Influence of Lanthanum on Stern Layer Conductance in the Nanochannel.

In this report, high-frequency electric impedance spectroscopy was performed to investigate ionic transport through nanochannels. Special attention was focused on (i) conductance behaviors depending on the role of cation valence in three background electrolytes (XCln): monovalent 1-1 (K+ and Cl-), divalent 2-1 (Mg2+ and 2Cl-), and trivalent 3-1 (La3+ and 3Cl-), (ii) the effects of proton and bicarbonate ions on bulk and surface conductance, and (iii) the connected microchannel dimension (surface/height ratio aspect) within the nanochannel apparent conductance. The results highlight a net quantitative increase in surface silanol density and a strong decrease in surface ionization degree when lanthanum cations are employed. The results also demonstrate that La3+ strongly interacts with the silica surface, leading to negative values of standard free energy for ion-site interactions and chemical potential for ion-ion correlations in the Stern layer of -0.8 and -10.2 kT, respectively. We ascribed the evolution of surface charge density to the balance between the mole ratios of water molecules and adsorbed cations at equilibrium. We found that La3+ behaves as an acidic cation (Lewis conceptualization) that neutralizes the negative silica surface accompanying water molecule expulsion due to steric hindrance. This study constitutes a new contribution to ion-site interactions and to ion-ion correlation phenomena on the planar silica surface to explain charge inversion observation in micro-nanofluidic devices.

Microfluidic Chip for the Electrochemical Detection of MicroRNAs: Methylene Blue Increasing the Specificity of the Biosensor.

MicroRNAs (miRNAs) are biomarkers involved in biological processes that are released by cells and found in biological fluids such as blood. The development of nucleic acid-based biosensors has significantly increased in the past 10 years because the detection of such nucleic acids can easily be applied in the field of early diagnosis. These biosensors need to be sensitive, specific, and fast in order to be effective. This work introduces a newly-built electrochemical biosensor that enables a fast detection in 30 min and, as a result of its integration in microfluidics, presents a limit of detection as low as 1 aM. The litterature concerning the specificity of electrochemical biosensors includes several studies that report one base-mismatch, with the base-mismatch located in the middle of the strand. We report an electrochemical nucleic acid biosensor integrated into a microfluidic chip, allowing for a one-base-mismatch specificity independently from the location of the mismatch in the strand. This specificity was improved using a solution of methylene blue, making it possible to discriminate a partial hybridization from a complete and complementary hybridization.

An Integrated Multiple Electrochemical miRNA Sensing System Embedded into a Microfluidic Chip.

In this article, we present the design, fabrication and characterization of a microfluidic device and a dedicated electronic system to perform 8 multiplexed electrochemical measurements of synthetic miRNA strands, as well as the biochemical protocols developed for the functionalization of the electrodes and the quantification experiments. The outcomes of this work highlight that the parallelization of eight microchannels containing 2-electrode cells driven by the dedicated electronics offers a solution as robust as a conventional 3-electrode cell and commercially available potentiostats. In addition, this solution presents the advantage of simultaneously reduce the microfabrication complexity, as well as offering an integrated; multiplexed and portable system for the quantification of miRNA. The results presented demonstrate that the system shows a linear response on concentrations down to 10-18 mol/L of perfect matched reporter and capture sequences of synthetic miRNA.

Magnetic Hyperthermia on γ-Fe2O3@SiO2 Core-Shell Nanoparticles for mi-RNA 122 Detection.

Magnetic hyperthermia on core-shell nanoparticles bears promising achievements, especially in biomedical applications. Here, thanks to magnetic hyperthermia, γ-Fe2O3 cores are able to release a DNA target mimicking the liver specific oncotarget miRNA-122. Our silica coated magnetic nanoparticles not only allow the grafting at their surface of a significant number of oligonucleotides but are also shown to be as efficient, by local heating, as 95 °C global heating when submitted to an alternative magnetic field, while keeping the solution at 28 °C, crucial for biological media and energy efficiency. Moreover, a slight modification of the silica coating process revealed an increased heating power, well adapted for the release of small oligonucleotides such as microRNA.

Electropreconcentration diagrams to optimize molecular enrichment with low counter pressure in a nanofluidic device.

Concentration polarization (CP)-based focusing electrokinetics nanofluidic devices have been developed in order to simultaneously detect and enrich highly diluted analytes on-a-chip. However, stabilization of focal points over long time under the application of the electric field remains as a technical bottleneck. If pressure-assisted preconcentration methods have been proposed to stabilize propagating modes at low inverse Dukhin number (1/Du≪1) , these recent protocols remain laborious for optimizing experimental parameters. In this paper, "electric field E/counter-pressure P" diagrams have been established during pressure-assisted electro-preconcentration of fluorescein as a model molecule. Such E/P diagram allows direct observation of the region for which the optimal counter-pressure P leads to a stable focusing regime. This region of stable focusing is shown to vary depending of the nanoslit length (100 µm < Lnanoslit < 500 µm) and the nature of the background electrolyte (KCl and NaCl). Longer nanoslits (500 µm) produce stabilization at low counter-pressure P, whereas NaCl offers a narrower region of stable focusing in the E/P diagram compared to KCl. Finally, the ability of such pressure-assisted protocol to concentrate negatively charged proteins has been tested with a more applicative protein, i.e., ovalbumin. The corresponding E/P diagram confirms the existence of the stable focusing regime at both low electric field E (≤20 V) and counter-pressure P (≤0.4 bar). With an enrichment factor as high as 70 after 2 min for ovalbumin at a concentration of 10 µM, such pressure-assisted nanofluidic electro-preconcentration protocol appears very promising to concentrate and detect biomolecules.

Release and Detection of microRNA by Combining Magnetic Hyperthermia and Electrochemistry Modules on a Microfluidic Chip.

The heating of a biologic solution is a crucial part in an amplification process such as the catalytic detection of a biological target. However, in many situations, heating must be limited in microfluidic devices, as high temperatures can cause the denaturation of the chip components. Local heating through magnetic hyperthermia on magnetic nano-objects has opened the doors to numerous improvements, such as for oncology where a reduced heating allows the synergy of chemotherapy and thermotherapy. Here we report on the design and implementation of a lab on chip without global heating of samples. It takes advantage of the extreme efficiency of DNA-modified superparamagnetic core-shell nanoparticles to capture complementary sequences (microRNA-target), uses magnetic hyperthermia to locally release these targets, and detects them through electrochemical techniques using ultra-sensitive channel DNA-modified ultramicroelectrodes. The combination of magnetic hyperthermia and microfluidics coupled with on-chip electrochemistry opens the way to a drastic reduction in the time devoted to the steps of extraction, amplification and nucleic acids detection. The originality comes from the design and microfabrication of the microfluidic chip suitable to its insertion in the millimetric gap of toric inductance with a ferrite core.

Logic digital fluidic in miniaturized functional devices: Perspective to the next generation of microfluidic lab-on-chips.

Microfluidics has emerged following the quest for scale reduction inherent to micro- and nanotechnologies. By definition, microfluidics manipulates fluids in small channels with dimensions of tens to hundreds of micrometers. Recently, microfluidics has been greatly developed and its influence extends not only the domains of chemical synthesis, bioanalysis, and medical researches but also optics and information technology. In this review article, we will shortly discuss an enlightening analogy between electrons transport in electronics and fluids transport in microfluidic channels. This analogy helps to master transport and sorting. We will present some complex microfluidic devices showing that the analogy is going a long way off toward more complex components with impressive similarities between electronics and microfluidics. We will in particular explore the vast manifold of fluidic operations with passive and active fluidic components, respectively, as well as the associated mechanisms and corresponding applications. Finally, some relevant applications and an outlook will be cited and presented.

Determination of the isomeric forms proportion of fluorogenic naphthalene-2,3-dicarboxaldehyde in a binary mixture of water:methanol using electrochemical methods.

The electrochemical response of the fluorogenic label naphthalene-2,3-dicarboxyaldehyde (NDA) in a binary mixture of water/methanol was characterized with cyclic voltammetry (CV) and differential pulse voltammetry (DPV) electrochemical techniques. Naphthalene-2,3-dicarboxyaldehyde does exist in three isomeric forms in aqueous solution: the unhydrated dialdehyde (DA), the acyclic monohydrated (MA) and the cyclic hemiacetal (HAC). The study underlines that the proportion of each of them varies according to the working pH. At low and high pH, the dialdehyde form is in larger proportion than the acyclic monohydrated form. Conversely at intermediate pH, the concentration of the acyclic form is in greater proportion than the dialdehyde form. These results allowed us to determine the optimal pH of 9 for which the labeling of biomolecules could be more efficient due to the base catalyzed regeneration of the unhydrated form. At this pH, the data processing from the analysis of measured currents and estimation of diffusion coefficients of each form according to the semi-empirical models of Wilke-Chang, Scheibel, Reddy-Doraiswamy and Lusis-Ratcliff allowed us to obtain the concentration of dialdehyde (0.28 mM), acyclic monohydrated (0.57 mM) and cyclic hemiacetal monohydrated (0.15 mM) forms starting from 1mM naphthalene-2,3-dicarboxyaldehyde.

Study of Surface Charge Instabilities by EOF Measurements on a Chip: A Real-Time Hysteresis and Peptide Adsorption Based Methodology.

This paper describes the measurement of the electroosmotic mobility (EOF) in a Wheatstone fluidic bridge (µFWB) as a direct probe of the surface instability. The variation of EOF known as one major contribution of the electrokinetic migration has been determined with a real-time measurement platform after different conditionings on chips. We also scan the pH of the background electrolytes with three different ionic strengths to evaluate the dependencies of the EOF as a function of the pH. A hysteresis methodology has been developed for probing the surface charge instabilities. EOF mobility has been recorded during on-a-chip electrophoresis to estimate the effect of such instability on the analytical performance. As expected, our experimental curves show that a decrease in the ionic strength increases the surface charge stability of the hybrid microchip. This result demonstrates that ionic exchanges between the surface and the fluid are clearly involved in the stability of the surface charge. With this original method based on real-time EOF measurement, the surface state can be characterized after hydrodynamic and electrophoresis sequences to mimic any liquid conditioning and separation steps. Finally, as a demonstrative application, isotherms of the adsorption of insulin have been recorded showing the change in surface charge by unspecific adsorption of this biomolecule onto the microfluidic channel's wall. These methodologies and findings could be particularly relevant to investigating various analytical pathways and to understanding the molecular mechanisms at solid/liquid interfaces.

Microchannel conductivity measurements in microchip for on line monitoring of dephosphorylation rates of organic phosphates using paramagnetic-beads linked alkaline phosphatase.

This paper presents the use of polymer coated microelectrodes for the realtime conductivity monitoring in a microchannel photoablated through the polymer without contact. Based on this strategy, a small conductometry sensor has been developed to record in time conductivity variation when an enzymatic reaction occurs through the channel. The rate constant determination, k2, for the dephosphorylation of organic phosphate-alkaline phosphatase-superparamagnetic beads complex using chemically different substrates such as adenosine monoesterphosphate, adenosine diphosphate and adenosine triphosphate was taken as an example to demonstrate selectivity and sensivity of the detection scheme. The k2 value measured for each adenosine phosphate decreases from 39 to 30 s(-1) in proportion with the number (3, 2 and 1) of attached phosphate moiety, thus emphasizing the steric hindrance effect on kinetics.

Electrochemiluminescence on-a-chip: towards a hand-held electrically powered optofluidic source.

We report a microfluidic platform that integrates several parallel optical sources based on electrochemiluminescence (ECL) of 9,10-diphenylanthracene (DPA) as luminophore agent. The annihilation of DPA radicals provides a low wavelength emission at λ=430 nm in the blue-visible range. By varying the distance between electrodes for each ECL integrated source, this glass/PDMS/glass platform enabled a systematic investigation of the main electrochemical parameters involved in ECL. These parameters have been studied either in a static mode or in a dynamic one. Even at slow flow rate (~2 µl s(-1)), the renewal of electroactive species could be easily promoted inside the microfluidic channel which gives rise to a stable optical intensity for several minutes. Compared with traditional optically pumped dye sources, this microfluidic system demonstrates that ECL can be easily implemented on chip for producing much compact optofluidic sources. Such simply electrically powered system-on-chip would surely encourage the future of hand-held µTAS devices with integrated fast detection and embedded electronics.

Improved electrochemical detection of a transthyretin synthetic peptide in the nanomolar range with a two-electrode system integrated in a glass/PDMS microchip.

An alternative to a three-electrode set-up for electrochemical detection and analysis in microfluidic chips is described here. The design of the electrochemical sensor consists of the surface of the glass substrate covered with a PDMS block which bears the microfluidic channels. A band microelectrode which acts as a working electrode surrounded by a large counter electrode is obtained at the micrometric level to propose a simple and efficient sensing area for on-a-chip analysis. The counter-electrode with a surface area about 22-fold greater than the working-microelectrode can also be considered as a pseudo reference since its current density is low and thus limits the potential variations around the rest potential. To this purpose, the [Fe(III)(CN)6]³â»/[Fe(II)(CN)6]4⁻ redox couple was used in order to set a reference potential at 0 V since both electrodes were platinum. The electrochemical microchip performance was characterized using differential pulse voltammetric (DPV) detection and quantification of the optically multi-labelled transthyretin synthetic peptide mimicking a tryptic fragment of interest for the diagnosis of familial transthyretin amyloidosis (ATTR). The limit of detection of the peptide by the working microelectrode was 25 nM, a value 100-fold lower than the one reported with conventional capillary electrophoresis coupled with laser-induced fluorescence under the same analytical conditions.

A real time affinity biosensor on an insulated polymer using electric impedance spectroscopy in dielectric microchips.

This paper presents development of real time monitoring of binding events on flexible plastic in microchips. Two planar carbon microelectrodes are integrated into an insulated polyethylene terephthalate microchip without direct electrical contact with the solution in the microchannel. It has been possible to probe the electric impedance changes through the interface constituted by the microelectrode/PET microchannel/solution when a biomolecular interaction takes place on the polymer surface. This new transduction for biosensing was demonstrated for the molecular recognition of BSA immobilized on the polymer microchannel surface using the corresponding rabbit anti-BSA antibodies as an analyte in the flow microchannel at the nanomolar range concentration. The equilibrium association constant was determined for the affinity reaction between both ligands and was obtained equal to 5 × 10(7) M(-1). The promising results obtained with this new device make it a competitive biosensor.

Investigating the kinetics of paramagnetic-beads linked alkaline phosphatase enzyme through microchannel resistance measurement in dielectric microchip.

Real time monitoring of electrolyte resistance changes during hydrolysis of 4-nitrophenylphosphate (pNPP) by alkaline phosphatase (ALP) bound on paramagnetic-beads was performed into a small dielectric channel. The reaction kinetic fit with a non-competitive substrate-inhibition equation. Michaelis-Menten apparent constant, KM(app), was determined as 0.33±0.06mM and the maximum apparent rate, Vmax(app) as 98±5pMs(-1). The detection limits were 15fM for ALP and 0.75mM for pNPP. This miniaturized device constitutes a powerful tool for analysis of interaction between ligands.

Dynamics of BSA adsorption onto a photoablated polymer surface in a dielectric microchip.

Dielectric impedance spectroscopy in a microchip was used for monitoring the adsorption of biomolecules onto a heterogeneous polymer surface obtained after the photoablation process. The sensor comprises a thin dielectric layer with two parallel carbon microband electrodes on the one side, and the photoablated surface on the other. The biomolecules need not to be labelled, as in an optical biosensor, even if they need to be attached to the polymer surface coupled with the microelectrodes when a biomolecular interaction occurs. Based on this principle, a flow sensor has been developed to record the adsorption dynamics illustrated with BSA coating on the heterogeneous surface in a linear dynamic range from 1 picomolar to 1 nanomolar at a fixed low frequency. Modeling the dielectric interface using an appropriate equivalent circuit permits extraction of the values of the interfacial impedance for ultralow protein concentrations. The promising results obtained with this methodology make it a competing method in comparison with optical or electrochemical transduction for biosensor development.

Investigating of labelling and detection of transthyretin synthetic peptide derivatized with naphthalene-2,3-dicarboxaldehyde.

Labelling and detection of a synthetic peptide (PN) mimicking a tryptic fragment of interest for the diagnosis of familial amyloidal polyneuropathy have been investigated optically and electrochemically. We decided to covalently label naphtalene-2,3-dicarboxyaldehyde (NDA), a fluorogenic and electroactive molecule on PN. First, the optimization of the labelling chemical reaction was performed by capillary electrophoresis coupled with laser induced fluorescence detection (CE-LIF). The analytical parameters such as separation efficiency and peak area were considered to propose this optimized derivatization reaction. The results obtained allowed us to establish the pH and ionic strength of the derivatization buffer, the molar ratio between NDA and PN and the reaction time of the labelling. Optimal conditions are obtained when [NDA]/[PN]=40, buffer pH of 9, buffer ionic strength of 70 mM and reaction time of 15 min. Second, differential pulse voltammetry (DPV) and cyclic voltammetry (CV) were also used to characterize NDA-labelled PN and different electroinactive amino acids (histidine, lysine, serine, threonine) which are in the PN sequence. The electrochemical detection experiments demonstrated that the labelled biomolecules could be also easily detected at low concentration. Moreover, the derivatization reaction could be followed to describe more precisely the labelling process of these biomolecules. Optimal conditions for labelling are obtained when [NDA]total/[CN(-)] ratio =1 and [NDA]total/[amino acid or peptide]=100 with a buffer having a pH=9 on a glassy carbon electrode. In all cases, an obvious oxidation peak for the N-2-substituted-1-cyanobenz-[f]-isoindole derivative (CBI) has been observed at 0.5-0.7 V/SCE. The multi-labelling of PN and lysine were shown with DPV. We presumed this result to occur because of the shouldered shape of the DPV peak shape. These experiments confirm that NDA can be used as a derivative agent for PN, allowing for electrochemical and fluorescence detections with a limit of detection of labelled PN estimated at 0.2 µM and 5 µM, respectively.

Rotating disk electrodes to assess river biofilm thickness and elasticity.

The present study examined the relevance of an electrochemical method based on a rotating disk electrode (RDE) to assess river biofilm thickness and elasticity. An in situ colonisation experiment in the River Garonne (France) in August 2009 sought to obtain natural river biofilms exhibiting differentiated architecture. A constricted pipe providing two contrasted flow conditions (about 0.1 and 0.45 m s(-1) in inflow and constricted sections respectively) and containing 24 RDE was immersed in the river for 21 days. Biofilm thickness and elasticity were quantified using an electrochemical assay on 7 and 21 days old RDE-grown biofilms (t(7) and t(21), respectively). Biofilm thickness was affected by colonisation length and flow conditions and ranged from 36 ± 15 µm (mean ± standard deviation, n = 6) in the fast flow section at t(7) to 340 ± 140 µm (n = 3) in the slow flow section at t(21). Comparing the electrochemical signal to stereomicroscopic estimates of biofilms thickness indicated that the method consistently allowed (i) to detect early biofilm colonisation in the river and (ii) to measure biofilm thickness of up to a few hundred µm. Biofilm elasticity, i.e. biofilm squeeze by hydrodynamic constraint, was significantly higher in the slow (1300 ± 480 µm rpm(1/2), n = 8) than in the fast flow sections (790 ± 350 µm rpm(1/2), n = 11). Diatom and bacterial density, and biofilm-covered RDE surface analyses (i) confirmed that microbial accrual resulted in biofilm formation on the RDE surface, and (ii) indicated that thickness and elasticity represent useful integrative parameters of biofilm architecture that could be measured on natural river assemblages using the proposed electrochemical method.

Polycarbonate microchannel network with carpet of gold nanowires as SERS-active device.

A polycarbonate (PC) microchannel network supporting gold nanowires was developed to be a SERS-active microchip. Observations of large increases in a Raman cross-section, allowed us to collect vibrational signatures which are not easily detectable by Raman techniques due to the high fluorescence level of bare PC. Compared to other SERS experiments, this study relies on the use of dielectric polymer/metal surfaces which are well defined at microscale and nanoscale levels. This device seems a promising tool for sensing the adsorption of biomolecules.

Nanomosaic network for the detection of proteins without direct electrical contact.

A nanomosaic network of metallic nanoparticles for the detection of ultralow concentrations of proteins is reported, which uses two planar microelectrodes embedded in a microchip that permit generation of capacitive coupling to the nanomosaic system without the need for direct electrical contact with the channel. By tailoring the microchannel surface using a sandwich configuration of polyethylene terephthalate/gold nanoparticles/poly(L-lysine), the surface charge can be modified following biomolecular interactions and monitored using a noncontact admittance technique. This nanodevice system behaves like a tunable capacitor and can be employed for the detection of any kind of molecule. The femtomolar detection of an anionic protein, such as beta-lactoglobulin in phosphate-buffered saline medium, is taken as an example.

Electroacoustic polymer microchip as an alternative to quartz crystal microbalance for biosensor development.

Laser photoablation of poly(ethylene terephthalate) (PET), a flexible dielectric organic polymer, was used to design an acoustic miniaturized DNA biosensor. The microchip device includes a 100-microm-thick PET layer, with two microband electrodes patterned in photoablated microchannels on one side and a depressed photoablated disk decorated by gold sputtered layer on the other side. Upon application of an electric signal between the two electrodes, an electroacoustic resonance phenomenon at approximately 30 MHz was established through the microelectrodes/PET/ gold layer interface. The electroacoustic resonance response was fitted with a series RLC motional arm in parallel with a static Co arm of a Buttlerworth-Van Dyke equivalent circuit: admittance spectra recorded after successive cycles of DNA hybridization on the gold surface showed reproducible changes on R, L, and C parameters. The same hybridizations runs were performed concomitantly on a 27-MHz (9 MHz, third overtone) quartz crystal microbalance in order to validate the PET device developed for bioanalysis applications. The electroacoustic PET device, approximately 100 times smaller than a microbalance quartz crystal, is interesting for the large-scale integration of acoustic sensors in biochips.