Microchip for continuous DNA analysis based on gel electrophoresis coupled with co-injection of size markers and in-channel staining.

A continuous-flow microchip enabling high-accuracy DNA analysis was developed. Serial consecutive analysis for multiple amplified DNA samples was demonstrated. The sample segments were continuously introduced to the microchip from the PCR device which was interfaced to the microchip through capillary tubing. Electrokinetic co-injection of the DNA samples with size marker enabled reproducible and reliable injection of the DNAs into the gel-filled separation channel providing accurate size determination of the DNA samples. Cross-contamination between serially introduced DNA samples was minimized by plugging a washing solution segment following the previous sample segment between two sample plugs. Using this microchip, continuous separation of multiple samples was performed without any inconvenient and labor-intensive sample preparation steps such as sample mixing, staining, and gel loading which are necessary for conventional gel electrophoresis. It has taken about 4 min to separate single DNA sample and taken 37 min for three serially injected samples which implies that this microchip can be a platform device for fast as well as highly accurate DNA analysis.

Carbon dots stabilized silver-lipid nano hybrids for sensitive label free DNA detection.

Carbon dots have been extensively used for the development of fluorescent based molecular affinity sensors. However, label free DNA sensing by electrochemical method is not reported so far. Herein, we report carbon dots stabilized silver nanoparticles (CD-AgNPs) lipid nano hybrids as a sensitive and selective platform for label free electrochemical DNA sensing. The CD-AgNPs were synthesized by wet chemical method and then characterized by UV-visible, Fourier-transform Infra-red (FT-IR), dynamic light scattering (DLS) and high resolution transmission electron microscopy (HR-TEM) techniques. These CD-AgNPs were used for decorating the binary lipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and N-[1-(2,3-Dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTAP) surface (named as lipid) and tethered on self-assembled monolayer of 3-mercaptopropionic acid (MPA) (MPA-lipid-CD-AgNPs). The formation of array of MPA-lipid-CD-AgNPs on Au electrode was confirmed by atomic force microscopy (AFM). Electrochemical behavior of MPA- lipid-CD-AgNPs was monitored in the presence of 1 mM potassium ferri/ferrocyanide (K3/K4 [Fe(CN)6]). The formation of layer-by-layer MPA-lipid-CD-AgNPs is indicated by increased anodic and cathodic peak (ΔEp) separation with decreased redox peak current of K3/K4 [Fe(CN)6]. Short chain DNA (30 mer oligonucleotide, representing the lung cancer) was used as a model system for label free DNA sensing. Un-hybridized (single stranded DNA), hybridized (complementary hybridized), single, double and triple base mismatched target DNA hybridized surfaces were efficiently discriminated at 1 µM target DNA concentration at the Au/MPA-lipid-CD-AgNPs electrode by change in the charge transfer resistance from impedance technique. Further, the modified electrode was successfully used to determine target DNA in a wide linear range from 10-16 to 10-11 M. The present work open doors for the utilization of CDs in molecular affinity based electrochemical sensor design and development.

A hydrophobic ionic liquid compartmentalized sampling/labeling and its separation techniques in polydimethylsiloxane microchip capillary electrophoresis.

This paper demonstrates a novel compartmentalized sampling/labeling method and its separation techniques using a hydrophobic ionic liquid (IL)-1-butyl-3-methylimidazolium bis(trifluoromethylsulfonyl)-imidate (BmimNTf2 )-as the immiscible phase, which is capable of minimizing signal losses during microchip capillary electrophoresis (MCE). The MCE device consists of a silica tube connected to a straight polydimethylsiloxane (PDMS) separation channel. Poly(diallyldimethylammonium chloride) (PDDAC) was coated on the inner surface of channel to ease the introduction of IL plugs and enhance the IL wetting on the PDMS surface for sample releasing. Electroosmotic flow (EOF)-based sample compartmentalization was carried out through a sequenced injection into sampling tubes with the following order: leading IL plug/sample segment/terminal IL plug. The movement of the sample segment was easily controlled by applying an electrical voltage across both ends of the chip without a sample volume change. This approach effectively prevented analyte diffusion before injection into MCE channels. When the sample segment was manipulated to the PDDAC-modified PDMS channel, the sample plug then was released from isolation under EOF while IL plugs adsorbed onto channel surfaces owing to strong adhesion. A mixture of flavin adenine nucleotides (FAD) and flavin mononucleotides (FMN) was successfully separated on a 2.5 cm long separation channel, for which the theoretical numbers of plates were 15 000 and 17 000, respectively. The obtained peak intensity was increased 6.3-fold over the corresponding value from conventional electrokinetic injection with the same sampling time. Furthermore, based on the compartmented sample segment serving as an interim reactor, an on-chip fluorescence labeling is demonstrated.

Acupuncture injection for field amplified sample stacking and glass microchip-based capillary gel electrophoresis.

Acupuncture sample injection is a simple method to deliver well-defined nanoliter-scale sample plugs in PDMS microfluidic channels. This acupuncture injection method in microchip CE has several advantages, including minimization of sample consumption, the capability of serial injections of different sample solutions into the same microchannel, and the capability of injecting sample plugs into any desired position of a microchannel. Herein, we demonstrate that the simple and cost-effective acupuncture sample injection method can be used for PDMS microchip-based field amplified sample stacking in the most simplified straight channel by applying a single potential. We achieved the increase in electropherogram signals for the case of sample stacking. Furthermore, we present that microchip CGE of ΦX174 DNA-HaeⅢ digest can be performed with the acupuncture injection method on a glass microchip while minimizing sample loss and voltage control hardware.

Parallel-processing continuous-flow device for optimization-free polymerase chain reaction.

A parallel-processing four-station polymerase chain reaction (PCR) device has been developed, which performs continuous-flow PCR without optimization of the annealing temperature. Since the annealing temperature of each station can be controlled independently, the device covers an annealing temperature range of 50-68 °C, which is wide enough to perform PCR for any DNA fragment regardless of its optimum annealing condition. This arrangement lets us continuously obtain an amplified amount of a DNA fragment at least from one of the stations. The device consists of four identical cylindrical stations (diameter 20 mm, height 55 mm). A polytetrafluoroethylene capillary reactor (length 2 m, I.D. 100 µm, O.D. 400 µm) is wound helically up around each station. The whole assembly is designed to minimize the number of heating blocks (for providing temperatures of denaturation, annealing, and extension) to be seven and to shape a compact cube (height 55 mm, base 60 mm × 60 mm). The reproducibility for continuous-flow PCR is reasonably high (run-to-run and station-to-station relative standard deviation of their amplification is lower than 6 % and about 4 %, respectively). Performance on the optimization-free DNA amplification has been evaluated with four DNA samples with different annealing conditions and product sizes (323, 608, 828, and 1101 bp), which has demonstrated that in all cases, PCR is successful at least on one station. In addition, three DNA fragments with different lengths (323, 1101, and 2836 bp) have been successfully amplified in a segmented-flow mode without the carry-over contamination between segments. This result suggests that this device could serve as the PCR module of a continuous-flow high-throughput on-line total DNA analysis system integrating all necessary modules from cell lysis/DNA extraction to PCR product analysis.

Acupuncture Sample Injection for Microchip Capillary Electrophoresis and Electrokinetic Chromatography.

A simple nanoliter-scale injection technique was developed for polydimethylsiloxane (PDMS) microfluidic devices to form the well-defined sample plugs in microfluidic channels. Sample injection was achieved by performing acupuncture on a channel with a needle and applying external pressure to a syringe. This technique allowed us to achieve reproducible injection of a 3-nL segment into a microchannel for PDMS microchip-based capillary electrophoresis (CE). Capillary zone electrophoresis (CZE) and capillary electrochromatography (CEC) with bead packing were successfully performed by applying a single potential in the most simplified straight channel. The advantages of this acupuncture injection over the electrokinetic injection in microchip CE include capability of minimizing sample loss and voltage control hardware, capability of serial injections of different sample solutions into a same microchannel, capability of injecting sample plugs into any position of a microchannel, independence on sample solutions during the loading step, and ease in making microchips due to the straight channel, etc.

Standard addition/absorption detection microfluidic system for salt error-free nitrite determination.

A continuous-flow microfluidic chip-based standard addition/absorption detection system has been developed for accurate determination of nitrite in water of varying salinity. The absorption detection of nitrite is made via color development using the Griess reaction. We have found the yield of the reaction is significantly affected by salinity (e.g., -12% error for 30‰ NaCl, 50.0 µg L(-1)N-NO2(-) solution). The microchip has been designed to perform standard addition, color development, and absorbance detection in sequence. To effectively block stray light, the microchip made from black poly(dimethylsiloxane) is placed on the top of a compact housing that accommodates a light-emitting diode, a photomultiplier tube, and an interference filter, where the light source and the detector are optically isolated. An 80-mm liquid-core waveguide mounted on the chip externally has been employed as the absorption detection flow cell. These designs for optics secure a wide linear response range (up to 500 µg L(-1)N-NO2(-)) and a low detection limit (0.12 µg L(-1)N-NO2(-) = 8.6 nM N-NO2(-), S/N = 3). From determination of nitrite in standard samples and real samples collected from an estuary, it has been demonstrated that our microfluidic system is highly accurate (<1% RSD, n = 3) and precise (<1% RSD, n = 3).

Formation of lipid bilayer membrane in a poly(dimethylsiloxane) microchip integrated with a stacked polycarbonate membrane support and an on-site nanoinjector.

This paper describes a new and facile approach for the formation of pore-spanning bilayer lipid membranes (BLMs) within a poly(dimethylsiloxane) (PDMS) microfluidic device. Commercially, readily available polycarbonate (PC) membranes are employed for the support of BLMs. PC sheets with 5 µm, 2 µm, and 0.4 µm pore diameters, respectively, are thermally bonded into a multilayer-stack, reducing the pore density of 0.4 µm-pore PC by a factor of 200. The BLMs on this support are considerably stable (a mean lifetime: 17 h). This multilayer-stack PC (MSPC) membrane is integrated into the PDMS chip by an epoxy bonding method developed to secure durable bonding under the use of organic solvents. The microchip has a special channel for guiding a micropipette in the proximity of the MSPC support. With this on-site injection technique, tens to hundreds of nanoliters of solutions can be directly dispensed to the support. Incorporating gramicidin ion channels into BLMs on the MSPC support has confirmed the formation of single BLMs, which is based on the observation from current signals of 20 pS conductance that is typical to single channel opening. Based on the bilayer capacitance (1.4 pF), about 15% of through pores across the MSPC membrane are estimated to be covered with BLMs.

Polypyrrole/Agarose-based electronically conductive and reversibly restorable hydrogel.

Conductive hydrogels are a class of composite materials that consist of hydrated and conducting polymers. Due to the mechanical similarity to biointerfaces such as human skin, conductive hydrogels have been primarily utilized as bioelectrodes, specifically neuroprosthetic electrodes, in an attempt to replace metallic electrodes by enhancing the mechanical properties and long-term stability of the electrodes within living organisms. Here, we report a conductive, smart hydrogel, which is thermoplastic and self-healing owing to its unique properties of reversible liquefaction and gelation in response to thermal stimuli. In addition, we demonstrated that our conductive hydrogel could be utilized to fabricate bendable, stretchable, and patternable electrodes directly on human skin. The excellent mechanical and thermal properties of our hydrogel make it potentially useful in a variety of biomedical applications such as electronic skin.

Nanoband electrode for high-performance in-channel amperometric detection in dual-channel microchip capillary electrophoresis.

A nanoband electrode detector integrated with a dual-channel polydimethylsiloxane microchip is proposed for in-channel amperometric detection in microchip capillary electrophoresis. Gold nanoband electrodes, which were fabricated on SU-8 substrates with a 100-nm-width gold layer, were introduced into the dual-channel microchip to be an electrochemical detector. Due to the nano-sized width of the detector, the noise of the amperometric detection was significantly reduced, and a high separation resolution was achieved for monitoring the analytes. The detection sensitivity of the system was improved by high signal-to-noise ratio, and a low detection limit on microchip was obtained for p-aminophenol (2.09 nM). Because of the high resolution in measuring half-peak width, the plate number that is used to evaluate the separation efficiency was 1.5-fold higher than that using 50-µm-width electrochemical detector. The effect of sample injection time and data acquisition time on separation efficiency was investigated, and an attractive separation efficiency was achieved with a plate number up to 17,500.

Quantitative analysis of a prostate-specific antigen in serum using fluorescence immunochromatography.

A quantitative analysis of prostate-specific antigen (PSA) in samples of human blood serum by fluorescence immunochromatography using monoclonal antibodies to PSA was developed. The fluorescence immunochromatographic analysis system is composed of anti-PSA-monoclonal antibody (mAb), fluorescence conjugates in detection solution, a immunochromatographic assay strip, and a laser fluorescence scanner. A fluorescence immunochromatographic analysis system was employed to detect PSA on the basis of the area ratio between the control line and the test line of the strip. Under optimal conditions, the area ratio was proportional to PSA concentration ranging from 0.72 to 46.0 ng/mL with a detection limit of 0.72 ng/mL.

Determination of trace amount of cyanobacterial toxin in water by microchip based enzyme-linked immunosorbent assay.

Routine monitoring of microcystin in natural waters is difficult because the concentration of the toxin is usually lower than the detection limits. As a more sensitive detection method for microcystin, we developed a microchip based enzyme-linked immunosorbent assay (ELISA) based on monoclonal antibodies. New monoclonal antibodies against the microcystin leucine-arginine variant (MCLR), a cyclic peptide toxin of the freshwater cyanobacterium Microcystis aeruginosa, were prepared from cloned hybridoma cell lines. We used keyhole limpet hemocyanin(KLH)-conjugated MCLR as an immunogen for the production of mouse monoclonal antibody. The immunization, cell fusion, and screening of hybridoma cells producing anti-MCLR antibody were conducted. Since the ELISA test was highly sensitive, the newly developed microchip based ELISA can be suitable for the trace analysis of cyanobacterial hepatotoxins, microcystins in water. The linear responses of monoclonal antibodies with different concentrations of microcystin LR were established between 0.025 and 0.3 ng/mL.

Label free electrochemical DNA hybridization discrimination effects at the binary and ternary mixed monolayers of single stranded DNA/diluent/s in presence of cationic intercalators.

Electrochemical label free DNA hybridization discrimination of the brain tumor sequence CK20 has been made at the gold-thiol and thiol diluent binary and ternary mixed monolayer interfaces in presence of the [Fe(CN)6](3-) and double stranded DNA (dsDNA) specific cationic intercalators, proflavine (PF) and methylene blue (MB), respectively. Thiol hexane labeled single stranded DNA (HS-ssDNA) and thiol diluents such as 6-mercapto-1-hexanol (MCH) and 3-mercaptopropionic acid (MPA) are used to construct the mixed monolayers. Change in the peak-to-peak separation (Delta Ep) for the [Fe(CN)6](3-) redox reaction indicates the efficiency of the diluents in removing the randomly oriented HS-ssDNA. Smaller Delta Ep 248 mV noticed for the HS-ssDNA/MPA compared to the HS-ssDNA/MCH mixed monolayers (812 mV) indicates the less influence of the MCH diluent on the arrangement of HS-ssDNA layer. However, the hybridization discrimination effect negotiated in presence of both the [Fe(CN)6](3-) and PF intercalator showed zero effect for the HS-ssDNA/MPA interface, and approximately 20-30% effect for the HS-ssDNA/MCH interface. The discrimination effect at the HS-ssDNA/MPA interface further increased to 80% by inserting the MCH at the local defects to form a multicomponent ternary HS-ssDNA/MPA/MCH layer interface. These differential discrimination effects are attributed to the formation of compact and/or defective layer structures, evidenced from their reductive desorption voltammetry in 0.5M KOH. The presence of single base (C-A) mismatch in the hybrid is diagnosed by a decrease in coulometric charge compared to the perfect dsDNA. The target concentration of 10 pM is detected selectively and sensitively.

Dual-channel method for interference-free in-channel amperometric detection in microchip capillary electrophoresis.

A novel in-channel amperometric detection method for microchip capillary electrophoresis (CE) has been developed to avoid the interference from applied potential used in the CE separation. Instead of a single separation channel as in conventional CE microchips, we use a dual-channel configuration consisting of two different parallel separation and reference channels. A working electrode (WE) and a reference electrode (RE) are placed equally at a distance 200 microm from its outlet on each channel. Running buffer flows through the reference channel. Our dual-channel CE microchips consist of a poly(dimethylsiloxane) (PDMS) upper plate and a glass lower plate to form a PDMS/glass hybrid chip. Amperometric signals are measured without any potential shift and interference from the applied CE potential, and CE separation maintains its high resolution because this in-channel configuration does not allow additional band broadening that is notorious in end-channel and off-channel configurations. The high performance of this new in-channel electrochemical detection methodology for CE has been demonstrated by analyzing a mixture of electrochemically active biomolecules: dopamine (DA), norepinephrine, and catechol. We have achieved a 0.1 pA detectability from the analysis of DA, which corresponds to a 1.8 nM concentration.

Precolumn diastereomerization and micellar electrokinetic chromatography on a plastic microchip: rapid chiral analysis of amino acids.

Precolumn derivatization and chiral separation of DL-amino acids based on diastereomerization have been performed on an integrated poly(dimethylsiloxane) microchip. Diastereomeric derivatives were formed in a microfabricated precolumn reactor by the reaction of amino acid enantiomers with o-phthaldialdehyde/2,3,4,6-tetra-O-acetyl-1-thio-beta-D-glucopyranose (OPA/TATG), and separated by MEKC in an achiral environment without chiral selectors in the running buffer. Optimized precolumn reactions and chiral separations of amino acids were achieved within 2.5 min. Resolutions of diastereomers of OPA/TATG-amino acids were in the range of 2.5-6.1 at optimized separation conditions. Simultaneous separation of a mixture of five chiral amino acids was successfully performed in a single run in less than 100 s.

Synthesis of cage-type molecules with pi-cavity and selective gas-phase cation complexation.

[structure: see text] Cage-type molecules composed of phenyl walls and caps were synthesized as hosts for the binding of ammonium and alkali metal cations through cation-pi interactions. The synthesis involved a key cyclization step, which was markedly dependent on the capping component. Binding studies by electrospray ionization mass spectrometry toward lithium, sodium, potassium, and ammonium cations showed that the cage-type molecules selectively form a 1:1 complex. A competitive binding study showed that cage 3c (R = Et, R' = OMe) has a preference toward lithium cation while cage 4b (R = Me, R' = OMe) has a similar preference toward both lithium and ammonium ion in the presence of others. This selectivity pattern was tentatively explained by the gate size of the cage-type compounds, not by their cavity size.

Integrated light collimating system for extended optical-path-length absorbance detection in microchip-based capillary electrophoresis.

We have developed an integrated light collimating system with a microlens and a pair of slits for extended optical path length absorbance detection in a capillary electrophoresis (CE) microchip. The collimating system is made of the same material as the chip, poly(dimethylsiloxane) (PDMS), and it is integrated into the chip during the molding of the CE microchannels. In this microchip, the centers of an extended 500-microm detection cell and two optical fibers are self-aligned, and a planoconvex microlens (r = 50 microm) for light collimation is placed in front of a light-delivering fiber. To block stray light, two rectangular apertures, realized by a specially designed three-dimensional microchannel, are made on each end of the detection cell. In comparison to conventional extended detection cell having no collimator, the percentage of stray radiation readout fraction in the collimator integrated detection cell is significantly reduced from 31.6 to 3.8%. The effective optical path length is increased from 324 to 460 microm in the collimator integrated detection cell. The detection sensitivity is increased by 10 times in the newly developed absorbance detection cell as compared to an unextended, 50-microm-long detection cell. The concentration detection limit (S/N = 3) for fluorescein in the collimator integrated detection cell is 1.2 microM at the absorbance detection limit of 0.001 AU.

Structural characterization of the molten globule state of apomyoglobin by limited proteolysis and HPLC-mass spectrometry.

A method to characterize the structural conformation of an acidic molten globule apomyoglobin (apoMb) at pH 4.2 was developed using limited proteolysis and HPLC-mass spectrometry (HPLC-MS). Endoproteinase Glu-C, which has a double maximum activity at pH 4.0 and pH 7.8 toward glutamic acid (Glu), was used as a proteolytic enzyme. Using this method enabled us to compare the proteolytic cleavages of native apoMb (at pH 8.0) and molten globule (at pH 4.2) directly. Only the first cleavage event in each molecule was considered as reflecting original structural information since the original structure of the protein can be altered after the fist cleavage. Structural changes of apoMb in various pH conditions were studied here to elucidate the local helicity of molten globule apoMb. Among 13 Glu sites, only Glu83 and Glu85 in the F-helix were cleaved at pH 8.0, which confirms that only helix F is frayed upon removal of heme group. At acidic molten globule state, rapid cleavages at Glu38, Glu52, Glu54, Glu85, and Glu148 were detected, while the remaining eight sites were protected. Glu6 and Glu18 in the A-helix, and Glu105 in the G-helix were protected due to the helicity of the secondary structures. The cleavage at Glu38 and the protection at Glu41 in the C-helix indicate that the first half of the C-helix is frayed and the second half of the C-helix is structured. Cleavage at both Glu52 and Glu54 in the D-helix proves that the D-helix is disordered. The N-terminal end of the E-helix at Glu59 was protected, and the beginning of the F-helix was protected by aid of the pH-induced C-cap of the E-helix. The cleavage at Glu148 in H suggests that the C-terminal end of the H-helix is disordered. The A-helix and the first half of the B-helix were highly stable.

Electrochemical sensing of DNA hybridization based on duplex-specific charge compensation.

A nonlabeling voltammetric detection method for DNA hybridization has been developed, in which [Fe(CN)(6)](3-) in solution can readily approach an electrode surface covered with a charge-compensated DNA duplex layer and thus provides a strong redox-sensing current. Charge compensation for negative charges on the DNA backbone has been specifically accomplished on DNA duplexes by discouraging nonspecific binding of positively charged intercalating molecules with single strands. A pretreatment of DNA-modified electrodes with sodium dodecyl sulfate before the intercalator binding process is essential in preventing the nonspecific binding. Since ferricyanide, the only electrochemically active species, is present in the voltammetric solution, the detection signal can be amplified by increasing its concentration. Combination of the duplex-specific charge compensation with the signal amplification has achieved a remarkable signal difference: in 30 mM [Fe(CN)(6)](3-), the area ratio between cyclic voltammograms of the hybridized and unhybridized electrodes is approximately 200 when 3,6-diaminoacridine is used as the intercalator. High sensitivity of the method has been demonstrated by detecting 10 fM (100 zmol in amount) of a target probe DNA.