Microchip capillary electrophoresis with an integrated indium tin oxide electrode-based electrochemiluminescence detector

This paper describes an indium tin oxide (ITO) electrode-based Ru(bPY)(3)(2+) electrochemiluminecence (ECL) detector for a microchip capillary electrophoresis (CE). The microchip CE-ECL system described in this article consists of a poly(dimethylsiloxane) (PDMS) layer containing separation and injection channels and an electrode plate with an ITO electrode fabricated by a photolithographic method. The PDMS layer was reversibly bound to the ITO electrode plate, which greatly simplified the alignment of the separation channel with the working electrode and enhanced the photon-capturing efficiency. In our study, the high separation electric field had no significant influence on the ECL detector, and decouplers for isolating the separation electric field were not needed in the microchip CE-ECL system. The ITO electrodes employed in the experiments displayed good durability and stability in the analytical procedures. Proline was selected to perform the microchip device with a limit of detection of 1.2 muM (S/N = 3) and a linear range from 5 to 600 muM.

Highly sensitive determination of the methylated p16 gene in cancer patients by microchip electrophoresis

The p16 tumor suppressor gene is inactivated by promoter region hypermethylation in many types of tumor. Recent studies showed that aberrant methylation of the p16 gene is an early event in many tumors, especially in lung cancer, and may constitute a new biomarker for early detection and monitoring of prevention trials. We detected tumor-associated aberrant hypermethylation of the p16 gene in plasma and tissue DNA from 153 specimens using a modified semi-nested methylation-specific PCR (MSP) combining plastic microchip electrophoresis or slab gel electrophoresis, respectively. Specimens were from 79 lung cancer patients, 15 abdominal tumor patients, 30 positive controls and 30 negative controls. The results showed that the positive rate obtained by microchip electrophoresis was more than 26.6% higher and the same speciticity was kept when compared with slab gel electrophoresis. The microchip electrophoresis can rapidly and accurately analyze the PCR products of methylated DNA and obviously improve the positive rate of diagnosis of cancer patients when compared with gel electrophoresis. This method with the high assay sensitivity might be used for detection of methylation of p16 gene and even to facilitate early diagnosis of cancer patients. (C) 2004 Elsevier B.V. All rights reserved.

Modification of a poly(methyl methacrylate) injection-molded microchip and its use for high performance analysis of DNA

Inexpensive and permanently modified poly(methyl methacrylate)(PMMA) microchips were fabricated by an injection-molding process. A novel sealing method for plastic microchips at room temperature was introduced. Run-to-run and chip-to-chip reproducibility was good, with relative standard deviation values between 1-3% for the run-to-run and less than 2.1% for the chip-to-chip comparisons. Acrylonitrile-butadiene-styrene (ABS) was used as an additive in PMMA substrates. The proportions of PMMA and ABS were optimized. ABS may be considered as a modifier, which obviously improved some characteristics of the microchip, such as the hydrophilicity and the electro-osmotic flow (EOF). The detection limit of Rhodamine 6G dye for the modified microchip on the home-made microchip analyzer showed a dramatic 100-fold improvement over that for the unmodified PMMA chip. A detection limit of the order of 10(-20) mole has been achieved for each injected phiX-174/HaeIII DNA fragment with the baseline separation between 271 and 281 bp, and fast separation of 11 DNA restriction fragments within 180 seconds. Analysis of a PCR product from the tobacco ACT gene was performed on the modified microchip as an application example.

Rapid authentication of ginseng species using microchip electrophoresis with laser-induced fluorescence detection

Ginseng is one of the most expensive Chinese herbal medicines and the effectiveness of ginseng depends strongly on its botanical sources and the use of different parts of the plants. In this study, a microchip electrophoresis method coupled with the polymerase chain reaction (PCR)-short tandem repeats (STR) technique was developed for rapid authentication of ginseng species. A low viscosity hydroxypropyl methylcellulose (HPMC) solution was used as the sieving matrix for separation of the amplified STR fragments. The allele sizing of the amplified PCR products could be detected within 240 s or less. Good reproducibility and accuracy of the fragment size were obtained with the relative standard deviation for the allele sizes less than 1.0% (n = 11). At two microsatellite loci (CT 12, CA 33), American ginseng had a different allele pattern on the electropherograms compared with that of the Oriental ginseng. Moreover, cultivated and wild American ginseng can be distinguished on the basis of allele sizing. This work establishes the feasibility of fast genetic authentication of ginseng species by use of microchip electrophoresis.

Simultaneous and ultrarapid determination of reactive oxygen species and reduced glutathione in apoptotic leukemia cells by microchip electrophoresis

A microchip electrophoresis method coupled with laser-induced fluorescence (LIF) detection was established for simultaneous determination of two kinds of intracellular signaling molecules (reactive oxygen species, ROS, and reduced glutathione, GSH) related to apoptosis and oxidative stress. As the probe dihydrorhodamine-123 (DHR123) can be converted intracellularly by ROS to the fluorescent rhodamine-123 (Rh123), and the probe naphthalene-2,3-dicarboxaldehyde (NDA) can react quickly with GSH to produce a fluorescent adduct, rapid determination of Rh-123 and GSH was achieved on a glass microchip within 27 s using a 20 mm borate buffer (pH 9.2). The established method was tested to measure the intracellular ROS and GSH levels in acute promyelocytic leukemia (APL)-derived NB4 cells. An elevation of intracellular ROS and depletion of GSH were observed in apoptotic N134 cells induced by arsenic trioxide (AS(2)O(3)) at low concentration (1-2 mu m). Buthionine sulfoximine (BSO), in combination with AS(2)O(3) enhanced the decrease of reduced GSH to a great extent. The combined treatment of AS(2)O(3) and hydrogen peroxide (H2O2) led to an inverse relationship between the concentrations of ROS and GSH obtained, showing the proposed method can readily evaluate the generation of ROS, which occurs simultaneously with the consumption of the inherent antioxidant.

Mini-electrochemical detector for microchip electrophoresis

This paper presents the development of a mini-electrochemical detector for microchip electrophoresis. The small size (3.6 x 5.0 cm(2), W x L) of the detector is compatible with the dimension of the microchip. The use of universal serial bus (USB) ports facilitates installation and use of the detector, miniaturizes the detector, and makes it ideal for lab-on-a-chip applications. A fixed 10 M Omega feedback resistance was chosen to convert current of the working electrode to voltage with second gain of 1, 2, 4, 8, 16, 32, 64 and 128 for small signal detection instead of adopting selectable feedback resistance. Special attention has been paid to the power support circuitry and printed circuit board (PCB) design in order to obtain good performance in such a miniature size. The working electrode potential could be varied over a range of +/-2.5 V with a resolution of 0.01 mV. The detection current ranges from -0.3 x 10(-7) A to 2.5 x 10(-7) A and the noise is lower than 1 pA. The analytical performance of the new system was demonstrated by the detection of epinephrine using an integrated PDMS/glass microchip with detection limit of 2.1 mu M (S/N = 3).

Rapid separation of biologically important low molecular weight compounds by microchip electrophoresis and ultra-fast performance liquid chromatography

The research work in this thesis reports rapid separation of biologically important low molecular weight compounds by microchip electrophoresis and ultrahigh liquid chromatography. Chapter 1 introduces the theory and principles behind capillary electrophoresis separation. An overview of the history, different modes and detection techniques coupled to CE is provided. The advantages of microchip electrophoresis are highlighted. Some aspects of metal complex analysis by capillary electrophoresis are described. Finally, the theory and different modes of the liquid chromatography technology are presented. Chapter 2 outlines the development of a method for the capillary electrophoresis of (R, S) Naproxen. Variable parameters of the separation were optimized (i.e. buffer concentration and pH, concentration of chiral selector additives, applied voltage and injection condition).The method was validated in terms of linearity, precision, and LOD. The optimized method was then transferred to a microchip electrophoresis system. Two different types of injection i.e. gated and pinched, were investigated. This microchip method represents the fastest reported chiral separation of Naproxen to date. Chapter 3 reports ultra-fast separation of aromatic amino acid by capillary electrophoresis using the short-end technique. Variable parameters of the separation were optimized and validated. The optimized method was then transferred to a microchip electrophoresis system where the separation time was further reduced. Chapter 4 outlines the use of microchip electrophoresis as an efficient tool for analysis of aluminium complexes. A 2.5 cm channel with linear imaging UV detection was used to separate and detect aluminium-dopamine complex and free dopamine. For the first time, a baseline, separation of aluminium dopamine was achieved on a 15 seconds timescale. Chapter 5 investigates a rapid, ultra-sensitive and highly efficient method for quantification of histamine in human psoriatic plaques using microdialysis and ultrahigh performance liquid chromatography with fluorescence detection. The method utilized a sub-two-micron packed C18 stationary phase. A fluorescent reagent, 4-(1-pyrene) butyric acid N-hydroxysuccinimide ester was conjugated to the primary and secondary amino moieties of histamine. The dipyrene-labeled histamine in human urine was also investigated by ultrahigh pressure liquid chromatography using a C18 column with 1.8 μm particle diameter. These methods represent one of the fastest reported separations to date of histamine using fluorescence detection.

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-mu m 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.

Dynamic Solid Phase DNA Extraction and PCR Amplification in Polyester-Toner Based Microchip

A variety of substrates have been used for fabrication of microchips for DNA extraction, PCR amplification, and DNA fragment separation, including the more conventional glass and silicon as well as alternative polymer-based materials. Polyester represents one such polymer, and the laser-printing of toner onto polyester films has been shown to be effective for generating polyester-toner (PeT) microfluidic devices with channel depths on the order of tens of micrometers. Here, we describe a novel and simple process that allows for the production of multilayer, high aspect-ratio PeT microdevices with substantially larger channel depths. This innovative process utilizes a CO(2) laser to create the microchannel in polyester sheets containing a uniform layer of printed toner, and multilayer devices can easily be constructed by sandwiching the channel layer between uncoated cover sheets of polyester containing precut access holes. The process allows the fabrication of deep channels, with similar to 270 mu m, and we demonstrate the effectiveness of multilayer PeT microchips for dynamic solid phase extraction (dSPE) and PCR amplification. With the former, we found that (i) more than 65% of DNA from 0.6 mu L of blood was recovered, (ii) the resultant DNA was concentrated to greater than 3 ng/mu L., (which was better than other chip-based extraction methods), and (iii) the DNA recovered was compatible with downstream microchip-based PCR amplification. Illustrative of the compatibility of PeT microchips with the PCR process, the successful amplification of a 520 bp fragment of lambda-phage DNA in a conventional thermocycler is shown. The ability to handle the diverse chemistries associated with DNA purification and extraction is a testimony to the potential utility of PeT microchips beyond separations and presents a promising new disposable platform for genetic analysis that is low cost and easy to fabricate.

Evaluación de dos anestésicos y metodología de marcaje individual con microchip subcutáneo en Octopus vulgaris

[EN]The present work evaluates, on one hand, two anaesthetic agents to facilitate octopus handling concluding that a 1.5% of ethanol (96%) in sea water (22ºC) shows rapid anaesthetic-recovery time, indicating its suitability for this species. On the other hand, a protocol for implanting Octopus vulgaris with subcutaneous PIT tags is established and after 10 weeks a 94% retention was found with 100% survival. Retention rises to 98,6% when tagging animals weighing more than 500 g.

Implementazione di protocolli automatici per la caratterizzazione di microchip mediante interfaccia labview

Uno dei principali passi della catena di produzione di circuiti integrati è quello di testare e valutare una serie di chip campione per verificare che essi rientrino nei valori e nelle specifiche scelte. Si tratta di un passaggio molto importante che determina le caratteristiche del prodotto nella realtà, mostrando le proprie capacità o i propri limiti, permettendo così di valutare un’eventuale produzione su larga scala. Ci permette inoltre di stimare quali dei chip rispetto agli altri presi in esame è migliore in alcuni aspetti, oppure quale risulta più lontano dalle specifiche volute. Il lavoro alle spalle di questa tesi è proprio questo: si è cercato di caratterizzare un microchip chiamato Carbonio, nato nei laboratori della II Facoltà di Ingegneria di Cesena, creando un banco di misura automatico, tramite l’ausilio del software Labview e di una scheda hardware realizzata ad hoc, che desse la possibilità di eseguire alcuni test consecutivi su ogni singolo circuito integrato in modo da caratterizzarlo estrapolando tutte le informazioni cercate e verificandone il funzionamento. Tutti i valori estratti sono stati poi sottoposti a una breve analisi statistica per stabilire per esempio quale circuito integrato fosse meno immune ai disturbi dovuti al rumore elettrico oppure per eseguire un’indagine al fine di vedere come i valori dei parametri scelti si disponessero rispetto ai lori rispettivi valori medi.

Diagnostic microchip to assay 3D colony-growth potential of captured circulating tumor cells

Microfluidic technology has been successfully applied to isolate very rare tumor-derived epithelial cells (circulating tumor cells, CTCs) from blood with relatively high yield and purity, opening up exciting prospects for early detection of cancer. However, a major limitation of state-of-the-art CTC-chips is their inability to characterize the behavior and function of captured CTCs, for example to obtain information on proliferative and invasive properties or, ultimately, tumor re-initiating potential. Although CTCs can be efficiently immunostained with markers reporting phenotype or fate (e.g. apoptosis, proliferation), it has not yet been possible to reliably grow captured CTCs over long periods of time and at single cell level. It is challenging to remove CTCs from a microchip after capture, therefore such analyses should ideally be performed directly on-chip. To address this challenge, we merged CTC capture with three-dimensional (3D) tumor cell culture on the same microfluidic platform. PC3 prostate cancer cells were isolated from spiked blood on a transparent PDMS CTC-chip, encapsulated on-chip in a biomimetic hydrogel matrix (QGel™) that was formed in situ, and their clonal 3D spheroid growth potential was assessed by microscopy over one week in culture. The possibility to clonally expand a subset of captured CTCs in a near-physiological in vitro model adds an important element to the expanding CTC-chip toolbox that ultimately should improve prediction of treatment responses and disease progression.

Modeling of electroosmotic and electrophoretic mobilization in capillary and microchip isoelectric focusing

Our dynamic capillary electrophoresis model which uses material specific input data for estimation of electroosmosis was applied to investigate fundamental aspects of isoelectric focusing (IEF) in capillaries or microchannels made from bare fused-silica (FS), FS coated with a sulfonated polymer, polymethylmethacrylate (PMMA) and poly(dimethylsiloxane) (PDMS). Input data were generated via determination of the electroosmotic flow (EOF) using buffers with varying pH and ionic strength. Two models are distinguished, one that neglects changes of ionic strength and one that includes the dependence between electroosmotic mobility and ionic strength. For each configuration, the models provide insight into the magnitude and dynamics of electroosmosis. The contribution of each electrophoretic zone to the net EOF is thereby visualized and the amount of EOF required for the detection of the zone structures at a particular location along the capillary, including at its end for MS detection, is predicted. For bare FS, PDMS and PMMA, simulations reveal that EOF is decreasing with time and that the entire IEF process is characterized by the asymptotic formation of a stationary steady-state zone configuration in which electrophoretic transport and electroosmotic zone displacement are opposite and of equal magnitude. The location of immobilization of the boundary between anolyte and most acidic carrier ampholyte is dependent on EOF, i.e. capillary material and anolyte. Overall time intervals for reaching this state in microchannels produced by PDMS and PMMA are predicted to be similar and about twice as long compared to uncoated FS. Additional mobilization for the detection of the entire pH gradient at the capillary end is required. Using concomitant electrophoretic mobilization with an acid as coanion in the catholyte is shown to provide sufficient additional cathodic transport for that purpose. FS capillaries dynamically double coated with polybrene and poly(vinylsulfonate) are predicted to provide sufficient electroosmotic pumping for detection of the entire IEF gradient at the cathodic column end.

High depth-resolution laser ablation chemical analysis of additive-assisted Cu electroplating for microchip architectures

Laser ablation/ionisation mass spectrometry with a vertical resolution at a nanometre scale was applied for the quantitative characterisation of the chemical composition of additive-assisted Cu electroplated deposits used in the microchip industry. The detailed chemical analysis complements information gathered by optical techniques and allows new insights into the metal deposition process.