High throughput fabrication of disposable nanofluidic lab-on-chip devices for single molecule studies

An easy method is introduced allowing fast polydimethylsiloxane (PDMS) replication of nanofluidic lab-on-chip devices using accurately fabricated molds featuring cross-sections down to 60 nm. A high quality master is obtained through proton beam writing and UV lithography. This master can be used more than 200 times to replicate nanofluidic devices capable of handling single DNA molecules. This method allows to fabricate nanofluidic devices through simple PDMS casting. The extensions of YOYO-1 stained bacteriophage T4 and λ−DNA inside these nanochannels have been investigated using fluorescence microscopy and follow the scaling prediction of a large, locally coiled polymer chain confined in nanochannels.     

Flow manipulation and cell immobilization for biochemical applications using thermally responsive fluids

A flow redirection and single cell immobilization method in a microfluidic chip is presented. Microheaters generated localized heating and induced poly(N-isopropylacrylamide) phase transition, creating a hydrogel that blocked a channel or immobilized a single cell. The heaters were activated in sets to redirect flow and exchange the fluid in which an immobilized cell was immersed. A yeast cell was immobilized in hydrogel and a 4′,6-diamidino-2-phenylindole (DAPI) fluorescent stain was introduced using flow redirection. DAPI diffused through the hydrogel and fluorescently labelled the yeast DNA, demonstrating in situ single cell biochemistry by means of immobilization and fluid exchange.The ability to control microfluidic flow is central to nearly all lab-on-a-chip processes. Recent developments in microfluidics either include microchannel based flow control in which microvalves are used to control the passage of fluid,¹ or are based on discrete droplet translocation in which electric fields or thermal gradients are used to determine the droplet path.^{2, 3} Reconfigurable microfluidic systems have certain advantages, including the ability to adapt downstream fluid processes such as sorting to upstream conditions and events. This is especially relevant for work with individual biomolecules and high throughput cell sorting.⁴ Additionally, reconfigurable microfluidic systems allow for rerouting flows around defective areas for high device yield or lifetime and for increasing the device versatility as a single chip design can have a variety of applications.Microvalves often form the basis of flow control systems and use magnetic, electric, piezoelectric, and pneumatic actuation methods.⁵ Many of these designs require complicated fabrication steps and can have large complex structures that limit the scalability or feasability of complex microfluidic systems. Recent work has shown how phase transition of stimuli-responsive hydrogels can be used to actuate a simple valve design.⁶ Beebe et al. demonstrated pH actuated hydrogel valves.⁷ Phase transition of thermosensitive poly(N-isopropylacrylamide) (PNIPAAm) using a heater element was demonstrated by Richter et al.⁸ Phase transition was also achieved by using light actuation by Chen et al.⁹ Electric heating has shown a microflow response time of less than 33 ms.¹¹ Previous work¹⁰ showed the use of microheaters to induce a significant shift in the viscosity of thermosensitive hydrogel to block microchannel flow and deflect a membrane, stopping flow in another microchannel. Additionally, Yu et al.¹² demonstrated thermally actuated valves based on porous polymer monoliths with PNIPAAm. Krishnan and Erickson¹³ showed how reconfigurable optically actuated hydrogel formation can be used to dynamically create highly viscous areas and thus redirect flow with a response time of  ～ 2?s. This process can be used to embed individual biomolecules in hydrogel and suppress diffusion as also demonstrated by others.^{15, 16} Fiddes et al.¹⁴ demonstrated the use of hydrogels to transport immobilized biomolecules in a digital microfluidic system. While the design of Krishnan and Erickson is highly flexible, it requires the use of an optical system and absorption layer to generate a geometric pattern to redirect flow.This paper describes the use of an array of gold microheaters positioned in a single layer polydimethylsiloxane (PDMS) microfluidic network to dynamically control microchannel flow of PNIPAAm solution. Heat generation and thus PNIPAAm phase transition were localized as the microheaters were actuated using pulse width modulation (PWM) of an applied electric potential. Additionally, hydrogel was used to embed and immobilise individual cells, exchange the fluid parts of the microfluidic system in order to expose the cells to particular reagents to carry out an in situ biochemical process. The PDMS microchannel network and the microheater array are shown in Figure ​Figure11.Open in a separate windowFigure 1A sketch of the electrical circuit and a microscope image of the gold microheaters and the PDMS microchannels. The power to the heaters was modulated with a PWM input through a H-bridge. For clarity, the electrical circuit for only the two heaters with gelled PNIPAAm is shown (H1 and V2). There are four heaters (V1-V4) in the “vertical channels” and three heaters (H1-H3) in the “horizontal” channel.The microchannels were fabricated using a patterned mould on a silicon wafer to define PDMS microchannels, as described by DeBusschere et al.¹⁷ and based on previous work.¹⁰ A 25 × 75 mm glass microscope slide served as the remaining wall of the microchannel system as well as the substrate for the microheater array. The gold layer had a thickness of 200 nm and was deposited and patterned using E-beam evaporation and photoresist lift-off.²¹ The gold was patterned to function as connecting electrical conductors as well as the microheaters.It was crucial that the microheater array was aligned with an accuracy of  ～ 20μm with the PDMS microchannel network for good heat localization. The PDMS and glass lid were treated with plasma to activate the surface and alignment was carried out by mounting the microscope slide onto the condenser lens of an inverted microscope (TE-2000 Nikon Instruments). While imaging with a 4× objective, the x, y motorized stage aligned the microchannels to the heaters and the condenser lens was lowered for the glass substrate to contact the PDMS and seal the microchannels.Local phase transition of 10% w/w PNIPAAm solution in the microchannels was achieved by applying a 7 V potential through a H-bridge that received a PWM input at 500 Hz which was modulated using a USB controller (Arduino Mega 2650) and a matlab (Mathworks) GUI. The duty cycle of the PWM input was calibrated for each microheater to account for differences in heater resistances (25?Ω to 52?Ω) due to varying lengths of on-chip connections and slight fabrication inconsistencies, as well as for different flow conditions during device operation. Additionally, thermal cross-talk between heaters required decreasing the PWM input significantly when multiple heaters were activated simultaneously. This allowed confining the areas of cross-linked PNIPAAm to the microheaters, allowing the fluid in other areas to flow freely.By activating the heaters in sets, it was possible to redirect the flow and exchange the fluid in the central area. Figure ​Figure22 demonstrates how the flow direction in the central microchannel area was changed from a stable horizontal flow to a stable vertical flow with a 3 s response time, using only PNIPAAm phase transition. Constant pressures were applied to the inlets to the horizontal channel and to the vertical channels. Activating heaters V1-4 (Figure ​(Figure2,2, left) resulted in flow in the horizontal channel only. Likewise, activating heaters H1 and H2 allowed for flow in the vertical channel only. In this sequence, the fluid in the central microchannel area from one inlet was exchanged with fluid from the other inlet. Additionally, by activating heater H3, a particle could be immobilised during the exchange of fluid as shown in Figure ​Figure33 (top).Open in a separate windowFigure 2Switching between fluid from the horizontal and the vertical channel using hydrogel activation and flow redirection with a response time of 3 s. A pressure of 25 mbar was applied to the inlet of the horizontal channel and a pressure of 20 mbar to the vertical channel. The flow field was determined using particle image velocimetry, in which the displacement of fluorescent seed particles was determined from image pairs generated by laser pulse exposure. Processing was carried out with davis software (LaVision).Open in a separate windowFigure 3A series of microscope images near heater H3 showing: (1a)-(1c) A single yeast cell captured by local PNIPAAm phase transition and immobilized for 5 min before being released. (2a) A single yeast cell was identified for capture by embedding in hydrogel. (2b) The cell as well as the hydrogel displayed fluorescence while embedded due to the introduction of DAPI in the surrounding region. (2c) The diffusion of DAPI towards the cell as the heating power of H3 is reduced after 15 min, showing a DAPI stained yeast cell immobilized.Particle immobilisation in hydrogel and fluid exchange in the central area of the microfluidic network were used to carry out an in situ biochemical process in which a yeast cell injected through one inlet was stained in situ with a 4′,6-diamidino-2-phenylindole (DAPI) solution (Invitrogen), which attached to the DNA of the yeast cell.¹⁸ A solution of yeast cells with a concentration of 5 × 10⁷cells/ml suspended in a 10% w/w PNIPAAm solution was injected through the horizontal channel. A solution of 2μg/l DAPI in a 10% w/w PNIPAAm solution was injected through the vertical channel. A single yeast cell was identified and captured near the central heater, and by deactivating the heaters in the vertical channel, DAPI solution was introduced in the microchannels around the hydrogel. After immobilising the cell for 15 min, the heater was deactivated, releasing the cell in the DAPI solution. This process is shown in Figure ​Figure33 (bottom). The sequence of the heater activation and deactivation in order to immobilize the cell and exchange the fluid is outlined in the supplementary material.²¹Eriksen et al.¹⁵ demonstrated the diffusion of protease K in the porous hydrogel matrix,¹⁹ and it was therefore expected that DAPI fluorescent stain (molecular weight of 350 kDa, Ref. ²⁰) would also diffuse. DAPI diffusion is shown in Figure 3(2b) in which the yeast cell shows fluorescence while embedded in the hydrogel. The yeast cell was released by deactivating the central heater and activating all the others to suppress unwanted flow in the microchannel. As a result, the single cell was fully immersed in the DAPI solution. Immobilization of a single cell allows for selection of a cell that exhibits a certain trait and introduction of a new fluid while maintaining the cell position in the field of view of the microscope such that a biochemical response can be imaged continuously.In summary, a microfluidic chip capable of local heating was used to induce phase transition of PNIPAAm to hydrogel, blocking microchannel flow, and thereby allowing for reconfigurable flow. Additionally, the hydrogel was used to embed and immobilise a single yeast cell. DAPI fluorescent stain was introduced using flow redirection, and it stained the immobilized cell, showing diffusion into the hydrogel. The versatile design of this microfluidic chip permits flow redirection, and is suitable to carry out in situ biochemical reactions on individual cells, demonstrating the potential of this technology for forming large-scale reconfigurable microfluidic networks for biochemical applications.     

Is the occurrence of the sticking region the result of diminishing potentiation in bench press?

In this study we investigated if the occurrence of the sticking region was a result of diminishing potentiation (coinciding delayed muscle activation) or the result of a mechanically poor region in which the muscles can produce less force. A regular one-repetition maximum (1RM) free-weight bench press was compared with isometric bench presses performed at 12 different positions. A lower force at the sticking region compared to the other regions in the isometric bench presses would confirm the mechanically-poor-position hypothesis. Twelve resistance-trained males (age 21.7 ± 1.3 years, mass 78 ± 5.8 kg, height 1.81 ± 0.05 m) were tested in 1RM and in isometric contractions in bench press in 12 different positions, indicated by the vertical distance between barbell and sternum, covering the whole range of motion during the concentric phase. Barbell kinematics and muscle activity were registered. In both types of executions a region of lower force output was observed, which supports the mechanically-poor-position hypothesis. Electromyographic activity of four muscles showed the same pattern in the isometric and 1RM attempts. It was concluded that diminishing effect potentiation could not explain the existence of the sticking region.     

Study of C-Reactive Protein and Myocardial Infarction in the Indian Population

To analyse the association of high sensitivity C-reactive (hsCRP) protein levels and −717A/G single nucleotide polymorphism of CRP with acute myocardial infarction (AMI) in the Indian population. Study population included 100 MI cases wherein 32 patients had experienced previous MI (MI-Group-1), 68 MI cases were recruited at presentation (MI-Group-2) and equal number of age and gender matched healthy individuals. hsCRP levels were determined by ELISA and genotyping of −717A/G was carried out by polymerase chain reaction-based restriction digestion method. The −717A/G genotypes did not influence hsCRP level and their distribution did not differ between groups. However, in the present study hsCRP demonstrated significant correlation with BMI in controls of both the genders and with triglycerides in females of AMI at presentation who otherwise are with low risk profile. Identifying traditional risk factors associated with inflammation may help in controlling the acute event.     

A Comparative Study of Bone Scan Findings and Serum Levels of Tumor Marker CA15-3 in Patients with Breast Carcinoma

Breast cancer is one of the most frequent malignancies in the world. Available staging procedures to detect breast cancer are bone scan, chest X-ray, liver ultrasonography, computerized tomography, estimation of tumor markers like carbohydrate antigen (CA15-3) and carcino embryonic antigen. These procedures are expensive and may not be required in all cases. Out of 70 patients studied, 55 had normal CA15-3 and 15 had elevated levels of Ca15-3. Eight (14.5%) of the 55 patients with normal CA15-3 had abnormal bone scan. Fifteen patients had CA15-3 levels above the normal range and among these 9 (60%) had abnormal bone scan. While prime facie it would appear that a high level of CA15-3 correlate with abnormal bone scan, it is also true that the numbers are small at present and conclusions about the validity of CA15-3 as marker of bone metastasis may be premature.     

Current status and development of percutaneous coronary intervention in China

##正##Dr.Gruntzig et al.(1979) successfully completed the world's first percutaneous coronary intervention (PCI),a percutaneous transluminal coronary     

Mathematical models of canine right and left atria cardiomyocytes

The aim of this study is to build two mathematical models of canine ionic currents specific to right atria and left atria.The canine left atria mathematical model was firstly modified from the Ramirez-Nattel-Courtemanche(RNC) model using the recently available experimental data of ionic currents and was further developed based on our own experimental data.A model of right atria was then built by considering the differences between right atria and left atria.The two developed models well reproduced the exper...     

Neurochip based on light-addressable potentiometric sensor with wavelet transform de-noising

Neurochip based on light-addressable potentiometric sensor(LAPS),whose sensing elements are excitable cells,can monitor electrophysiological properties of cultured neuron networks with cellular signals well analyzed.Here we report a kind of neurochip with rat pheochromocytoma(PC12) cells hybrid with LAPS and a method of de-noising signals based on wavelet transform.Cells were cultured on LAPS for several days to form networks,and we then used LAPS system to detect the extracellular potentials with signals d...     

Congenital atresia of the left main coronary artery in an infant

Congenital atresia of the left main coronary artery is a rare occurrence, and surgical revascularizationbypass graft is required. We here report a rare case of congenital coronary anomaly in an infant. A 10-month-old male infant was admitted to the hospital with heart failure symptoms. Echocardiographic examinations revealed mitral valve regurgitation and ischemic changes of the anterolateral papillary muscle and chordae. Coronary angiography showed atresia of the left main coronary artery with a severe hypoplastic left anterior descending artery and a circumflex coronary artery. Unfortunately, sudden cardiac arrest occurred after catheterization and the infant did not recover despite of immediate cardiopulmonary resuscitation. Further studies are needed to find a newer diagnostic method to detect coronary anomaly in an infant, and coronary angiography, if necessary, has to be performed very carefully.     

Electromagnetic effects on the biological tissue surrounding a transcutaneous transformer for an artificial anal sphincter system

This paper reports on the electromagnetic effects on the biological tissue surrounding a transcutaneous transformer for an artificial anal sphincter. The coupling coils and human tissues, including the skin, fat, muscle, liver, and blood, were considered. Specific absorption rate (SAR) and current density were analyzed by a finite-length solenoid model. First, SAR and current density as a function of frequency (10–10⁷ Hz) for an emission current of 1.5 A were calculated under different tissue thickness. Then relations between SAR, current density, and five types of tissues under each frequency were deduced. As a result, both the SAR and current density were below the basic restrictions of the International Commission on Non-Ionizing Radiation Protection (ICNIRP). The results show that the analysis of these data is very important for developing the artificial anal sphincter system.