Capillary Electrophoresis Chip Research Articles

On-chip Paper Electrophoresis for Ultrafast Screening of Infectious Diseases

The outbreak of new viral strains promotes advances in universal diagnostic techniques for detecting infectious diseases with unknown viral sequence. Long double-stranded RNA (dsRNA), a hallmark of infections, serves as a virus marker for prompt detection of viruses with unknown genomes. Here, we report on-chip paper electrophoresis for ultrafast screening of infectious diseases. Negatively charged RNAs pass through the micro and nanoscale pores of cellulose in order of size under an external electric field applied to the paper microfluidic channel. Quantitative separation of long dsRNA mimicking poly I:C was analyzed from 1.67 to 33 ng·μL−1, which is close to the viral dsRNA concentration in infected cells. This paper-based capillary electrophoresis chip (paper CE chip) can provide a new diagnostic platform for ultrafast viral disease detection at the point-of-care (POC) level.

Modular-Based Integrated Microsystem with Multiple Sample Preparation Modules for Automated Forensic DNA Typing from Reference to Challenging Samples.

The realization of an automated short tandem repeat (STR) analysis for forensic investigations is facing a unique challenge, that is DNA evidence with wide disparities in sample types, quality, and quantity. We developed a fully integrated microsystem in a modular-based architecture to accept and process various forensic samples in a "sample-in-answer-out" manner for forensic STR analysis. Two sample preparation modules (SPMs), the direct and the extraction SPM, were designed to be easily assembled with a capillary array electrophoresis (CAE) chip using a chip cartridge to efficiently achieve an adequate performance to different samples at a low cost. The direct SPM processed buccal swabs to produce STR profiles without DNA extraction in about 2 h. The extraction SPM analyzed more challenging blood samples based on chitosan-modified quartz filter paper for DNA extraction. This newly developed quartz filter provided a 90% DNA extraction efficiency and the "in situ" PCR capability, which enabled DNA extraction and PCR performed within a single chamber with all the DNA concentrated in the filter. We demonstrated that minute amounts of blood (0.25 μL), highly diluted blood (0.5 μL blood in 1 mL buffer), and latent bloodstains (5-μL bloodstain on cloth washed with detergent) can be automatically analyzed using our microsystem, reliably producing full STR profiles with a 100% calling of all the alleles. This modular-based microsystem with the capability of analyzing a wide range of samples should be able to play an increasing role in both urgent situations and routine forensic investigations, dramatically extending the applications and utility of automated DNA typing.

A fully integrated microchip system for automated forensic short tandem repeat analysis.

We have successfully developed an integrated microsystem that combines two plastic microchips for DNA extraction and PCR amplification with a glass capillary array electrophoresis chip together in a compact control and detection instrument for automated forensic short tandem repeat (STR) analysis. DNA extraction followed by an "in situ PCR" was conducted in a single reaction chamber of the microchip based on a filter paper-based extraction methodology. PCR products were then mixed with sizing standards by an injection electrode and injected into the electrophoresis chip for four-color confocal fluorescence detection. The entire STR analysis can be completed in about two hours without any human intervention. Since the 15-plex STR system has a more stringent requirement for PCR efficiency, we optimized the structure of the plastic DNA extraction and amplification chip, in which the reaction chamber was formed by sandwiching a hollow structure layer with two blank cover layers, to reduce the adsorption of PCR reagents to the surfaces. In addition, PCR additives, bovine serum albumin, poly(ethylene glycol), and more magnesium chloride were included into the on-chip multiplex STR system. The limit-of-detection study demonstrated that our microsystem was able to produce full 15-plex STR profiles from 3.75 ng standard K562 DNA. Buccal swab and whole blood samples were also successfully typed by our system, validating the feasibility of performing rapid DNA typing in a "sample-in-answer-out" manner for on-site forensic human identification.

Capillary Electrophoresis Chips for Fingerprinting Endotoxin Chemotypes and Subclasses.

Endotoxins (lipopolysaccharides, LPS; lipooligosaccharides, LOS) are components of the envelope of Gram-negative bacteria. These molecules, responsible for both advantageous and harmful biological activity of these microorganisms, are highly immunogenic and directly involved in numerous bacterial diseases in humans, such as Gram-negative sepsis. The characterization of endotoxins is of importance, since their physiological and pathophysiological effects depend on their chemical structure. The differences among the LPS from different bacterial serotypes and their mutants include variations mainly within the composition and length or missing of their O-polysaccharide chains. Microchip electrophoretic methodology enables the structural characterization of LPS molecules from several bacteria and the quantitative evaluation of components of endotoxin extracts. The improved microchip electrophoretic method is based on the direct labeling of endotoxins by covalent binding of a fluorescent dye. The classification of the S-type LPSs can be done according to their electrophoretic profiles, which are characteristics of the respective bacterial strains. According to the number, distribution, and the relative amounts of components in an endotoxin extract, it is possible to differentiate between the S-type endotoxins from different Gram-negative bacterial strains. The microchip electrophoresis affords high-resolution separation of pure and partially purified (e.g., obtained from whole-cell lysate) S and R endotoxins. This microchip technique provides a new, standardizable, fast, and sensitive method for the detection of endotoxins and for the quantitative evaluation of components of an endotoxin extract.

Development of a Capillary Electrophoresis Chip with Integrated Electrochemical Sensors

Capillary electrophoresis (CE) chips with amperometric integration offers improved portability, high detection throughput, small sample and reagent volumes, low driving voltages and sample injection and separation. This article reviews recent CE chip developments and applications in the integration of amperometric sensors. We describe the development of three different configurations (in-channel, off-channel and end-channel). CE chips with integrated EC detectors have great promise in applications including diagnostics, environmental monitoring and biological assays

Roll-to-plate fabrication of microfluidic devices with rheology-modified thiol-ene resins

In this paper, the replication possibilities of microfluidic channels by UV-roll-to-plate fabrication were investigated and a study of rheology-modified thiol-ene for the application in such a UV-roll-to-plate setup was conducted. The system allows the manufacture of channels with aspect ratios of 2:1 and a maximal channel depth of 90 μm as well as the sealing of the finished devices with patterning and sealing speeds of up to 19 m min−1. By adding fumed silica nanoparticles to the uncured resins, it was possible to alter the rheological behavior of the resin system to fabricate shallow microfluidic channels with 40 × 95 μm cross-sectional dimensions. Moreover, deeper (90 μm) channels can be fabricated with highly viscous resins based on thiol-terminated oligomers. As a demonstration, capillary electrophoresis chips were prepared and tested for a simple separation of two fluorescent dyes.

Thermal lens spectrometry in electromigration methods of analysis

We consider the basic principles, instruments, problems, and examples of application of thermal lens spectroscopy and microscopy, highly sensitive force methods of molecular absorption spectroscopy, in various embodiments of electromigration methods of analysis, particularly, capillary electrophoresis and microfluidic chip electrophoresis. Examples illustrating the sensitivity and selectivity of the combined methods are presented.

A fully automated microfluidic micellar electrokinetic chromatography analyzer for organic compound detection

An integrated microfluidic chemical analyzer utilizing micellar electrokinetic chromatography (MEKC) is developed using a pneumatically actuated Lifting-Gate microvalve array and a capillary zone electrophoresis (CZE) chip. Each of the necessary liquid handling processes such as metering, mixing, transferring, and washing steps are performed autonomously by the microvalve array. In addition, a method is presented for automated washing of the high resistance CZE channel for device reuse and periodic automated in situ analyses. To demonstrate the functionality of this MEKC platform, amino acids and thiols are labeled and efficiently separated via a fully automated program. Reproducibility of the automated programs for sample labeling and periodic in situ MEKC analysis was tested and found to be equivalent to conventional sample processing techniques for capillary electrophoresis analysis. This platform enables simple, portable, and automated chemical compound analysis which can be used in challenging environments.

Universal Microfluidic Automaton for Autonomous Sample Processing: Application to the Mars Organic Analyzer

A fully integrated multilayer microfluidic chemical analyzer for automated sample processing and labeling, as well as analysis using capillary zone electrophoresis is developed and characterized. Using lifting gate microfluidic control valve technology, a microfluidic automaton consisting of a two-dimensional microvalve cellular array is fabricated with soft lithography in a format that enables facile integration with a microfluidic capillary electrophoresis device. The programmable sample processor performs precise mixing, metering, and routing operations that can be combined to achieve automation of complex and diverse assay protocols. Sample labeling protocols for amino acid, aldehyde/ketone and carboxylic acid analysis are performed automatically followed by automated transfer and analysis by the integrated microfluidic capillary electrophoresis chip. Equivalent performance to off-chip sample processing is demonstrated for each compound class; the automated analysis resulted in a limit of detection of ~16 nM for amino acids. Our microfluidic automaton provides a fully automated, portable microfluidic analysis system capable of autonomous analysis of diverse compound classes in challenging environments.

Simple Boric Acid-Based Fluorescent Focusing for Sensing of Glucose and Glycoprotein via Multipath Moving Supramolecular Boundary Electrophoresis Chip

Boric acid-based fluorescent complex probe of BBV-HPTS (boronic acid-based benzyl viologen (BBV) and hydroxypyrene trisulfonic acid trisodium salt (HPTS)) was rarely used for sensitive sensing of saccharide (especially glycoprotein) via electrophoresis. We proposed a novel model of moving supramolecular boundary (MSB) formed with monosaccharide or glycoprotein in microcolumn and the complex probe of BBV-HPTS in the cathodic injection tube, developed a method of MSB fluorescent focusing for sensitive recognition of monosaccharide and glycoprotein, and designed a special multipath capillary electrophoresis (CE) chip for relative experiments. As a proof of concept, glucose and hemoglobin A1c (HbA1c) were respectively used as the mode saccharide and glycoprotein for the relevant demonstration. The experiments revealed that (i) the complex of BBV-HPTS could interact with free glucose or bound one in glycoprotein; (ii) the fluorescent signal was a function of glucose or glycoprotein content approximately; and (iii) interestingly the fluorescent band motion was dependent on glucose content. The developed method had the following merits: (i) low cost; (ii) low limit of detection (down to 1.39 pg/mL for glucose and 2.0 pg per capillary HbA1c); and (iii) high throughput (up to 12 runs or more per patch) and speed (less than 5 min). The developed method has potential use for sensitive monitoring of monosaccharide and glycoprotein in biomedical samples.

Integrated slidable and valveless polymerase chain reaction–capillary electrophoresis microdevice for pathogen detection

An integrated slidable and valveless polymerase chain reaction (PCR) and capillary electrophoresis (CE) microdevice has been developed for pathogen detection on a portable genetic analysis microsystem. The proposed microdevice does not need any microvalve or external pneumatic actuators, thereby simplifying the chip fabrication and the chip operation process. The slidable PCR–CE chip consists of three glass layers: a channel wafer that includes a sample injection and CE microchannel, a Ti/Pt electrode-patterned resistance temperature detector (RTD) wafer, and a slidable plate in which a PCR chamber was fabricated. First, the slidable PCR plate was placed in the sample injection zone to load a PCR cocktail, and then moved to the PCR zone for thermal cycling. After PCR, the slidable PCR chamber was switched to the CE region for separation, the resultant amplicons were then analyzed by a miniaturized laser-induced fluorescence detector. Non-specific DNA adsorption was prevented by treating the glass surface with hydrophobic decyltrichlorosilane, and the leakage and evaporation of the PCR cocktail during the movement and PCR could be minimized by surrounding the slidable chamber with mineral oil. The target protein A gene (101 bp) of Staphylococcus aureus (S. aureus) was detected with a limit of detection (LOD) of 6 copies, even with a background of 1000-fold excess of Escherichia coli K12 (E. Coli K12) templates. This demonstrates a highly sensitive and selective genetic analysis was achieved on our slidable and valveless PCR–CE microdevice.

Signal Detection of Multi-Channel Capillary Electrophoresis Chip Based on CCD

A kind of multi-channel capillary electrophoresis (CE) chip signal detection system based on CCD was developed. The output signal of the CCD sensor was processed by a series of pre-processing circuits and ADC, and then it was collected by the Field Programmable Gate Array (FPGA) chip which communicated with a host computer. The core in FPGA was designed to control the signal flow of the CCD and transfer the data to PC based on a Nios II embedded soft-processor. The application of PC was used to store the data and demonstrate the curve. The measurement of the fluorescent signals for different concentration Rhodamine B dyes is presented and the comparison with other detection systems is also discussed.

Assessment of chemokine profiles in human skin biopsies by an immunoaffinity capillary electrophoresis chip

Atopic dermatitis is a skin condition resulting in a skin rash from exposure to environmental factors. Skin biopsies taken from patients suffering from atopic dermatitis were micro-dissected and analyzed using a microchip-based immunoaffinity CE system for the presence of CXCL1, CXCL5 and CXCL8 and CCL1, CCL3 and CCL5 chemokines. Disposable immunoaffinity disks with immobilized antibodies were used to capture the CXC and CC chemokines from the homogenized skin samples. The captured analytes were then labeled with AlexaFluor 633, eluted from the disk and separated by CE. The labeled chemokines were identified and quantified by laser induced fluorescence. The total analysis time was less than 40min, including the biopsy microdissection, pre-analysis preparation of the sample and the ICE-CHIP analysis, which took less than 10min with inter- and intra-assay CV’s below 6.4%. Microchip-based immunoaffinity CE could distinguish between normal skin biopsies and those with inflammation. Patients with neutrophil cellular infiltrates by histopathology showed increased concentrations of CXCL1, CXCL5 and CXCL8 while increases of CCL1, CCL3 and CCL5 corresponded to the patient group demonstrating monocytic and T-lymphocyte infiltration by histopathology. This system demonstrates the ability to identify and quantify immunochemical analytes in frozen sections taken from clinical histopathology samples.

Digitally synchronized LCD projector for multi-color fluorescence excitation in parallel capillary electrophoresis detection

A simple method is proposed for modulating the excitation light used for multi-color fluorescence detection in a single capillary electrophoresis (CE) channel. In the proposed approach, a low-cost commercial liquid crystal device (LCD) projector with digitally-modulated LCD switches is used to provide the illumination light source and the fluorescence emitted from the CE chip is synchronously detected using an ultraviolet–visible–near infrared (UV–vis–NIR) spectrometer. The modulated light source enables the detection of multiple fluorescence signals within a single CE channel without the need of mechanically switching optical components. In order to enhance the sensing performance of the proposed system, two short-pass filters and one band-pass filter are inserted into the LCD projector to modify the wavelength spectra for fluorescence excitation. With this simple approach, the signal-to-noise (SN) ratio of the fluorescence detection signals is greatly improved by a factor of approximately 22 when detecting Atto647N fluorescent dye. The feasibility of the proposed multi-color CE detection approach is demonstrated by detecting two different samples including a mixed sample comprising FITC, Rhodamine B and Atto647N fluorescent dyes and a bio-sample composed of two ssDNAs labeled with FITC and Cy3, respectively. Results confirm that the digitally-modulated excitation system proposed in this study has significant potential for the parallel analysis of fluorescently-labeled bio-samples using a multi-color detection scheme.

Auto Focusing Confocal Laser Induced Fluorescence Detection System

Laser induced fluorescence (LIF) detection is one of the main means of Capillary Electrophoresis (CE) chip detection, in which the confocal detecting device is commonly used for its higher sensitivity and signal-to-noise ratio (SNR). Based on confocal LIF detection principle, the confocal laser induced fluorescence detecting system, which could realize the auto focusing, and auto tracking was presented, and it contained the confocal optical system, the microprocessor control system and the computer process system. This device can acquire the fluorescence data by PMT or the chip images by CCD, and 3-dimensional electric moving stage could be controlled to accomplish the auto focusing and auto tracking by image process. The device could detect or observe the CE chip data in real time.

Fast and Selective Microfluidic Chips for Electrochemical Antioxidant Sensing in Complex Samples

In this work, capillary electrophoresis chips with electrochemical detection have been assessed as creative and selective microfluidic platforms to integrate and simplify on a microscale the traditional methods for complex natural antioxidants determination. Depending on the acid-base properties of the analytes, two approaches (class-selective electrochemical index determination (CSEID) and individual antioxidant determination (IAD)) were investigated for the analysis of nine antioxidants ((+)-catechin, rutin, quercetin, chlorogenic, ferulic, caffeic, protocatechuic, vanillic, and gallic acids) in food samples. First, the novel concept of a class-selective electrochemical index is proposed allowing a fast and reliable determination of the main antioxidant classes (flavonoids and phenolic acids) in less than 100 s. In addition, an impressive separation of nine antioxidants is also offered in less than 260 s with the individual antioxidant determination approach. Qualitative and quantitative performances of both approaches were studied. The analytical figures of merit (i.e., electroosmotic flow (EOF) precision as relative standard deviation (RSD), resolution, signal precision as RSD, limit of detection, limit of quantification, and accuracy as recovery) of both approaches were <4%, approximately 1, < or = 5%, <8 microM, 30 microM, between 91% and 104%, and <4%, < or = 2%, <9%, < or = 6 microM, < or = 20 microM (with the exception of protocatechuic acid, which shows values of 40 and 130 microM, respectively), between 80% and 107% for the CSEID and IAD concepts, respectively, which are excellent for food samples analysis. A set of representative samples was analyzed including apple and pear skins and pulps, red and white wines, and green tea tablets. High agreement was observed between the results of the sample analyses from the two microchip-based approaches, and good correlation was observed with results obtained from traditional methods. Although the prominent phenolic antioxidant classes and compounds were successfully determined, some nonprominent peaks were not detected in the samples when applying the IAD approach. Different integration strategies on microchip platforms were further explored, looking for a simplification of the overall analytical process without losing the excellent analytical characteristics obtained in both approaches. The results are promising and indicative of the progress of analytical microfluidics toward the "plateau of productivity" and the routine laboratory application.

Ceramic capillary electrophoresis chip for the measurement of inorganic ions in water samples

We present a microchip capillary electrophoresis (CE) device build-up in low temperature co-fired ceramics (LTCC) multilayer technology for the analysis of major inorganic ions in water samples in less than 80 s. Contactless conductivity measurement is employed as a robust alternative to direct-contact conductivity detection schemes. The measurement electrodes are placed in a planar way at the top side of the CE chip and are realized by screen printing. Laser-cutting of channel and double-T injector structures is used to minimize irregularities and wall defects, elevating plate numbers per meter up to values of 110,000. Lowest limit of detection is 6 microM. The cost efficient LTCC module is attractive particularly for portable instruments in environmental applications because of its chemical inertness, hermeticity and easy three-dimensional integration capabilities of fluidic, electrical and mechanical components.

Electromagnetic induction detector with a vertical coil for capillary electrophoresis and microfluidic chip

We report a new electromagnetic induction detector with two vertical inductors for capillary electrophoresis and microfluidic chip capillary electrophoresis. Compared with currently available detection methods, this new detector has several advantages, including simple construction, no complicated elements, ease of assembly and operation, and potential for universal applications. The conditions affecting the response of the detector, including the number of coil turns, the frequency and the effective voltage, were optimized. Its feasibility and performance were demonstrated by analyzing inorganic ions. The new detector showed a well-defined correlation between the ion concentrations and the responses (r=0.993–0.998), with a detection limit of 5.6μmolL−1 for Mn2+, as well as good reproducibility and stability for both CE and microfluidic chip CE.

Enhanced electrophoretic DNA separation in photonic crystal fiber

Joule heating generated by the electrical current in capillary electrophoresis leads to a temperature gradient along the separation channel and consequently affects the separation quality. We describe a method of reducing the Joule heating effect by incorporating photonic crystal fiber into a micro capillary electrophoresis chip. The photonic crystal fiber consists of a bundle of extremely narrow hollow channels, which ideally work as separation columns. Electrophoretic separation of DNA fragments was simultaneously but independently carried out in 54 narrow capillaries with a diameter of 3.7 microm each. The capillary bundle offers more efficient heat dissipation owing to the high surface-to-volume ratio. Under the same electrical field strength, notable improvement in resolution was obtained in the capillary bundle chip.

Laser patterning and packaging of CCD-CE-Chips made of PMMA

The rapid manufacturing of functional capillary electrophoresis (CE) chips made of polymethylmethacrylate (PMMA) based on laser processing technologies including structuring, surface functionalization and packaging was investigated. CO 2 laser-assisted micro-patterning was demonstrated to have a great potential for the fabrication of micro-channel devices and the production capability of CO 2 laser processes has been extended to micro-channels less than 30 μm width. A high reproducibility of micro-channel geometry was attained. The average deviation between the fabricated cross-section areas and the desired cross-section areas of micro-channels was better than 3%. The packaging of these micro-structured transparent polymers was successfully established with a laser transmission welding technique using high-power diode laser radiation. CE-chips with channel widths of 150, 100 and 50μm were fabricated and electro-analytically characterized. For this purpose high frequency contactless conductivity detection (CCD) was established. Detection of small ions (Li +, Na +, and K +) with concentrations down to 0.1 × 10 −3 mol/l was performed. Analytical results compare well to those obtained by conventional hot embossing and thermal welding. Furthermore, UV-assisted surface modification can be used to influence the micro-analytical performance. The pattern qualities and analytical results show that laser-based technologies have a great potential for application in flexible and cost-efficient production of polymeric microfluidic devices.