A simple and reliable new microchip electrophoresis method for fast measurements of imidazole dipeptides in meat from different animal species.

Microchip electrophoresis (ME) was applied for the separation of two physiologically important imidazole dipeptides-carnosine and anserine. The capacitively coupled contactless conductivity detector (C4D) was employed for quantification of both dipeptides after separation in a new home-built ME unit. The separation parameters were optimized as follows to enable quantitative, baseline separation of both dipeptides: injection time 16 s, injection voltage 900 V/cm, and separation voltage 377.1 V/cm. The C4D detector responded linearly to both imidazole dipeptides in the range 0-20 mg L-1. The known addition methodology was applied to test the accuracy of the measurement of imidazole dipeptides in a complex sample. The recoveries for measurement of carnosine in the mixture ranged from 96.1 to 105.0%, whereas those for anserine amounted to 96.6 to 102.0%. This method was also applied to real biological samples. The results exhibited a satisfactory agreement with a standard HPLC method. The proposed ME method represents a cheap, fast, and simple alternative to the existing, more complicated and expensive HPLC methods. This method does not demand either the optical detectors nor tedious derivatization of sample, which are unavoidable in HPLC methods. The method was succesfuly applied for animal species determination in unknown meat samples using the carnosine/anserine ratio, and subsequently, it could be used in a food fraud prevention process. Graphical abstract Microchip electrophoresis portable device with a C4D detector for determination of imidazole dipeptides in model samples and real meat samples from different animal species.

The pressing need for point-of-care diagnostics for sickle cell disease: A review of current and future technologies.

Sickle cell disease (SCD) is a common and life threatening inherited blood disorder, affecting over 300,000 newborns per year. Over 75% of SCD births occur in sub-Saharan Africa, where the lack of timely and accurate diagnosis results in premature death within the first few years of life for a majority of affected infants. Current methods to diagnosis SCD require expensive laboratory equipment and reagents, and adequately trained laboratory personnel. In addition, test results are often delayed due to transport and batching of samples in a central laboratory. Financial and technical limitations often preclude any form of SCD laboratory testing at the local level in regions where SCD is most prevalent. There has been a recent surge of interest in addressing the global burden of SCD, including improving and optimizing diagnostic capacities. Largely stimulated by a funding opportunity from the NIH, several point-of-care diagnostics have been developed for SCD with a focus on developing devices that are inexpensive, simple, and practical in limited resource settings. In this manuscript, we review the global burden of SCA, including the rationale for the development of POC assays, and carefully review the POC devices currently in development.

Rapid Isolation and Detection of Exosomes and Associated Biomarkers from Plasma.

Exosomes found in the circulation are a primary source of important cancer-related RNA and protein biomarkers that are expected to lead to early detection, liquid biopsy, and point-of-care diagnostic applications. Unfortunately, due to their small size (50-150 nm) and low density, exosomes are extremely difficult to isolate from plasma. Current isolation methods are time-consuming multistep procedures that are unlikely to translate into diagnostic applications. To address this issue, we demonstrate the ability of an alternating current electrokinetic (ACE) microarray chip device to rapidly isolate and recover glioblastoma exosomes from undiluted human plasma samples. The ACE device requires a small plasma sample (30-50 µL) and is able to concentrate the exosomes into high-field regions around the ACE microelectrodes within 15 min. A simple buffer wash removes bulk plasma materials, leaving the exosomes concentrated on the microelectrodes. The entire isolation process and on-chip fluorescence analysis is completed in less than 30 min which enables subsequent on-chip immunofluorescence detection of exosomal proteins, and provides viable mRNA for RT-PCR analysis. These results demonstrate the ability of the ACE device to streamline the process for isolation and recovery of exosomes, significantly reducing the number of processing steps and time required.

Fast high-throughput screening of angiotensin-converting enzyme insertion/deletion polymorphism by variable programmed electric field strength-based microchip electrophoresis.

An insertion (I)/deletion (D) polymorphism in angiotensin-converting enzyme (ACE) has been associated with susceptibility to various diseases in numerous studies. Traditionally, slab gel electrophoresis (SGE) after polymerase chain reaction (PCR) has been used to genotype this ACE I/D polymorphism. In this study, single- and multi-channel microchip electrophoresis (ME) methods based on variable programmed electric field strength (PEFS) (i.e., low constant, high constant, (+)/(-) staircase, and random electric field strengths) were developed for fast high-throughput screening of this specific polymorphism. The optimum PEFS conditions were set as 470V/cm for 0-9s, 129V/cm for 9-13s, 470V/cm for 13-13.9s, 294V/cm for 13.9-16s, and 470V/cm for 16-20s for single-channel ME, and 615V/cm for 0-22.5s, 231V/cm for 22.5-28.5s, and 615V/cm for 28.5-40s for multi-channel ME, respectively. In the multi-channel PEFS-ME, target ACE I/D polymorphism DNA fragments (D=190bp and I=490bp) were identified within 25s without loss of resolving power, which was ∼300 times faster than conventional SGE. In addition, PCR products of the ACE gene from human blood samples were detected after only 10 cycles by multi-channel PEFS-ME, but not by SGE. This parallel detection multichannel-based PEFS-ME method offers a powerful tool for fast high-throughput ACE I/D polymorphism screening with high sensitivity.

Ultrafast immunoassays by coupling dielectrophoretic biomarker enrichment in nanoslit channel with electrochemical detection on graphene.

Heterogeneous immunoassays usually require long incubation times to promote specific target binding and several wash steps to eliminate non-specific binding. Hence, signal saturation is rarely achieved at detection limit levels of analyte, leading to significant errors in analyte quantification due to extreme sensitivity of the signals to incubation time and methodology. The poor binding kinetics of immunoassays at detection limit levels can be alleviated through creating an enriched analyte plug in the vicinity of immobilized capture probes to enable signal saturation at higher levels and at earlier times, due to higher analyte association and its faster replenishment at the binding surface. Herein, we achieve this by coupling frequency-selective dielectrophoretic molecular dam enrichment of the target biomarker in physiological media to capture probes immobilized on graphene-modified surfaces in a nanoslit to enable ultrafast immunoassays with near-instantaneous (<2 minutes) signal saturation at dilute biomarker levels (picomolar) within ultra-low sample volumes (picoliters). This methodology is applied to the detection of Prostate Specific Antigen (PSA) diluted in serum samples, followed by validation against a standard two-step immunoassay using three de-identified patient samples. Based on the ability of dielectrophoretic molecular dam analyte enrichment methods to enable the detection of PSA at 1-5 pg mL(-1) levels within a minute, and the relative insensitivity of the signals to incubation time after the first two minutes, we envision its application for improving the sensitivity of immunoassays and their accuracy at detection limit levels.

A non-invasive genomic diagnostic method for bladder cancer using size-based filtration and microchip electrophoresis.

Bladder cancer (BC) cells spontaneously exfoliated in the urine of patients with BC. Detection of exfoliated tumor cells has clinical significance in cancer therapy because it would enable earlier non-invasive screening, diagnosis, or prognosis of BC. In this research, a method for analyzing genetic abnormalities of BC cells collected from urine samples was developed. Target BC cells were isolated by filtration. To find conditions that achieve high cell recovery, we investigated the effects of filter type, concentration of fixative, and flow rate. Cells captured on the filter membrane were completely retrieved within 15s. Selected genes for genomic analysis, mutated genes (FGFR3, TERT and HRAS) and methylated genes (ALX4, RALL3, MT1A, and RUNX3) were amplified by polymerase chain reaction (PCR), and subsequently, were identified by microchip electrophoresis (MCE). Analysis by MCE reduces the risk of contamination, sample consumption, and analysis time. Our developed approach is economical, effectively isolates cancer cells, and permits flexible molecular characterization, all of which make this approach a promising method for non-invasive BC detection.

Simple and low cost integration of highly conductive three-dimensional electrodes in microfluidic devices.

This work presents a fast, simple, and cost-effective technique for fabricating and integrating highly conductive 3D microelectrodes into microfluidic devices. The 3D electrodes are made of low cost, commercially available conductive adhesive and carbon powder. The device can be fabricated by a single-step soft lithography and controllable injections of a conductive composite into microchannels. Functioning of the microfluidic device with 3D electrodes was demonstrated through DEP particle switching as an example for a wide range of microfluidic applications.

Fast quantification of amino acids by microchip electrophoresis-mass spectrometry.

A fast microchip electrophoresis-nano-electrospray ionization-mass spectrometric method (MCE-nanoESI-MS) was developed for analysis of amino acids in biological samples. A glass/poly(dimethylsiloxane) hybrid microchip with a monolithic nanoESI emitter was used in the platform. The proposed MCE-nanoESI-MS analytical method showed high separation efficiency for amino acids. Baseline separation of an amino acid mixture containing Lys, Arg, Val, Tyr, and Glu was completed within 120 s with theoretical plate numbers of >7,500. The method was applied to study cellular release of excitatory amino acids (i.e., aspartic acid (Asp) and glutamic acid (Glu)) under chemical stimulations. Linear calibration curves were obtained for both Asp and Glu in a concentration range from 1.00 to 150.0 µM. Limits of detection were found to be 0.37 µM for Asp and 0.33 µM for Glu (S/N = 3). Assay repeatability (relative standard deviation, n = 6) was 4.2 and 4.5%, for Asp and Glu at 5.0 µM, respectively. In the study of cellular release, PC-12 nerve cells were incubated with alcohol at various concentrations for 1 h. Both extra- and intracellular levels of Asp and Glu were measured by the proposed method. The results clearly indicated that ethanol promoted the release of both Asp and Glu from the cells.

In-plane alloy electrodes for capacitively coupled contactless conductivity detection in poly(methylmethacrylate) electrophoretic chips.

A simple method for producing PMMA electrophoresis microchips with in-plane electrodes for capacitively coupled contactless conductivity detection is presented. One PMMA plate (channel plate) is embossed with the microfluidic and electrode channels and lamination bonded to a blank PMMA cover plate of equal dimensions. To incorporate the electrodes, the bonded chip is heated to 80 °C, above the melting point of the alloy (≈ 70 °C) and below the glass transition temperature of the PMMA (≈ 105 °C), and the molten alloy drawn into the electrode channels with a syringe before being allowed to cool and harden. A 0.5 mm diameter stainless steel pin is then inserted into the alloy filled reservoirs of the electrode channels to provide external connection to the capacitively coupled contactless conductivity detection detector electronics. This advance provides for a quick and simple manufacturing process and negates the need for integrating electrodes using costly and time-consuming thin film deposition methods. No additional detector cell mounting structures were required and connection to the external signal processing electronics was achieved by simply slipping commercially available shielded adaptors over the pins. With a non-optimised electrode arrangement consisting of a 1 mm detector gap and 100 µm insulating distance, rapid separations of ammonium, sodium and lithium (<22 s) yielded LODs of approximately 1.5-3.5 ppm.

Packed multi-channels for parallel chromatographic separations in microchips.

Here we report on a simple method to fabricate microfluidic chip incorporating multi-channel systems packed by conventional chromatographic particles without the use of frits. The retaining effectivities of different bottlenecks created in the channels were studied. For the parallel multi-channel chromatographic separations several channel patterns were designed. The obtained multipackings were applied for parallel separations of dyes. The implementation of several chromatographic separation units in microscopic size makes possible faster and high throughput separations.

Multi-dimension microchip-capillary electrophoresis device for determination of functional proteins in infant milk formula.

To improve resolution of important minor proteins and eliminate time-consuming precipitation of major protein with associated analyte co-precipitation risk, a multi-dimension strategy is adopted in the 2D microchip-CE device to isolate major proteins on-chip, enrich minor proteins in capillary before their separation in CE for UV quantitation. A standard fluorescent protein mixture containing FITC-BSA, myoglobin and cytochrome as specific pI markers has prepared to demonstrate capability of the device to fractionate minor proteins by IEF. The results using a standard protein mixture with profile resembling infant milk formula show a complete isolation of high abundance proteins by a 2-min 1D IEF run. The subsequent t-ITP/CZE run by on-chip high voltage switching delivers a high stacking ratio, realizing 60 folds enrichment of isolated protein fractions. All five important functional proteins (LF, IgG, α-LA, ß-LgA and ß-LgB) known to fortify infant milk formula are isolated and determined using two consecutive t-ITP-CZE runs within a 18-min total assay time, a significant saving compared to several hours conventional pretreatment. For a 100g infant milk formula sample, working ranges of 20-8000mg, repeatability 3.8-5.3% and detection limits 2.3-10mg have been achieved to meet government regulations. Method reliability is established by 100% recoveries and agreeable results within expected ranges and labeled values. The capability of the device for field operation, rapid assay with quick results, label-free universal detection, simple operation by aqueous dissolution before injection, and the demanding matching in 2D separation based on isolated fractions at specified pI ranges, closely matched migration time and baseline-resolved peak shape makes the device a general tool to detect unknown proteins and determine known minor proteins in protein-rich samples with interfering constituents.

Rapid direct PCR for forensic genotyping in under 25 min.

In this paper, a rapid thermal cycling procedure is combined with a direct amplification from a paper punch, permitting a high-speed amplification of a 7-locus multiplex that requires no extraction step. When coupled with a short 1.8 cm microfluidic electrophoresis system, the entire procedure from paper punch to genotype can be completed in under 25 min. The paper describes selection and optimization of enzyme, direct amplification conditions, the reproducibility of the procedure, and concordance with standard forensic genotyping methods. The procedure utilizes a small high-speed thermal cycler and microfluidic device along with a small laptop and is highly portable. Overall, this technique should provide a useful and reliable procedure for rapid determination of identity of individuals retained at checkpoints as well as a quick method for preliminary identification of individuals at remote locations following mass disasters.

Simple detection of surface antigens on living cells by applying distinct cell positioning with negative dielectrophoresis.

We report the fabrication of two different cell patterns based on negative dielectrophoresis (n-DEP) and apply it to simple and rapid distinction of cells with specific surface antigens from a cell population. The DEP device for cell manipulation comprised a microfluidic channel with an upper indium tin oxide (ITO) electrode and a lower ITO-interdigitated band array (ITO-IDA) electrode modified with an antibody. Cells immediately accumulated on the surface in the gap area between both bands of the ITO-IDA electrode by n-DEP upon AC voltage between the upper ITO and both lower bands. Switching of the applied band electrode voltage resulted in the removal of accumulated cells to form another pattern because of the formation of a different electric field pattern in the device. Modifying the ITO-IDA surface with the antibody inhibited the removal of the cells with a specific surface antigen for irreversible capture by immunoreactions during the first accumulation. In this study, we targeted the CD33 surface antigen expressed on human promyelocytic leukemia cells (HL-60). The time required for the assay was substantially short: 60 s for forcing and 60 s for separating the unbound cells. Furthermore, the present method does not require pretreatment such as target labeling or washing of unbound cells. Moreover, the use of the swing technique considerably improved cell binding to the antibody-modified surface for cells with a specific surface antigen. The distinct integration of cells with n-DEP in the high conductivity medium provided higher cell binding efficiency compared to that obtained in our previous study (Hatanaka, H.; Yasukawa, T.; Mizutani, F. Anal. Chem., 2011, 83, 7207-7212) without loss of rapidity and simplicity.

Application of microfluidic technology to the BIOMED-2 protocol for detection of B-cell clonality.

The BIOMED-2 protocol is widely used for detecting clonality in lymphoproliferative disorders. The protocol requires multiple PCR reactions, which are analyzed by either capillary electrophoresis (GeneScan analysis) or heteroduplex PAGE analysis. We tested a microfluidic chip-based electrophoresis device (Agilent 2100 Bioanalyzer) for the analysis of B-cell clonality using PCR for the three framework subregions (FR) of the Ig heavy chain gene (IGH) and PCR for two rearrangements occurring in the Ig κ chain gene (IGK-VJ and IGK-DE). We analyzed 62 B-cell lymphomas (33 follicular and 29 nonfollicular) and 16 reactive lymph nodes. Chip-based electrophoresis was conclusive for monoclonality in 59/62 samples; for 20 samples, it was compared with GeneScan analysis. Concordant results were obtained in 45/55 IGH (FR1, FR2, and FR3) gene rearrangements, and in 34/37 IGK gene rearrangements. However, when the chip device was used to analyze selected IGK gene rearrangements (biallelic IGK rearrangements or IGK rearrangements in a polyclonal background), its performance was not completely accurate. We conclude, therefore, that this microfluidic chip-based electrophoresis device is reliable for testing cases with dominant PCR products but is less sensitive than GeneScan in detecting clonal peaks in a polyclonal background for IGH PCR, or with complex IGK rearrangement patterns.

Generation of a miniaturized free-flow electrophoresis chip based on a multi-lamination technique--isoelectric focusing of proteins and a single-stranded DNA fragment.

Free-flow electrophoresis techniques have been applied for separations in various areas of chemistry and biochemistry. Here we focus on the generation of a free-flow electrophoresis chip and direct monitoring of the separation of different molecules in the separation bed of the miniaturized chip. We demonstrate a fast and efficient way to generate a low-cost micro-free-flow electrophoresis (µFFE) chip with a filling capacity of 9.5 µL based on a multi-lamination technique. Separating webs realized by two transfer-adhesive tapes avoid the problem of gas bubbles entering the separation area. The chip is characterized by isoelectric focusing markers (IEF markers). The functionality of the chip is demonstrated by free-flow isoelectric focusing (FFIEF) of the proteins BSA (bovine serum albumin) and avidin and a single-stranded DNA (ssDNA) fragment in the pH range 3 to 10. The separation voltage ranges between 167 V cm(-1) and 422 V cm(-1), depending on the application.

A simple and rapid approach for measurement of dissociation constants of DNA aptamers against proteins and small molecules via automated microchip electrophoresis.

Automated microchip electrophoresis was used as a simple and rapid method to measure effective dissociation constants (K(d,eff)) of aptamers against both large and small molecule targets. Human thrombin, immunoglobulin E (IgE), and adenosine triphosphate (ATP) were selected as model analytes to validate the method, with four ligands including two DNA aptamers for thrombin (two distinct epitopes), an IgE aptamer, and an ATP aptamer. The approach is based on a microchip version of a DNA mobility shift assay. Non-denaturing microchip gel electrophoresis separations of DNA could resolve and quantify unbound from target-bound aptamers when using large molecules as targets. To extend the technique to small molecule targets such as ATP, an aptamer/competitor strategy was used, in which a DNA competitor complementary to the aptamer could be displaced by ATP and electrophoretically resolved. Using an automated microchip electrophoresis platform, parallel separations of 11 titration samples were completed in ~0.5 h. Analytical performance comparisons show that our approach provides significant advantages in minimized reagent consumption (typically tens of pmol of aptamer and target), reduced analysis time, and minimized user interaction when compared to previously reported methods for aptamer K(d) measurement. Moreover, the flexibility and ease of K(d,eff) measurement for aptamers against large and small targets make this a unique and valuable approach that should find widespread use. Finally, the feasibility of using this method during aptamer selection processes (e.g. SELEX) was shown by accurate bulk K(d,eff) measurement of a known thrombin aptamer (THRaptA) spiked into a random-sequence DNA pool at as low as 5.0% (molar %) of the total pool; only ~825 fmol of total binding sequences were needed for an 11-point titration curve.

A low cost solution for the fabrication of dielectrophoretic microfluidic devices and embedded electrodes.

The versatility of a simple method for producing microfluidic devices with embedded electrodes is demonstrated through the fabrication and operation of two dielectrophoretic devices; one employing interdigitated electrode structures on glass and the other employing contactless electrode reservoirs. Device manufacture is based on the precipitation of silver and subsequent photolithography of thin film resists conducted outside of a cleanroom environment. In current experiments, minimum channel widths of 50 microns and electrode widths of 25 microns are achieved when the distance between features is 40 microns or greater. These results illustrate this technique's potential to produce microfluidic devices with embedded electrodes for lab on chip applications while significantly reducing fabrication expense.

A rapid and reliable bonding process for microchip electrophoresis fabricated in glass substrates.

In this report, we describe a rapid and reliable process to bond channels fabricated in glass substrates. Glass channels were fabricated by photolithography and wet chemical etching. The resulting channels were bonded against another glass plate containing a 50-microm thick PDMS layer. This same PDMS layer was also used to provide the electrical insulation of planar electrodes to carry out capacitively coupled contactless conductivity detection. The analytical performance of the proposed device was shown by using both LIF and capacitively coupled contactless conductivity detection systems. Efficiency around 47,000 plates/m was achieved with good chip-to-chip repeatability and satisfactory long-term stability of EOF. The RSD for the EOF measured in three different devices was ca. 7%. For a chip-to-chip comparison, the RSD values for migration time, electrophoretic current and peak area were below 10%. With the proposed approach, a single chip can be fabricated in less than 30 min including patterning, etching and sealing steps. This fabrication process is faster and easier than the thermal bonding process. Besides, the proposed method does not require high temperatures and provides excellent day-to-day and device-to-device repeatability.

In-line extraction employing functionalized magnetic particles for capillary and microchip electrophoresis.

An approach to performing in-line extraction employing functionalized magnetic particles for CE and microchip electrophoresis is presented. Silica-coated iron oxide particles were synthesized and used as the solid support. The particles were functionalized with octadecylsilane and used as reverse-phase sorbents for in-line SPE followed by electrophoresis. Magnets were used to locally immobilize these sorbents inside the capillary or microchip. Extraction, elution, and detection of the analytes were performed sequentially without interruption or need for sample handling. Mixtures of hydrophobic analytes were successfully extracted from solution using the synthesized magnetic sorbents. CE was able to extract and separate mixture of parabens within 10 min. In-line extraction was also carried out on a disposable PMMA microfluidic device with LIF detection. Electrophoretic separation of fluorescent dyes, Rhodamine 110 and SulfoRhodamine B, was completed in under a minute. The results demonstrated the feasibility of performing the in-line extraction/separation technique in a microchip platform enabling rapid analysis, low sorbent consumption, and increased analyte recovery (relative to the capillary format).

High-resolution electrophoretic separation and integrated-waveguide excitation of fluorescent DNA molecules in a lab on a chip.

By applying integrated-waveguide laser excitation to an optofluidic chip, fluorescently labeled DNA molecules of 12 or 17 different sizes are separated by CE with high operating speed and low sample consumption of approximately 600 pL. When detecting the fluorescence signals of migrating DNA molecules with a PMT, the LOD is as low as 2.1 pM. In the diagnostically relevant size range (approximately 150-1000 base-pairs) the molecules are separated with reproducibly high sizing accuracy (> 99%) and the plug broadening follows Poissonian statistics. Variation of the power dependence of migration time on base-pair size--probably with temperature and condition of the sieving gel matrix--indicates that the capillary migration cannot be described by a simple physical law. Integrated-waveguide excitation of a 12-microm narrow microfluidic segment provides a spatio-temporal resolution that would, in principle, allow for a 20-fold better accuracy than the currently supported by state-of-the-art electrophoretic separation in microchips, thereby demonstrating the potential of this integrated optical approach to fulfill the resolution demands of future electrophoretic microchips.