Triblock copolymer matrix‐based capillary electrophoretic microdevice for high‐resolution multiplex pathogen detection

Rapid and simple analysis for the multiple target pathogens is critical for patient management. CE‐SSCP analysis on a microchip provides high speed, high sensitivity, and a portable genetic analysis platform in molecular diagnostic fields. The capability of separating ssDNA molecules in a capillary electrophoretic microchannel with high resolution is a critical issue to perform the precise interpretation in the electropherogram. In this study, we explored the potential of poly(ethyleneoxide)‐poly(propyleneoxide)‐poly(ethyleneoxide) (PEO‐PPO‐PEO) triblock copolymer as a sieving matrix for CE‐SSCP analysis on a microdevice. To demonstrate the superior resolving power of PEO‐PPO‐PEO copolymers, 255‐bp PCR amplicons obtained from 16S ribosomal RNA genes of four bacterial species, namely Proteus mirabilis, Haemophilus ducreyi, Pseudomonas aeruginosa, and Neisseria meningitidis, were analyzed in the PEO‐PPO‐PEO matrix in comparison with 5% linear polyacrylamide and commercial GeneScan? gel. Due to enhanced dynamic coating and sieving ability, PEO‐PPO‐PEO copolymer displayed fourfold enhancement of resolving power in the CE‐SSCP to separate same‐sized DNA molecules. Fivefold input of genomic DNA of P. aeruginosa and/or N. meningitidis produced proportionally increased corresponding amplicon peaks, enabling correct quantitative analysis in the pathogen detection. Besides the high‐resolution sieving capability, a facile loading and replenishment of gel in the microchannel due to thermally reversible gelation property makes PEO‐PPO‐PEO triblock copolymer an excellent matrix in the CE‐SSCP analysis on the microdevice.     

A hybrid microdevice for electrophoresis and electrochromatography using UV detection

We have designed a new class of microdevices composed of a supporting plastic (polyvinyl chloride, PVC) plate integrated with a groove for a piece of fused silica capillary (the separation channel), a slit for on-tube detection, an "islet" for the application of sample, electrode vessels and platinum electrodes. The design permits electrophoretic, electrochromatographic and chromatographic separations with on-tube UV detection. The efficient heat dissipation allows relatively high field strengths. This article is the first one dealing with microdevices where polymer solutions are replaced by homogeneous gels. A new type of gels synthesized from acrylamide and 2-hydroxy-3-allyloxy-propyl-beta-cyclodextrin (allyl-beta-CD) as a cross-linker was employed for electrophoresis and electrochromatography. 2-Acrylamido-2-methylpropanesulfonic acid was added to the monomer solution to create a high electroendosmotic flow in electrochromatographic runs. These gels have excellent electrochromatographic and electrophoretic properties for low-molecular-weight compounds and DNA, as shown previously, namely high resolution combined with high stability. The unique cross-linker can be used for specific interaction with the alkyl and phenyl groups. The tripeptide glutathione (gamma-L-glutamyl-L-cysteinyl-glycine) and its benzyl conjugates were selected as model compounds to study the resolving power of the gel because they are difficult to separate by free zone electrophoresis. The limit of detection (LOD) for S-benzyl-glutathione was determined (ca. 7 microM). Run-by-run reproducibility was high (the separation factor of glutathione in the gel was 0.3 with 2.5% coefficient of variation, CV). Neutral compounds (acetone, acetophenone, propiophenone and butyrophenone) were separated electrochromatographically in the gel. The influence of organic solvent (acetonitrile) on the electroendosmotic mobility was similar to that in reversed-phase separations, although the separation mechanism is different. ATP, ADP and AMP were separated in less than 10 s by free-zone electrophoresis.     

Segmented continuous-flow multiplex polymerase chain reaction microfluidics for high-throughput and rapid foodborne pathogen detection

High-throughput and rapid identification of multiple foodborne bacterial pathogens is vital in global public health and food industry. To fulfill this need, we propose a segmented continuous-flow multiplex polymerase chain reaction (SCF-MPCR) on a spiral-channel microfluidic device. The device consists of a disposable polytetrafluoroethylene (PTFE) capillary microchannel coiled on three isothermal blocks. Within the channel, n segmented flow regimes are sequentially generated, and m-plex PCR is individually performed in each regime when each mixture is driven to pass three temperature zones, thus providing a rapid analysis throughput of m × n. To characterize the performance of the microfluidic device, continuous-flow multiplex PCR in a single segmented flow has been evaluated by investigating the effect of key reaction parameters, including annealing temperatures, flow rates, polymerase concentration and amount of input DNA. With the optimized parameters, the genomic DNAs from Salmonella enterica, Listeria monocytogenes, Escherichia coli O157:H7 and Staphylococcus aureus could be amplified simultaneously in 19 min, and the limit of detection was low, down to 10² copies μL⁻¹. As proof of principle, the spiral-channel SCF-MPCR was applied to sequentially amplify four different bacterial pathogens from banana, milk, and sausage, displaying a throughput of 4 × 3 with no detectable cross-contamination.     

Electrochemical biosensors for rapid pathogen detection

Rapid pathogen detection is an emerging issue in clinical, environmental, and food industry sectors. Biosensors can represent a solution to culture-based and molecular methods as they respond to sensitivity, specificity, and rapidity needs. Screen-printed electrodes have been used in association with nanoparticles to increase the signal and improve sensitivity reaching low numbers of the targets. Antibodies, DNA probes, and aptamers are mainly used to functionalize the working electrodes to ensure high specific pathogen detection by the use of voltammetry, impedance spectroscopy, amperometry, and conductivity. Electrochemical biosensors can be miniaturized to construct portable devices useful for in situ assays.     

Microfluidics-based technologies for the analysis of extracellular vesicles at the single-cell level and single-vesicle level

Extracellular vesicles(EVs) are membrane vesicles secreted by cells, playing critical roles in mediating intercellular communications for various physiological and pathological processes. Most of the EV analysis is currently performed at the bulk level, obscuring the origin of the EVs and diverse characteristics of the individual extracellular vesicle. Technologies to analyze the extracellular vesicles at the single-cell and single-vesicle levels are needed to evaluate EV comprehensively and dec...     

Microfluidic electroporation for selective release of intracellular molecules at the single-cell level

Analysis of intracellular materials at the single-cell level presents opportunities for probing the heterogeneity of a cell population. Lysis by electroporation has been gaining popularity as a rapid method for disruption of the cell membrane and release of intracellular contents. In this report, we selectively released specific intracellular molecules for interrogation at the single-cell level by tuning the parameters of electroporation. We examined the release of a small molecule, calcein (MW approximately 600), and a 72-kDa protein kinase, Syk, tagged by enhanced green fluorescent protein (EGFP) from chicken B cells during electroporation at the single-cell level. We studied the effects of the field intensity and the field duration on the release of the two molecules. We found that calcein in general was released at lower field intensities and shorter durations than did SykEGFP. By tuning the electrical parameters, we were able to deplete calcein from the cells before SykEGFP started to release. This approach potentially provides a high-throughput alternative for probing different intracellular molecules at the single-cell level compared to chemical cytometry by eliminating complete disruption of the cell membrane.     

Highly sensitive and quantitative detection of rare pathogens through agarose droplet microfluidic emulsion PCR at the single-cell level

Genetic alternations can serve as highly specific biomarkers to distinguish fatal bacteria or cancer cells from their normal counterparts. However, these mutations normally exist in very rare amount in the presence of a large excess of non-mutated analogs. Taking the notorious pathogen E. coli O157:H7 as the target analyte, we have developed an agarose droplet-based microfluidic ePCR method for highly sensitive, specific and quantitative detection of rare pathogens in the high background of normal bacteria. Massively parallel singleplex and multiplex PCR at the single-cell level in agarose droplets have been successfully established. Moreover, we challenged the system with rare pathogen detection and realized the sensitive and quantitative analysis of a single E. coli O157:H7 cell in the high background of 100?000 excess normal K12 cells. For the first time, we demonstrated rare pathogen detection through agarose droplet microfluidic ePCR. Such a multiplex single-cell agarose droplet amplification method enables ultra-high throughput and multi-parameter genetic analysis of large population of cells at the single-cell level to uncover the stochastic variations in biological systems.     

Microchip capillary electrophoresis with amperometric detection for rapid separation and detection of phenolic acids

A microchip capillary-electrophoresis protocol for rapid and effective measurements of food-related phenolic acids (including chlorogenic, gentisic, ferulic, and vanillic acids) is described. Relevant parameters of the chip separation and amperometric detection are examined and optimized. Under optimum conditions, the analytes could be separated and detected in a 15 mM borate buffer (pH 9.5, with 10% of methanol) within 300 s using a separation voltage of 2000 V and a detection voltage of +1.0 V. Linear calibration plots are observed for micromolar concentrations of the phenolic acid compounds. The negligible sample volumes used in the microchip procedure obviates surface fouling common to amperometric measurements of phenolic compounds. The new microchip protocol offers great promise for a wide range of food applications requiring fast measurements and negligible sample consumption. An application on a commercial red wine was performed with minimal sample preparation and promising results.     

Fluorescent polymeric transducer for the rapid, simple, and specific detection of nucleic acids at the zeptomole level

We report the specific detection of a few hundred molecules of genetic material using a fluorescent polythiophene biosensor. Such recognition is based on simple electrostatic interactions between a cationic polymeric optical transducer and the negatively charged nucleic acid target and can be done in less than 1 h, simply and affordably, and without any chemical reaction. This simple system is versatile enough to detect nucleic acids of various lengths, including a segment from the RNA genome of the Influenza virus.     

An integrated light emitting diode-induced fluorescence detector for capillary electrophoresis

An integrated light emitting diode (LED)-induced fluorescence detector was described and evaluated. The LED and its related components including lens and interference filter, the optical fiber used to collect fluorescence, and the capillary column are integrated into a substrate block, which eliminates the need of align procedure of the fiber and the capillary. Forty-fold enhancement of sensitivity was obtained compared with our previous work and the detection limit for fluorescein was 5 nM. Application of the detector for the analysis of FITC-labeled Ephedrine extract was demonstrated.     

Mono- and bis-intercalating dyes for multiplex fluorescence lifetime detection of DNA restriction fragments in capillary electrophoresis

The use of novel intercalating dyes as labels in DNA restriction fragment analysis by capillary electrophoresis with frequency-domain fluorescence lifetime detection is described. The dyes, including one mono-intercalating dye with three positive charges and three bis-intercalating, homodimeric dyes with four positive charges, were excited by the 488 nm line of an argon ion laser and exhibited lifetimes in the range of 1-3 ns. The separations were performed using a gel containing 1% high-molecular-weight (HMW) hydroxyethylcellulose (HEC) (90,000-105,000) and 0.3% low-molecular-weight (LMW) HEC (24,000-27,000) in Tris-borate-EDTA buffer (TBE). Multiplex lifetime detection of mixtures of dye-labeled DNA restriction fragment digests and size standard fragments was achieved. Compared to previous results obtained with several mono-intercalating dyes of lesser charge (McIntosh, S. L., Nunnally, B. K., Nesbit, A. R., Deligeorgiev, T. G., Gadjev, N. I., McGown, L. B., Anal. Chem. 2000, 72, 5444-5449), the present dyes provided a wider range of lifetimes and better lifetime discrimination in multiplex detection. There was no evidence of dye exchange during the capillary electrophoresis experiment.     

A microchip electrophoresis system with integrated in-plane electrodes for contactless conductivity detection

We present a new approach for contactless conductivity detection for microchip-based capillary electrophoresis (CE). The detector integrates easily with well-known microfabrication techniques for glass-based microfluidic devices. Platinum electrodes are structured in recesses in-plane with the microchannel network after glass etching, which allows precise positioning and batch fabrication of the electrodes. A thin glass wall of 10-15 microm separates the electrodes and the buffer electrolyte in the separation channel to achieve the electrical insulation necessary for contactless operation. The effective separation length is 34 mm, with a channel width of 50 microm and depth of 12 microm. Microchip CE devices with conductivity detection were characterized in terms of sensitivity and linearity of response, and were tested using samples containing up to three small cations. The limit of detection for K+ (18 microM) is good, though an order of magnitude higher than for comparable capillary-based systems and one recently reported example of contactless conductivity on chip. However, an integrated field-amplified stacking step could be employed prior to CE to preconcentrate the sample ions by a factor of four.     

3D打印便携式凝胶电泳装置用于蛋白质的快速检测

随着现场分析对于快速、便携和经济型检测的需求,分析仪器的便携化和微型化备受关注。3D打印技术的不断发展,将会极大推动小型化、便携式实验设备的开发和研制。分析仪器的微型化有助于促进资源不足地区在医疗现场、食品安全和环境污染等方面的现场监测。目前,用于蛋白质分离的凝胶电泳装置多为实验室用小型化分析仪器,可用于现场快速分离蛋白质的小型化仪器尚未见报道。该研究设计加工了一款便携式凝胶电泳装置,用于蛋白质的快速分离检测。首先,通过3D打印加工的凝胶电泳装置可在实验室内方便、快捷、低成本的复制。其次,通过对预染蛋白质相对分子质量标准的分离测试,对该系统结构进行优化。优化后该凝胶电泳装置电泳槽的尺寸仅为15 mm×20 mm×17 mm,采用3D打印技术可在5 h内加工完成,耗费打印材料10 mL。正负极所用电泳缓冲液共需4 mL,所使用的25 V锂电池可实现100 h左右的工作时间。装置优化后可实现蛋白质的快速高效分离。随后,在5种常用蛋白质相对分子质量标准的分离中,该装置与商业化平板凝胶电泳分离效果相当,同时具备更快的分离速度。该研究在便携式凝胶电泳装置的开发及其在蛋白质快速分离方面取得了初步成...     

GUcal: An integrated application for capillary electrophoresis based glycan analysis

Recent emergence in the use of monoclonal antibody therapeutics and other glycoprotein biopharmaceuticals requires high‐throughput, robust, and automated techniques for their glycosylation analysis. Capillary electrophoresis is one of the high‐performance methods of choice; however, while the necessary instrumentation is well developed, the related bioinformatics tools are lacked behind. In this paper, we introduce an integrated toolset dubbed as GUcal, to automatically calculate the glucose unit (GU) values for all sample components of interest in an electropherogram with a concomitant database search for structural assignment. The database comprises CE GUs and suggested structures of N‐glycans released from human IgG. The app is freely available online ( www.lendulet.uni‐pannon.hu/gucal ) and readily facilitates CE‐based glycan analysis.     

An integrated fluorescence detection system for lab-on-a-chip applications

We present a low-cost miniaturized fluorescence detection system for lab-on-a-chip applications with a sensitivity in the low nanomolar range; a built-in lock-in amplifier enables measurements under ambient light.     

An integrated solid-phase extraction system for sub-picomolar detection

A microchip structure etched on a glass substrate for packed column solid-phase extraction (SPE) and capillary electrochromatography (CEC) is described. A 200 microm long, octadecylsilane (ODS) packed column was secured using two different approaches: solvent lock or polymer entrapment. The former method was utilized for SPE while the latter approach was applied for CEC. In SPE, the ODS packed chamber gave a detection limit of 70 fM for a nonpolar BODIPY (493/503) dye when concentrated for 3 min at an electroosmotic flow rate of 4.14 nL/min, compared to 30 pM for this detector without the SPE step. SPE beds showed reproducible, linear calibration curves (R(2) = 0.9989) between 1 and 100 pM BODIPY at fixed preconcentration times. Breakthrough curves for the 330 pL (ODS-packed) bed indicated a capacity for BODIPY dye of 8.1 x 10(-14) mmol, or 0.25 mmol dye per liter of bed. The ODS-chamber could also be used to analyze dilute amino acid and peptide solutions. In the CEC format, two neutral dyes (BODIPY and acridine orange) were baseline-separated in an isocratic run with a theoretical plate count of 84 (420 000 plates/m) and a reduced plate height of about 1. A labeled peptide was also analyzed by CEC, using the acidic eluent (84% acetonitrile, and 26% aqueous trifluoroacetic acid (0.05%)) preferred for peptide separations on ODS-coated silica particles.