Paper-Based Analytical Devices for Accurate Assessment of Transferrin Saturation in Diagnosed Clinical Samples from Ischemic Stroke Patients.

For the first time, a paper-based analytical device (PAD) was developed for the assessment of transferrin saturation (TSAT), which is defined as the ratio between iron bound to transferrin (Tf) and the total iron-binding capacity (TIBC) of Tf. Both parameters were simultaneously measured on the same PAD using ferrozine as a chromophore and a smartphone as the color reader. To this end, Tf was first isolated from serum using anti-Tf immunomagnetic beads to ensure that only the Tf-bound iron was measured, improving the selectivity and accuracy of TSAT assessment. To demonstrate the practical utility of the device, it was validated by analyzing a certified reference material, showing excellent accuracy (Er < 4%) and good precision (RSD ≤ 6%). Finally, 18 diagnosed serum samples from ischemic stroke patients were analyzed by this approach, and the results were compared with those obtained by urea-PAGE, showing not only an excellent correlation (r = 0.93, p < 0.05) but that the PAD approach has become statistically identical to the free-interference urea-PAGE. In comparison with the slow, tedious, and non-miniaturized-PAGE, this PAD approach exhibited attractive characteristics such as low cost, disposability, and connectivity, showing great potential for future point-of-care testing, especially in developing countries and/or remote areas, where access to medical or clinical facilities is limited.

Integrated calibration and serum iron in situ analysis into an array microfluidic paper-based analytical device with smartphone readout.

In this work, a colorimetric microfluidic paper-based analytical device (µPAD) combined with a smartphone readout was proposed for the determination of serum iron (Fe3+), which is linked to transferrin. Firstly, Fe3+ was selectively isolated and preconcentrated from serum by using anti-transferrin immunomagnetic beads (anti-Tf-MBs). Secondly, Fe3+ is reduced to Fe2+ by a hydroxylamine solution (pH 4.8) and then measured in the µPAD, which contains the colorimetric reagent ferrozine. Finally, the intensity of the purple color formed in the µPAD was measured by a smartphone. The approach exhibited an excellent linear correlation (r = 0.996) and good limit of detection (0.3 µg mL-1). Moreover, a certified reference material (human serum) was analyzed by this approach, showing an excellent accuracy (Er < 4%) and inter-device reproducibility (RSD = 1%, n = 3). Interestingly, the µPAD array-design allowed the simultaneous analysis of different samples, improving the sample throughput (up to 5 samples in 130 min, using 100 µL each), and the integration of calibration and analysis into the same device, simplifying the analysis without losing accuracy or sensitivity, and avoiding inter-device variability, which constituted an added value to this approach. These disposable µPADs meet several requirements of point-of-care testing (POCT) because it is cheap, portable, easy-to-use, sensitive, and specific. Therefore, it may be an interesting way for measuring patients' serum iron levels in situ with reliability, especially, in developing countries, where the prevalence of iron deficiency and iron-deficiency anemia is higher and there are lower health resources than in developed countries.

3D-Printed SARS-CoV-2 RNA Genosensing Microfluidic System.

Additive manufacturing technology, referred as 3D printing technology, is a growing research field with broad applications from nanosensors fabrication to 3D printing of buildings. Nowadays, the world is dealing with a pandemic and requires the use of simple sensing systems. Here, the strengths of fast screening by a lab-on-a-chip device through electrochemical detection using 3D printing technology for SARS-CoV-2 sensing are combined. This system comprises a PDMS microfluidic channel integrated with an electrochemical cell fully 3D-printed by a 3D printing pen (3D-PP). The 3D-PP genosensor is modified with an ssDNA probe that targeted the N gene sequence of SARS-CoV-2. The sensing mechanism relies on the electro-oxidation of adenines present in ssDNA when in contact with SARS-CoV-2 RNA. The hybridization between ssDNA and target RNA takes a place and ssDNA is desorbed from the genosensor surface, causing a decrease of the sensor signal. The developed SARS-CoV-2/3D-PP genosensor shows high sensitivity and fast response.

Effect of nanocellulose polymorphism on electrochemical analytical performance in hybrid nanocomposites with non-oxidized single-walled carbon nanotubes.

Two cellulose nanocrystals/single-walled carbon nanotube (CNC/SW) hybrids, using two cellulose polymorphs, were evaluated as electrochemical transducers: CNC type I (CNC-I/SW) and CNC type II (CNC-II/SW). They were synthesized and fully characterized, and their analytical performance as electrochemical sensors was carefully studied. In comparison with SWCNT-based and screen-printed carbon electrodes, CNC/SW sensors showed superior electroanalytical performance in terms of sensitivity and selectivity, not only in the detection of small metabolites (uric acid, dopamine, and tyrosine) but also in the detection of complex glycoproteins (alpha-1-acid glycoprotein (AGP)). More importantly, CNC-II/SW exhibited 20 times higher sensitivity than CNC-I/SW for AGP determination, yielding a LOD of 7 mg L-1.These results demonstrate the critical role played by nanocellulose polymorphism in the electrochemical performance of CNC/SW hybrid materials, opening new directions in the electrochemical sensing of these complex molecules. In general, these high-active-surface hybrids smartly exploited the preserved non-oxidized SW conductivity with the high aqueous dispersibility of the CNC, avoiding the use of organic solvents or the incorporation of toxic surfactants during their processing, making the CNC/SW hybrids promising nanomaterials for electrochemical detection following greener approaches.

Pump-Free Microfluidic Device for the Electrochemical Detection of α1-Acid Glycoprotein.

α1-Acid glycoprotein (AGP) is a glycoprotein present in serum, which is associated with the modulation of the immune system in response to stress or injuries, and a biomarker for inflammatory diseases and cancers. Here, we propose a pump-free microfluidic device for the electrochemical determination of AGP. The microfluidic device utilizes capillary-driven flow and a passive mixing system to label the AGP with the Os (VI) complex (an electrochemical tag) inside the main channel, before delivering the products to the electrode surface. Furthermore, thanks to the resulting geometry, all the analytical steps can be carried out inside the device: labeling, washing, and detection by adsorptive transfer stripping square wave voltammetry. The microfluidic device exhibited a linear range from 500 to 2000 mg L-1 (R2 = 0.990) and adequate limit of detection (LOD = 231 mg L-1). Commercial serum samples were analyzed to demonstrate the success of the method, yielding recoveries around 83%. Due to its simplicity, low sample consumption, low cost, short analysis time, disposability, and portability, the proposed method can serve as a point-of-care/need testing device for AGP.

Electrochemical sensor for the assessment of carbohydrate deficient transferrin: Application to diagnosis of congenital disorders of glycosilation.

Carbohydrate deficient transferrin (CDT) is used as biomarker of different health problems as, for example, congenital disorders of glycosylation (CDG). We propose a screen-printed-based electrochemical sensor for the determination of carbohydrate deficient transferrin using an Os (VI) tag-based electrochemistry. When transferrin is labeled with Os (VI) complex, it generates two voltammetric signals: one from carbohydrates (electrochemical signal of osmium (VI) complex at -0.9 V/Ag) and one from the amino acids present in glycoprotein (intrinsic electrochemical signal of glycoprotein at +0.8 V/Ag). The relationship between the two analytical signals (carbohydrate signal/protein signal) is an indicator of the degree of glycosylation (electrochemical index of glycosylation), which has shown an excellent correlation (r = 0.990) with the official parameter %CDT obtained by CE-UV. The suitability of this approach was demonstrated by analyzing serum samples from CDG patients.

Electrochemically Reduced Graphene Oxide-Based Screen-Printed Electrodes for Total Tetracycline Determination by Adsorptive Transfer Stripping Differential Pulse Voltammetry.

Disposable electrochemically reduced graphene oxide-based (ERGO) screen-printed electrodes (SPE) were developed for the determination of total tetracyclines as a sample screening approach. To this end, a selective adsorption-detection approach relied on adsorptive transfer stripping differential pulse voltammetry (AdTDPV) was devised, where the high adsorption capacity and the electrochemical properties of ERGO were simultaneously exploited. The approach was very simple, fast (6 min.), highly selective by combining the adsorptive and the electrochemical features of tetracyclines, and it used just 10 µL of the sample. The electrochemical sensor applicability was demonstrated in the analysis of environmental and food samples. The not-fully explored AdTDPV analytical possibilities on disposable nanostructured transducers become a new tool in food and environmental fields; drawing new horizons for "in-situ" analysis.

Determination of Glycoproteins by Microchip Electrophoresis Using Os(VI)-Based Selective Electrochemical Tag.

Glycoproteins are excellent biomarkers for diagnosis and prognosis of several illnesses. α-1-Acid glycoprotein (AGP) and transferrin (Tf) are the main glycoproteins present in the serum, whose levels are increased during disease or injury. In this work, a selective detection of these glycoproteins using a microchip electrophoresis-electrochemical detection (ME-ED) platform is proposed for the first time. Because of the reduced sensitivity of glycoproteins, they were labeled with an electrochemical tag (osmium(VI) complex), which binds only to glycans, increasing the amperometric signal. Interestingly, oxidation potential of glycoprotein-Os(VI) adducts started at +0.50 V (vs Ag/AgCl) while nontagged glycoproteins started at +0.60 V. So, when the detection potential is set at +0.50 V, only glycoproteins tagged with Os(VI) complex are detected, avoiding the interference of the rest of the proteins. Determination of AGP and Tf was successfully demonstrated in the analysis of a certified human serum reference material yielding excellent accuracy (Er ≤ 4%) in just 400 s. This work offers new possibilities for ED for glycoprotein analysis in microfluidic systems, which has been dominated by fluorescence and MS detection until now. It is worth mentioning that ED has two interesting advantages with respect to others in the point-of-care testing field: easy miniaturization (bedside devices) and low cost.

Monitorization of α1-Acid Glycoprotein Deglycosylation Using SU-8 Microchips Electrophoresis with LIF Detection.

In the last few years, biopharmaceuticals-therapeutic drugs which are generally obtained by using molecular biology techniques-have become a major growing sector in pharmaceutical industry. A large part of these biopharmaceuticals are therapeutic glycoproteins. The production of these drugs and their purification process are implying the development of efficient analytical methods, which allow quick and reliable control of the manufacturing process and ensuring the regulatory compliance about the quality of these drugs. Capillary gel electrophoresis (CGE) in the presence of sodium dodecyl sulfate (SDS) is becoming a method of choice in the quality control of these biopharmaceuticals. On the other hand, CGE can be improved if analyses are carried out in microchip format.This chapter reports a detailed microchips gel electrophoresis (MGE) method to separate glycosylated and deglycosylated forms of α1-acid glycoprotein (AGP) labeled with Chromeo P540, using SU-8 microchips and laser induced fluorescence detection. Due to the analogy between AGP and some therapeutic glycoproteins, we have selected AGP as a model system to illustrate the potential of MGE in the analysis of this type of biopharmaceutical compounds.

Disposable carbon nanotube scaffold films for fast and reliable assessment of total α1-acid glycoprotein in human serum using adsorptive transfer stripping square wave voltammetry.

Alpha-1-acid glycoprotein (AGP) is a serum glycoprotein whose levels are increased two or three times during disease or injury. This makes it a potential biomarker for inflammatory bowel diseases and sepsis. Consequently, fast, simple, and cheap analytical methods for prognosis, diagnosis, and follow-up of these diseases are demanded. In this work, we propose a simple electrochemical approach based on carbon nanotubes scaffold films (CNSFs) for total AGP determination in serum samples. Firstly, AGP is labeled with an electrochemical tag (osmium(VI) complex), and then the total amount of AGP is quantified by adsorptive transfer stripping square wave voltammetry (AdTSWV). Multi-walled carbon nanotubes scaffold films (MWSFs) yielded the best analytical performance in terms of sensitivity with a good limit of detection of 0.6 mg L-1 for AGP determination in serum samples, in less than 20 min. A simplified AGP calibration and its sequential serum sample analysis strategy with good accuracy (81%) and excellent reproducibility (RSD < 1%) was additionally proposed to meet the point-of-care/needs requirements. Graphical abstract Multi-walled carbon nanotubes scaffold films for total AGP determination on disposable platforms integrating single-point calibration and sequential sample analysis.

Electrochemical detection based on nanomaterials in CE and microfluidic systems.

Electrochemical detection has a great potential in microfluidic systems due to its easy miniaturization without losing analytical performance. In addition, the use of nanomaterials in electroanalysis improves sensitivity, selectivity, and reproducibility. The topic of this review is the use of nanomaterials (nanoparticles, nanotubes, graphene) in electrochemical detection for capillary electrophoresis and microfluidic systems (microchips and paper based analytical devices). This review covers from 2015 up to now and it is a continuation of our previous review, also published in Electrophoresis journal. The following aspects of the surveyed articles are mainly addressed: type of nanomaterial, protocol of working electrode preparation (composite, drop casting and others), advantages of nanomaterial employment and application field (clinical, food, environmental and home security). The use of nanomaterials is still an interesting approach to improve the analytical performance of electrochemical detection based on microfluidic devices. Along the review, readers will find new protocols for working electrode modification, new carbon nanomaterials and promising applications in the aforementioned fields.

Extraction-free colorimetric determination of thymol and carvacrol isomers in essential oils by pH-dependent formation of gold nanoparticles.

An extraction-free method is described for the colorimetric determination of thymol (TY) and carvacrol (CA) isomers in essential oils by making use of the pH-dependent formation of gold nanoparticles (AuNPs). In solutions of pH 12, TY and CA form gold nanoparticles, while at pH ≤ 11 only CA does so. By taking advantage of this finding, two different approaches based on colorimetric assay (absorption at 550 nm) were developed: one at pH 12 for the determination of total CA and TY, and other at pH 9 and pH 12 for differential quantification of TY and CA. The former agrees with the well-established Folin-Ciocalteu method, and the latter provides a simple way for calculation of TY/CA ratio. The linear ranges are from 100 to 1000 µM at pH 9, and from 50 to 200 µM at pH 12. The limits of detection are 0.09 µM at pH 9, and 0.02 µM at pH 12. These features make this method simple, fast and reliable. Conceivably, it can be used to assess the quality of essential oils and may become a valuable alternative to more sophisticated, laborious and high time-consuming methods. Graphical abstract Schematic of the assay: At pH 12 (blue color), thymol and carvacrol form gold nanoparticles (Au), while at pH 9 (red color) only carvacrol does so. This finding resulted in a colorimetric method for the differential quantification of both compounds in essential oils.

Total α1-acid glycoprotein determination in serum samples using disposable screen-printed electrodes and osmium (VI) as electrochemical tag.

Alpha-1-acid glycoprotein (AGP) or Orosomucoid is a serum glycoprotein which belongs to the group of acute phase proteins. It is a potential biomarker for inflammatory bowel diseases. In this sense, there is a need for developing fast and cheap analytical methods for diagnosis, prognosis and follow-up of these diseases. In this work, we propose a simple and cheap electrochemical method for total AGP determination using disposable carbon screen-printed electrodes (SPE) and using a selective acidic precipitation of the rest of proteins. This method avoids the use of biological components, decreasing dramatically the analysis cost. Firstly, AGP is labeled with an electrochemical tag (osmium (VI) complex) and then the total amount of AGP is quantified by adsorptive transfer stripping square wave voltammetry. The method optimized showed a good linear correlation (r = 0.9992) and limit of detection of 1.6mgl-1. The methodology was successfully applied to quantify AGP in a commercial serum sample. This methodology could be useful in clinical diagnosis because of AGP levels increase two or three times when inflammatory processes happen. Moreover, the inherent advantages of SPE technology (low sample consumption, low cost and point of care testing) make this methodology very attractive in this field.

Derivatization agents for electrochemical detection in amino acid, peptide and protein separations: The hidden electrochemistry?

In proteomics and clinical analysis, LIF and MS are the most used detectors coupled to separation techniques. MS offers good sensitivity and analyte identification ability but, as counterbalance, it is expensive and it needed background electrolytes compatible with mass spectrometers. LIF detection offers the highest sensitivity but, in general, proteins, peptides and amino acids are not fluorescents so it is necessary the use of fluorescence tags. This fact hindered the potential sensitivity of LIF detection due to the difficulty of labeling analytes at extremely low concentrations. In the context of micro- and nanofluidics, electrochemical detection (ED) is an excellent alternative to LIF and MS because it offers several advantages such as easy miniaturization, simpler methods, lower cost and high sensitivity in spite of decreasing the electrode size and sample volume. Among ED techniques, amperometric detection (AD and PAD) are the most used. The problem is that the sensitivity is not enough for the majority of proteomics and clinical applications. To overcome this handicap, electrochemical derivatization reagents have been employed with excellent results allowing limits of detection in nanomolar range. This review surveys all papers related to electrochemical derivatization reagents employed for amino acids, peptides and proteins in separation techniques. Additionally, we highlight the possibilities of this electrochemical tags in the context of micro- and nano- separation techniques and showed potential electrochemical tags which could be employed in the near future for proteomics applications.

Development of an SDS-gel electrophoresis method on SU-8 microchips for protein separation with LIF detection: Application to the analysis of whey proteins.

This work describes the development of an SDS-gel electrophoresis method for the analysis of major whey proteins (α-lactalbumin, ß-lactoglobulin, and BSA) carried out in SU-8 microchips. The method uses a low-viscosity solution of dextran as a sieving polymer. A commercial coating agent (EOTrol LN) was added to the separation buffer to control the EOF of the chips. The potential of this coating agent to prevent protein adsorption on the walls of the SU-8 channels was also evaluated. Additionally, the fluorescence background of the SU-8 material was studied to improve the sensitivity of the method. By selecting an excitation wavelength of 532 nm at which the background fluorescence remains low and by replacing the mercury arc lamp by a laser in the detection system, an LOD in the nanomolar range was achieved for proteins derivatized with the fluorogenic reagent Chromeo P540. Finally, the method was applied to the analysis of milk samples, demonstrating the potential of SU-8 microchips for the analysis of proteins in complex food samples.

The preferential electrocatalytic behaviour of graphite and multiwalled carbon nanotubes on enediol groups and their analytical implications in real domains.

Carbon nanotubes (CNTs) possess preferential 'electrocatalytic' properties that affect the oxidation of enediol groups, establishing a relationship between electrocatalysis and chemical structure. Since this chemical structure occurs in analytes involved in high impact areas such as the pharmaceutical, cosmetic, and food safety industries, this preferential electrochemical behaviour was demonstrated using both standard and selected real-world samples. The oxygen-containing species present on the surface of CNTs and generated during acid treatment were responsible for an enhanced electron transfer reaction for these structures using a proton-assisted electron transfer mechanism, thus confirming their crucial role during the surface preparation process of electrocatalysis. The analytical benefits were that the inherent selectivity and sensitivity from these nanomaterials could be exploited for the direct detection of analytes in complex matrices, revealing their crucial role in the simplification of analytical processes.

Towards lab-on-a-chip approaches in real analytical domains based on microfluidic chips/electrochemical multi-walled carbon nanotube platforms.

"Lab-on-a-chip" approaches based on the novel marriage between an electrokinetic microfluidic platform and nanotechnology is proposed for analytical domains. Conceptually, the analytical challenges are linked with the analytical promises offered from the integration of lab-on-a-chip and nanotechnologies. The analytical suitability of the electrokinetic microfluidic platform with multi-walled carbon nanotubes as detectors is proposed based on its dual format/use as a flow and separation system, independently. Two relevant applications of high significance, determination of total isoflavones and fast detection of antioxidant profiles were chosen to demonstrate their analytical potential. For both analytical uses, the target challenges, the strategy proposed, the expected role of microfluidics and carbon nanotubes and future prospects are discussed and demonstrated. A good analytical performance of the proposed microfluidic platform in terms of reliability, versatility and fast analytical solutions is demonstrated.

Carbon nanotube disposable detectors in microchip capillary electrophoresis for water-soluble vitamin determination: analytical possibilities in pharmaceutical quality control.

In this work, the synergy of one mature example from "lab-on-chip" domain, such as CE microchips with emerging miniaturized carbon nanotube detectors in analytical science, is presented. Two different carbon electrodes (glassy carbon electrode (GCE) 3 mm diameter, and screen-printed electrode (SPE) 0.3 mm x 2.5 mm) were modified with multiwalled carbon nanotubes (MWCNTs) and their electrochemical behavior was evaluated as detectors in CE microchip using water-soluble vitamins (pyridoxine, ascorbic acid, and folic acid) in pharmaceutical preparations as representative examples. The SPE modified with MWCNT was the best electrode for the vitamin analysis in terms of analytical performance. In addition, accurate determination of the three vitamins in four different pharmaceuticals was obtained (systematic error less than 9%) in only 400 s using a protocol that combined the sample analysis and the methodological calibration.

Microchips for CE: breakthroughs in real-world food analysis.

The well-known complexity of food matrices is approached using CE microchips with different strategies to improve the selectivity and sensitivity of the analysis by avoiding and/or making the sample preparation as simple as possible: (i) enhancing the peak capacity in order to perform direct injection, (ii) using the microchip platform to measure one target analyte/group of analytes with or without separating other related interferences, (iii) integrating sample preparation steps on the microchip platform, and (iv) integrating new analytical tools from nanotechnology in the detection stage. New analyte separations of food significance involving DNA probes, biogenic amines, vanilla flavors, and dyes have been reported as successfully breaking new barriers in areas of high impact in the market, such as transgenic food analysis, as well as the detection of frauds and toxins. Simple microchip layouts are still the most common designs used, though sophisticated new ones are emerging. In contrast to other application areas, electrochemical detection continues to be the most common detection route, followed by LIF, though non-conventional detection routes are also emerging, such as chemiluminescence or UV. In terms of analytical performance, the integration of calibration and quality control on a microchip platform, and remarkable accuracy and precision are being obtained using creative analytical methodologies that enhance the analytical potency of microfluidic chips for their future commercialization. This review critically states the most important advances derived from work done in the field over the past 2-3 years.