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An analytical method based on capillary electrophoresis (CE) using capacitively coupled contactless conductivity detection (C 4 D) was developed and validated for fast, straightforward, and reliable determination of lactate in artificial and human sweat samples. The background electrolyte was composed of equimolar concentrations (10 mmol/L) of 2-(N-morpholino)ethanesulfonic acid and histidine, with 0.2 mmol/L of cetyltrimethylammonium bromide as electroosmotic flow inverter. The limit of detection and quantification were 3.1 and 10.3 µmol/L, respectively. Recoveries in the 97 to 118% range were obtained using sweat samples spiked with lactate at three concentration levels, indicating an acceptable accuracy. The intraday and interday precisions were 1.49 and 7.08%, respectively. The proposed CE-C 4 D method can be a starting point for monitoring lactate concentrations in sweat samples for diagnostics, physiological studies, and sports performance assessment applications., (© 2023 Wiley-VCH GmbH.)

The study of neoplastic cells enabled the discovery of important tumor-related biomarkers which resulted in new forms of early diagnosis, therapeutic options, and prognostic markers. Thus, immunofluorescence (IF), a high throughput imaging technology, represents a valuable method that enables the virtual characterization and localization of diverse cell types and targets, preserving tissue architecture and spatial surroundings. IF staining and analysis of formalin-fixed paraffin-embedded (FFPE) tissues are considered a challenge due to several difficulties, such as tissue autofluorescence, non-specific antibody binding, and image acquisition and quality. This study aimed to develop a multiplex-fluorescence staining technique with high-contrast and high-quality multiple-color images to enrich the investigation of important biomarkers. We present a robust optimized multiple-immunofluorescence procedure that reduced sample autofluorescence, enabled the use of simultaneous antibodies on the same sample, and showed super-resolution imaging through precise antigen localization. We illustrated the utility of this powerful method in FFPE neoplastic appendix, lymph node and bone marrow biopsies, and a 3D-coculture system, in which cells are enabled to grow and interact with their surroundings in all 3D dimensions. Our optimized multiple-immunofluorescence method represents a powerful tool for better understanding the complexity of tumor cells, characterizing cell populations and spatial localization, revealing predictive and prognostic biomarkers, and identifying immunologic phenotypes in a single and limited sample. This valuable IF protocol successfully enables tumor microenvironment profiling that could contribute to the study of cellular crosstalk and the niche, and to the identification of predictive biomarkers for neoplasms., (© 2023. The International CCN Society.)

Devices that integrate field effect transistors into microfluidic channels are becoming increasingly promising in the medical, environmental, and food realms, among other applications. The uniqueness of this type of sensor lies in its ability to reduce the background signals existing in the measurements, which interfere in obtaining good limits of detection for the target analyte. This and other advantages intensify the development of selective new sensors and biosensors with coupling configuration. This review work focused on the main advances in the fabrication and application of field effect transistors integrated into microfluidic devices as a way of identifying the potentialities that exist in these systems when used in chemical and biochemical analyses. The emergence of research on integrated sensors is not a recent study, although more recently the progress of these devices is more accentuated. Among the studies that used integrated sensors with electrical and microfluidic parts, those that investigated protein binding interactions seem to be the ones that expanded the most due, among other things, to the possibility of obtaining several physicochemical parameters involved in protein-protein interactions. Studies in this area have a great possibility of advancing innovations in sensors with electrical and microfluidic interfaces in new designs and applications.

Myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) are characterized by risk of relapses, poor survival, unwanted side effects and high toxicity with the current therapies. In light of these facts, there are efforts to develop new drugs specific for deregulated molecules that participate in leukemia pathogenesis. Hematopoietic cell kinase (HCK), an Src kinase family member, is overexpressed on hematopoietic stem cells of MDS and de novo AML patients and involved in the oncogenic process. Thus, we investigated in vitro, ex vivo and in vivo effects of a novel chemical compound targeting HCK inhibition (iHCK-37), in combination with the most used drugs for the treatment of MDS and de novo AML, 5-Azacytidine and Cytarabine. Herein, the combination treatment with iHCK-37 and 5-Azacytidine or Cytarabine demonstrated additive effects against leukemia cells, compared to either drug alone. iHCK-37 plus 5-Azacytidine or Cytarabine treatment was able to reduce the activation of oncogenic pathways, MAPK/ERK and PI3K/AKT, leading to reduction of ERK and AKT phosphorylation, and increased BAX and decreased BCL-XL protein expression. Moreover, treatment with iHCK-37 reduced MDS and AML CD34-positive cell numbers inside a 3D-structure but did not affect normal CD34-positive cell numbers. In vivo analysis showed that leukemic mice treated with iHCK-37 had reduced ERK and AKT proteins phosphorylation levels and leukocyte numbers. In conclusion, the iHCK-37 inhibitor has anti-neoplastic activity in leukemia cells without altering apoptosis and survival rate of normal cells, suggesting on-target malignant cell killing activity as a single agent or in combination with 5-Azacytidine or Cytarabine., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

In this work, we demonstrate for the first time the design and fabrication of microchip electrophoresis devices containing cross-shaped channels and spiral electrodes around the separation channel for microchip electrophoresis and capacitively coupled contactless conductivity detection. The whole device was prepared in a digital light processing-based 3D printer in poly(ethylene glycol) diacrylate resin. Outstanding X-Y resolution of the customized 3D printer ensured the fabrication of 40-μm cross section channels. The spiral channels were filled with melted gallium to form conductive electrodes around the separation channel. We demonstrate the applicability of the device on the separation of sodium, potassium, and lithium cations by microchip electrophoresis. Graphical abstract., (© 2021. Springer-Verlag GmbH Germany, part of Springer Nature.)

DNA-protein interactions are essential for several molecular and cellular mechanisms, such as transcription, transcriptional regulation, DNA modifications, among others. For many decades scientists tried to unravel how DNA links to proteins, forming complex and vital interactions. However, the high number of techniques developed for the study of these interactions made the choice of the appropriate technique a difficult task. This review intends to provide a historical context and compile the methods that describe DNA-protein interactions according to the purpose of each approach, summarise the respective advantages and disadvantages and give some examples of recent uses for each technique. The final aim of this work is to help in deciding which technique to perform according to the objectives and capacities of each research team. Considering the DNA-binding proteins characterisation, filter binding assay and EMSA are easy in vitro methods that rapidly identify nucleic acid-protein binding interactions. To find DNA-binding sites, DNA-footprinting is indeed an easier, faster and reliable approach, however, techniques involving base analogues and base-site selection are more precise. Concerning binding kinetics and affinities, filter binding assay and EMSA are useful and easy methods, although SPR and spectroscopy techniques are more sensitive. Finally, relatively to genome-wide studies, ChIP-seq is the desired method, given the coverage and resolution of the technique. In conclusion, although some experiments are easier and faster than others, when designing a DNA-protein interaction study several concerns should be taken and different techniques may need to be considered, since different methods confer different precisions and accuracies., (© 2021. The Author(s).)

We report in this communication a ready-to-use fused deposition modeling (FDM) based 3D-printed spectroelectrochemical cell to perform for the first time voltammetry of immobilized microparticles (VIMP) and Raman spectroscopy in situ using acrylonitrile butadiene styrene (ABS) as the filament material for printing. The 3D-printed cell was applied to evaluate solid state electrochemical behavior of tadalafil as a proof-of-concept. Several advantages were achieved in the use of the developed device, such as less manipulation of the working electrode, monitoring the same region of the solid microparticles before and after electrochemical measurements, better control of the laser incidence, low-cost and low-time production. Furthermore, the device was printed in a single-step, without handling to assembly and it has an estimated material cost of approximately 2 $. The use of 3D-printing technology was significantly important to integrate Raman spectroscopic method with VIMP measurements and to support mechanism elucidation and characterization of the compounds with less manipulation of the working electrode, avoiding loss of solid products formed from electrochemical reactions., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Arginine (ARG), ascorbic acid (ASC) and aspartic acid (ASP) are very popular and widely consumed active ingredients used for fatigue treatment or improvement of physical performance. For this reason, these compounds are usually available in the same pharmaceutical formulation. In the current paper, we describe for the first-time methods for simultaneous determination of ARG, ASC and ASP using capillary electrophoresis with capacitively coupled contactless conductivity detection (CE-C 4 D) and with UV spectrophotometric detection (CE-UV). The proposed methods are simple, rapid (78 and 23 injections h -1 , respectively) and have low environmental impact (minimal waste generation). The separation by CE-C 4 D was achieved with a background electrolyte (BGE) composed by 20 mmol L -1 N-tris(hydroxymethyl)- methyl]-3-aminopropanesulfonic acid (TAPS) and 10 mmol L -1 of NaOH (pH 8.7). The limits of detection (LOD) were 0.01, 0.02 and 0.04 mmol L -1 for ARG, ASC and ASP, respectively. The proposed CE-UV method was optimized with a BGE composed by 10 mmol L -1 sodium tetraborate (pH 9.4). The limits of detection (LOD) were 0.03, 0.02 and 0.04 mmol L -1 for ARG, ASC and ASP, respectively. No statistically significant differences were observed (95% confidence level) between the results obtained by the developed CE methods and reference procedures (HPLC for ARG, iodometry for ASC and, acid-base titration for ASP)., (Copyright © 2019 Elsevier B.V. All rights reserved.)

This study reports the separation of fructose, galactose, glucose, lactose and sucrose on glass microchip electrophoresis (ME) devices using a microfluidic platform adapted with external reservoirs for controlling the electrolysis phenomenon. The connections between external reservoirs and microfluidic platform were performed by saline bridges created using silicone tubing filled with BGE. The separation conditions were optimized and the best results were achieved using a BGE containing 75 mmol/L NaOH and 15 mmol/L trisodium phosphate. Electrophoretic separations were monitored using a capacitively coupled contactless conductivity detection system. The controlled electrolysis has successfully allowed the application of a higher voltage on the separation channel promoting the baseline separation of five carbohydrates within 180 s with great run-to-run repeatability (RSD < 1%). The achieved efficiencies ranged from 45 000 ± 6000 to 70 000 ± 3000 plates/m demonstrating a performance better than ME devices without controlled electrolysis. The proposed system offered good linearity from 1 to 10 mmol/L and LODs between 150 and 740 μmol/L. The use of external tubes for controlling the electrolysis phenomenon on ME devices has solved common problems associated to run-to-run repeatability and analytical reliability required for routine and quantitative analysis., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Microchip electrophoresis is a versatile separation technique. Electrochemical detection is suitable to apply to microdevices due to its easy integration to the fabrication process and good sensitivity and selectivity. Here we describe the procedures to prepare Pt band electrodes deposited on glass to couple to polydimethylsiloxane (PDMS) microchips aiming the separation and detection of nitrite using an isolated potentiostat.

We present an on-line, single step coupling between liquid-liquid extraction and capillary electrophoresis with capacitively coupled contactless conductivity detection, which allows an efficient analysis of complex food matrices with high sodium content. The sodium depletion was demonstrated using an aqueous two-phase system. The aqueous two-phase system enables the electrically driven extraction of the target compounds. The sample was prepared in Dextran-rich phase (8% w/v 500 kDa Dextran, DEX). The background electrolyte (acetic acid 5.0 mol/L) contained 6% w/v of 6 kDa PEG. As proof of applicability, we employed the developed method for glutamic acid quantification on soy sauces. The peak area of glutamic acid presents no significant difference (α = 0.05), while the peak area of the sodium presented a reduction of 11.7 ± 0.2 and 19 ± 3% for premium and low-cost soy sauce samples analyzed. The glutamic acid concentration for premium soy sauce sample was 2.7 ± 0.8 and 4.8 ± 0.4 g/L, and for low-cost soy sauce sample, the concentration was 9.9 ± 0.9 g/L, which agreed with those obtained by other analytical techniques., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

In this paper, we further investigate the properties of off-stoichiometry thiol-ene polymers (OSTE) aiming its application in microchip electrophoresis for bioanalytical applications. The proportion of 1.3:1 (allyl:thiol) and 1:2.5 (allyl:thiol) presented the best results in terms of sealing. Raman imaging mapping of the polymers surfaces revealed an outstanding homogeneity. Water contact angle were measured as 68° ± 6° and 71° ± 5° for 1.3:1 allyl and 1:2.5 thiol, respectively. Substrates prepared with OSTE demonstrated to be less prone to sorption of nonpolar compounds. The electroosmotic flow measured for this OSTE composition was 3.8 ± 0.3·10 -4 cm 2 s -1 V -1 , 1.5 times higher than the one found for polydimethylsiloxane microchips under the same conditions. As a proof-of-concept for the applicability of OSTE microchips in bioanalysis the immobilization of α-amylase on the polymer surface and the implementation of a Saccharomyces cerevisiae cell counter using contactless conductivity detection are demonstrated.

We investigated a strategy for the chemotaxonomy study of Chrysobalanus icaco Linnaeus (Chrysobalanaceae) based on ultra-high performance liquid chromatography coupled with diode array detection fingerprint in combination with multivariate analysis. Two models using principal component analysis and partial least squares discriminant analysis were developed, and the samples could be successfully classified into two classes: Class 1 (red morphotype) and Class 2 (white and black morphotypes). Furthermore, ultra-high performance liquid chromatography coupled with diode array and electrospray ionization tandem mass spectrometry was used to identify the main compounds responsible for class separation. The partial least squares discriminant analysis model accurately classified the C. icaco samples using an external validation subset with prediction ability of 100% and revealed the existence of two chemotypes. The most important finding obtained in this study is that the three morphotypes distinguished by the mature fruit color (white, red, and black) are not all phytoequivalent to each other., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The separation and detection of common mono- and disaccharides by capillary electrophoresis (CE) with contactless conductivity detection (CCD) is presented. At high values of pH, the sugars are converted to anionic species that can be separated by CE and indirectly detected by CCD. The main anionic species present in the running electrolytes are hydroxide and phosphate, which have greater mobility than the ionized sugars, and, thus, the indirect detection is possible. The method was applied to analysis of glucose, fructose, and sucrose in soft drinks, isotonic beverages, fruit juice, and sugarcane spirits. Galactose was used as internal standard in all cases. Plate numbers range from ca. 70,700 to 168,200 and the limits of detection from 13 to 31 microM.