On-Chip Magnetic Extraction of Circulating Cell-Free DNA from Biological Samples.

In recent years, the analysis of circulating cell-free DNA (cfDNA) containing tumor-derived DNA has emerged as a noninvasive means for cancer monitoring and personalized medicine. However, the isolation of cfDNA from peripheral blood has remained a challenge due to the low abundance and high fragmentation of these molecules. Here, we present a dynamic Magnetic ExTRactiOn (METRO) protocol using microfluidic fluidized bed technology to isolate circulating cfDNA from raw biological materials such as undiluted serum. This protocol maximizes the surface area for DNA binding within the chip in order to capture short DNA fragments. It uses only a few µL of sample and reagents. The protocol can be automated, and it is fully compatible with sensitive DNA amplification methods such as droplet-based digital PCR (ddPCR).

Cryogenic Pretreatment Enhances Drying Rates in Whole Berries.

The impact of cryogenic pretreatments on drying performance was studied in blueberries, seabuckthorn fruits and green grapes. The fruits were immersed in liquid nitrogen in 2 min freezing/thawing cycles (one to five). Untreated samples were used as the control. Drying experiments were carried out on treated and non-treated berries at 50 °C and 1 m/s (hot-air-drying), 50 °C and 25″ Hg vacuum (vacuum-drying), 30 mTorr total pressure and 25 °C shelf temperature (freeze-drying). The weight loss evolution of the foodstuffs was measured as a function of time. Microscopic (SEM and optical) determinations of the epicarp were performed. A visual inspection was performed and color changes and volume reductions were assessed before and after dehydration. The thickness of the berries' epicarp decreased between 20 and 50% (depending on the fruit) after 3-5 immersions in liquid N2. The drying kinetics was accelerated significantly for the three tested drying processes (i.e., drying time decreased from 48 to 16 h for blueberry freeze-drying). The best quality of dried berries was observed for pretreated blueberries after freeze-drying, keeping their volume, shape and color after the process. This work shows that "tailor-made" dried berry products with desired properties can be achieved and drying performance can be improved by the application of ultra-low temperature pretreatments.

Acoustic detection of a mutation-specific Ligase Chain Reaction based on liposome amplification.

Single nucleotide variants (SNVs) play a crucial role in understanding genetic diseases, cancer development, and personalized medicine. However, existing ligase-based amplification and detection techniques, such as Rolling Circle Amplification and Ligase Detection Reaction, suffer from low efficiency and difficulties in product detection. To address these limitations, we propose a novel approach that combines Ligase Chain Reaction (LCR) with acoustic detection using highly dissipative liposomes. In our study, we are using LCR combined with biotin- and cholesterol-tagged primers to produce amplicons also modified at each end with a biotin and cholesterol molecule. We then apply the LCR mix without any purification directly on a neutravidin modified QCM device Au-surface, where the produced amplicons can bind specifically through the biotin end. To improve sensitivity, we finally introduce liposomes as signal enhancers. For demonstration, we used the detection of the BRAF V600E point mutation versus the wild-type allele, achieving an impressive detection limit of 220 aM of the mutant target in the presence of the same amount of the wild type. Finally, we combined the assay with a microfluidic fluidized bed DNA extraction technology, offering the potential for semi-automated detection of SNVs in patients' crude samples. Overall, our LCR/acoustic method outperforms other LCR-based approaches and surface ligation biosensing techniques in terms of detection efficiency and time. It effectively overcomes challenges related to DNA detection, making it applicable in diverse fields, including genetic disease and pathogen detection.

High-throughput extraction on a dynamic solid phase for low-abundance biomarker isolation from biological samples.

Liquid biopsy, in particular circulating tumor DNA (ctDNA) analysis, has paved the way for a new noninvasive approach to cancer diagnosis, treatment selection and follow-up. As a crucial step in the analysis, the extraction of the genetic material from a complex matrix needs to meet specific requirements such as high specificity and low loss of target. Here, we developed a new generation of microfluidic fluidized beds (FBs) that enable the efficient extraction and preconcentration of specific ctDNA sequences from human serum with flow rates up to 15 µL/min. We first demonstrated that implementation of a vibration system inducing flow rate fluctuations combined with a mixture of different bead sizes significantly enhanced bead homogeneity, thereby increasing capture efficiency. Taking advantage of this new generation of high-throughput magnetic FBs, we then developed a new method to selectively capture a double-stranded (dsDNA) BRAF mutated DNA sequence in complex matrices such as patient serum. Finally, as proof of concept, ligation chain reaction (LCR) assays were performed to specifically amplify a mutated BRAF sequence, allowing the detection of concentrations as low as 6 × 104 copies/µL of the mutated DNA sequence in serum.

Straightforward extraction and selective bioconversion of high purity chitin from Bombyx eri larva: Toward an integrated insect biorefinery.

Chitins of different purity grades (45%, 89.7% and 93.3%) were efficiently extracted from Bombyx eri larva and fully physico-chemically characterized. Compared to commercially available and extracted α-chitin from shrimp shell, the collected data showed that insect chitins had similar characteristics in terms of crystallographic structures (α-chitin), thermal stability and degree of acetylation (>87%). The major differences lay in the crystallinity indexes (66% vs 75% for shrimp chitin) and in the morphological structures. Furthermore, low ash contents were determined for the insect chitins (1.90% vs 21.73% for shrimp chitin), making this chitin extraction and purification easier, which is highly valuable for an industrial application. Indeed, after only one step (deproteinization), the obtained chitin from Bombyx eri showed higher purity grade than the one extracted from shrimp shells under the same conditions. Insect chitins were then subjected to room temperature ionic liquid (RTIL) pretreatment prior to enzymatic degradation and presented a higher enzymatic digestibility compared to commercial one whatever their purity grade and would be thus a more relevant source for the selective production of N-acetyl-D-glucosamine (899.2 mg/g of chitin-2 stepsvs 760 mg/g of chitin com). Moreover, for the first time, the fermentescibility of chitin hydrolysates was demonstrated with Scheffersomyces stipitis used as ethanologenic microorganism.

Wheat Bran Pretreatment by Room Temperature Ionic Liquid-Water Mixture: Optimization of Process Conditions by PLS-Surface Response Design.

Room Temperature Ionic Liquids (RTILs) pretreatment are well-recognized to improve the enzymatic production of platform molecules such as sugar monomers from lignocellulosic biomass (LCB). The conditions for implementing this key step requires henceforth optimization to reach a satisfactory compromise between energy saving, required RTIL amount and hydrolysis yields. Wheat bran (WB) and destarched wheat bran (DWB), which constitute relevant sugar-rich feedstocks were selected for this present study. Pretreatments of these two distinct biomasses with various 1-ethyl-3-methylimidazolium acetate ([C2mim][OAc])-water mixtures prior to hydrolysis catalyzed by hemicellulolytic cocktail (Cellic CTec2) were finely investigated. The main operating conditions such as pretreatment temperature (25-150°C), time (40-180 min), WB and DWB loading (2-5% w/v) and concentration of [C2mim][OAc] in water [10-100% (v/v)] were screened through glucose and xylose yields and then optimized through a Partial Least Square (PLS)-Second Order Design. In an innovative way, the PLS results showed that the four factors and their interactions could be well-fitted by a second-order model (p < 0.05). The quadratic PLS models were used to predict optimal pretreatment conditions. Thus, maximum glucose (83%) and xylose (95%) yields were obtained from enzymatic hydrolysis of WB pretreated at 150°C for 40 min with 10% of [C2mim][OAc] in water and 5% of WB loading. For DWB, maximum glucose (100%) and xylose (57%) yields were achieved for pretreatment temperatures of 150°C and 25°C, respectively. The required duration was still 40 min, with 20% of [C2mim][OAc] in water and a 5% DWB loading. Then, Multiple Response Optimization (MRO) performed by Nelder-Mead Simplex Method displayed sugar yields similar to those obtained by individual PLS optimization. This complete statistical study confirmed that the established models were appropriate to predict the sugar yields achieved after different pretreatment conditions from WB and DWB biomasses. Finally, Scanning Electron microscopy (SEM) studies allowed us to establish clearer link between structural changes induced by pretreatment and the best enzymatic performances obtained.

Solid supports for extraction and preconcentration of proteins and peptides in microfluidic devices: A review.

Determination of proteins and peptides is among the main challenges of today's bioanalytical chemistry. The application of microchip technology in this field is an exhaustively developed concept that aims to create integrated and fully automated analytical devices able to quantify or detect one or several proteins from a complex matrix. Selective extraction and preconcentration of targeted proteins and peptides especially from biological fluids is of the highest importance for a successful realization of these microsystems. Incorporation of solid structures or supports is a convenient solution employed to face these demands. This review presents a critical view on the latest achievements in sample processing techniques for protein determination using solid supports in microfluidics. The study covers the period from 2006 to 2015 and focuses mainly on the strategies based on microbeads, monolithic materials and membranes. Less common approaches are also briefly discussed. The reviewed literature suggests future trends which are discussed in the concluding remarks.

A lab-on-a-chip for monolith-based preconcentration and electrophoresis separation of phosphopeptides.

A microdevice combining online preconcentration and separation of phosphopeptides was developed in a glass microchip. An ethylene glycol methacrylate phosphate (EGMP), acrylamide (AM) and bisacrylamide (BAA) based monolith was synthesized within microchannels through a photo-driven process. Morphological investigations revealed a homogeneous monolithic structure composed of uniform nodules (∼0.8 µm), with a large pore volume (0.62 cm3 g-1) and sufficiently high specific surface area (34.1 m2 g-1). These features make the monolith particularly interesting for preconcentration purposes. Immobilization of Zr4+ ions on the phosphate groups present at the poly(EGMP-co-AM-co-BAA) monolith surface leads to immobilized metal affinity chromatography support. This monolith-Zr4+ showed a great capacity to capture phosphopeptides. Successful preconcentration and separation of a mixture of ERK2 derived peptides differing only by their phosphorylation degree and sites could be achieved with signal enhancement factors between 340 and 910 after only 7 min of preconcentration. This integrated microdevice represents a novel approach for phosphoproteomic applications.

Microscope-assisted UV-initiated preparation of well-defined porous polymer monolithic plugs in glass microchips for peptide preconcentration.

Herein, highly defined monolithic beds were prepared in glass microchips by photopolymerization of ethylene glycol methacrylate phosphate (EGMP), acrylamide, and N,N'-methylenebisacrylamide (BAA) using an epifluorescence microscope as UV-irradiation source. Such a fast and easy method allowed precise control of (i) the edge shape, (ii) the location along the microchannel, and (iii) the length of the monolithic plugs within glass microchips. The addition of hydroquinone, a polymerization inhibitor, to the prepolymerization mixture was beneficial for achieving local and robust incorporation of monoliths with sharp edges within microchannels. The monolith length was easily tuned from 160 to 400 µm through simple change in the magnification of the objective and was found to be repeatable (relative standard deviation <7.5%). Further application for on-chip monolith-assisted solid - phase extraction is demonstrated for fluorescently labeled peptide. Both binding and subsequent elution behaviors were found to fully agree with a cation-exchange mechanism in concordance with the presence of phosphate groups at the monolith surface. Graphical abstract In-chip microscope-UV-synthesis of monolithic plugs with sharp edges.

Rapid HPLC-MS method for the simultaneous determination of tea catechins and folates.

An effective and rapid HPLC-MS method for the simultaneous separation of the eight most abundant tea catechins, gallic acid, and caffeine was developed. These compounds were rapidly separated within 9 min by a linear gradient elution using a Zorbax SB-C18 packed with sub 2 µm particles. This methodology did not require preparative and semipreparative HPLC steps. In fact, diluted tea samples can be easily analyzed using HPLC-MS as described in this study. The use of mass spectrometry detection for quantification of catechins ensured a higher specificity of the method. The percent relative standard deviation was generally lower than 4 and 7% for most of the compounds tested in tea drinks and tea extracts, respectively. Furthermore, the method provided excellent resolution for folate determination alone or in combination with catechins. To date, no HPLC method able to discriminate catechins and folates in a quick analysis has been reported in the literature.

A two-stage, single-arm, phase II study of EGCG-enriched green tea drink as a maintenance therapy in women with advanced stage ovarian cancer.

OBJECTIVES: A two-stage, single-arm, phase II study was conducted to assess the effectiveness and safety of an epigallocatechin gallate (EGCG)-enriched tea drink, the double-brewed green tea (DBGT), as a maintenance treatment in women with advanced stage serous or endometrioid ovarian cancer (clinicaltrials.gov, NCT00721890). METHODS: Eligible women had FIGO stage III-IV serous or endometrioid ovarian cancer. They had to undergo complete response after debulking surgery followed by 6 to 8 cycles of platinum/taxane chemotherapy at the Centre Hospitalier Universitaire de Québec. They all had to drink the DBGT, 500 mL daily until recurrence or during a follow-up of 18 months. The primary endpoint was the absence of recurrence at 18 months. Statistical analyses were done according to the principle of intention to treat. Using a two-stage design, the first stage consisted of 16 enrolled patients. At the end of the follow-up, if 7 or fewer patients were free of recurrence, the trial stopped. Otherwise, accrual would continue to a total of 46 patients. RESULTS: During the first stage of the study, only 5 of the 16 women remained free of recurrence 18 months after complete response. Accordingly, the clinical trial was terminated. Women's adherence to DBGT was high (median daily intake during intervention, 98.1%, interquartile range: 89.7-100%), but 6 women discontinued the intervention before the end of their follow-up. No severe toxicity was reported. CONCLUSIONS: DBGT supplementation does not appear to be a promising maintenance intervention in women with advanced stage ovarian cancer after standard treatment.

Redox properties of catechins and enriched green tea extracts effectively preserve L-5-methyltetrahydrofolate: assessment using cyclic voltammetry analysis.

A cyclic voltammetry (CV) study was performed in pH 5.5 Britton-Robinson buffer at room temperature to study the stability of 1mM l-5-methyltetrahydrofolate (l-5-MTHF) in combination with epigallocatechin-gallate-enriched extract (EGCGe) and epigallocatechin-enriched extract (EGCe). The combination of l-5-MTHF with enriched catechin extracts provided enhanced stability of l-5-MTHF over a period of 12h under ambient air conditions at pH 5.5. CV experiments showed that increasing the concentrations of EGCGe or EGCe extracts from 80 to 400mg/L produced a decrease in the second oxidation peak of l-5-MTHF. Thus, we calculated that l-5-MTHF remained at nearly 90% when in the presence of enriched tea extracts, compared to 74% without the tea antioxidants. The catechins responsible for this preservation were EGCG and C, confirmed by LC-MS. Compared to covalent link only low interaction (hydrogen bonds) between the different catechins present in the tea extract would stabilise l-5-MTHF. Rather, it was hypothesised that EGCGe and EGCe were effective agents to preserve l-5-MTHF, through a mechanism that also involved the redox potential of catechins to maintain l-5-MTHF in its reduced form.

Effect of calcium and carbonate concentrations on anionic membrane fouling during electrodialysis.

A previous study on electrodialysis of calcium and carbonate high concentration solutions demonstrated that calcium migrated through the cation-exchange membrane (CEM) was blocked by the anion-exchange membrane (AEM) where it formed another fouling. The aim of the present work was to complete the identification of the deposit formed on AEM during electrodialysis and to characterize its physical structure at the interface of the membrane. No fouling was found on the anionic membranes treated without calcium chloride in presence of sodium carbonate, while membranes used during ED process of solutions containing calcium chloride and sodium carbonate were slightly fouled. A thin layer of precipitates was observed on the anionic membrane surface. The appearance of precipitates was typical of a crystalline substance. The size and form of crystal increased in proportion to the concentration of calcium chloride in solution. Large and cubic crystals were the best defined on the membrane treated at 1600 mg/L of CaCl2. The precipitate was identified as calcium hydroxide. However, this fouling was not found to affect significantly the electrical conductivity and the thickness of the membranes. Furthermore, the fouling formed was reversible.

Electrodialysis of calcium and carbonate high concentration solutions and impact on composition in cations of membrane fouling.

Fouling, which is the accumulation of undesired solid materials at the phase interfaces of permselective membranes, is one of the major problems in electrodialysis. The objectives of the present work were to investigate the effect of the composition in calcium and carbonate of a model solution to be treated by conventional electrodialysis on their migration kinetics and the composition in cations of the membrane fouling. In the absence of sodium carbonate in the solution, no fouling was visually observed on anion-exchange membranes (AEM) and fouling was observed only at 1600 mg/L CaCl2 on cation-exchange membrane (CEM), while at only 800 mg/L CaCl2 with sodium carbonate, a deposit was observed on both membranes. This difference could be explained by the fact that carbonate has a high buffer capacity, and the time to reach pH 4.0 was then longer than the one without carbonate. Consequently, the migration of the ionic species was carried out over a longer period of time during ED treatment with sodium carbonate addition and in extent the demineralization rates were higher: 43 vs 86%. For treatment with sodium carbonate and 1600 mg/L CaCl2, the higher migration during ED treatment, increased the concentration of calcium, from 14.24 to 93.38 mg/g dry membrane and from 0.74 to 10.27 mg/g dry membrane for CEM and AEM, respectively. Due to the basic pH on the side of the membrane in contact with the NaCl solution, the calcium would precipitate to form calcium hydroxide on CEM while the calcium migrated through the CEM was blocked by the AEM where it formed another fouling.

Effect of calcium and carbonate concentrations on cationic membrane fouling during electrodialysis.

Fouling, which is the accumulation of undesired solid materials at the phase interfaces of permselective membranes, is one of the major problems in electrodialysis. The aim of the present work was to investigate the effect on the fouling of cation-exchange membranes of the composition in calcium and carbonate of a model solution to be treated by electrodialysis. No fouling was observed at 400 and 800 mg/L of CaCl(2) in the absence of carbonate, while at only 400 mg/L CaCl(2) with carbonate, a deposit was observed. This difference could be explained by the buffering capacity of the carbonate, which affects the treatment duration with and without sodium carbonate. Since the duration was longer with carbonate, more calcium ions were able to migrate across the CMX-S membrane, which explained the higher deposit on its surface. Furthermore, whether there was carbonate in the solution treated by electrodialysis or not, the deposit on the surface of the cationic membrane was calcium hydroxide. However, this fouling formed during conventional ED was easily cleaned by an acid procedure.