Strategies for capillary electrophoresis: Method development and validation for pharmaceutical and biological applications-Updated and completely revised edition.

This review is in support of the development of selective, precise, fast, and validated capillary electrophoresis (CE) methods. It follows up a similar article from 1998, Wätzig H, Degenhardt M, Kunkel A. "Strategies for capillary electrophoresis: method development and validation for pharmaceutical and biological applications," pointing out which fundamentals are still valid and at the same time showing the enormous achievements in the last 25 years. The structures of both reviews are widely similar, in order to facilitate their simultaneous use. Focusing on pharmaceutical and biological applications, the successful use of CE is now demonstrated by more than 600 carefully selected references. Many of those are recent reviews; therefore, a significant overview about the field is provided. There are extra sections about sample pretreatment related to CE and microchip CE, and a completely revised section about method development for protein analytes and biomolecules in general. The general strategies for method development are summed up with regard to selectivity, efficiency, precision, analysis time, limit of detection, sample pretreatment requirements, and validation.

Rapid Detection of Staphylococcus aureus Using Paper-Derived Electrochemical Biosensors.

Several groups have recently explored the idea of developing electrochemical paper-based wearable devices, specifically targeting metabolites in sweat. While these sensors have the potential to provide a breadth of analytical information, there are several key challenges to address before these sensors can be widely adopted for clinical interventions. Toward this goal, we describe the development of a paper-based electrochemical sensor for the detection of Staphylococcus aureus. Enabling the application, this report describes the use of paper-derived carbon electrodes, which were modified with a thin layer of sputtered gold (that minimizes lateral resistivity and significantly improves the electron transfer process) and with chitosan (used as a binder, to offer flexibility). The resulting material was laser-patterned and applied for the development of an electrochemical biosensor controlled (via a wireless connection) by a custom-built, portable potentiostat. As no interference was observed when exposed to other bacteria or common metabolites, this wearable system (paper-derived electrodes + potentiostat) has the potential to detect the presence of S. aureus in the skin, a commonly misdiagnosed and mistreated infection.

Dielectric Spectroscopy Can Predict the Effect of External AC Fields on the Dynamic Adsorption of Lysozyme.

This report describes the application of dielectric spectroscopy as a simple and fast way to guide protein adsorption experiments. Specifically, the polarization behavior of a layer of adsorbed lysozyme was investigated using a triangular-wave signal with frequencies varying from 0.5 to 2 Hz. The basic experiment, which can be performed in less than 5 min and with a single sample, not only allowed confirming the susceptibility of the selected protein towards the electric signal but also identified that this protein would respond more efficiently to signals with lower frequencies. To verify the validity of these observations, the adsorption behavior of lysozyme onto optically transparent carbon electrodes was also investigated under the influence of an applied alternating potential. In these experiments, the applied signal was defined by a sinusoidal wave with an amplitude of 100 mV and superimposed to +800 mV (applied as a working potential) and varying the frequency in the 0.1-10000 Hz range. The experimental data showed that the greatest adsorbed amounts of lysozyme were obtained at the lowest tested frequencies (0.1-1.0 Hz), results that are in line with the corresponding dielectric features of the protein.

Fluorescent patterning of paper through laser engraving.

While thermal treatment of paper can lead to the formation of aromatic structures via hydrothermal treatment (low temperature) or pyrolysis (high temperature), neither of these approaches allow patterning the substrates. Somewhere in between these two extremes, a handful of research groups have used CO2 lasers to pattern paper and induce carbonization. However, none of the previously reported papers have focused on the possibility to form fluorescent derivatives via laser-thermal engraving. Exploring this possibility, this article describes the possibility of using a CO2 laser engraver to selectively treat paper, resulting in the formation of fluorescent compounds, similar to those present on the surface of carbon dots. To determine the most relevant variables controlling this process, 3 MM chromatography paper was treated using a standard 30 W CO2 laser engraver. Under selected experimental conditions, a blue fluorescent pattern was observed when the substrate was irradiated with UV light (365 nm). The effect of various experimental conditions (engraving speed, engraving power, and number of engraving steps) was investigated to maximize the fluorescence intensity. Through a comprehensive characterization effort, it was determined that 5-(hydroxymethyl)furfural and a handful of related compounds were formed (varying in amount) under all selected experimental conditions. To illustrate the potential advantages of this strategy, that could complement those applications traditionally developed from carbon dots (sensors, currency marking, etc.), a redox-based optical sensor for sodium hypochlorite was developed.

A Multi-Pump Magnetohydrodynamics Lab-On-A-Chip Device for Automated Flow Control and Analyte Delivery.

This article shows the development of a computer-controlled lab-on-a-chip device with three magnetohydrodynamic (MHD) pumps and a pneumatic valve. The chip was made of a stack of layers of polymethylmethacrylate (PMMA), cut using a laser engraver and thermally bonded. The MHD pumps were built using permanent magnets (neodymium) and platinum electrodes, all of them controlled by an Arduino board and a set of relays. The implemented pumps were able to drive solutions in the open channels with a flow rate that increased proportionally with the channel width and applied voltage. To address the characteristic low pressures generated by this kind of pump, all channels were interconnected. Because the electrodes were immersed in the electrolyte, causing electrolysis and pH variations, the composition and ionic strength of the electrolyte solution were controlled. Additionally, side structures for releasing bubbles were integrated. With this multi-pump and valve solution, the device was used to demonstrate the possibility of performing an injection sequence in a system that resembles a traditional flow injection analysis system. Ultimately, the results demonstrate the possibility of performing injection sequences using an array of MHD pumps that can perform fluid handling in the 0-5 µL s-1 range.

Patterning and Modeling Three-Dimensional Microfluidic Devices Fabricated on a Single Sheet of Paper.

This work describes a method to fabricate three-dimensional paper microfluidic devices in one step, without the need of stacking layers of paper, glue, or tape. We used a nontransparent negative photoresist that allows patterning selectively (vertically) the paper, creating systems of two or three layers, including channels. To demonstrate the capabilities of this methodology, we designed, fabricated, and tested a six-level diluter. The performance of the device was also simulated using a simple numerical model implemented in the program PETSc-FEM. The resulting µPAD is small (1.6 cm × 2.2 cm), inexpensive, requires low volumes of sample (5 µL), and is able to perform mixing and dilution in 2 min.

Photochemical and photocatalytic degradation of 1-propanol using UV/H2 O2 : Identification of malonate as byproduct.

1-propanol is a primary alcohol extensively used in the pharmaceutical, chemical, and food industries. It has been also found as a contaminant in the atmosphere and is considered a model compound to mimic the behavior and fate of aliphatic alcohols exposed to environmental conditions. In order to understand that role of relevant variables, this paper presents results obtained with a simple experimental set-up to investigate the reactivity of 1-propanol under mild oxidizing conditions. Coupling this system with CE-C4 D allowed the quantification of the carboxylic acids formed. For the described experiments, aqueous solutions of 1-propanol were placed inside a photoreactor and oxidized upon the addition of TiO2 and/or H2 O2 . According to the described results, the addition of H2 O2 (0.1% w/w) was the most significant variable, roughly tripled the amount of carboxylic acids generated and led to the conversion of up to 70% of the initially available 1-propanol (1 mmol/L). More importantly, the reaction yielded the formation (within 10 min) of propionate (50 µmol/L), acetate (400 µmol/L), formate (50 µmol/L), and malonate (200 µmol/L). The latter is critically important because it represents the first example of the photochemical oxidation of both terminal carbons of the C3 -chain of 1-propanol under mild conditions, and opens new avenues for the production of this important chemical building block.

Analysis of inorganic cations and amino acids in high salinity samples by capillary electrophoresis and conductivity detection: Implications for in-situ exploration of ocean worlds.

With growing interest in exploring ocean worlds, such as Europa and Enceladus, there is a fundamental need to develop liquid-based analytical techniques capable of handling high salinity samples while performing both bulk and trace species measurements. In this context, CE with capacitively coupled contactless conductivity detection (CE-C4 D) has tremendous potential. One of its advantages is that this combination allows the detection of a wide number of charged species (both organic and inorganic) without the need of derivatization. Amino acids are an example of organic targets that are powerful biosignatures in the search for life beyond Earth. Simultaneous information on the inorganic cations in a sample helps with assessing the habitability of an extraterrestrial environment, as well as providing sample context for any measurements of trace amino acids. In this work, we present a series of flight-compatible methods capable of simultaneously measuring inorganic cations and amino acids in samples of varying salinity by CE-C4 D. Regardless of the sample total salinity, 5.0 M acetic acid was selected as the optimum BGE. The methods were evaluated by analyzing natural samples of low and high salinity from Hot Creek Gorge, Mono Lake, and Santa Monica beach. Prospects for mission implementation are also discussed.

Determination of topiramate by capillary electrophoresis with capacitively-coupled contactless conductivity detection: A powerful tool for therapeutic monitoring in epileptic patients.

Topiramate (TPM) is the main antiepileptic drug used for the control of partial and generalized seizures in both adults and children. In association with clinical observations, the analysis of plasmatic concentration of TPM is of utmost importance for the individual adjustment of the administered dose to the patient. In the present work, a bioanalytical method was developed and validated for TPM analysis in plasma samples by capillary electrophoresis with capacitively-coupled contactless conductivity detection (CE-C4 D). A simple background electrolyte composed of 15 mmol/L triethylamine, hydrodynamic injections (0.8 psi for 5 s) and a moderate separation voltage (20 kV) were used, rendering relatively short analysis times (<3 min). The sample pre-treatment was carried out by liquid-liquid extraction using methyl terc-butyl ether as solvent and 200 µL of plasma. The method was validated according to the official guidelines from the European Medicine Agency and showed linearity in plasmatic concentration range from 1 to 30 µg/mL, which covers the clinically-relevant interval. The lower limit of quantification of 1 µg/mL obtained also allows following patients with low dosage of the drug. The method was successfully applied to analysis of plasma samples and allowed the identification of 80% under-medicated patients in the analyzed patient pool.

Analysis of Methanol in the Presence of Ethanol, Using a Hybrid Capillary Electrophoresis Device with Electrochemical Derivatization and Conductivity Detection.

Concurrently with ethanol, many other compounds can be formed during the fermentation of grains and fruits. Among those, methanol is particularly important (because of its toxicity) and is typically formed at concentrations much lower than ethanol, presenting a particular challenge that demands the implementation of separation techniques. Aiming to provide an alternative to traditional chromatographic approaches, a hybrid electrophoresis device with electrochemical preprocessing and contactless conductivity detection (hybrid EC-CE-C4D) is herein described. The device was applied to perform the electro-oxidation of primary alcohols, followed by the separation and detection of the respective carboxylates. According to the presented results, the optimum conditions were obtained when the sample was diluted with 2 mmol L-1 HNO3 and then electro-oxidized by applying a potential of 1.4 V for 60 s. The oxidation products were then electrokinetically injected by applying a potential of 3 kV for 4 s and separated using a potential of 3 kV and a background running electrolyte (BGE) consisting of 10 mmol L-1 N-cyclohexyl-2-aminoethanesulfonic acid (CHES) and 5 mmol L-1 sodium hydroxide (NaOH). n-Propanol was used as an internal standard and the three carboxylate peaks were resolved with baseline separation within <3 min, defining linear calibration curves in the range of 0.10-5.0 mmol L-1. Limits of detection (LODs) of 20, 40, and 50 µmol L-1 were obtained for ethanol, n-propanol, and methanol, respectively. To demonstrate the applicability of the proposed strategy, a laboratory-made sample (moonshine) was used. Aliquots collected along the beginning of the fractional distillation presented a decreasing methanol ratio (from 4% to <0.5%) and a growing ethanol ratio (from 80% to 100%) in the collected volume.

Addressing the distribution of proteins spotted on µPADs.

Adsorption is the most common approach to immobilize biorecognition elements on the surface of paper-based devices. Adsorption is also the route selected to coat the substrate with albumin, therefore minimizing the interaction of other proteins. While similar in nature, the structure of the selected proteins as well as the conditions selected from the immobilization have a significant effect on the amount and distribution of the resulting composites. To illustrate these differences and provide general guidelines to efficiently prepare these devices, this article explores the interaction (adsorption and desorption) of BSA with 3MM chromatography paper. The experimental conditions investigated were the protein concentration, the interaction time, the number of times the protein was spotted, the pH of buffer solution, and the ionic strength of the buffer solution. The proposed approach mimics the steps involved in the fabrication (adsorption) and use (rinsing induced by the sample) of paper-based microfluidic devices. To identify the protein location following the rinsing step, the protein was fixed by dehydration in a convection oven and then stained using Coomassie Blue. The color intensity, which was found to be proportional to the amount of protein immobilized, was determined using a desktop scanner. To highlight the importance of understanding the adsorption process to the rational development of µPADs, results were complemented by experiments performed with lysozyme and immunoglobulin G.

Spectroscopic ellipsometry as a complementary tool to characterize coatings on PDMS for CE applications.

This paper describes the use of spectroscopic ellipsometry to investigate the adsorption process of model polyelectrolytes (PDDAC and PSS) to thin-films of PDMS. A description of the information collected by ellipsometry as well as complementary information obtained by atomic force microscopy and contact angle measurements is discussed. Upon identification of the driving forces and optimum experimental conditions required for the adsorption, multilayer constructs were fabricated (ranging from 1 to 20 nm in thickness) and used to evaluate their effect on the separation of phenolic compounds by capillary electrophoresis. According to the presented results, polyelectrolyte layers of approximately 10 nm thick provided the best conditions for the separation of the selected phenolic compounds.

Fast production of microfluidic devices by CO2 laser engraving of wax-coated glass slides.

Glass is one of the most convenient materials for the development of microfluidic devices. However, most fabrication protocols require long processing times and expensive facilities. As a convenient alternative, polymeric materials have been extensively used due their lower cost and versatility. Although CO2 laser ablation has been used for fast prototyping on polymeric materials, it cannot be applied to glass devices because the local heating causes thermal stress and results in extensive cracking. A few papers have shown the ablation of channels or thin holes (used as reservoirs) on glass but the process is still far away from yielding functional glass microfluidic devices. To address these shortcomings, this communication describes a simple method to engrave glass-based capillary electrophoresis devices using standard (1 mm-thick) microscope glass slides. The process uses a sacrificial layer of wax as heat sink and enables the development of both channels (with semicircular shape) and pass-through reservoirs. Although microscope images showed some small cracks around the channels (that became irrelevant after sealing the engraved glass layer to PDMS) the proposed strategy is a leap forward in the application of the technology to glass. In order to demonstrate the capabilities of the approach, the separation of dopamine, catechol and uric acid was accomplished in less than 100 s.

Synthesis of CuNP-Modified Carbon Electrodes Obtained by Pyrolysis of Paper.

A one-step approach for the synthesis and integration of copper nanoparticles (CuNPs) onto paper-based carbon electrodes is herein reported. The method is based on the pyrolysis (1000 °C under a mixture of 95% Ar / 5% H2 for 1 hour) of paper strips modified with a saturated solution of CuSO4 and yields to the formation of abundant CuNPs on the surface of carbonized cellulose fibers. The resulting substrates were characterized by a combination of scanning electron microscopy, EDX, Raman spectroscopy as well as electrical and electrochemical techniques. Their potential application, as working electrodes for nonenzymatic amperometric determination of glucose, was then demonstrated (linear response up to 3 mM and a sensitivity of 460 ± 8 µA·cm-2·mM-1). Besides being a simple and inexpensive process for the development of electrochemically-active substrates, this approach opens new possibilities for the in-situ synthesis of metallic nanoparticles without the traditional requirements of solutions and adjuvants.

Adsorption of soft and hard proteins onto OTCEs under the influence of an external electric field.

The adsorption behavior of hard and soft proteins under the effect of an external electric field was investigated by a combination of spectroscopic ellipsometry and molecular dynamics (MD) simulations. Optically transparent carbon electrodes (OTCE) were used as conductive, sorbent substrates. Lysozyme (LSZ) and ribonuclease A (RNase A) were selected as representative hard proteins, whereas myoglobin (Mb), α-lactalbumin (α-LAC), bovine serum albumin (BSA), glucose oxidase (GOx), and immunoglobulin G (IgG) were selected to represent soft proteins. In line with recent publications from our group, the experimental results revealed that while the adsorption of all investigated proteins can be enhanced by the potential applied to the electrode, the effect is more pronounced for hard proteins. In contrast with the incomplete monolayers formed at open-circuit potential, the application of +800 mV to the sorbent surface induced the formation of multiple layers of protein. These results suggest that this effect can be related to the intrinsic polarizability of the protein (induction of dipoles), the resulting surface accessible solvent area (SASA), and structural rearrangements induced upon the incorporation on the protein layer. The described experiments are critical to understand the relationship between the structure of proteins and their tendency to form (under electric stimulation) layers with thicknesses that greatly surpass those obtained at open-circuit conditions.

Fast and versatile fabrication of PMMA microchip electrophoretic devices by laser engraving.

This paper describes the effects of different modes and engraving parameters on the dimensions of microfluidic structures produced in PMMA using laser engraving. The engraving modes included raster and vector, while the explored engraving parameters included power, speed, frequency, resolution, line-width, and number of passes. Under the optimum conditions, the technique was applied to produce channels suitable for CE separations. Taking advantage of the possibility to cut-through the substrates, the laser was also used to define solution reservoirs (buffer, sample, and waste) and a PDMS-based decoupler. The final device was used to perform the analysis of a model mixture of phenolic compounds within 200 s with baseline resolution.

Getting started with open-hardware: development and control of microfluidic devices.

Understanding basic concepts of electronics and computer programming allows researchers to get the most out of the equipment found in their laboratories. Although a number of platforms have been specifically designed for the general public and are supported by a vast array of on-line tutorials, this subject is not normally included in university chemistry curricula. Aiming to provide the basic concepts of hardware and software, this article is focused on the design and use of a simple module to control a series of PDMS-based valves. The module is based on a low-cost microprocessor (Teensy) and open-source software (Arduino). The microvalves were fabricated using thin sheets of PDMS and patterned using CO2 laser engraving, providing a simple and efficient way to fabricate devices without the traditional photolithographic process or facilities. Synchronization of valve control enabled the development of two simple devices to perform injection (1.6 ± 0.4 µL/stroke) and mixing of different solutions. Furthermore, a practical demonstration of the utility of this system for microscale chemical sample handling and analysis was achieved performing an on-chip acid-base titration, followed by conductivity detection with an open-source low-cost detection system. Overall, the system provided a very reproducible (98%) platform to perform fluid delivery at the microfluidic scale.

Rational selection of substrates to improve color intensity and uniformity on microfluidic paper-based analytical devices.

A systematic investigation was conducted to study the effect of paper type on the analytical performance of a series of microfluidic paper-based analytical devices (µPADs) fabricated using a CO2 laser engraver. Samples included three different grades of Whatman chromatography paper, and three grades of Whatman filter paper. According to the data collected and the characterization performed, different papers offer a wide range of flow rate, thickness, and pore size. After optimizing the channel widths on the µPAD, the focus of this study was directed towards the color intensity and color uniformity formed during a colorimetric enzymatic reaction. According to the results herein described, the type of paper and the volume of reagents dispensed in each detection zone can determine the color intensity and uniformity. Therefore, the objective of this communication is to provide rational guidelines for the selection of paper substrates for the fabrication of µPADs.

Modification of microfluidic paper-based devices with silica nanoparticles.

This paper describes a silica nanoparticle-modified microfluidic paper-based analytical device (µPAD) with improved color intensity and uniformity for three different enzymatic reactions with clinical relevance (lactate, glucose, and glutamate). The µPADs were produced on a Whatman grade 1 filter paper and using a CO2 laser engraver. Silica nanoparticles modified with 3-aminopropyltriethoxysilane were then added to the paper devices to facilitate the adsorption of selected enzymes and prevent the washing away effect that creates color gradients in the colorimetric measurements. According to the results herein described, the addition of silica nanoparticles yielded significant improvements in color intensity and uniformity. The resulting µPADs allowed for the detection of the three analytes in clinically relevant concentration ranges with limits of detection (LODs) of 0.63 mM, 0.50 mM, and 0.25 mM for lactate, glucose, and glutamate, respectively. An example of an analytical application has been demonstrated for the semi-quantitative detection of all three analytes in artificial urine. The results demonstrate the potential of silica nanoparticles to avoid the washing away effect and improve the color uniformity and intensity in colorimetric bioassays performed on µPADs.

Predicting the formation of NADES using a transformer-based model.

The application of natural deep eutectic solvents (NADES) in the pharmaceutical, agricultural, and food industries represents one of the fastest growing fields of green chemistry, as these mixtures can potentially replace traditional organic solvents. These advances are, however, limited by the development of new NADES which is today, almost exclusively empirically driven and often derivative from known mixtures. To overcome this limitation, we propose the use of a transformer-based machine learning approach. Here, the transformer-based neural network model was first pre-trained to recognize chemical patterns from SMILES representations (unlabeled general chemical data) and then fine-tuned to recognize the patterns in strings that lead to the formation of either stable NADES or simple mixtures of compounds not leading to the formation of stable NADES (binary classification). Because this strategy was adapted from language learning, it allows the use of relatively small datasets and relatively low computational resources. The resulting algorithm is capable of predicting the formation of multiple new stable eutectic mixtures (n = 337) from a general database of natural compounds. More importantly, the system is also able to predict the components and molar ratios needed to render NADES with new molecules (not present in the training database), an aspect that was validated using previously reported NADES as well as by developing multiple novel solvents containing ibuprofen. We believe this strategy has the potential to transform the screening process for NADES as well as the pharmaceutical industry, streamlining the use of bioactive compounds as functional components of liquid formulations, rather than simple solutes.