Integration of High-Resolution Radiation Detector for Hybrid Microchip Electrophoresis.

For decades, there has been immense progress in miniaturizing analytical methods based on electrophoresis to improve sensitivity and to reduce sample volumes, separation times, and/or equipment cost and space requirements, in applications ranging from analysis of biological samples to environmental analysis to forensics. In the field of radiochemistry, where radiation-shielded laboratory space is limited, there has been great interest in harnessing the compactness, high efficiency, and speed of microfluidics to synthesize short-lived radiolabeled compounds. We recently proposed that analysis of these compounds could also benefit from miniaturization and have been investigating capillary electrophoresis (CE) and hybrid microchip electrophoresis (hybrid-MCE) as alternatives to the typically used high-performance liquid chromatography (HPLC). We previously showed separation of the positron-emission tomography (PET) imaging tracer 3'-deoxy-3'-fluorothymidine (FLT) from its impurities in a hybrid-MCE device with UV detection, with similar separation performance to HPLC, but with improved speed and lower sample volumes. In this paper, we have developed an integrated radiation detector to enable measurement of the emitted radiation from radiolabeled compounds. Though conventional radiation detectors have been incorporated into CE systems in the past, these approaches cannot be readily integrated into a compact hybrid-MCE device. We instead employed a solid-state avalanche photodiode (APD)-based detector for real-time, high-sensitivity ß particle detection. The integrated system can reliably separate [18F]FLT from its impurities and perform chemical identification via coinjection with nonradioactive reference standard. This system can quantitate samples with radioactivity concentrations as low as 114 MBq/mL (3.1 mCi/mL), which is sufficient for analysis of radiochemical purity of radiopharmaceuticals.

Fully Automated Microchip Electrophoresis Analyzer for Potential Life Detection Missions.

There are a variety of complementary observations that could be used in the search for life in extraterrestrial settings. At the molecular scale, patterns in the distribution of organics could provide powerful evidence of a biotic component. In order to observe these molecular biosignatures during spaceflight missions, it is necessary to perform separation science in situ. Microchip electrophoresis (ME) is ideally suited for this task. Although this technique is readily miniaturized and numerous instruments have been developed over the last 3 decades, to date, all lack the automation capabilities needed for future missions of exploration. We have developed a portable, automated, battery-powered, and remotely operated ME instrument coupled to laser-induced fluorescence detection. This system contains all the necessary hardware and software interfaces for end-to-end functionality. Here, we report the first application of the system for amino acid analysis coupled to an extraction unit in order to demonstrate automated sample-to-data operation. The system was remotely operated aboard a rover during a simulated Mars mission in the Atacama Desert, Chile. This is the first demonstration of a fully automated ME analysis of soil samples relevant to planetary exploration. This validation is a critical milestone in the advancement of this technology for future implementation on a spaceflight mission.

Voltage control for microchip capillary electrophoresis analyses.

This paper presents an inexpensive and easy-to-implement voltage sequencer instrument for use in microchip capillary electrophoresis (MCE) actuation. The voltage sequencer instrument takes a 0-5 V input signal from a microcontroller and produces a reciprocally proportional voltage signal with the capability to achieve the voltages required for MCE actuation. The unit developed in this work features four independent voltage channels, measures 105 × 143 × 45 mm (width × length × height), and the cost to assemble is under 60 USD. The system is controlled by a peripheral interface controller and commands are given via universal serial bus connection to a personal computer running a command line graphical user interface. The performance of the voltage sequencer is demonstrated by its integration with a fluorescence spectroscopy MCE sensor using pinched sample injection and electrophoretic separation to detect ciprofloxacin in samples of milk. This application is chosen as it is particularly important for the dairy industry, where fines and health concerns are associated with the shipping of antibiotic-contaminated milk. The voltage sequencer instrument presented represents an effective low-cost instrumentation method for conducting MCE, thereby making these experiments accessible and affordable for use in industries such as the dairy industry.

Development of a microchip electrophoresis-based, high-throughput PCR-RFLP method to type Tax 233 variants of bovine leukemia virus in Japan.

Bovine leukemia virus (BLV) is the causative agent of enzootic bovine leukosis (EBL). We used microchip electrophoresis in combination with automatic image analysis to develop a novel high-throughput PCR-RFLP to type the gene sequences that encode BLV Tax 233. This method revealed that 233L-Tax is more prevalent than 233P-Tax in cattle in Japan. The proportion infected with BLV carrying the gene encoding 233L-Tax was significantly higher in Holstein cattle than in Japanese Black cattle. Holsteins infected with BLV encoding 233L-Tax had higher proviral loads than did Holsteins infected with BLV encoding 233P-Tax and Japanese Blacks infected with BLV encoding 233L-Tax or 233P-Tax. The novel method developed in this study will be a useful tool for identifying cattle harboring BLV with a higher risk of EBL and viral transmission.

A thiol-ene microfluidic device enabling continuous enzymatic digestion and electrophoretic separation as front-end to mass spectrometric peptide analysis.

One of the most attractive aspects of microfluidic chips is their capability of integrating several functional units into one single platform. In particular, enzymatic digestion and chemical separation are important steps in processing samples for many biochemical assays. This study presents the development and application of a free-flow electrophoresis microfluidic chip, and its upstream combination with an enzyme microreactor with immobilized pepsin in the same miniaturized platform. The whole microfluidic chip was fabricated by making use of thiol-ene click chemistry. As a proof of concept, different fluorescent dyes and labeled amino acids were continuously separated in the 2D electrophoretic channel. The protease pepsin was immobilized using a covalent linkage with ascorbic acid onto a high-surface monolithic support, also made of thiol-ene. To show the potential of the microfluidic chip for continuous sample preparation and analysis, an oligopeptide was enzymatically digested, and the resulting fragments were separated and collected in a single step (prior to mass spectrometric detection), without the need of further time-consuming liquid handling steps.

Rapid and inexpensive method for the simple fabrication of PDMS-based electrochemical sensors for detection in microfluidic devices.

The fabrication of PDMS microfluidic structures through soft lithography is widely reported. While this well-established method gives high precision microstructures and has been successfully used for many researchers, it often requires sophisticated instrumentation and expensive materials such as clean room facilities and photoresists. Thus, we present here a simple protocol that allows the rapid molding of simple linear microchannels in PDMS substrates aiming microfluidics-based applications. It might serve as an alternative to researchers that do not have access to sophisticated facilities such as clean rooms. The method developed here consists on the use of pencil graphite leads as template for the molding of PDMS channels. It yields structures that can be used for several applications, such as housing support for electrochemical sensors or channels for flow devices. Here, the microdevices produced through this protocol were employed for the accommodation of carbon black paste, which was utilized for the first time as amperometric sensor in microchip electrophoresis. This platform was successfully used for the separation and detection of model analytes. Ascorbic acid and iodide were separated within 45 s with peak resolution of 1.2 and sensitivities of 198 and 492 pA/µM, respectively. The background noise was ca. 84 pA. The analytical usefulness of the system developed was successfully tested through the quantification of iodide in commercial pharmaceutical formulations. It demonstrates good efficiency of the microfabrication protocol developed and enables its use for the easy and rapid prototyping of PDMS structures over a low fabrication cost.

A microchip device to enhance free flow electrophoresis using controllable pinched sample injections.

Micro free flow electrophoresis (µFFE) is a valuable technique capable of high throughput rapid microscale electrophoretic separation along with mild operating conditions. However, the stream flow separation nature of free flow electrophoresis affects its separation performance with additional stream broadening due to sample stream deflection. To reduce stream broadening and enhance separation performance of µFFE, we presented a simple microfluidic device that enables injection bandwidth control. A pinched injection was formed in the reported µFFE system using operating buffer at sample flow rate ratio (r) setting. Initial bandwidth at the entrance of separation chamber can be shrunk from 800 to 30 µm when r increased from 1 to 256. Stream broadening at the exit of separation chamber can be reduced by about 96% when r increased from 4 to 128, according to both theoretical and experimental results. Moreover, the separation resolution for a dye mixture was enhanced by a factor of 4 when r increased from 16 to 128, which corresponded to an 80% reduction in sample initial bandwidth. Furthermore, a similar enhancement on amino acids separation was obtained by using injection control in the reported µFFE device and readily integrated into online/offline sample preparation and/or downstream analysis procedures.

Rapid quantification of quinine by multi-stacking in a portable microchip electrophoresis system.

A new multi-stacking pre-concentration procedure based on field-enhanced sample injection (FESI), field-amplified sample stacking, and transient isotachophoresis was developed and implemented in a compact microchip electrophoresis (MCE) with a double T-junction glass chip, coupled with an on-chip capacitively coupled contactless conductivity detection (C4 D) system. A mixture of the cationic target analyte and the terminating electrolyte (TE) from the two sample reservoirs was injected under FESI conditions within the two sample-loading channels. At the double T-junction, the stacked analyte zones were further concentrated under field-amplified stacking conditions and then subsequently focused by transient-isotachophoresis and separated along the separation channels. The proposed multi-stacking strategy was verified under a Universal Serial Bus (USB) fluorescence microscope employing Rhodamine 6G as the model analyte. This developed approach was subsequently used to monitor the target quinine present in human plasma samples. The total analysis time for quinine was approximately 200 s with a sensitivity enhancement factor of approximately 61 when compared to the typical gated injection. The detection and quantification limits of the developed approach for quinine were 3.0 µg/mL and 10 µg/mL, respectively, with intraday and interday repeatability (%RSDs, n = 5) of 3.6 and 4.4%. Recoveries in spiked human plasma were 98.1-99.8%.

On-line sample preconcentration by polarity switching in floating electrode-integrated microchannel.

In this study, we found that the polarity switching was effective to enrich and separate fluorescent analytes which have weakly-dissociated groups in a floating platinum electrode (width, 50 µm; thickness, 2.5 µm)-integrated straight-channel in microchip electrophoresis (MCE). In the straight channel filled with an Alexa Flour 488 (AF488) solution, a sharp peak was observed after the polarity inversion with a 530-fold enhancement of the sensitivity relative to the conventional MCE analysis. By using a fluorescent pH indicator, we verified that a sharp high-pH zone was generated nearby the floating electrode and moved toward the anode with maintaining the high pH, which induced the sample enrichment like a dynamic pH junction mechanism. In the floating electrode-embedded channel, the mixture of AF488-labeled proteins was also well concentrated and separated within 100 s.

Biomimetic preparation of core-shell structured surface-enhanced Raman scattering substrate with antifouling ability, good stability, and reliable quantitative capability.

The fouling and stability are two most critical limiting factors for practical applications of surface-enhanced Raman scattering (SERS)-based microfluidic electrophoresis device. Herein, we present a novel biomimetic nanoengineering strategy to achieve a SERS substrate featuring antifouling ability, good stability, and reliable quantitative capability. Typically, by employing tea polyphenol as the reducing agent, the substrate made of silver core-gold shell nanostructures in situ grown on silicon wafer surface is fabricated. The core-shell nanostructures are further embedded with internal standard molecules. Remarkably, the fabricated substrate preserves distinct SERS effects, adaptable reproducibility, and reliable quantitative ability even if the substrate is incubated with 15% H2 O2 , 13% HNO3 , or 108  CFU/mL bacteria, or suffered from 12-day continuous vibration at 250 rpm/min in PBS buffer. As a proof-of-concept application, the DNA-functionalized substrate is capable of precise quantification of Hg2+ with a limit of detection down to ca. 1 pM even in sewage water.

Droplet sample introduction to microchip gel and zone electrophoresis for rapid analysis of protein-protein complexes and enzymatic reactions.

Electrophoresis has demonstrated utility as tool for screening of small molecule modulators of protein-protein interactions and enzyme targets. Screening of large chemical libraries requires high-throughput separations. Such fast separation can be accessed by microchip electrophoresis. Here, microchip gel electrophoresis separations of proteins are achieved in 2.6 s with 1200 V/cm and 3-mm separation lengths. However, such fast separations can still suffer from limited overall throughput from sample introduction constraints. Automated introduction of microfluidic droplets has been demonstrated to overcome this limitation. Most devices for coupling microfluidic droplets to microchip electrophoresis are only compatible with free-solution separations. Here, we present a device that is compatible with coupling droplets to gel and free-solution electrophoresis. In this device, automated sample introduction is based on a novel mechanism of carrier phase separation using the difference in density of the carrier phase and the running buffer. This device is demonstrated for microchip gel electrophoresis and free-solution electrophoresis separations of protein-protein interaction and enzyme samples, respectively. Throughputs of about 10 s per sample are achieved and over 1000 separations are demonstrated without reconditioning of the device. Graphical abstract.

Development of a Microchip CE-HPMS Platform for Cell Growth Monitoring.

Improvements were made to a previously developed platform coupling microchip capillary electrophoresis (CE) with high pressure mass spectrometry (HPMS). The RF drive frequency was increased to over 30 MHz from less than 10 MHz, and the ion trap was scaled down to 100 µm critical dimensions. A stretched length ion trap was used to improve sensitivity, and a tube lens was used to improve ion transmission. Detection of the 20 common amino acids was demonstrated, resulting in an average improvement of signal-to-noise of 28 times and an average improvement in peak width of 2.6 times over those obtained in previous work. Consumption of amino acids by cells in growth media was monitored over time using the improved CE-HPMS platform, and several amino acids were shown to be consumed at different rates, demonstrating the potential for real-time bioreactor monitoring.

PDMS/glass hybrid device with a reusable carbon electrode for on-line monitoring of catecholamines using microdialysis sampling coupled to microchip electrophoresis with electrochemical detection.

On-line separations-based sensors employing microdialysis (MD) coupled to microchip electrophoresis (ME) enable the continuous monitoring of multiple analytes simultaneously. Electrochemical detection (EC) is especially amenable to on-animal systems employing MD-ME due to its ease of miniaturization. However, one of the difficulties in fabricating MD-ME-EC systems is incorporating carbon working electrodes into the device. In this paper, a novel fabrication procedure is described for the production of a PDMS/glass hybrid device that is capable of integrating hydrodynamic MD flow with ME-EC using a flow-gated interface and a pyrolyzed photoresist film carbon electrode. This fabrication method enables the reuse of carbon electrodes on a glass substrate, while still maintaining a good seal between the PDMS and glass to allow for pressure-driven MD flow. The on-line MD-ME-EC device was characterized in vitro and in vivo for monitoring analytes in the dopamine metabolic pathway. The ultimate goal is to use this device and associated instrumentation to perform on-animal, near-real time in vivo monitoring of catecholamines.

Toward miniaturized analysis of chemical identity and purity of radiopharmaceuticals via microchip electrophoresis.

Miniaturized synthesis of positron emission tomography (PET) tracers is poised to offer numerous advantages including reduced tracer production costs and increased availability of diverse tracers. While many steps of the tracer production process have been miniaturized, there has been relatively little development of microscale systems for the quality control (QC) testing process that is required by regulatory agencies to ensure purity, identity, and biological safety of the radiotracer before use in human subjects. Every batch must be tested, and in contrast with ordinary pharmaceuticals, the whole set of tests of radiopharmaceuticals must be completed within a short-period of time to minimize losses due to radioactive decay. By replacing conventional techniques with microscale analytical ones, it may be possible to significantly reduce instrument cost, conserve lab space, shorten analysis times, and streamline this aspect of PET tracer production. We focus in this work on miniaturizing the subset of QC tests for chemical identity and purity. These tests generally require high-resolution chromatographic separation prior to detection to enable the approach to be applied to many different tracers (and their impurities), and have not yet, to the best of our knowledge, been tackled in microfluidic systems. Toward this end, we previously explored the feasibility of using the technique of capillary electrophoresis (CE) as a replacement for the "gold standard" approach of using high-performance liquid chromatography (HPLC) since CE offers similar separating power, flexibility, and sensitivity, but can readily be implemented in a microchip format. Using a conventional CE system, we previously demonstrated the successful separation of non-radioactive version of a clinical PET tracer, 3'-deoxy-3'-fluorothymidine (FLT), from its known by-products, and the separation of the PET tracer 1-(2'-deoxy-2'-fluoro-ß-D-arabinofuranosyl)-cytosine (D-FAC) from its α-isomer, with sensitivity nearly as good as HPLC. Building on this feasibility study, in this paper, we describe the first effort to miniaturize the chemical identity and purity tests by using microchip electrophoresis (MCE). The fully automated proof-of-concept system comprises a chip for sample injection, a separation capillary, and an optical detection chip. Using the same model compound (FLT and its known by-products), we demonstrate that samples can be injected, separated, and detected, and show the potential to match the performance of HPLC. Addition of a radiation detector in the future would enable analysis of radiochemical identity and purity in the same device. We envision that eventually this MCE method could be combined with other miniaturized QC tests into a compact integrated system for automated routine QC testing of radiopharmaceuticals in the future. Graphical abstract Miniaturized quality control (QC) testing of batches of radiopharmaceuticals via microfluidic analysis. The proof-of-concept hybrid microchip electrophoresis (MCE) device demonstrated the feasibility of achieving comparable performance to conventional analytical instruments (HPLC or CE) for chemical purity testing.

Cellular dielectrophoresis coupled with single-cell analysis.

In this review, recent advances that leverage dielectrophoretic approaches to accomplish single-cell analysis (both "on-chip" and "off-chip") are discussed with special emphasis on eukaryotic cells. Dielectrophoresis as an electric-field-induced force utilized for cell manipulation can confer selectivity without labeling. Recent technical improvements have increased the volumetric throughput of the separation of cells from complex mixtures, introduced new strategies for massively parallel single-cell confinement for subsequent on-chip analysis, made possible selective transport of individual cells off-chip, and integrated preconcentration and prefocusing steps to enhance dielectrophoretic performance. Collectively, these studies potentiate all-in-one platforms capable of taking as their input complex mixtures of cells and accomplishing single-cell analysis. Assays requiring small reaction volumes (e.g., enzymatic assays, fluorescent in situ hybridization, and immunostaining) have been demonstrated. Still greater opportunities to unravel cell-to-cell variations and for point-of-care applications can be realized by making possible on-chip gene amplification, live-cell assays, and either dielectrophoretic manipulation in native media or on-chip exchange of media. We therefore conclude with a discussion of emerging capabilities in these areas.

Automated microfluidic devices integrating solid-phase extraction, fluorescent labeling, and microchip electrophoresis for preterm birth biomarker analysis.

We have developed multichannel integrated microfluidic devices for automated preconcentration, labeling, purification, and separation of preterm birth (PTB) biomarkers. We fabricated multilayer poly(dimethylsiloxane)-cyclic olefin copolymer (PDMS-COC) devices that perform solid-phase extraction (SPE) and microchip electrophoresis (µCE) for automated PTB biomarker analysis. The PDMS control layer had a peristaltic pump and pneumatic valves for flow control, while the PDMS fluidic layer had five input reservoirs connected to microchannels and a µCE system. The COC layers had a reversed-phase octyl methacrylate porous polymer monolith for SPE and fluorescent labeling of PTB biomarkers. We determined µCE conditions for two PTB biomarkers, ferritin (Fer) and corticotropin-releasing factor (CRF). We used these integrated microfluidic devices to preconcentrate and purify off-chip-labeled Fer and CRF in an automated fashion. Finally, we performed a fully automated on-chip analysis of unlabeled PTB biomarkers, involving SPE, labeling, and µCE separation with 1 h total analysis time. These integrated systems have strong potential to be combined with upstream immunoaffinity extraction, offering a compact sample-to-answer biomarker analysis platform. Graphical abstract Pressure-actuated integrated microfluidic devices have been developed for automated solid-phase extraction, fluorescent labeling, and microchip electrophoresis of preterm birth biomarkers.

Microfluidic electrophoretic non-enzymatic kanamycin assay making use of a stirring bar functionalized with gold-labeled aptamer, of a fluorescent DNA probe, and of signal amplification via hybridization chain reaction.

The authors describe an enzyme-free aptamer-based assay for the determination of the model antibiotic kanamycin (Kana). The method is making use of (a) microfluidic chip electrophoresis; (b) a stirring bar carrying a gold-labeled aptamer probe, and (c) the hybridization chain reaction (HCR) for signal amplification. Firstly, a stirring bar (length: 1 cm; diameter: 0.2 mm) was modified with a large amount of duplex DNA and then hybridized with aptamer and its partially complementary chains (cDNA). In the presence of Kana, the binding between the Kana and aptamer unwinds the duplex structures and releases a corresponding amount of cDNA into the supernatant. The released cDNA triggers the HCR in the presence of H1 and H2 DNA hairpin to produce a large amount of duplex DNA chains with different lengths. At the same time, the amounts of H1 and H2 are reduced. The decreased signal of H1/H2 after several HCR cycles can be used to quantify kana in the 1 pg·mL-1 to 10 ng·mL-1, with a detection limit of 0.29 pg·mL-1. The signal is generated by reading the fluorescence, best at excitation/emission maxima of 470/525 nm. The whole detection process takes 3 min only. The assay was employed to the detection of Kana in spiked milk and fish samples. Results are consistent with those of an enzyme linked immunosorbent assay. The assay has high throughput, high selectivity, and high amplification capability. Graphical abstract Schematic of a stirring bar functionalized with gold-labeled aptamer acting as the capture probe. It can capture the target and release primer simultaneously. The primer triggers the hybridization chain reaction inducing the consumption of H1 and H2. After a certain reaction time, the mixture is injected into the MCE platform for microfluidic electrophoretic separation and fluorometric detection.

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.

Combination of large-volume sample stacking with an electroosmotic flow pump with field-amplified sample injection on cross-channel chips.

A combination of two online sample concentration techniques, large-volume sample stacking with an electroosmotic flow (EOF) pump (LVSEP) and field-amplified sample injection (FASI), was investigated in microchip electrophoresis (MCE) to achieve highly sensitive analysis. By applying reversed-polarity voltages on a cross-channel microchip, anionic analytes injected throughout a microchannel were first concentrated on the basis of LVSEP, followed by the electrokinetic stacking injection of the analytes from a sample reservoir by the FASI mechanism. As well as the voltage application, a pressure was also applied to the sample reservoir in LVSEP-FASI. The applied pressure generated a counter-flow against the EOF to reduce the migration velocity of the stacked analytes, especially around the cross section of the microchannel, which facilitated the FASI concentration. At the hydrodynamic pressure of 15 Pa, 4520-fold sensitivity increase was obtained in the LVSEP-FASI analysis of a standard dye, which was 33-times higher than that obtained with a normal LVSEP. Furthermore, the use of the sharper channel was effective for enhancing the sensitivity, e.g., 29 100-fold sensitivity increase was achieved with the 75-µm wide channel. The developed method was applied to the chiral analysis of amino acids in MCE, resulting in the sensitivity enhancement factor of 2920 for the separated d-leucine.

Electric field mediated spraying of miniaturized droplets inside microchannel.

We report a facile and noninvasive way to disintegrate a microdroplet into a string of further miniaturized ones under the influence of an external electrohydrodynamic field inside a microchannel. The deformation and breakup of the droplet was engendered by the Maxwell's stress originating from the accumulation of induced and free charges at the oil-water interface. While at smaller field intensities, for example less than 1 MV/m, the droplet deformed into a plug, at relatively higher field intensities, e.g. ∼1.16 MV/m, a pair of droplets having opposite surface charge was formed. The charged droplets showed an interesting periodic bridging and breakup during their translation motion across the channel. For even higher field intensities, for example more than 1.2 MV/m, the entire droplet underwent dielectrophoresis toward one of the electrodes before experiencing a strong attractive force from the other electrode to deform into a shape of a Taylor cone. With progress in time, mimicking the electrospraying phenomenon, the cone tip periodically ejected a string of miniaturized water droplets to form a microemulsion inside the channel. The frequency and size of the droplet ejection could be tuned by varying the applied field intensity. A water droplet of ∼214 µm diameter could continuously eject droplets of size ∼10 µm or even smaller to form a microemulsion inside the channel.