Capillary-driven microfluidics: impacts of 3D manufacturing on bioanalytical devices.

Over decades, decentralized diagnostics continues to move towards rapid and cost-effective testing at the point-of-care (POC). Although microfluidics has become a key enabling technology for POC testing, the need for robust peripheral equipment has been a key limiting factor in reaching an ideal device. Manufacturing technologies are now reaching a level of maturity that allows the definition of 3D features down to the sub-millimeter scale. Employing three-dimensional (3D) features and surface chemistry allows the possibility to pre-program sophisticated control of the capillary flow avoiding bulky peripheral equipment. By designing a sequence of steps, like elution of reagents, washing, mixing, and sensing, capillary valves have become a powerful tool for POC applications. These valves use capillary force to stop and then release flows within pre-programmed capillary circuits without any moving part. Without their 3D structure, the feasibility of creating pre-programmed bioanalytical devices would be nearly impossible. Besides, the advent of smart materials and their variety of surface properties permitted the unprecedented ability to fabricate reliable flow control with a range of capillary driving forces. The classification of such capillary elements is presented in two functional steps - stop and actuation. This review includes the advances in 3D microfabrication, design, and surface chemistry for manufacturing bioanalytical devices. These developments are critically reviewed, focusing on the process and considering phenomena such as timing, reproducibility, unwanted diffusion, and cross-contaminations.

Stalk cell polar ion transport provide for bladder-based salinity tolerance in Chenopodium quinoa.

Chenopodium quinoa uses epidermal bladder cells (EBCs) to sequester excess salt. Each EBC complex consists of a leaf epidermal cell, a stalk cell, and the bladder. Under salt stress, sodium (Na+ ), chloride (Cl- ), potassium (K+ ) and various metabolites are shuttled from the leaf lamina to the bladders. Stalk cells operate as both a selectivity filter and a flux controller. In line with the nature of a transfer cell, advanced transmission electron tomography, electrophysiology, and fluorescent tracer flux studies revealed the stalk cell's polar organization and bladder-directed solute flow. RNA sequencing and cluster analysis revealed the gene expression profiles of the stalk cells. Among the stalk cell enriched genes, ion channels and carriers as well as sugar transporters were most pronounced. Based on their electrophysiological fingerprint and thermodynamic considerations, a model for stalk cell transcellular transport was derived.

Thread-based isoelectric focusing coupled with desorption electrospray ionization mass spectrometry.

The combination of a thread-based electrofluidic analytical device and desorption electrospray ionization mass-spectrometry (DESI-MS) was investigated for the separation and concentration of proteins. The combination delivered a low-cost novel approach for sample pretreatment and target focusing, with direct "on-thread" ambient mass spectrometry detection. For this purpose, a platform for thread-based isoelectric focusing (TB-IEF) was 3D-printed, optimised, and applied to the separation and focusing of three model proteins. Successful separation and focusing was achieved within 30 min. The TB-IEF device was coupled with DESI-MS by direct exposure of the focused solutes on the dried thread to the DESI source. As a proof-of-concept, a 10-fold increase in the DESI-MS response for insulin was achieved following the TB-IEF preconcentration, whilst simultaneously isolating the target solutes from their sample matrix.

Recent advances in enhancing the sensitivity of electrophoresis and electrochromatography in capillaries and microchips (2016-2018).

One of the most cited limitations of capillary and microchip electrophoresis is the poor sensitivity. This review continues to update this series of biannual reviews, first published in Electrophoresis in 2007, on developments in the field of online/in-line concentration methods in capillaries and microchips, covering the period July 2016-June 2018. It includes developments in the field of stacking, covering all methods from field-amplified sample stacking and large-volume sample stacking, through to isotachophoresis, dynamic pH junction, and sweeping. Attention is also given to online or in-line extraction methods that have been used for electrophoresis.

Rapid screening of inorganic and organic anions in liquid by-products from hydrothermal treatment of biomass by capillary electrophoresis.

A simple and rapid capillary electrophoresis method with capacitively coupled contactless conductivity detection (CE-C4 D) for the simultaneous determination of inorganic and organic anions in liquid product obtained from the hydrothermal treatment of biomass residues is presented. Under the optimal analytical conditions, limits of detection ranged from 1.8 to 9.4 µM for most target solutes and 53 µM for citrate. Relative standard deviations were below 0.5% for migration times and within 0.6-6.5% for peak areas for all solutes. The proposed method was successfully applied for the rapid determination and screening of inorganic and organic anions in liquid product produced following differing hydrothermal treatment temperatures for banana and pineapple biomass, and the contribution of organic acid formation to acidity in the liquid was evaluated. CE-C4 D could be a suitable method for the optimization or tailoring of HTT conditions for desired liquid product composition, and additionally for determination of the best variety(s) of biomass to use in such processes.

Isotachophoretic Fluorescence in Situ Hybridization of Intact Bacterial Cells.

A counter-pressure-assisted capillary isotachophoresis method in combination with a sieving matrix and ionic spacer was used to perform in-line fluorescence in situ hybridization (FISH) of bacterial cells. A high concentration of sieving matrix (1.8% w/v HEC) was introduced at one end of the capillary, and the bacterial cells were suspended in the spacer electrolyte for injection. Using a 2 min injection with 18 psi counter-pressure, 50% of the cells injected into the capillary were hybridized with the fluorescently labeled oligonucleotide, and the excess unhybridized probe was separated from the hybridized cell-probe complexes in a two-stage ITP method. With an LOD (6.0 × 104 cells/mL) comparable with the CE analysis of a sample processed using an off-line FISH protocol, the total analysis time was reduced from 2.5 h to 30 min. Provided the appropriate probe is selected, this approach can be used for specific detection of bacterial cells in aqueous samples.

Comparing Microfluidic Performance of Three-Dimensional (3D) Printing Platforms.

Three-dimensional (3D) printing has emerged as a potential revolutionary technology for the fabrication of microfluidic devices. A direct experimental comparison of the three 3D printing technologies dominating microfluidics was conducted using a Y-junction microfluidic device, the design of which was optimized for each printer: fused deposition molding (FDM), Polyjet, and digital light processing stereolithography (DLP-SLA). Printer performance was evaluated in terms of feature size, accuracy, and suitability for mass manufacturing; laminar flow was studied to assess their suitability for microfluidics. FDM was suitable for microfabrication with minimum features of 321 ± 5 µm, and rough surfaces of 10.97 µm. Microfluidic devices >500 µm, rapid mixing (71% ± 12% after 5 mm, 100 µL/min) was observed, indicating a strength in fabricating micromixers. Polyjet fabricated channels with a minimum size of 205 ± 13 µm, and a surface roughness of 0.99 µm. Compared with FDM, mixing decreased (27% ± 10%), but Polyjet printing is more suited for microfluidic applications where flow splitting is not required, such as cell culture or droplet generators. DLP-SLA fabricated a minimum channel size of 154 ± 10 µm, and 94 ± 7 µm for positive structures such as soft lithography templates, with a roughness of 0.35 µm. These results, in addition to low mixing (8% ± 1%), showed suitability for microfabrication, and microfluidic applications requiring precise control of flow. Through further discussion of the capabilities (and limitations) of these printers, we intend to provide guidance toward the selection of the 3D printing technology most suitable for specific microfluidic applications.

In Silico Screening of Two-Dimensional Separation Selectivity for Ion Chromatography × Capillary Electrophoresis Separation of Low-Molecular-Mass Organic Acids.

A prerequisite for ordered two-dimensional (2D) separations and full utilization of the enhanced 2D peak capacity is selective exploitation of the sample attributes, described as sample dimensionality. In order to take sample dimensionality into account prior to optimization of a 2D separation, a new concept based on construction of 2D separation selectivity maps is proposed and demonstrated for ion chromatography × capillary electrophoresis (IC×CE) separation of low-molecular-mass organic acids as test analytes. For this purpose, 1D separation selectivity maps were constructed based on calculation of pairwise separation factors and identification of critical pairs for four IC stationary phases and 28 levels of background electrolyte pH in CE. The derived IC and CE maps were then superimposed and the effectiveness of the respective 2D separations assessed using an in silico approach, followed by testing examples of one successful and one unsuccessful 2D combination experimentally. The results confirmed the efficacy of the predictions, which require a minimal number of experiments compared to the traditional one-at-a-time approach. Following the same principles, the proposed framework can also be adapted for optimization of separation selectivity in various 2D combinations and for other applications.

Recent advances in enhancing the sensitivity of electrophoresis and electrochromatography in capillaries and microchips (2014-2016).

One of the most cited limitations of capillary (and microchip) electrophoresis is the poor sensitivity. This review continues to update this series of biennial reviews, first published in Electrophoresis in 2007, on developments in the field of on-line/in-line concentration methods in capillaries and microchips, covering the period July 2014-June 2016. It includes developments in the field of stacking, covering all methods from field amplified sample stacking and large volume sample stacking, through to isotachophoresis, dynamic pH junction, and sweeping. Attention is also given to on-line or in-line extraction methods that have been used for electrophoresis.

Fibre-based electrofluidics on low cost versatile 3D printed platforms for solute delivery, separations and diagnostics; from small molecules to intact cells.

A novel and effective fibre-based microfluidic methodology was developed to move and isolate charged solutes, biomolecules, and intact bacterial cells, based upon a novel multi-functional 3D printed supporting platform, with potential applications in the fields of microfluidics and biodiagnostics. Various on-fibre electrophoretic techniques are demonstrated to separate, pre-concentrate, move, split, or cut and collect the isolated zones of target solutes, including proteins and live bacterial cells. The use of knotting to link different fibre materials, and the unique ability of this approach to physically concentrate solutes in different locations are shown such that the concentrated solutes can be physically isolated and easily transferred to other fibres. Application of this novel fibre-based technique within a potential diagnostic platform for urinary tract infection is shown, together with the post-electrophoretic incubation of live bacterial cells, demonstrating the cell survival following on-fibre electrophoretic concentration.

Characterisation of graphene fibres and graphene coated fibres using capacitively coupled contactless conductivity detector.

The use of capacitively coupled contactless conductivity detection (C(4)D) for the characterisation of thin conductive graphene fibres, graphene composite fibres, and graphene coated fibrous materials is demonstrated for the first time. Within a few seconds, the non-destructive C(4)D detector provides a profile of the longetudinal physical homogeneity of the fibre, as well as extra information regarding fibre mophology and composition. In addition to the theoretical considerations related to the factors affect the output signal, this work evaluates the properties of graphene fibres using scanning C(4)D following the manufacturing process of wet-spinning. Furthermore, conductive graphene-coated fibrous materials and the effectiveness of the coating and reduction procedures applied could be investigated. Apart from the application of C(4)D in the monitoring of such processes, the feasibility of this small, highly sensitive and rapidly-responsive detector to monitor strain and elasticity responses of conductive and elastomeric composite fibres for applications in motion sensing, biomedical monitoring, and stretchable electronics was also demonstrated.

Novel Instrument for Automated pK(a) Determination by Internal Standard Capillary Electrophoresis.

The internal standard capillary electrophoresis method (IS-CE) has been implemented in a novel sequential injection-capillary electrophoresis instrument for the high-throughput determination of acidity constants (pK(a)) regardless of aqueous solubility, number of pK(a) values, or structure. This instrument comprises a buffer creation system that automatically mixes within a few seconds four reagents for in situ creation of the separation electrolyte with a pH range of 2-13, ionic strength of 10-100 mM and organic solvent content from 0% to 40%. Combined with 1.2 kV/cm and a short effective length (15 cm to the UV detector) fast 20 s electrophoretic separations can be obtained. The low standard deviation of the replicates and the low variation compared to reference values show that this system can accurately determine acidity constants of drugs by IS-CE. A single pK(a) can be determined in 2 min and a set of 20 measurements in half an hour, allowing rapid, simple, and flexible determination of pK(a) values of pharmaceutical targets.

Determination of acidity constants of sparingly soluble drugs in aqueous solution by the internal standard capillary electrophoresis method.

A set of 33 drugs with different solubilities, ranging from soluble to very insoluble, has been chosen in order to evaluate the performance of the internal standard CE method to determine acidity constants of compounds with limited solubility. The set of drugs tested in this work has been chosen as a function of their intrinsic solubility. For the most insoluble compounds, several analytical conditions to overcome the insolubility in aqueous buffers have been tested. This paper assesses the compound solubility limits for the IS-CE method in aqueous pKa determinations, and also compares the determined pKa s with the results from the literature data obtained by other methods. It is proved that IS-CE method determines acidity constants of sparingly soluble drugs in aqueous media (compounds with logS down to around -6), whereas other reference methods require the use of aqueous-organic solvent buffers and extrapolation procedures to obtain the aqueous pKa for the same compounds.

Astrocyte S100ß expression and selective differentiation to GFAP and GS in the entorhinal cortex during ageing in the 3xTg-Alzheimer's disease mouse model.

The study of astrocytes and its role in the development and evolution of neurodegenerative diseases, including Alzheimer's disease (AD) is essential to fully understand their aetiology. The aim if this study is to deepen into the concept of the heterogeneity of astrocyte subpopulations in the EC and in particular the identification of differentially functioning astrocyte subpopulations that respond differently to AD progression. S100ß protein belongs to group of small calcium regulators of cell membrane channels and pumps that are expressed by astrocytes and is hypothesised to play and have a relevant role in AD development. We analysed the selective differentiation of S100ß-positive astrocytes into Glutamine synthetase (GS) and Glial fibrillary acidic protein (GFAP)-positive sub-groups in the entorhinal cortex (EC) of AD triple transgenic animal model (3xTg-AD). EC is the brain region earliest affected in humans AD but also in this closest animal model regarding their pathology and time course. We observed no changes in the number of S100ß-positive astrocytes between 1 and 18 months of age in the EC of 3xTg-AD mice. However, we identified relevant morphological changes in S100ß/GFAP positive astrocytes showing a significant reduction in their surface and volume whilst an increase in number and percentage. Furthermore, the percentage of S100ß/GS positive astrocyte population was also increased in 18 months old 3xTg-AD mice compared to the non-Tg mice. Our findings reveal the presence of differentially controlled astrocyte populations that respond differently to AD progression in the EC of 3xTg-AD mice. These results highpoints the major astrocytic role together with its early and marked affection in AD and arguing in favour of its importance in neurogenerative diseases and potential target for new therapeutic approaches.

Diffusion-free valve for preprogrammed immunoassay with capillary microfluidics.

By manipulating the geometry and surface chemistry of microfluidic channels, capillary-driven microfluidics can move and stop fluids spontaneously without external instrumentation. Furthermore, complex microfluidic circuits can be preprogrammed by synchronizing the capillary pressures and encoding the surface tensions of microfluidic chips. A key component of these systems is the capillary valve. However, the main concern for these valves is the presence of unwanted diffusion during the valve loading and activation steps that can cause cross-contamination. In this study, we design and validate a novel diffusion-free capillary valve: the π-valve. This valve consists of a 3D structure and a void area. The void acts as a spacer between two fluids to avoid direct contact. When the valve is triggered, the air trapped within the void is displaced by pneumatic suction induced from the capillary flow downstream without introducing a gas bubble into the circuit. The proposed design eliminates diffusive mixing before valve activation. Numerical simulation is used to study the function and optimize the dimensions of the π-valve, and 3D printing is used to fabricate either the mould or the microfluidic chip. A comparison with a conventional valve (based on a constriction-expansion valve) demonstrates that the π-valve eliminates possible backflow into the valve and reduces the mixing and diffusion during the loading and trigger steps. As a proof-of-concept, this valve is successfully implemented in a capillary-driven circuit for the determination of benzodiazepine, achieving the successive release of 3 solutions in a 3D-printed microfluidic chip without external instrumentation. The results show a 40% increase in the fluorescence intensity using the π-valve relative to the conventional value. Overall, the π-valve prevents cross-contamination, minimizes sample use, and facilitates a sophisticated preprogrammed release of fluids, offering a promising tool for conducting automated immunoassays applicable at point-of-care testing.

Prominent and conspicuous astrocyte atrophy in human sporadic and familial Alzheimer's disease.

Pathophysiology of sporadic Alzheimer's disease (SAD) and familial Alzheimer's disease (FAD) remains poorly known, including the exact role of neuroglia and specifically astroglia, in part because studies of astrocytes in human Alzheimer's disease (AD) brain samples are scarce. As far as we know, this is the first study of a 3-D immunohistochemical and microstructural analysis of glial fibrillary acidic protein (GFAP)- and glutamine synthetase (GS)-positive astrocytes performed in the entorhinal cortex (EC) of human SAD and FAD samples. In this study, we report prominent atrophic changes in GFAP and GS astrocytes in the EC of both SAD and FAD characterised by a decrease in area and volume when compared with non-demented control samples (ND). Furthermore, we did not find neither astrocytic loss nor astrocyte proliferation or hypertrophy (gliosis). In contrast with the astrogliosis classically accepted hypothesis, our results show a highly marked astrocyte atrophy that could have a major relevance in AD pathological processes being fundamental and key for AD mnesic and cognitive alterations equivalent in both SAD and FAD.

Coupling Capillary-Driven Microfluidics with Lateral Flow Immunoassay for Signal Enhancement.

Microfluidics has emerged as a versatile technology that is applied to enhance the performance of analytical techniques, among others. Pursuing this, we present a capillary-driven microfluidic device that improves the sensitivity of lateral flow immunoassay rapid tests thanks to offering an automated washing step. A novel multilevel microfluidic chip was 3D-printed with a photocurable black resin, sealed by an optically clear pressure-sensitive adhesive, and linked to the lateral flow strip. To depict the efficacy of microfluidics and the washing step, cortisol was measured quantitatively within the proposed device. Measuring cortisol levels is a way to capture physiological stress responses. Among biofluids, saliva is less infectious and easier to sample than others. However, higher sensitivity is demanded because the salivary cortisol concentrations are much lower than in blood. We carried out a competitive lateral flow immunoassay protocol with the difference that the microfluidic device applies an automated washing step after the sample is drained downstream. It washes the trapped quantum-dot-labeled antibodies out from nitrocellulose, diminishing background noise as these are bonded to cortisols and not to the immobilized receptors. Fluorescence spectroscopy, as a high-precision analysis, was successfully applied to determine clinically relevant salivary cortisol concentrations within a buffer quantitatively. The microfluidic design relied on a 3D valve that avoids reagent cross-contamination. This cross-contamination could make the washing buffer impure and undesirably dilute the sample. The proposed device is cost-effective, self-powered, robust, and ideal for non-expert users.

Reduced Graphene Oxide Electrolyte-Gated Transistor Immunosensor with Highly Selective Multiparametric Detection of Anti-Drug Antibodies.

The advent of immunotherapies with biological drugs has revolutionized the treatment of cancers and auto-immune diseases. However, in some patients, the production of anti-drug antibodies (ADAs) hampers the drug efficacy. The concentration of ADAs is typically in the range of 1-10 pm; hence their immunodetection is challenging. ADAs toward Infliximab (IFX), a drug used to treat rheumatoid arthritis and other auto-immune diseases, are focussed. An ambipolar electrolyte-gated transistor (EGT) immunosensor is reported based on a reduced graphene oxide (rGO) channel and IFX bound to the gate electrode as the specific probe. The rGO-EGTs are easy to fabricate and exhibit low voltage operations (≤ 0.3 V), a robust response within 15 min, and ultra-high sensitivity (10 am limit of detection). A multiparametric analysis of the whole rGO-EGT transfer curves based on the type-I generalized extreme value distribution is proposed. It is demonstrated that it allows to selectively quantify ADAs also in the co-presence of its antagonist tumor necrosis factor alpha (TNF-α), the natural circulating target of IFX.

Determination of the aqueous pKa of very insoluble drugs by capillary electrophoresis: Internal standards for methanol-water extrapolation.

A fast determination of acidity constants (pKa) of very insoluble drugs has become a necessity in drug discovery process because it often produces molecules that are highly lipophilic and sparingly soluble in water. In this work the high throughput internal standard capillary electrophoresis (IS-CE) method has been adapted to the determination of pKa of water insoluble compounds by measurement in methanol/aqueous buffer mixtures. For this purpose, the reference pKa values for a set of 46 acid-base compounds of varied structure (internal standards) have been established in methanol-water mixtures at several solvent composition levels (with a maximum of 40% methanol). The IS-CE method has been successfully applied to seven test drugs of different chemical nature with intrinsic solubilities lower than 10-6 M. pKa values have been determined at different methanol/aqueous buffer compositions and afterwards Yasuda-Shedlovsky extrapolation method has been applied to obtain the aqueous pKa. The obtained results have successfully been compared to literature ones obtained by other methods. It is concluded that the IS-CE method allows the determination of aqueous pKa values using low proportions of methanol, becoming then more accurate in the extrapolation procedure than other reference methods.

Thread-based isotachophoresis for DNA extraction and purification from biological samples.

A rapid, low-cost, and disposable microfluidic thread-based isotachophoresis method was developed for the purification and preconcentration of nucleic acids from biological samples, prior to their extraction and successful analysis using quantitative polymerase chain reaction (qPCR). This approach extracts and concentrates protein-free DNA from the terminating electrolyte buffer, via a continuous sampling approach, resulting in significant focussing of the extracted DNA upon a 6 cm length nylon thread. The platform was optimised using the preconcentration of a fluorescent dye, showing a 600-fold concentration capacity within <5 min. The system was then applied to the one-step extraction of lambda DNA - an E. coli bacteriophage - spiked into whole blood, exhibiting the exclusion of PCR inhibitors. The extraction efficiency from the thread material following concentration was consistent, between 94.4-113.9%. The determination of lambda DNA in whole blood was achieved within a linear range of 1.0-1 × 105 fg µL-1 (20-2 × 106 copies per µL). This technique demonstrates great potential for the development of thread-based affordable analytical and diagnostic devices based upon DNA and RNA isolation.