High electric field strength two-dimensional peptide separations using a microfluidic device

New instrumentation has been developed to improve the resolution, efficiency, and speed of microfluidic 2D separations using MEKC coupled to high field strength CE. Previously published 2D separation instrumentation [Ramsey, J. D. et al., Anal. Chem. 2003, 75, 3758-3764] from our group was limited to a maximum potential difference of 8.4 kV, resulting in an electric field strength of only approximately 200 V/cm in the first dimension. The circuit described in this report has been designed to couple a higher voltage supply with a rapidly switching, lower voltage supply to utilize the best features of each. Voltages applied in excess of 20 kV lead to high electric field strength separations in both dimensions, increasing the separation resolution, efficiency, and peak capacity while reducing the required analysis time. Detection rates as high as six peptides per second (based on total analysis time) were observed for a model protein tryptic digest separation. Additionally, higher applied voltages used in conjunction with microfluidic chips with longer length channels maintained higher electric field strengths and produced peak capacities of over 4000 for some separations. Total separation time in these longer channel devices was comparable to that obtained in short channels at low field strength; however, resolving power improved approximately threefold.     

Development of a gel monolithic column polydimethylsiloxane microfluidic device for rapid electrophoresis separation

A β-cyclodextrin (β-CD)-bonded gel monolithic column polydimethylsiloxane (PDMS) microfluidic device was developed in a simple and feasible way. Before preparation of gel monolithic column in PDMS microchannel, PDMS surface was activated by UV light to create silanol groups, which is an active molecule to covalently bond 3-(trimethoxysilyl)-propyl methacrylate (Bind-Silane) and seal microfluidic device. By the way, Bind-Silane is a bifunctional molecule to link polyacrylamide (PAA) gel and inner wall of PDMS microchannel covalently. Allyl-β-CD was used not only as a multifunctional crosslinker in PAA gel to control the size of the pores, but also as a chiral selector for the enantioseparation. The stability, transferring heat and optical characteristic of the microfluidic device were examined. The separation capability of the gel monolithic column was confirmed by the successful separation of fluorescein isothiocyanate (FITC)-labeled arginine (Arg), glutamine acid (Glu), tryptophan (Try), cysteine (Cysteine) and phenylalanine (Phe) in the PDMS microfluidic device less than 100 s at 36 mm effective separation length. A maximum of 2.06 × 10⁵ theoretical plates was obtained by the potential strength of 490 V/cm. A pair of FITC-labeled dansyl-d,l-threonine (Dns-Thr) was separated absolutely.     

A single-bead analysis on a disk-shaped microfluidic device using an antigen-immobilized bead.

A disk-shaped microfluidic device (lab-on-a-Disk) was developed to allow the evaluation of mental stress. As a standard sample, secretory immunoglobulin A (sIgA), which is a candidate marker of mental stress, was measured by a heterogeneous enzyme immunoassay (EIA) on the lab-on-a-Disk. Centrifugal force provided a microfluidic control on the lab-on-a-Disk. We examined the relationship between the rotational speed, the channel profile, and the position of the microfluidic chambers from the center of rotation to manipulate sample solutions into each reaction reservoir through microchannels sequentially, i.e., retain in a reservoir or flow into a subsequent reservoir. A single glass bead with immobilized sIgA on its surface was injected into a reservoir for a competitive antigen-antibody reaction, and applied to a specific surface in a heterogeneous assay. It is expected that the lab-on-a-Disk would be suitable for miniaturization and automation of the processes in EIA compared with a conventional EIA using a titer plate.     

Imaging of peptide adsorption to microfluidic channels in a plastic compact disc using a positron emitting radionuclide

A method for studying peptide-surface interactions within microfluidic channels by radionuclide imaging is described. With the high surface area-to-volume ratio of channels in miniaturised devices, combined with low amounts of analyte, non-specific peptide adsorption is a critical issue. The objective of the study was therefore to develop a method capable of direct detection of adsorbed peptide within microfluidic channels. A micro-device consisting of channels moulded in a plastic compact disc was chosen for the study, together with two selected peptides of different lengths and isoelectric point (pI) values. A bifunctional chelator, DOTA, was attached to the peptide by conjugation and labelled with the short-lived positron emitting radionuclide 68Ga. Quantitative images of radiotracer distribution within the microfluidic channels were obtained using a PhosphorImager system. The power of the method was demonstrated by the ability to clearly measure changes in adsorption when varying a number of parameters that typically affect peptide adsorption. These included surface modifications, analyte concentration, pH, and ionic strength. Additionally, two quantification methods were developed and compared. Radionuclide imaging also permitted visualisation of adsorption and release processes in microchannel chromatographic columns. The results suggest that radionuclide imaging is a suitable tool not only for the study of peptide adsorption to the microchannels presented in this study but also as a versatile tool to measure peptide-surface interactions in a wide variety of miniaturised structures and devices.     

Quantitative analysis of Caenorhabditis elegans chemotaxis using a microfluidic device

Caenorhabditis elegans, one of the widely studied model organisms, sense external chemical cues and perform relative chemotaxis behaviors through its simple chemosensory neuronal system. To study the mechanism underlying chemosensory behavior, a rapid and reliable method for quantitatively analyzing the worms' behaviors is essential. In this work, we demonstrated a microfluidic approach for investigating chemotaxis responses of worms to chemical gradients. The flow-based microfluidic chip was consisted of circular tree-like microchannels, which was able to generate eight flow streams containing stepwise chemical concentrations without the difference in flow velocity. Worms' upstream swimming into microchannels with various concentrations was monitored for quantitative analysis of the chemotaxis behavior. By using this microfluidic chip, the attractive and repellent responses of C. elegans to NaCl were successfully quantified within several minutes. The results demonstrated the wild type-like repellent responses and severely impaired attractive responses in grk-2 mutant animals with defects in calcium influx. In addition, the chemotaxis analysis of the third stage larvae revealed that its gustatory response was different from that in the adult stage. Thus, our microfluidic method provided a useful platform for studying the chemosensory behaviors of C. elegans and screening of chemosensation-related chemical drugs.     

Effects of separation length and voltage on isoelectric focusing in a plastic microfluidic device

This paper describes the investigation on the effects of separation length and voltage on IEF in a plastic microfluidic device. A LIF, whole-channel imaging detection (WCID) system was developed to monitor proteins while they were moving under an electric field. IEF was carried out in a separation medium consisting of carrier ampholytes and a mixture of linear polymers (hydroxyethylcellulose and hydroxypropylcellulose). We found that the IEF separation resolution is essentially independent of separation length when the same voltage is applied, which agrees with the theory. This result supports the notion that IEF in a microfabricated device leads to more rapid analysis without sacrificing the resolving power. A higher separation voltage also brought about more rapid analysis and superior separation resolution. IEF of two proteins (green fluorescence protein and R-phycoerythrin) was achieved in 1.5 min when 500 V was applied across a 1.9-cm channel. We found that a linear relationship exists between the focusing time and the inverse of the electrical field strength. In addition, we confirmed the phenomenon in which the pH gradient was compressed to the middle of a channel, and we found that the relative amount of the gradient compression decreased with the channel length.     

Label-free quantitation of peptide release from neurons in a microfluidic device with mass spectrometry imaging

Microfluidic technology allows the manipulation of mass-limited samples and when used with cultured cells, enables control of the extracellular microenvironment, making it well suited for studying neurons and their response to environmental perturbations. While matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) provides for off-line coupling to microfluidic devices for characterizing small-volume extracellular releasates, performing quantitative studies with MALDI is challenging. Here we describe a label-free absolute quantitation approach for microfluidic devices. We optimize device fabrication to prevent analyte losses before measurement and then incorporate a substrate that collects the analytes as they flow through a collection channel. Following collection, the channel is interrogated using MS imaging. Rather than quantifying the sample present via MS peak height, the length of the channel containing appreciable analyte signal is used as a measure of analyte amount. A linear relationship between peptide amount and band length is suggested by modeling the adsorption process and this relationship is validated using two neuropeptides, acidic peptide (AP) and α-bag cell peptide [1-9] (αBCP). The variance of length measurement, defined as the ratio of standard error to mean value, is as low as 3% between devices. The limit of detection (LOD) of our system is 600 fmol for AP and 400 fmol for αBCP. Using appropriate calibrations, we determined that an individual Aplysia bag cell neuron secretes 0.15 ± 0.03 pmol of AP and 0.13 ± 0.06 pmol of αBCP after being stimulated with elevated KCl. This quantitation approach is robust, does not require labeling, and is well suited for miniaturized off-line characterization from microfluidic devices.     

Development of a method for the direct analysis of peptide AM336 in monkey cerebrospinal fluid using liquid chromatography/electrospray ionization mass spectrometry with a mixed-function column

A liquid chromatography/mass spectrometry (LC/MS) analytical procedure, using a single column for sample clean-up, enrichment and separation, has been developed for the determination of the peptide AM336 in monkey cerebrospinal fluid (CSF). CSF samples were injected and analyzed using a polymer-coated mixed-function high-performance liquid chromatography (HPLC) column with gradient elution and application of a timed valve-switching event. The mass spectrometer was operated in the positive electrospray ionization (ESI(+)) mode with single ion recording (SIR) at m/z 920. The method was validated, yielding calibration curves with correlation coefficients greater than 0.9892. Assay precision and accuracy were evaluated by direct injection of AM336-fortified CSF samples at three concentration levels. Analyzed concentrations ranged from 99.93 to 113.1% of their respective theoretical concentrations with coefficients of variation below 9.0%. An evaluation of the signal-to-noise (S/N) ratio for a 200 ng/mL calibration standard, considered to be the lower limit of quantitation (LLOQ), resulted in an estimated limit of detection (LOD) of 31.2 ng/mL. Preliminary data suggest the possibility of using this method to analyze AM336 also in plasma samples, pending the successful outcome of additional investigations.     

Rapid screening of phenylketonuria using a CD microfluidic device

We herein present a compact disc (CD) microfluidic chip based hybridization assay for phenylketonuria (PKU) screening. This CD chip is composed of a polydimethylsiloxane (PDMS) top layer containing 12 DNA hybridization microchannels, and a glass bottom layer with hydrogel pad conjugated DNA oligonucleotides. Reciprocating flow was generated on the CD chip through a simple rotation-pause operation to facilitate rapid DNA hybridization. When rotated the CD chip, the sample solution was driven into the hybridization channel by centrifugal force. When stopped the CD chip, the sample plug was pulled backward through the channel by capillary force. The hybridization assay was firstly validated with control samples and was then used to analyze 30 clinical samples from pregnant women with suspected PKU fetus. The on-chip DNA hybridization was completed in 15 min with a sample consumption as low as 1.5μL, and the limit-of-detection (LOD) of DNA template was 0.7ng/μL. Among the 30 samples tested, V245V mutation was identified in 4 cases while R243Q mutation was detected in one case. Results of the hybridization assay were confirmed by DNA sequencing. This CD-chip based hybridization assay features short analysis time, simple operation and low cost, thus has the potential to serve as the tool for PKU screening.     

Spherical phospholipid polymer hydrogels for cell encapsulation prepared with a flow-focusing microfluidic channel device

To prepare spherical polymer hydrogels, we used a flow-focusing microfluidic channel device for mixing aqueous solutions of two water-soluble polymers. Continuous encapsulation of cells in the hydrogels was also examined. The polymers were bioinspired 2-methacryloyloxyethyl phosphorylcholine polymer bearing phenyl boronic acid groups (PMBV) and poly(vinyl alcohol) (PVA), which spontaneously form a hydrogel in aqueous medium via specific molecular complexation upon mixing, even when they were in cell culture medium. The microfluidic device was prepared with polydimethylsiloxan, and the surface of the channel was treated with fluoroalkyl compound to prevent sticking of the polymers on the surface. The microfluidic channel process could control the diameter of the spherical hydrogels in the range of 30-90 μm and generated highly monodispersed diameter spherical hydrogels. We found that the polymer distribution in the hydrogel was influenced by the PVA concentration and that the hydrogel could be dissociated by the addition of d-sorbitol to the suspension. The single cells could be encapsulated and remain viable in the hydrogels. The localized distribution of polymers in the hydrogel may provide an environment for modulating cell function. It is concluded that the spontaneous hydrogel formation between PMBV and PVA in the flow-focusing microfluidic channel device is applicable for continuous preparation of a spherical hydrogel-encapsulating living cell.     

Activity monitoring of functional OprM using a biomimetic microfluidic device

This paper describes the fabrication and use of a biomimetic microfluidic device for the monitoring of a functional porin reconstituted within a miniaturized suspended artificial bilayer lipid membrane (BLM). Such a microfluidic device allows for (1) fluidic and electrical access to both sides of the BLM and (2) reproducible membrane protein insertion and long-term electrical monitoring of its conductance (G(i)), thanks to the miniaturization of the BLM. We demonstrate here for the first time the feasibility to insert a large trans-membrane protein through its β-barrel, and monitor its functional activity for more than 1 hour (limited by buffer evaporation). In this paper, we specifically used our device for the monitoring of OprM, a bacterial efflux channel involved in the multidrug resistance of the bacteria Pseudomonas aeruginosa. Sub-steps of the OprM channel conductance were detected during the electrical recordings within our device, which might be due to oscillations between several structural conformations (sub-states) adopted by the protein, as part of its opening mechanism. This work is a first step towards the establishment of a genuine platform dedicated to the investigation of bacterial proteins under reconstituted conditions, a very promising tool for the screening of new inhibitors against bacterial channels involved in drug resistance.     

Characterization of cell lysis events on a microfluidic device for high-throughput single cell analysis

A microfluidic device capable of rapidly analyzing cells in a high-throughput manner using electrical cell lysis is further characterized. In the experiments performed, cell lysis events were studied using an electron multiplying charge coupled device camera with high frame rate (>100 fps) data collection. It was found that, with this microfluidic design, the path that a cell follows through the electric field affects the amount of lysate injected into the analysis channel. Elimination of variable flow paths through the electric field was achieved by coating the analysis channel with a polyamine compound to reverse the electroosmotic flow (EOF). EOF reversal forced the cells to take the same path through the electric field. The improved control of the cell trajectory will reduce device-imposed bias on the analysis and maximizes the amount of lysate injected into the analysis channel for each cell, resulting in improved analyte detection capabilities.     

Aptamer-based fluorometric determination of norovirus using a paper-based microfluidic device

Microchimica Acta - The authors describe a rapid and highly sensitive point-of-care device for rapid determination of noroviruses, a leading cause of acute gastroenteritis. The assay is based on...     

Spreadsheet analysis of the field-driven start-up flow in a microfluidic channel

We discuss, in this article, the solution method of the unsteady electroosmotic flow of Newtonian fluid in a square microfluidic channel cross-section in the framework of spreadsheet analysis. We demonstrate the implementation of the finite difference scheme, which is used for the discretization of the transport equations governing the flow dynamics of the present problem, in the spreadsheet tool. Also, we have shown the implementation details of different boundary conditions, which are typically used for the underlying electrohydrodynamics in a microfluidic channel, in the spreadsheet analysis tool. We show that the results obtained from the spreadsheet analysis match accurately with the numerical solutions for both the electrostatic potential distribution and the flow velocity. Our results of this analysis justify the credibility of the spreadsheet tool for capturing the intricate details of the electrically actuated microflows during the initial transiences, that is, for the start-up flows and the phenomenon due to the electrical double layer effect, quite effectively. The inferences of this analysis will open up a new research paradigm of microfluidics and microscale transport processes by providing the potential applicability of the spreadsheet tools to obtain the flow physics of our interest in a very intuitive and less expensive manner.     

Novel microfluidic device for the continuous separation of cancer cells using dielectrophoresis

We describe the design, microfabrication, and testing of a microfluidic device for the separation of cancer cells based on dielectrophoresis. Cancer cells, specifically green fluorescent protein‐labeled MDA‐MB‐231, are successfully separated from a heterogeneous mixture of the same and normal blood cells. MDA‐MB‐231 cancer cells are separated with an accuracy that enables precise detection and counting of circulating tumor cells present among normal blood cells. The separation is performed using a set of planar interdigitated transducer electrodes that are deposited on the surface of a glass wafer and slightly protrude into the separation microchannel at one side. The device includes two parts, namely, a glass wafer and polydimethylsiloxane element. The device is fabricated using standard microfabrication techniques. All experiments are conducted with low conductivity sucrose‐dextrose isotonic medium. The variation in response between MDA‐MB‐231 cancer cells and normal cells to a certain band of alternating‐current frequencies is used for continuous separation of cells. The fabrication of the microfluidic device, preparation of cells and medium, and flow conditions are detailed. The proposed microdevice can be used to detect and separate malignant cells from heterogeneous mixture of cells for the purpose of early screening for cancer.     

Simple 2D-HPLC using a monolithic silica column for peptide separation

Separation of peptides by fast and simple two-dimensional (2D)-HPLC was studied using a monolithic silica column as a second-dimension (2nd-D) column. Every fraction from the first column, 5 cm long (2.1 mm ID) packed with polymer-based cation exchange beads, was subjected to separation in the 2nd-D using an octadecylsilylated (C18) monolithic sillica column (4.6 mm ID, 2.5 cm). A capillary-type monolithic silica C18column (0.1 mm ID, 10 cm) was also employed as a 2nd-D column with split flow/injection. Effluentof the first dimension (1st-D) was directly loaded into an injector loop of 2nd-D HPLC. UV and MS detection were successfully carried out at high linear velocity of mobile phase at 2nd-D using flow splitting for the 4.6 mm ID 2nd-D column, or with directconnection of the capillary column to the MS interface. Two-minute fractionation inthe 1st-D, 118-second loading, and 2-second injection by the 2nd-D injector, allowed one minute for gradient separation in the 2nd-D, resulting in a maximum peak capacity of about 700 within 40 min. The use of a capillary column in solvent consumption and better MS detectability compared to a larger-sized column. This kind of fast and simple 2D-HPLC utilizing monolithic silica columns will be useful for the separation of complex mixtures in a short time.     

Colorimetric determination of acidity constant using a paper-based microfluidic analytical device

Acid dissociation constant is an important chemical characteristic of organic and inorganic compounds and it affects both chemical properties and biological activities of the molecules. Herein, a very simple, fast and cost-effective method based on microfluidic technology has been reported for colorimetric determination of acidity constants. The designed device works based on pH-metric titration of colorful indicators followed by colorimetric measurements by a smart phone mobile device or a flatbed scanner. So, it does not need sophisticated instrumentation and is accomplished in a very short time (about 1 min). All titration steps are transferred on a star-like designed µPAD device: (1) spotting 0.3 µL buffers of different pHs at the end of the channels (reaction zones), (2) spotting a 30 µL portion of the indicator on the center of µPAD followed by movement of indicator solution toward the reaction zone by capillary action of the paper. The measured color change of the indicators at the reaction zone is fitted to the Henderson–Hasselbalch equation, through which acidity constants are calculated. The performance of the device was evaluated by measuring acidity constant of 4 indicators including bromothymol blue, bromocresol green, bromocresol purple and phenolphthalein. A very close agreement was achieved between those measured by the suggested device here and the previously reported values. The reproducibility of this method was lower than 5% for relative standard deviation of three replicate measurements.     

Real time culture and analysis of embryo metabolism using a microfluidic device with deformation based actuation

We report a computerized microfluidic real time embryo culture and assay device that can perform automated periodic analyses of embryo metabolism. This automated program uses a modified "gated injection" scheme (sample injection, reagent mixing, enzyme reaction of 15 min incubation, and sample detection) to sequentially measure fluorescence from sample, reference, and background (without any analyte) every hour. Measurements assessed with reference solutions demonstrated the stability of these microfluidic measurements over a 24 h period. Furthermore, this system was able to measure time dependent nutrient consumption by single or multiple (10) live mouse blastocyst-stage embryos with pmol h(-1) sensitivity. Mechanical deformation-based microfluidic actuation created by computerized movement of Braille pins enables automated fluid pumping and valving sequences without unwanted gravity-driven backflow or exposure to electrical fields as would be required in electrokinetic schemes. The convenient, non-invasive, and automated nature of these assays open the way for the development of integrated microfluidic platforms for practical single embryo culture and real time biochemical analysis to assess embryo viability and select embryos with the greatest implantation potential, thus improving success in clinical assisted reproductive technology laboratories.     

Ultra-sensitive trace analysis of cyanide water pollutant in a PDMS microfluidic channel using surface-enhanced Raman spectroscopy

Rapid and highly sensitive trace analysis of cyanide water pollutant in an alligator teeth-shaped PDMS microfluidic channel was investigated using surface-enhanced Raman spectroscopy. Compared with previously reported analytical methods, the detection sensitivity was enhanced by several orders of magnitude.     

Classification of cell types using a microfluidic device for mechanical and electrical measurement on single cells

This paper presents a microfluidic system for cell type classification using mechanical and electrical measurements on single cells. Cells are aspirated continuously through a constriction channel with cell elongations and impedance profiles measured simultaneously. The cell transit time through the constriction channel and the impedance amplitude ratio are quantified as cell's mechanical and electrical property indicators. The microfluidic device and measurement system were used to characterize osteoblasts (n=206) and osteocytes (n=217), revealing that osteoblasts, compared with osteocytes, have a larger cell elongation length (64.51 ± 14.98 μm vs. 39.78 ± 7.16 μm), a longer transit time (1.84 ± 1.48 s vs. 0.94 ± 1.07 s), and a higher impedance amplitude ratio (1.198 ± 0.071 vs. 1.099 ± 0.038). Pattern recognition using the neural network was applied to cell type classification, resulting in classification success rates of 69.8% (transit time alone), 85.3% (impedance amplitude ratio alone), and 93.7% (both transit time and impedance amplitude ratio as input to neural network) for osteoblasts and osteocytes. The system was also applied to test EMT6 (n=747) and EMT6/AR1.0 cells (n=770, EMT6 treated by doxorubicin) that have a comparable size distribution (cell elongation length: 51.47 ± 11.33 μm vs. 50.09 ± 9.70 μm). The effects of cell size on transit time and impedance amplitude ratio were investigated. Cell classification success rates were 51.3% (cell elongation alone), 57.5% (transit time alone), 59.6% (impedance amplitude ratio alone), and 70.2% (both transit time and impedance amplitude ratio). These preliminary results suggest that biomechanical and bioelectrical parameters, when used in combination, could provide a higher cell classification success rate than using electrical or mechanical parameter alone.