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Microfluidic platforms are an attractive option for incorporating complex fluid handling into low-cost and rapid diagnostic tests. A persistent challenge for microfluidics, however, is the mismatch in the "world-tochip" interface - it is challenging to detect analytes present at low concentrations in systems that can only handle small volumes of sample. Here we describe a new technique termed pre-concentration by liquid intake by paper (P-CLIP) that addresses this mismatch, allowing digital microfluidics to interface with volumes on the order of hundreds of microliters. In P-CLIP, a virtual microchannel is generated to pass a large volume through the device; analytes captured on magnetic particles can be isolated and then resuspended into smaller volumes for further processing and analysis. We characterize this method and demonstrate its utility with an immunoassay for Plasmodium falciparum lactate dehydrogenase, a malaria biomarker, and propose that the P-CLIP strategy may be useful for a wide range of applications that are currently limited by low-abundance analytes.

Digital microfluidics (DMF) is a platform that enables highly reconfigurable and automated fluidic operations using a generic device architecture. A unique hallmark of DMF is its "flexibility": a generic device design can be used and reused for many different, divergent fluidic operations. The flexibility of DMF is compromised when devices are permanently modified with embedded sensors. Here we introduce a solution to the "flexibility gap" between fluidic operations in digital microfluidics and embedded sensors: "plug-n-play DMF" (PnP-DMF). In PnP-DMF, devices are designed to allow for rapid and seamless exchange of sensors depending on the application needs. This paper provides "proof of concept" for PnP-DMF using commercial biosensors for glucose and β-ketone, a custom paper-based electrochemical sensor for lactate, and a generic screen-printed electroanalytical cell. We demonstrate that hot-swapping sensors between experiments allows for convenient implementation of complex processes such as automated analysis of blood samples by standard addition. Finally, we explored the suitability for using PnP sensors in tandem with other sensing modalities, combining biosensor-based electrochemical measurement of glucose with a chemiluminescent magnetic bead-based sandwich immunoassay for insulin. The latter is notable, as it constitutes the first report of an analysis of different analytes in both the supernatant and precipitate from a single sample-aliquot in a microfluidic device. The results presented here highlight the versatility of PnP-DMF, illustrating how it may be useful for a wide range of applications in diagnostics and beyond.

The legend in Figure 4a in the previously published version of this article contained erroneous units. The correct units are given in the caption–that is, 1 µM (black), 500 nM (red), 100 nM (blue), 50 nM (cyan), 10 nM (pink), and 0 nM (1× PBS solution, brown).

Homeostasis of dopamine, a classical neurotransmitter, is a key indicator of neuronal health. Dysfunction in the regulation of dopamine is implicated in a long list of neurological disorders, including addiction, depression, and neurodegeneration. The existing methods used to evaluate dopamine homeostasis in vitro are inconvenient and do not allow for continuous non-destructive measurement. In response to this challenge, we introduce an integrated microfluidic system that combines dopaminergic cell culture and differentiation with electroanalytical measurements of extracellular dopamine in real-time at any point during an assay. We used the system to examine the behavior of differentiated SH-SY5Y cells upon exposure to four dopamine transporter ant/agonists (cocaine, ketamine, epigallocatechin gallate, and amphetamine) and study their pharmacokinetics. The IC50 values of cocaine, ketamine, and epigallocatechin gallate were determined to be (average ± standard deviation) 3.7 ± 1.1 µM, 51.4 ± 17.9 µM, and 2.6 ± 0.8 µM, respectively. Furthermore, we used the new system to study amphetamine-mediated dopamine release to probe the related phenomena of dopamine transporter-mediated reverse-transport and dopamine release from vesicles. We propose that this platform, which is the first platform to simultaneously evaluate uptake and release, could be useful to screen for drugs and other agents that target dopaminergic neurons and the function of the dopamine transporter. More broadly, this platform should be adaptable for any application that could benefit from high-temporal resolution electroanalysis combined with multi-day cell culture using small numbers of cells. A microfluidic-based setup permits real-time in-vitro assessment of cellular dopamine uptake. Regulation of dopamine in the body is crucial to the health of the nervous system; ineffective regulation can result in damage to neurons and a range of neurological conditions, including addiction and Parkinson’s Disease. A key process in the regulation of dopamine is its release/uptake from cells. Now, a team led by Aaron Wheeler from the University of Toronto report a digital microfluidic device that integrates in-vitro cell culture with electrochemical analysis, in order to detect dopamine uptake and release in real-time during an assay. This new system might also be useful for screening libraries of reagents for the identification of new drugs.

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 cm2 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.

Microfluidic platforms are an attractive option for incorporating complex fluid handling into low-cost and rapid diagnostic tests. A persistent challenge for microfluidics, however, is the mismatch in the "world-to-chip" interface - it is challenging to detect analytes present at low concentrations in systems that can only handle small volumes of sample. Here we describe a new technique termed pre-concentration by liquid intake by paper (P-CLIP) that addresses this mismatch, allowing digital microfluidics to interface with volumes on the order of hundreds of microliters. In P-CLIP, a virtual microchannel is generated to pass a large volume through the device; analytes captured on magnetic particles can be isolated and then resuspended into smaller volumes for further processing and analysis. We characterize this method and demonstrate its utility with an immunoassay for Plasmodium falciparum lactate dehydrogenase, a malaria biomarker, and propose that the P-CLIP strategy may be useful for a wide range of applications that are currently limited by low-abundance analytes.

In this work is presented a method for the modification of native PDMS surface in order to improve its applicability as a substrate for microfluidic devices, especially in the analysis of nonpolar analytes. Therefore, poly(ethylene glycol) divinyl ether modified PDMS substrate was obtained by surface modification of native PDMS. The modified substrate was characterized by attenuated total reflectance infrared spectroscopy, water contact angle measurements, and by evaluating the adsorption of rhodamine B and the magnitude of the EOF mobility. The reaction was confirmed by the spectroscopic evaluation. The formation of a well-spread water film over the surface immediately after the modification was an indicative of the modified surface hydrophilicity. This characteristic was maintained for approximately ten days, with a gradual return to a hydrophobic state. Fluorescence assays showed that the nonpolar adsorption property of PDMS was significantly decreased. The EOF mobility obtained was 3.6 × 10(-4) cm(2) V(-1) s(-1) , higher than the typical values found for native PDMS. Due to the better wettability promoted by the modification, the filling of the microchannels with aqueous solutions was facilitated and trapping of air bubbles was not observed.

Excess nitric oxide (NO) production occurs in several pathological states, including neurodegeneration, ischemia, and inflammation, and is generally accompanied by increased oxidative/nitrosative stress. Carnosine [β-alanine-histidine (β-Ala-His)] has been reported to decrease oxidative/nitrosative stress-associated cell damage by reducing the amount of NO produced. In this study, we evaluated the effect of carnosine on NO production by murine RAW 264.7 macrophages stimulated with lipopolysaccharides + interferon-γ. Intracellular NO and intracellular and extracellular nitrite were measured by microchip electrophoresis with laser-induced fluorescence and by the Griess assay, respectively. Results showed that carnosine causes an apparent suppression of total NO production by stimulated macrophages accompanied by an unexpected simultaneous drastic increase in its intracellular low toxicity endproduct, nitrite, with no inhibition of inducible nitric oxide synthase (iNOS). ESI-MS and NMR spectroscopy in a cell-free system showed the formation of multiple adducts (at different ratios) of carnosine-NO and carnosine-nitrite, involving both constituent amino acids (β-Ala and His) of carnosine, thus providing a possible mechanism for the changes in free NO and nitrite in the presence of carnosine. In stimulated macrophages, the addition of carnosine was also characterized by changes in the expression of macrophage activation markers and a decrease in the release of IL-6, suggesting that carnosine might alter M1/M2 macrophage ratio. These results provide evidence for previously unknown properties of carnosine that modulate the NO/nitrite ratio of stimulated macrophages. This modulation is also accompanied by changes in the release of pro-inflammatory molecules, and does not involve the inhibition of iNOS activity.

Superoxide, a naturally produced reactive oxygen species (ROS) in the human body, is involved in many pathological and physiological signaling processes. However, if superoxide formation is left unregulated, overproduction can lead to oxidative damage to important biomolecules, such as DNA, lipids, and proteins. Superoxide can also lead to the formation of peroxynitrite, an extremely hazardous substance, through its reaction with endogenously produced nitric oxide. Despite its importance, quantitative information regarding superoxide production is difficult to obtain due to its high reactivity and low concentrations in vivo. MitoHE, a fluorescent probe that specifically reacts with superoxide, was used in conjunction with microchip electrophoresis (ME) and laser-induced fluorescence (LIF) detection to investigate changes in superoxide production by RAW 264.7 macrophage cells following stimulation with phorbol 12-myristate 13-acetate (PMA). Stimulation was performed in the presence and absence of the superoxide dismutase (SOD) inhibitors, diethyldithiocarbamate (DDC) and 2-metoxyestradiol (2-ME). The addition of these inhibitors resulted in an increase in the amount of superoxide specific product (2-OH-MitoE(+)) from 0.08 ± 0.01 fmol (0.17 ± 0.03 mM) in native cells to 1.26 ± 0.06 fmol (2.5 ± 0.1 mM) after PMA treatment. This corresponds to an approximately 15-fold increase in intracellular concentration per cell. Furthermore, the addition of 3-morpholino-sydnonimine (SIN-1) to the cells during incubation resulted in the production of 0.061 ± 0.006 fmol (0.12 ± 0.01 mM) of 2-OH-MitoE(+) per cell on average. These results demonstrate that indirect superoxide detection coupled with the use of SOD inhibitors and a separation method is a viable method to discriminate the 2-OH-MitoE(+) signal from possible interferences.

Peroxynitrite (ONOO−) is a powerful oxidant produced from the reaction of nitric oxide (NO) and superoxide (O2˙−). In healthy cells, a balance exists between endogenous antioxidants and pro-oxidants to prevent oxidation, nitrosation, and nitration reactions with cellular macromolecules, and reactions of peroxynitrite with small molecules, such as carbon dioxide. However, when this system is not in homeostasis, ONOO− can cause cytotoxicity by reacting with important biomolecules such as proteins, nucleic acids, and lipids, resulting in inhibited or altered function. For this reason, peroxynitrite has been linked to a number of disease states including cancer, stroke, myocardial infarction, atherosclerosis, hypertension, chronic heart failure, Alzheimer's disease, Parkinson's disease, multiple sclerosis, and amyotrophic lateral sclerosis. This chapter reviews the electrophoretic methods that have been developed for the separation and subsequent detection of peroxynitrite as well as its metabolites and degradation products.

Methacryloxypropyl-modified poly(dimethylsiloxane) rubbers were obtained from poly(dimethylsiloxane), PDMS, and methacryloxypropyltrimethoxysilane, MPTMS, by polycondensation reactions. The modified rubbers, prepared with 20 and 30% (v/v) of MPTMS, were used as substrates for microchannel fabrication by the CO(2) laser ablation technique. Raman imaging spectroscopy was used for the surface characterization, showing the homogeneity of the rubbery material, with uniform distribution of the crosslinking centers. Under the experimental conditions used, damage to the rubber from the CO(2) laser radiation used for the channel engraving was not observed. Correlation maps of the surface were obtained in order to spatially evaluate the modification inside and outside the channels. The correlations between the methacryloxypropyl-modified poly(dimethylsiloxane) rubbers and MPTMS (spectral range of 1800-1550 cm(-1)) and PDMS (spectral range of 820-670 cm(-1)) precursors were higher than 0.95 and 0.99, respectively. In addition, Raman imaging spectroscopy allows monitoring the topography of the fabricated microchannel.