Microfluidic Volumes Research Articles

Microfluidic volume optical monitoring system based on functional channels integrated by hollow cylindrical waveguide

Real-time monitoring and manipulating various liquids in the microflow channels have great potentials in micro total analysis system. A novel microfluidics volume optical monitoring system (MVOMS) is proposed. Functional channels embedded with hollow cylindrical waveguide (HCW) are integrated in MVOMS, which is convenient to realize the real-time monitoring and manipulating microfluidic. The microfluidic volume can be monitored in real time and the sensitivity can reach 20.0 dB/μL. In addition, the MVOMS can realize the monitoring of various liquid for the designed sensor structure is insensitive to the refractive index of liquid. The temperature cross and the pressure cross are tested that can be ignored. Microfluidic volume monitoring, delivering and mixing can be achieved in MVOMS. On this basis, we design an integrated light-driven microfluidic quantitative sampling system, which can sample a specific volume of microfluidic in microchips automatically and accurately.

High-Throughput and Automated Acoustic Trapping of Extracellular Vesicles to Identify microRNAs With Diagnostic Potential for Prostate Cancer.

Molecular profiling of extracellular vesicles (EVs) offers novel opportunities for diagnostic applications, but the current major obstacle for clinical translation is the lack of efficient, robust, and reproducible isolation methods. To bridge that gap, we developed a microfluidic, non-contact, and low-input volume compatible acoustic trapping technology for EV isolation that enabled downstream small RNA sequencing. In the current study, we have further automated the acoustic microfluidics-based EV enrichment technique that enables us to serially process 32 clinical samples per run. We utilized the system to enrich EVs from urine collected as the first morning void from 207 men referred to 10-core prostate biopsy performed the same day. Using automated acoustic trapping, we successfully enriched EVs from 199/207 samples (96%). After RNA extraction, size selection, and library preparation, a total of 173/199 samples (87%) provided sufficient materials for next-generation sequencing that generated an average of 2 × 106 reads per sample mapping to the human reference genome. The predominant RNA species identified were fragments of long RNAs such as protein coding and retained introns, whereas small RNAs such as microRNAs (miRNA) accounted for less than 1% of the reads suggesting that partially degraded long RNAs out-competed miRNAs during sequencing. We found that the expression of six miRNAs was significantly different (Padj < 0.05) in EVs isolated from patients found to have high grade prostate cancer [ISUP 2005 Grade Group (GG) 4 or higher] compared to those with GG3 or lower, including those with no evidence of prostate cancer at biopsy. These included miR-23b-3p, miR-27a-3p, and miR-27b-3p showing higher expression in patients with GG4 or high grade prostate cancer, whereas miR-1-3p, miR-10a-5p, and miR-423-3p had lower expression in the GG4 PCa cases. Cross referencing our differentially expressed miRNAs to two large prostate cancer datasets revealed that the putative tumor suppressors miR-1, miR-23b, and miR-27a are consistently deregulated in prostate cancer. Taken together, this is the first time that our automated microfluidic EV enrichment technique has been found to be capable of enriching EVs on a large scale from 900 μl of urine for small RNA sequencing in a robust and disease discriminatory manner.

A fast technique to measure the dewpoint pressure of a retrograde condensate gas using a microfluidic volume.

A new method to determine the dewpoint pressure of a retrograde condensate from a fast, non-equilibrium measurement performed in a microfluidic optical cell is presented. The inflection point of the optical transmission recorded during depressurization agrees well with the dewpoint pressure of the sample, determined by conventional laboratory techniques. With this new technique, a measurement can be performed in less than 5 min and requires far less than a milliliter of the sample. Benchmarking of this technique is presented using four retrograde condensate samples, which were created in the laboratory using multi-component compositions that are based on oilfield samples. Each sample was characterized at three different temperatures, and their maximum relative liquid volumes (maximum liquid volume/total system volume at the dewpoint pressure) ranged from 1.3% to 13.5% for these temperatures. The dewpoint pressure measured by this technique differs by no more than 100 psi from that measured in a conventional laboratory for samples of a richness of 4% or higher, while leaner samples display a difference of ∼200 psi.

Novel PDMS-Based Sensor System for MPWM Measurements of Picoliter Volumes in Microfluidic Devices.

In order for automatic microinjection to serve biomedical and genetic research, we have designed and manufactured a PDMS-based sensor with a circular section channel using the microwire molding technique. For the very precise control of microfluidic transport, we developed a microfluidic pulse width modulation system (MPWM) for automatic microinjections at a picoliter level. By adding a computer-aided detection and tracking of fluid-specific elements in the microfluidic circuit, the PDMS microchannel sensor became the basic element in the automatic control of the microinjection sensor. With the PDMS microinjection sensor, we precise measured microfluidic volumes under visual detection, assisted by very precise computer equipment (with precision below 1 μm) based on image processing. The calibration of the MPWM system was performed to increase the reproducibility of the results and to detect and measure microfluidic volumes. The novel PDMS-based sensor system for MPWM measurements of microfluidic volumes contributes to the advancement of intelligent control methods and techniques, which could lead to new developments in the design, control, and in applications of real-time intelligent sensor system control.

Design and analysis of microfluidic cell counter using spice simulation

Microfluidic cytometers based on Coulter principle have recently shown a great potential for point of care biosensors for medical diagnostics. In this study, the design and characterization of Coulter-based microfluidic cytometer are investigated through electrical circuit simulations considering an equivalent electrical model for the biological cell. We explore the effects related to microelectrode dimensions, microfluidic detection volume, suspension medium, size/morphology of the target cells, and the impedance of the external readout circuit, on the output response of the sensor. We show that the effect of microelectrodes’ surface area and the dielectric properties of the suspension medium should be carefully considered when characterizing the output response of the sensor. In particular, the area of the microelectrodes can have a significant effect on cell’s electrical opacity (the ratio of cell impedance at high to low frequency) which is commonly used to distinguish between subpopulations of the target cells (e.g., lymphocytes vs. monocytes when counting white blood cells). Moreover, we highlight that the opacity response versus frequency can significantly vary based upon whether the absolute cell impedance or the differential output impedance is used in its calculation. These insights could provide valuable guidelines for the design and characterization of Coulter-based microfluidic sensors.

Measuring Single-Cell Phenotypic Growth Heterogeneity Using a Microfluidic Cell Volume Sensor

An imaging-integrated microfluidic cell volume sensor was used to evaluate the volumetric growth rate of single cells from a Saccharomyces cerevisiae population exhibiting two phenotypic expression states of the PDR5 gene. This gene grants multidrug resistance by transcribing a membrane transporter capable of pumping out cytotoxic compounds from the cell. Utilizing fluorescent markers, single cells were isolated and trapped, then their growth rates were measured in two on-chip environments: rich media and media dosed with the antibiotic cycloheximide. Approximating growth rates to first-order, we assessed the fitness of individual cells and found that those with low PDR5 expression had higher fitness in rich media whereas cells with high PDR5 expression had higher fitness in the presence of the drug. Moreover, the drug dramatically reduced the fitness of cells with low PDR5 expression but had comparatively minimal impact on the fitness of cells with high PDR5 expression. Our experiments show the utility of this imaging-integrated microfluidic cell volume sensor for high-resolution, single-cell analysis, as well as its potential application for studies that characterize and compare the fitness and morphology of individual cells from heterogeneous populations under different growth conditions.

An Assay System for Point-of-Care Diagnosis of Tuberculosis using Commercially Manufactured PCB Technology

Rapid advances in clinical technologies, detection sensitivity and analytical throughput have delivered a significant expansion in our knowledge of prognostic and diagnostic biomarkers in many common infectious diseases, such as Tuberculosis (TB). During the last decade, a significant number of approaches to TB diagnosis have been attempted at Point-of-Care (PoC), exploiting a large variation of techniques and materials. In this work, we describe an electronics-based Enzyme-Linked ImmunoSorbent Assay (eELISA), using a Lab-on-a-Printed Circuit Board (LoPCB) approach, for TB diagnosis based on cytokine detection. The test relies upon an electrochemical (amperometric) assay, comprising a high-precision bioinstrumentation board and amperometric sensors, produced exclusively using standard PCB manufacturing processes. Electrochemical detection uses standard Au and Ag electrodes together with a bespoke, low-power, multichannel, portable data-acquisition system. We demonstrate high-performance assay chemistry performed at microfluidic volumes on Au pads directly at the PCB surface with improved limit of detection (~10 pg/mL) over standard colorimetric ELISA methods. The assay has also been implemented in plasma, showing the utility of the system for medical applications. This work is a significant step towards the development of a low-cost, portable, high-precision diagnostic and monitoring technology, which once combined with appropriate PCB-based microfluidic networks will provide complete LoPCB platforms.

Micro Device for Bio-Particle Positioning in a Confined Microfluidic Volume Using Carbon MEMS and Dielectrophoretic Forces

Experiments on micron sized bio particles (bio compatible micro particles, cells) are limited by the manipulation techniques used to position particles in a zone of interest, like the focus point of an optical system, a micro electrode for an electrical measurement, a tridimensional cellular scaffold or a reaction chamber for a chemical process. We introduce a device that would aid in the precise positioning of micron sized bio particles inside a microfluidic channel, using a combination of individually addressable 3D electrodes and planar electrodes based on carbon micro electro mechanical systems (C-MEMS) to induce dielectrophoretic (DEP) forces 1,2. The proposed device uses a series of carbon-based 3D electrodes and a matrix of planar electrodes produced by the pyrolysis of a common photoresist (SU-8) 3. The electrodes are built on a fused silica substrate to enable the view of experiments trough optical microscopy. Our hypothesis consist in that by using the electrodes to exert a pulsed DEP force, coplanar to the substrate by the 3D electrodes and orthogonal to the substrate by the planar electrodes, it is possible to precisely position a bio particle in a confined microfluidic volume at low flow rates 4. These types of electrodes are chosen because they can induce a DEP force by applying small electric potentials (~ 10 V) due to the fact that are immersed in the carrying fluid. Pulsed DEP was selected due to its low cost implementation and because it is able to affect bio particles positions while maintaining their viability 5. The manufacturing process requires the use of traditional techniques of photolithography to 3D pattern photoresist and a carefully tailored pyrolysis process to obtain the desired electrode features 6. In order to validate the device, the resistivity of the carbon films is measured, cyclic voltammetry is used to characterize the electrochemical behavior of the electrodes, electrode half-cell potential stability is tested over time, particle image velocimetry is used to analyze the flow inside the micro channel and the spatial position of a target particle is monitored using optical microscopy while it is manipulated using DEP force 7. Finally, these results are compared with those obtained in the simulation and theoretical design of the proposed micro-platform.

Tunable electrochemical pH modulation in a microchannel monitored via the proton-coupled electro-oxidation of hydroquinone.

Electrochemistry is a promising tool for microfluidic systems because it is relatively inexpensive, structures are simple to fabricate, and it is straight-forward to interface electronically. While most widely used in microfluidics for chemical detection or as the transduction mechanism for molecular probes, electrochemical methods can also be used to efficiently alter the chemical composition of small (typically <100 nl) microfluidic volumes in a manner that improves or enables subsequent measurements and sample processing steps. Here, solvent (H2O) electrolysis is performed quantitatively at a microchannel Pt band electrode to increase microchannel pH. The change in microchannel pH is simultaneously tracked at a downstream electrode by monitoring changes in the i-V characteristics of the proton-coupled electro-oxidation of hydroquinone, thus providing real-time measurement of the protonated forms of hydroquinone from which the pH can be determined in a straightforward manner. Relative peak heights for protonated and deprotonated hydroquinone forms are in good agreement with expected pH changes by measured electrolysis rates, demonstrating that solvent electrolysis can be used to provide tunable, quantitative pH control within a microchannel.

Quantifying the volume of single cells continuously using a microfluidic pressure-driven trap with media exchange

We demonstrate a microfluidic device capable of tracking the volume of individual cells by integrating an on-chip volume sensor with pressure-activated cell trapping capabilities. The device creates a dynamic trap by operating in feedback; a cell is periodically redirected back and forth through a microfluidic volume sensor (Coulter principle). Sieve valves are positioned on both ends of the sensing channel, creating a physical barrier which enables media to be quickly exchanged while keeping a cell firmly in place. The volume of individual Saccharomyces cerevisiae cells was tracked over entire growth cycles, and the ability to quickly exchange media was demonstrated.

Resonant light scattering spectroscopy of gold, silver and gold–silver alloy nanoparticles and optical detection in microfluidic channels

Dark field resonant light scattering by gold and silver nanoparticles enables the detection and spectroscopy of such particles with high sensitivity, down to the single-particle level, and can be used to implement miniaturised optical detection schemes for chemical and biological analysis. Here, we present a straightforward optical spectroscopic methodology for the quantitative spectrometric study of resonant light scattering (RLS) by nanoparticles. RLS spectroscopy is complementary to UV-visible absorbance measurements, and we apply it to the characterisation and comparison of different types of gold, silver and gold-silver alloy nanoparticles. The potential of gold and silver particles as alternatives for fluorescent probes in certain applications is discussed. RLS spectroscopy is shown to be useful for studying analyte-induced gold nanoparticle assembly and nanoparticle chemistry, which can induce radical changes in the plasmonic resonances responsible for the strong light scattering. Furthermore, the feasibility of dark field RLS detection and quantitation of metal nanoparticles in microfluidic volumes is demonstrated, opening interesting possibilities for the further development of microfluidic detection schemes.

The third decade of microfluidics

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Selective particle and cell clustering at air–liquid interfaces within ultrasonic microfluidic systems

In this study, we demonstrate particle and cell clustering in distinct patterns on the free surface of microfluidic volumes. Employing ultrasonic actuation, submersed microparticles are forced to two principal positions: nodal lines (pressure minima) of a standing wave within the liquid bulk, and distinct locations on the air–liquid interface (free surface); the latter of which has not been previously demonstrated using ultrasonic standing waves. As such, we unravel the fundamental mechanisms behind such patterns, showing that the contribution of fluid particle velocity variations on the free surface (acoustic radiation force) results in patterned particle clustering. In addition, by varying the size and density of the microparticles (3.5–31 μm polystyrene and 1–5 μm silica), acoustic streaming is found to increase the tendency for a smaller and lighter particle to cluster at the air–liquid interface. This selectivity is exploited for the isolation of multiple microparticle and cell types on the free surface from their nodally aligned counterparts. Free surface clustering is demonstrated in both an open microfluidic chamber and a sessile droplet, as well as using a range of biological species Escherichia coli, blood cells, Ragweed pollen and Paper Mulberry pollen). The ability to selectively cluster submersed microparticles and cells in distinct patterns on the free surface showcases the excellent suitability of this method to lab-on-a-chip systems.

Using the fringing electric field in microfluidic volume sensors to enhance sensitivity and accuracy

The particle trajectory above impedance-monitoring coplanar electrodes in a microfluidic channel dramatically influences the measured electric current change. We use finite element modeling to predict changes in ionic current for microspheres flowing in highly fringing fields, and validate these results by introducing a buoyancy-based particle focusing technique. Using 6 μm polystyrene particles in solutions of varying density, we control the height of the particle trajectories near the sensing electrodes and show that sensitivity can be increased by up to 3.5× when particles flow close to the electrodes compared to particles flowing further away, while simultaneously improving accuracy.

Microfluidic cell volume sensor with tunable sensitivity

We report the fabrication and validation of a microfluidic cell volume sensor integrated on a multi-layered polydimethylsiloxane (PDMS) microchip with a tunable detection volume for dynamic control of sensitivity, enabling the detection of individual Escherichia coli and microparticles.

Selective particle trapping using an oscillating microbubble

The ability to isolate and sort analytes within complex microfluidic volumes is essential to the success of lab-on-a-chip (LOC) devices. In this study, acoustically-excited oscillating bubbles are used to selectively trap particles, with the selectivity being a function of both particle size and density. The operating principle is based on the interplay between the strong microstreaming-induced drag force and the attractive secondary Bjerknes force. Depending upon the size of the bubble, and thus its resonant frequency, it is possible to cause one force to dominate over the other, resulting in either particle attraction or repulsion. A theoretical analysis reveals the extent of the contribution of each force for a given particle size; in close agreement with experimental findings. Density-based trapping is also demonstrated, highlighting that denser particles experience a larger secondary Bjerknes force resulting in their attraction. This study showcases the excellent applicability and versatility of using oscillating bubbles as a trapping and sorting mechanism within LOC devices.

Solid phase DNA extraction on PDMS and direct amplification

In this paper we report an innovative use of Poly(DiMethyl)Siloxane (PDMS) to design a microfluidic device that combines, for the first time, in one single reaction chamber, DNA purification from a complex biological sample (blood) without elution and PCR without surface passivation agents. This result is achieved by exploiting the spontaneous chemical structure and nanomorphology of the material after casting. The observed surface organization leads to spontaneous DNA adsorption. This property allows on-chip complete protocols of purification of complex biological samples to be performed directly, starting from cells lysis. Amplification by PCR is performed directly on the adsorbed DNA, avoiding the elution process that is normally required by DNA purification protocols. The use of one single microfluidic volume for both DNA purification and amplification dramatically simplifies the structure of microfluidic devices for DNA preparation. X-Ray Photoelectron Spectroscopy (XPS) was used to analyze the surface chemical composition. Atomic Force Microscopy (AFM) and Field Emission Scanning Electron Microscopy (FESEM) were employed to assess the morphological nanostructure of the PDMS-chips. A confocal fluorescence analysis was utilized to check DNA distribution inside the chip.

Development of a magnetic lab-on-a-chip for point-of-care sepsis diagnosis

We present design criteria, operation principles and experimental examples of magnetic marker manipulation for our magnetic lab-on-a-chip prototype. It incorporates both magnetic sample preparation and detection by embedded GMR-type magnetoresistive sensors and is optimized for the automated point-of-care detection of four different sepsis-indicative cytokines directly from about 5 μl of whole blood. The sample volume, magnetic particle size and cytokine concentration determine the microfluidic volume, sensor size and dimensioning of the magnetic gradient field generators. By optimizing these parameters to the specific diagnostic task, best performance is expected with respect to sensitivity, analysis time and reproducibility.

Contribution of Aquaporins to Cellular Water Transport Observed by a Microfluidic Cell Volume Sensor

Here we demonstrate that an impedance-based microfluidic cell volume sensor can be used to study the roles of aquaporin (AQP) in cellular water permeability and screen AQP-specific drugs. Human embryonic kidney (HEK-293) cells were transiently transfected with AQP3- or AQP4-encoding genes to express AQPs in plasma membranes. The swelling of cells in response to hypotonic stimulation was traced in real time using the sensor. Two time constants were obtained by fitting the swelling curves with a two-exponential function, a fast time constant associated with osmotic water permeability of AQP-expressing cells and a slow phase time constant associated mainly with water diffusion through lipid bilayers in the nontransfected cells. The AQP-expressing cells showed at least 10× faster osmotic water transport than control cells. Using the volume sensor, we examined the effects of Hg2+ and Ni2+ on the water transport via AQPs. Hg2+ inhibited the water flux in AQP3-expressing cells irreversibly, while Ni2+ blocked the AQP3 channels reversibly. Neither of the two ions blocked the AQP4 channels. The microfluidic volume sensor can sense changes in cell volume in real time, which enables perfusion of various reagents sequentially. It provides a convenient tool for studying the effect of reagents on the function and regulation mechanism of AQPs.

Dynamic Effects of Hg2+-induced Changes in Cell Volume

Using a microfluidic volume sensor, we studied the dynamic effects of Hg2+ on hypotonic stress-induced volume changes in CHO cells. A hypotonic challenge to control cells caused them to swell but did not evoke a significant regulatory volume decrease (RVD). Treatment with 100 muM HgCl2 caused a substantial increase in the steady-state volume following osmotic stress. Continuous hypotonic challenge following a single 10-min exposure to HgCl2 produced a biphasic volume increase with a steady-state volume 100% larger than control cells. Repeated hypotonic challenges to cells exposed once to Hg2+ resulted in a sequential approach to the same steady-state volume. Stimulation after reaching steady state caused a reduction in peak cell volume. Repeated stimulation was different than continuous stimulation resulting in a more rapid approach to steady state. Substituting extracellular Na+ with impermeant NMDG+ in the hypotonic solution produced a rapid RVD-like volume decrease and eliminated the Hg2+-induced excess swelling. The volume decrease in the presence of Hg2+ was inhibited by tetraethylammonium and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid disodium, blockers of K+ and Cl(-) channels, respectively, suggesting that part of the Hg2+ effect was increasing NaCl influx over KCl efflux. The presence of multiple phases of steady-state volume and their sensitivity to the stimulation history suggests that factors beyond solute fluxes, such as modification of mechanical stress within the cytoskeleton also plays a role in the response to hypotonic stress.