Vitro Metabolomics

Regulatory bodies have started to recognise the value of in vitro screening and metabolomics as two types of new approach methodologies (NAMs) for chemical risk assessments, yet few high-throughput in vitro toxicometabolomics studies have been reported. A significant challenge is to implement automated sample preparation of the low biomass samples typically used for in vitro screening. Building on previous work, we have developed, characterised and demonstrated an automated sample preparation and analysis workflow for in vitro metabolomics of HepaRG cells in 96-well microplates using a Biomek i7 Hybrid Workstation (Beckman Coulter) and Orbitrap Elite (Thermo Scientific) high-resolution nanoelectrospray direct infusion mass spectrometry (nESI-DIMS), across polar metabolites and lipids. The experimental conditions evaluated included the day of metabolite extraction, order of extraction of samples in 96-well microplates, position of the 96-well microplate on the instrument’s deck and well location within a microplate. By using the median relative standard deviation (mRSD (%)) of spectral features, we have demonstrated good repeatability of the workflow (final mRSD < 30%) with a low percentage of features outside the threshold applied for statistical analysis. To improve the quality of the automated workflow further, small method modifications were made and then applied to a large cohort study (4860 sample infusions across three nESI-DIMS assays), which confirmed very high repeatability of the whole workflow from cell culturing to metabolite measurements, whilst providing a significant improvement in sample throughput. It is envisioned that the automated in vitro metabolomics workflow will help to advance the application of metabolomics (as a part of NAMs) in chemical safety, primarily as an approach for high throughput screening and prioritisation.

Automated Chloroform-Methanol Protein Extraction on the Biomek-FX Liquid Handler System v2

This protocol details steps to extract protein from Gram-negative bacterial or fungal cells (that have been pretreated with zymolyase) in quantitative proteomic workflows by using a Biomek FX liquid handler system. It is a semi-automated protocol that includes several 'pause' steps for centrifugation steps that are conducted manually "off-deck". This protocol works best as part of an automated proteomic sample preparation workflow with: Automated Protein Quantitation with the Biomek-FX liquid handler system and Automated Protein Normalization and Tryptic Digestion on a Biomek-NX Liquid Handler System