Rapid fabrication of electrochemical enzyme sensor chip using polydimethylsiloxane microfluidic channel

Applicability of polydimethylsiloxane (PDMS) for easy and rapid fabrication of enzyme sensor chips, based on electrochemical detection, is examined. The sensor chip consists of PDMS substrate with a microfluidic channel fabricated in it, and a glass substrate with enzyme-modified microelectrodes. The two substrates are clamped together between plastic plates. The sensor chip has shown no leakage around the microelectrodes under continuous solution flow (34 μl/min). Amperometric response of the sensor chips developed in this work suggest that various types of enzyme sensors can be designed by using PDMS microfluidic channels.     

Distillation and detection of SO2 using a microfluidic chip

A miniaturized distillation system is presented for separating sulfurous acid (H(2)SO(3)) into sulfur dioxide (SO(2)) and water (H(2)O). The major components of the proposed system include a microfluidic distillation chip, a power control module, and a carrier gas pressure control module. The microfluidic chip is patterned using a commercial CO(2) laser and comprises a serpentine channel, a heating zone, a buffer zone, a cooling zone, and a collection tank. In the proposed device, the H(2)SO(3) solution is injected into the microfluidic chip and is separated into SO(2) and H(2)O via an appropriate control of the distillation time and temperature. The gaseous SO(2) is then transported into the collection chamber by the carrier gas and is mixed with DI water. Finally, the SO(2) concentration is deduced from the absorbance measurements obtained using a spectrophotometer. The experimental results show that a correlation coefficient of R(2) = 0.9981 and a distillation efficiency as high as 94.6% are obtained for H(2)SO(3) solutions with SO(2) concentrations in the range of 100-500 ppm. The SO(2) concentrations of two commercial red wines are successfully detected using the developed device. Overall, the results presented in this study show that the proposed system provides a compact and reliable tool for SO(2) concentration measurement purposes.     

Rapid DNA hybridization analysis using a PDMS microfluidic sensor and a molecular beacon.

A rapid DNA analysis has been developed based on a fluorescence intensity change of a molecular beacon in a PDMS microfluidic channel. Recently, we reported a new analytical method of DNA hybridization involving a PDMS microfluidic sensor using fluorescence energy transfer (FRET). However, there are some limitations in its application to real DNA samples because the target DNA must be labelled with a suitable fluorescent dye. To resolve this problem, we have developed a new DNA microfluidic sensor using a molecular beacon. By monitoring the change in the restored fluorescence intensity along the channel length, it is possible to rapidly detect any hybridization of the molecular beacon to the target DNA. In this case, the target DNA does not need to be labelled. Our experimental results demonstrate that this microfluidic sensor using a molecular beacon is a promising diagnostic tool for rapid DNA hybridization analysis.     

Rapid detection of a cocaine-binding aptamer using biological nanopores on a chip

This paper describes a methodology for the rapid and highly selective detection of cocaine using a membrane protein channel combined with a DNA aptamer. The DNA aptamer recognizes the cocaine molecule with high selectivity. We successfully detected a low concentration of cocaine (300 ng/mL, the drug test cutoff limit) within 60 s using a biological nanopore embedded in a microchip.     

Plant microRNA detection using the circular code information

In this paper we present a new computational method leading to the identification of a new property in the plant microRNAs. This property which is based on the circular code information is then used to detect microRNAs in plants. The common C(3) circular code X is a set of 20 trinucleotides identified in the reading frames of both eukaryotic and prokaryotic genes allowing retrieval of any frame in genes, locally anywhere in the three frames (reading frame and its two shifted frames) and automatically with the same window length of 13 nucleotides in each frame. This code X is detected around the beginning of microRNAs. This method based only on the internal structure of genes, i.e. the circular code, allows sensible and precise microRNA site identification in precursor microRNAs with a sliding window of only 14 nucleotides.     

Rapid detection of dysfunctional high-density lipoproteins using isoelectric focusing-based microfluidic device to diagnose senescence-related disease

Recently, we reported elevated levels of advanced glycated end products (AGEs) in human high-density lipoproteins (HDL), with fragmentation of apoA-I in an elderly group, compared with a younger group. More dysfunctional HDL from human plasma was demonstrated in the elderly group, including reconstituted HDL containing glycated apoA-I (gA-I-rHDL) with elevation of AGEs. Based on SDS-PAGE analysis, HDL(3) from the elderly group (E-HDL(3)) exhibited increased multimerization with increased smear band intensity compared to HDL(3) from the younger group (Y-HDL(3)). According to isoelectric focusing gel analysis, gA-I-rHDL and E-HDL(3) showed electromobility to the basic region of pH with a broader band range. In a microfluidic channel, E-HDL(3) had faster mobility with a broader range and a higher isoelectric point (pI, approximately 8.1), whereas Y-HDL(3) showed a narrow band range with a lower pI (approximately 6.9). In conclusion, gA-I-rHDL and E-HDL share several electrophoretic properties with multimerization and faster mobility in microfluidic channels, depending on the isoelectric point. These results can be applied to develop a rapid detection system for modified HDL to predict the extent of aging and aging-related metabolic diseases, such as cardiovascular disease and diabetes.     

Rapid point-of-care concentration of bacteria in a disposable microfluidic device using meniscus dragging effect

We report a low cost, disposable polymer microfluidic sample preparation device to perform rapid concentration of bacteria from liquid samples using enhanced evaporation targeted at downstream detection using surface enhanced Raman spectroscopy (SERS). The device is composed of a poly(dimethylsiloxane) (PDMS) liquid sample flow layer, a reusable metal airflow layer, and a porous PTFE (Teflon?) membrane sandwiched in between the liquid and air layers. The concentration capacity of the device was successfully demonstrated with fluorescently tagged Escherichia coli (E. coli). The recovery concentration was above 85% for all initial concentrations lower than 1 × 10(4) CFU mL(-1). In the lowest initial concentration cases, 100 μL initial volumes of bacteria solution at 100 CFU mL(-1) were concentrated into 500 nL droplets with greater than 90% efficiency in 15 min. Subsequent tests with SERS on clinically relevant Methicillin-Sensitive Staphylococcus aureus (MSSA) after concentration in this device proved more than 100-fold enhancement in SERS signal intensity compared to the signal obtained from the unconcentrated sample. The concentration device is straightforward to design and use, and as such could be used in conjunction with a number of detection technologies.     

Rapid hybridization at high salt concentration and detection of bacterial DNA using fluorescence polarization

The effects of NaCl concentration and temperature on the rate of hybridization of complementary single-stranded DNA (24-mers) were investigated. The single label of fluorescein was used for the probe DNA. The time courses of fluorescence polarization for the probe DNA were monitored. It was shown that detection of a specific DNA sequence (24-mer) was possible in less than 10 min using fluorescence polarization under the optimized conditions of 0.8 M NaCl at 46 degrees C in TE buffer. The effects of base-pair mismatches on DNA hybridization in the presence of NaCl or MgCl(2) were also investigated, and the specificity was considered by comparing the hybridization rate of the fluorescein-labeled probe. Determination of a specific DNA sequence was also possible in TE buffer containing 0.2 M MgCl(2). Moreover, in the presence of 0.2 M MgCl(2), there were no undesirable effects on hybridization and the presence of a single base pair mismatch could be identified. Rapid and specific determination of the DNA of enterohemorrhagic Escherichia coli, methicillin resistant Staphylococcus aureus and Legionella pneumophila, which had been multiplied by the asymmetric PCR, was performed under the optimized conditions for hybridization. It was confirmed that the conditions were also applicable to the hybridization between the probes and the amplified products of the actual bacterial genes. The combination of fluorescence polarization with the asymmetric PCR was quite effective. Moreover, the nested and asymmetric PCR product of bacterial gene could be detected effectively. The DNA detection method could also be used even if the specificity of the DNA amplification was not perfect and some unexpected bands were mixed with the target band during electrophoresis.     

Semiconductor sensor embedded microfluidic chip for protein biomarker detection using a bead-based immunoassay combined with deoxyribonucleic acid strand labeling

Two major issues need to be addressed in applying semiconductor biosensors to detecting proteins in immunoassays. First, the length of the antibody on the sensor surface surpasses the Debye lengths (approximately 1 nm, in normal ionic strength solution), preventing certain specifically bound proteins from being tightly attached to the sensor surface. Therefore, these proteins do not contribute to the sensor’s surface potential change. Second, these proteins carry a small charge and can be easily affected by the pH of the surrounding solution. This study proposes a magnetic bead-based immunoassay using a secondary antibody to label negatively charged DNA fragments for signal amplification. An externally imposed magnetic force attaches the analyte tightly to the sensor surface, thereby effectively solving the problem of the analyte protein’s distance to the sensor surface surpassing the Debye lengths. In addition, a normal ion intensity buffer can be used without dilution for the proposed method. Experiments revealed that the sensitivity can be improved by using a longer DNA fragment for labeling and smaller magnetic beads as solid support for the antibody. By using a 90 base pair DNA label, the signal was 15 times greater than that without labeling. In addition, by using a 120 nm magnetic bead, a minimum detection limit of 12.5 ng mL⁻¹ apolipoprotein A1 can be measured. Furthermore, this study integrates a semiconductor sensor with a microfluidic chip. With the help of microvalves and micromixers in the chip, the length of the mixing step for each immunoassay has been reduced from 1 h to 20 min, and the sample volume has been reduced from 80 μL to 10 μL. In practice, a protein biomarker in a urinary bladder cancer patient’s urine was successfully measured using this technique. This study provides a convenient and effective method to measure protein using a semiconductor sensor.     

Spatio-temporally resolved detection on a microfluidic chip for monitoring the dynamic processes of molecular events

Detection is an essential aspect in analytical approaches. In liquid phase separations, many attempts have been focused on the capability to detect a partial or an entire column. However, detection in both spatial and temporal resolutions has not gained much attention yet. Here we present the concept of spatio-temporally resolved detection (STRD) and a proof-of-the-concept microchip electrophoresis (MCE)-STRD system. The MCE-STRD system was mainly composed of a microchip and an STRD unit, which were designed completely based on the requirements for spatial and temporal resolutions. In the STRD unit, a linear light beam expanded from a UV LED light source was employed to illuminate the whole separation channel of the microchip while a linear CCD sensor that has an identical effective length as the separation channel and more pixels per unit length was used to detect the absorbance signals through the separation channel. As each pixel of the CCD sensor can detect a corresponding channel space in real time, the CCD provides both spatial and temporal resolutions. A significant advantage of STRD over conventional detection schemes is its capability for monitoring the dynamic processes of molecular events occurring in the separation channel. This was demonstrated through the monitoring of the dynamic processes of protein-DNA and protein-drug interactions in chip isoelectric focusing (chip IEF). The MCE-STRD system provided not only whole pictures of the entire dynamic processes at-a-glance but also quantitative kinetic information (dissociation rate constants) of the dynamic processes. With further development, we anticipate that STRD could be a promising tool for the characterization of biomolecular interactions and the observation of migration behaviours of analytes.     

Analysis of the paracrine loop between cancer cells and fibroblasts using a microfluidic chip

We use a microfluidic cell culture chip equipped with pneumatic microvalves to analyze the paracrine loop between lung cancer cells and fibroblasts. In order to assess the cellular responses in the paracrine loop, we measure the migration speeds of cancer cells and the aspect ratios of fibroblasts which reflect the phenotype of myofibroblasts. With well-controlled interaction sequences between these two types of cells, we verify that the cytokines from cancer cells effectively stimulate the fibroblasts into myofibroblasts. The cytokines from myofibroblasts, rather than fibroblasts, increase the migration speeds of cancer cells. We confirm that the transforming growth factor-β1 (TGF-β1) is involved in the interaction between cancer cells and fibroblasts, and we also interrupt this paracrine loop in the cell culture chip by inhibiting the TGF-β1 receptors on fibroblasts.     

A first step towards practical single cell proteomics: a microfluidic antibody capture chip with TIRF detection

We have developed a generic platform to undertake the analysis of protein copy number from single cells. The approach described here is 'all-optical' whereby single cells are manipulated into separate analysis chambers using an optical trap; single cells are lysed by a shock wave caused by laser-induced microcavitation, and the protein released from a single cell is measured by total internal reflection microscopy as it is bound to micro-printed antibody spots within the device. The platform was tested using GFP transfected cells and the relative precision of the measurement method was determined to be 88%. Single cell measurements were also made on a breast cancer cell line to measure the relative levels of unlabelled human tumour suppressor protein p53 using a chip incorporating an antibody sandwich assay format. These results suggest that this is a viable method for measuring relative protein levels in single cells.     

Early development drug formulation on a chip: fabrication of nanoparticles using a microfluidic spray dryer

Early development drug formulation is exacerbated by increasingly poor bioavailability of potential candidates. Prevention of attrition due to formulation problems necessitates physicochemical analysis and formulation studies at a very early stage during development, where the availability of a new substance is limited to small quantities, thus impeding extensive experiments. Miniaturization of common formulation processes is a strategy to overcome those limitations. We present a versatile technique for fabricating drug nanoformulations using a microfluidic spray dryer. Nanoparticles are formed by evaporative precipitation of the drug-loaded spray in air at room temperature. Using danazol as a model drug, amorphous nanoparticles of 20-60 nm in diameter are prepared with a narrow size distribution. We design the device with a geometry that allows the injection of two separate solvent streams, thus enabling co-spray drying of two substances for the production of drug co-precipitates with tailor-made composition for optimization of therapeutic efficiency.     

Digital droplet immunoassay based on a microfluidic chip with magnetic beads for the detection of prostate-specific antigen

Sensitive biomarker detection techniques are beneficial for both disease diagnosis and postoperative examinations. In this study, we report an integrated microfluidic chip designed for the immunodetection of prostate-specific antigens (PSAs). The microfluidic chip is based on the three-dimensional structure of quartz capillaries. The outlet channel extends to 1.8 cm, effectively facilitating the generation of uniform droplets ranging in size from 3 to 50 μm. Furthermore, we successfully immobilized the captured antibodies onto the surface of magnetic beads using an activator, and we constructed an immunosandwich complex by employing biotinylated antibodies. A key feature of this microfluidic chip is its integration of microfluidic droplet technology advantages, such as high-throughput parallelism, enzymatic signal amplification, and small droplet size. This integration results in an exceptionally sensitive PSA detection capability, with the detection limit reduced to 7.00 ± 0.62 pg/mL.     

On‐chip pressure generation using a gel membrane fabricated outside of the microfluidic network

On‐chip generation of pressure gradients via electrokinetic means can offer several advantages to microfluidic assay design and operation in a variety of applications. In this article, we describe a simple approach to realizing this capability by employing a polyacrylamide‐based gel structure fabricated within a fluid reservoir located at the terminating end of a microchannel. Application of an electric field across this membrane has been shown to block a majority of the electroosmotic flow generated within the open duct yielding a high pressure at the channel–membrane junction. Experiments show the realization of higher pressure‐driven velocities in an electric field‐free separation channel integrated to the micropump with this design compared to other similar micropumps described in the literature. In addition, the noted velocity was found to be less sensitive to the extent of Debye layer overlap in the channel network, and therefore more impressive when working with background electrolytes having higher ionic strengths. With the current system, pressure‐driven velocities up to 3.6 mm/s were realized in a 300‐nm‐deep separation channel applying a maximum voltage of 3 kV at a channel terminal. To demonstrate the separative performance of our device, a nanofluidic pressure‐driven ion‐chromatographic analysis was subsequently implemented that relied on the slower migration of cationic analytes relative to the neutral and anionic ones in the separation channel likely due to their strong electrostatic interaction with the channel surface charges. A mixture of amino acids was thus separated with resolutions greater than those reported by our group for a similar analysis previously.     

Sequence-selective DNA detection using multiple laminar streams: a novel microfluidic analysis method

On-site detection methods for DNA have been demanded in the pathophysiology field. Such analysis requires a simple and accurate method, rather than high-throughput. This report describes a novel microfluidic analysis method and its application for simple sequence-selective DNA detection. The method uses a microchannel device with a serpentine structure. Sequence-specific binding of probe DNA can be detected at one side of the microchannel. This method is capable of sequence-specific detection of DNA with high accuracy. Single base mutations can also be analyzed. Combination of laminar stream and laminar secondary flow in the microchannel enable specific detection of probe-bound DNA.     

Stable triplex formation using the strong stacking effect of consecutive thionucleoside moieties

In this study, it was found that the arrangement of consecutive thiocarbonyl groups of s(2)T and m(5)s(2)C remarkably stabilized the pre-protonated form of the triplex, and that the stabilization of the pre-protonated form increased the pKa value of a cytosine derivative in the triplex.     

Highly sensitive signal detection of duplex dye-labelled DNA oligonucleotides in a PDMS microfluidic chip: confocal surface-enhanced Raman spectroscopic study

Rapid and highly sensitive detection of duplex dye-labelled DNA sequences in a PDMS microfluidic channel was investigated using confocal surface enhanced Raman spectroscopy (SERS). This method does not need either an immobilization procedure or a PCR amplification procedure, which are essential for a DNA microarray chip. Furthermore, Raman peaks of each dye-labelled DNA can be easily resolved since they are much narrower than the corresponding broad fluorescence bands. To find the potential applicability of confocal SERS for sensitive bio-detection in a microfluidic channel, the mixture of two different dye-labelled (TAMRA and Cy3) sex determining Y genes, SRY and SPGY1, was adsorbed on silver colloids in the alligator teeth-shaped PDMS microfluidic channel and its SERS signals were measured under flowing conditions. Its major SERS peaks were observable down to the concentration of 10(-11) M. In the present study, we explore the feasibility of confocal SERS for the highly sensitive detection of duplex dye-labelled DNA oligonucleotides in a PDMS microfluidic chip.