Micro-fabricated perforated polymer devices for long-term drug delivery

Fabrication techniques have been developed to produce a perforated polymer microtube as a drug delivery device. The technique consists of first forming a silicon platform with trenches and alignment marks to hold the tubes for subsequent processing. Photolithography and reactive ion etching with an inductively coupled plasma source were used to fabricate micro holes on the surface of polyimide tubes. Several materials have been used to form the etching mask, including titanium film deposited by e-beam evaporation and SiO₂ and SiN_x films deposited by high-density plasma chemical vapor deposition (HDPCVD). Three equidistant holes of 20 μm in diameter were fabricated on polyimide tubes (I.D. = 125 μm). The perforated tubes were loaded with ethinyl estradiol and tested for drug release in phosphate buffered saline (pH = 7.1) at 37°C. Zero order release was observed over a period of 30 days with a potential to be extended to 4 years.     

Afferent arteriolopathy and glomerular collapse but not segmental sclerosis induce tubular atrophy in old spontaneously hypertensive rats

In chronic renal disease, the temporal and spatial relationship between vascular, glomerular and tubular changes is still unclear. Hypertension, an important cause of chronic renal failure, leads to afferent arteriolopathy, segmental glomerulosclerosis and tubular atrophy in the juxtamedullary cortex. We investigated the pathological changes of hypertensive renal disease in aged spontaneously hypertensive rats using a large number of serial sections, where we traced and analyzed afferent arteriole, glomerulus and proximal tubule of single nephrons. Our major finding was that both afferent arteriolopathy and glomerular capillary collapse were linked to tubular atrophy. Only nephrons with glomerular collapse (n = 13) showed tubules with reduced diameter indicating atrophy [21.66 ± 2.56 μm vs. tubules in normotensive Wistar Kyoto rats (WKY) 38.56 ± 0.56 μm, p < 0.05], as well as afferent arteriolar wall hypertrophy (diameter 32.74 ± 4.72 μm vs. afferent arterioles in WKY 19.24 ± 0.98 μm, p < 0.05). Nephrons with segmental sclerosis (n = 10) did not show tubular atrophy and tubular diameters were unchanged (35.60 ± 1.43 μm). Afferent arteriolar diameter negatively correlated with glomerular capillary volume fraction (r = −0.36) and proximal tubular diameter (r = −0.46) implying reduced glomerular and tubular flow. In line with this, chronically damaged tubules showed reduced staining for the ciliary protein inversin indicating changed ciliary signalling due to reduced urinary flow. This is the first morphological study on hypertensive renal disease making correlations between vascular, glomerular and tubular components of individual nephron units. Our data suggest that afferent arteriolopathy leads to glomerular collapse and reduced urinary flow with subsequent tubular atrophy.     

Micromachined optical fiber enclosed 4-electrode IPMC actuator with multidirectional control ability for biomedical application

The present paper examined a novel micromachined column structured Ion Polymer Metal Composite (IPMC) actuator with multidirectional control capability. The developed 4-electrode transducer enclosed a section of optical fiber, thereby allowing electronic directional control of conducted laser light. The fabricated device with IPMC actuator dimensions of 5 mm × 2 mm × 1 mm reached a maximum displacement of 400 μm when a square wave of 9 V was applied to the top-bottom electrode pair. Displacements in different directions and moving angles were characterized with side-side and top-right electrode pairs connected to the actuating signals. Furthermore, the generating moment per volt per second by the transducer was analyzed. The maximum value of approximately 200 μN*m/V/s was displayed when the device actuated with the side-side electrode pair. Controlling the developed IPMC actuator moved the laser beam in multiple directions. This device could be promising for biomedical applications such as microendoscopic ocular surgery.     

Designing of a Si-MEMS device with an integrated skeletal muscle cell-based bio-actuator

With the aim of designing a mechanical drug delivery system involving a bio-actuator, we fabricated a Micro Electro Mechanical Systems (MEMS) device that can be driven through contraction of skeletal muscle cells. The device is composed of a Si-MEMS with springs and ratchets, UV-crosslinked collagen film for cell attachment, and C2C12 muscle cells. The Si-MEMS device is 600 μm × 1000 μm in size and the width of the collagen film is 250 ~ 350 μm, which may allow the device to go through small blood vessels. To position the collagen film on the MEMS device, a thermo-sensitive polymer was used as the sacrifice-layer which was selectively removed with O₂ plasma at the positions where the collagen film was glued. The C2C12 myoblasts were seeded on the collagen film, where they proliferated and formed myotubes after induction of differentiation. When C2C12 myotubes were stimulated with electric pulses, contraction of the collagen film-C2C12 myotube complex was observed. When the edge of the Si-MEMS device was observed, displacement of ~8 μm was observed, demonstrating the possibility of locomotive movement when the device is placed on a track of adequate width. Here, we propose that the C2C12-collagen film complex is a new generation actuator for MEMS devices that utilize glucose as fuel, which will be useful in environments in which glucose is abundant such as inside a blood vessel.     

Design considerations in the development and application of microdisc electrode arrays (MDEAs) for implantable biosensors

The use of microlithographically fabricated Microdisc Electrode Arrays (MDEAs) in the development of implantable voltammetric biosensors necessitates design criteria that balances the overall footprint of the device with the advantages to be derived from large separation distances between non-interacting microdisc elements. Using the dynamic electroanalytical techniques of Multiple Scan Rate Cyclic Voltammetry (MSRCV) experiments with finite element simulations and Electrochemical Impedance Spectroscopy with equivalent circuit modeling, three unique MDEA designs; MDEA 050 (r = 25 μm, 5,184 discs), MDEA 100 (r = 50 μm, 1,296 discs) and MDEA 250 (r = 125 μm, 207 discs) of constant critical dimensions (center-to-center d/r = 4) and area (A = 0.1 cm²) were studied in 1.0 mM ferrocene monocarboxylic acid (FcCO₂H) solution (in 0.1 M Tris/0.1 M KCl buffer, pH = 7.2). The critical disc-to-disc spacing (d/r) required to archive 67% of maximal current response was defined as optimal. Based on the predictive model, new MDEA designs; MDEA 001 (r = 0.5 μm, 127,324 discs), MDEA 002.5 (r = 1.25 μm, 20,372 discs), MDEA 005 (r = 2.5 μm, 5,093 discs), MDEA 010 (r = 5 μm, 1,273 discs), MDEA 015 (r = 7.5 μm, 566 discs), MDEA 020 (r = 10 μm, 318 discs) were simulated at 10 and 100 mV/s. The final disc count of each MDEA was dictated by the need to maintain a comparable electroactive area between the MDEAs, which was chosen to be 0.001 cm², which in turn was dictated by the need to generate sufficient electrochemical current to be comfortably measured by common electrochemical detectors.     

A computational and experimental study inside microfluidic systems: the role of shear stress and flow recirculation in cell docking

In this paper, microfluidic devices containing microwells that enabled cell docking were investigated. We theoretically assessed the effect of geometry on recirculation areas and wall shear stress patterns within microwells and studied the relationship between the computational predictions and experimental cell docking. We used microchannels with 150 μm diameter microwells that had either 20 or 80 μm thickness. Flow within 80 μm deep microwells was subject to extensive recirculation areas and low shear stresses (<0.5 mPa) near the well base; whilst these were only presented within a 10 μm peripheral ring in 20 μm thick microwells. We also experimentally demonstrated that cell docking was significantly higher (p < 0.01) in 80 μm thick microwells as compared to 20 μm thick microwells. Finally, a computational tool which correlated physical and geometrical parameters of microwells with their fluid dynamic environment was developed and was also experimentally confirmed.     

A flexible polymer tube lab-chip integrated with microsensors for smart microcatheter

A flexible polymer tube lab-chip integrated with physical and biochemical sensor modules mounted on a flexible spiral structure for measuring physiological (temperature/flow rate) and metabolic data (glucose concentration) in a catheter application was designed, fabricated and characterized in this work. This new approach not only provides a unique way to assemble multiple sensors on both the inside and outside the flexible polymer tube using standard microfabrication methods while avoiding wiring and assembling problems associated with previous methods, but also maintains catheter inherent lumen potency for in situ drug delivery or insertion of medical tools. Three well-known sensors: temperature sensor (RTD), flow rate sensor (hot film anemometry) and glucose biosensor (amperometric sensor) have been successfully fabricated and fully integrated outside the spirally rolled polymer tube (ID = 500 μm, OD = 650 μm) of this demonstration device. The fabricated sensors showed good performances not only in a planar configuration but also in a spirally rolled configuration. This flexible micro tube lab-chip system provides a generic platform for developing patient-specific “smart” microcatheters that incorporate microsensors, microactuators, microfluidic devices and wireless signal communication modules that are tailored for the patients’ unique condition.     

Ice-lithographic fabrication of concave microwells and a microfluidic network

We describe a novel method to produce concave microwells utilizing solid–liquid phase change. This method, named ‘ice-lithography’, does not require any lithographic processes and consists of a few simple steps that yield multiple concave microwells. We demonstrated that the shape and size of the microwells can be controlled by varying substrates and vapor-collection time. Patterned wells with sizes in the range of 10μm to several millimeters in diameter could be produced. Additionally, we fabricated a uniformly aligned concave microwell pattern and a microfluidic network. Ice-lithography has potential biological and biomedical applications in areas such as the fabrication of cell docking devices and microbioreactors as well as the formation of uniformly sized embryoid bodies. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Exenatide enhances kaliuresis under conditions of hyperkalemia

Experiments on Wistar rats showed that exenatide (0.015–0.5 nmol per 100 g body weight) somewhat increased renal excretion of potassium from 7 ± 1 to 16 ± 1 μmol/h/100 g body weight (p < 0.05) in animals with normal serum concentration of glucose (4.6 ± 0.4 mM) and potassium (4.3 ± 0.1 mM). Exenatide dramatically enhanced excretion of potassium under conditions of hyperkalemia (11.4 ± 0.4 mM) produced by intraperitoneal injection of 1.25% KCl solution (5 ml per 100 g body weight). During the fi rst postinjection hour, potassium excretion increased 2-fold and attained 97 ± 11 μmol/h/100 g body weight in comparison with potassium load alone (47 ± 9 μmol/h/100 g body weight, p < 0.05). The data attest to a possible role of peptide regulators in normalization of potassium balance via renal mechanisms.     

A novel micropit device integrates automated cell positioning by dielectrophoresis and nuclear transfer by electrofusion

Nuclear transfer (NT) cloning involves manual positioning of individual donor-recipient cell couplets for electrofusion. This is time-consuming and introduces operator-dependent variation as a confounding parameter in cloning trials. In order to automate the NT procedure, we developed a micro-fluidic device that integrates automated cell positioning and electrofusion of isolated cell couplets. A simple two layer micro-fluidic device was fabricated. Thin film interdigitated titanium electrodes (300 nm thick, 250 μm wide and 250 μm apart) were deposited on a solid borosilicate glass substrate. They were coated with a film of electrically insulating photosensitive epoxy polymer (SU-8) of either 4 or 22 μm thickness. Circular holes (“micropits”) measuring 10, 20, 30, 40 or 80 μm in diameter were fabricated above the electrodes. The device was immersed in hypo-osmolar fusion buffer and manually loaded with somatic donor cells and recipient oocytes. Dielectrophoresis (DEP) was used to attract cells towards the micropit and form couplets on the same side of the insulating film. Fusion pulses between 80 V and 120 V were applied to each couplet and fusion scored under a stereomicroscope. Automated couplet formation between oocytes and somatic cells was achieved using DEP. Bovine oocyte-oocyte, oocyte-follicular cells and oocyte-fibroblast couplets fused with up to 69% (n = 13), 50% (n = 30) and 78% (n = 9) efficiency, respectively. Fusion rates were comparable to parallel plate or film electrodes that are conventionally used for bovine NT. This demonstrates proof-of-principle that a micropit device is capable of both rapid cell positioning and fusion.     

RNAi-mediated silencing of a novel Ascaris suum gene expression in infective larvae

In the present study, the potential of RNA interference (RNAi) as a gene silencing tool and the resultant effects on Ascaris suum larval development was examined by targeting a gene (represented by the EST 06G09) specifically expressed in the infective larvae of A. suum. BALB/c mice were infected with RNAi-treated larvae. The results showed that the target gene was silenced after soaking for 72 h, and the survival rate of the RNAi-treated larvae was reduced by 17.25% (P < 0.01). A significant difference (P < 0.05) was detected in the numbers of larvae collected from the livers and lungs of infected mice 4 days after infection with untreated larvae (164.29 ± 21.51) and RNAi-treated larvae (71.43 ± 14.35). Significant differences (P < 0.01) were also found in the body length and width between untreated larvae (480 ± 105.77 μm for length and 23.93 ± 3.72 μm for width) and RNAi-treated larvae (400.57 ± 71.31 μm for length and 20.20 ± 2.43 μm for width). These results show that the gene represented by EST 06G09 may play a role in the development of A. suum larvae.     

Controlled cellular orientation on PLGA microfibers with defined diameters

In this study, we investigated the effects of the diameter of microfibers on the orientation (angle between cells’ major axis and the substrate fiber long axis) of adhered cells. For this purpose, mouse fibroblast L929 cells were cultured on the surface of PLGA fibers of defined diameters ranging from 10 to 242 μm, and their adhesion and alignment was quantitatively analyzed. It was found that the mean orientation of cells and the spatial variation of cell alignment angle directly related to the microfiber diameter. Cells that were cultured on microfibrous scaffolds oriented along the long axis of the microfiber and the orientation increased as the fiber diameter decreased. For the fiber diameter of 10 μm, the mean orientation was 3.0 ± 0.2° (mean ± SE), whereas for a diameter of 242 μm, it decreased to 37.7 ± 2.1°. Using these studies we demonstrate that fibroblasts have a characteristic alignment on microscale fibers and that the microscale fiber diameter plays a critical role in cellular orientation. The ability to control cellular alignment on engineered tissue scaffold can be a potentially powerful approach to recreate the microscale architecture of engineered tissues. This may be important for engineering a variety of human tissues such as tendon, muscle and nerves as well as applications in 3D tissue culture and drug screening.     

In vitro antiplasmodial activity and cytotoxicity of crude extracts and compounds from the stem bark of <Emphasis Type="Italic">Kigelia africana</Emphasis> (Lam.) Benth (Bignoniaceae)

In order to assess the potential of the stem bark of Kigelia africana (Lam.) Benth as source of new anti-malarial leads, n-hexane and ethyl acetate (EtOAc) extracts and four compounds isolated from the stem bark were screened in vitro against the chloroquine-resistant W-2 and two field isolates of Plasmodium falciparum using lactate dehydrogenase assay. The products were also tested for their cytotoxicity on LLC/MK2 monkey kidney cells. The EtOAc extract exhibited a significant antiplasmodial activity (IC₅₀ = 11.15 μg/mL on W-2; 3.91 and 4.74 μg/mL on field CAM10 and SHF4 isolates, respectively), whereas the n-hexane fraction showed a weak activity (IC₅₀ = 73.78 μg/mL on W-2 and 21.85 μg/mL on SHF4). Three out of the four compounds showed good activity against all the three different parasite strains (IC₅₀ < 5 μM). Specicoside exhibited the highest activity on W-2 (IC₅₀ = 1.54 μM) followed by 2β, 3β, 19α-trihydroxy-urs-12-en-28-oic acid (IC₅₀ = 1.60 μM) and atranorin (IC₅₀ = 4.41 μM), while p-hydroxycinnamic acid was the least active (IC₅₀ = 53.84 μM). The EtOAc extract and its isolated compounds (specicoside and p-hydroxycinnamic acid) were non-cytotoxic (CC₅₀ > 30 μg/mL), whereas the n-hexane extract and two of its products, atranorin and 2β, 3β, 19α-trihydroxy-urs-12-en-28-oic acid showed cytotoxicity at high concentrations, with the last one being the most toxic (CC₅₀ = 9.37 μg/mL). These findings justify the use of K. africana stem bark as antimalaria by traditional healers of Western Cameroon, and could constitute a good basis for further studies towards development of new leads or natural drugs for malaria.     

Hypertrophy,gene expression,and beating of neonatal cardiac myocytes are affected by microdomain heterogeneity in 3D

Cardiac myocytes are known to be influenced by the rigidity and topography of their physical microenvironment. It was hypothesized that 3D heterogeneity introduced by purely physical microdomains regulates cardiac myocyte size and contraction. This was tested in vitro using polymeric microstructures (G′ = 1.66 GPa) suspended with random orientation in 3D by a soft Matrigel matrix (G′ = 22.9 Pa). After 10 days of culture, the presence of 100 μm-long microstructures in 3D gels induced fold increases in neonatal rat ventricular myocyte size (1.61 ± 0.06, p < 0.01) and total protein/cell ratios (1.43 ± 0.08, p < 0.05) that were comparable to those induced chemically by 50 μM phenylephrine treatment. Upon attachment to microstructures, individual myocytes also had larger cross-sectional areas (1.57 ± 0.05, p < 0.01) and higher average rates of spontaneous contraction (2.01 ± 0.08, p < 0.01) than unattached myocytes. Furthermore, the inclusion of microstructures in myocyte-seeded gels caused significant increases in the expression of beta-1 adrenergic receptor (β1-AR, 1.19 ± 0.01), cardiac ankyrin repeat protein (CARP, 1.26 ± 0.02), and sarcoplasmic reticulum calcium-ATPase (SERCA2, 1.59 ± 0.12, p < 0.05), genes implicated in hypertrophy and contractile activity. Together, the results demonstrate that cardiac myocyte behavior can be controlled through local 3D microdomains alone. This approach of defining physical cues as independent features may help to advance the elemental design considerations for scaffolds in cardiac tissue engineering and therapeutic microdevices.     

Effect of praziquantel prolonged administration on granuloma formation around Schistosoma japonicum eggs in lung of sensitized mice

Schistosomiasis remains a major public health problem and it is an immune disease. The schistosome egg is the primary parasite factor responsible for the overt disease. The eggs release the soluble antigen, which induces intensive tissue reaction, a granulomatous reaction to the eggs. If granuloma formation could be suppressed, overt disease might not develop. Praziquantel is an effective antischistosomal drug especially for adult worms. However, whether praziquantel has a suppressing effect on granuloma formation around schistosome eggs directly remains unclear. The purpose of the present study was to investigate the effect of praziquantel, especially administered persistently, on granuloma formation around Schistosoma japonicum eggs in the lung of sensitized mice. Thirty-six mice were divided into three groups averagely. Group A was a control group. First, the mice were injected with schistosomal eggs hypodermically in abdomen, and 10 days later injected with schistosomal eggs intravenously via a tail vein. Group B was a praziquantel short administration group. In addition to the injections of schistosomal eggs as the same of Group A, the mice were administered with praziquantel in a daily dose of 300 mg/kg for 3 days, from 1 day before the intravenous injection of the eggs. Group C was a praziquantel prolonged administration group. In addition to the injections of schistosomal eggs as the same of Group A, the mice were administered with praziquantel in a daily dose of 150 mg/kg for 5 days weekly until the mice were sacrificed. Three mice of each group were sacrificed on days 7, 14, 28, and 56, respectively after the intravenous injection of the eggs, and the lung tissues were fixed with formalin and the slices were HE stained. The granulomas containing eggs in their centers were selected, and 25–30 granulomas from the animals of each group were measured at each time period. The mean areas of egg granulomas of each group were calculated, and the neutrophilic granulocytes, eosinocytes, lymphocytes, fibroblasts, and macrophages within the egg granulomas were counted. The mean numbers of them of each group were calculated. All the data of each group were analyzed and compared statistically. On day 56 after the intravenous injection of the eggs, the mean area of schistosomal egg granulomas in group B was (227.4 ± 728.0) × 10³ μm², less than that of [(297.9 ± 153.3) × 10³ μm²] in group A, and the suppression rate was 23.7% (P < 0.05). On days 7, 14, 28, and 56, the mean areas of schistosomal egg granulomas in group C were (575.8 ± 155.6) × 10³ μm², (310.5 ± 854.0) × 10³ μm², (267.7 ± 513.3) × 10³ μm², and (214.9 ± 446.4) × 10³ μm², respectively, significantly less than those of [(692.7 ± 232.6) × 10³ μm², (439.4 ± 165.0) × 10³ μm², (385.7 ± 129.3) × 10³ μm², and (297.9 ± 153.3) × 10³ μm²] in group A. The suppression rates were 16.9%, 29.3%, 30.6%, and 27.9%, respectively (P values <0.05). On day 56, the mean numbers of neutrophilic granulocytes were 11.4 ± 5.0 in group A and 5.2 ± 3.1 in group C, respectively, with the suppression rate of 54.4% in group C (P < 0.05). On day 56, the mean numbers of eosinocytes within the egg granulomas were 2.3 ± 2.0, 0.1 ± 0.3, and 0.3 ± 0.6 in groups A, B, and C, respectively, with the suppression rate of 95.7% in group B and 87.0% in group C (P values <0.05). On day 56, the mean numbers of macrophages within egg granulomas were 14.3 ± 6.9 in group C, compared with 18.6 ± 8.2 in group A, the suppression rate was 23.1% (P < 0.05). On day 56, the mean numbers of fibroblasts within the egg granulomas were 6.6 ± 4.4 and 5.8 ± 2.6 in groups B and C, respectively, and compared with 14.3 ± 7.8 in group A, the increasing extents decreased by 53.8% and 59.4%, respectively (P values <0.05). Therefore, the administration of praziquantel, especially the prolonged administration, can suppress the formation of schistosomal egg granulomas, including reduction in the areas of granulomas and suppression of the inflammatory cells and the hyperplasia of fibroblasts within granulomas.     

Integrated siphon-based metering and sedimentation of whole blood on a hydrophilic lab-on-a-disk

In this paper, we present a novel and fully integrated centrifugal microfluidic “lab-on-a-disk” for rapid colorimetric assays in human whole blood. All essential steps comprising blood sampling, metering, plasma extraction and the final optical detection are conducted within t = 150 s in passive, globally hydrophilized structures which obviate the need for intricate local hydrophobic surface patterning. Our technology features a plasma extraction structure (V = 500 nL, CV < 5%) where the purified plasma (c _RBC < 0.11%) is centrifugally separated, metered by an overflow and subsequently extracted by a siphon-based principle through a hydrophilic extraction channel into the detection chamber.     

Ultrathin-wall vascular grafts for endovascular surgery

 The purposes of this study were to develop ultrathin-wall grafts suitable for stent-graft procedures and to examine the feasibility of the grafts from the physical point of view. We fabricated ultrathin-wall grafts with a wall thickness of 42 to 137 μm, using 50 denier polyester threads consisting of 72 polyester monofilaments. We studied the following physical properties of the ultrathin wall: the surface structure of the grafts, the longitudinal tensile strength of the grafts, the water permeability of the grafts, and the size of introducers through which the stent grafts can pass. In ultrathin-wall grafts with a wall thickness of 75 μm or more, a regular surface structure with zero planimetric porosity was recognized. In those with a thickness less than 64 μm, the porosity increased as the wall thickness decreased. The longitudinal tensile strengths of the 75 μm and 64 μm grafts were 13.1 ± 0.9 and 9.5 ± 0.9 kg, respectively. The water permeability of the 75-μm grafts was 380 ml/min, and that of the thinner grafts increased as the wall thickness decreased. Stents with a diameter of 40 mm covered with the ultrathin-wall grafts could pass through introducers with an inner diameter of 18 French. We conclude that the newly developed ultrathin-wall grafts are physiologically suitable for endovascular surgery. Received: December 4, 2000 / Accepted: August 13, 2001     

Multichannel oscillatory-flow multiplex PCR microfluidics for high-throughput and fast detection of foodborne bacterial pathogens

In the field of continuous-flow PCR, the amplification throughput in a single reaction solution is low and the single-plex PCR is often used. In this work, we reported a flow-based multiplex PCR microfluidic system capable of performing high-throughput and fast DNA amplification for detection of foodborne bacterial pathogens. As a demonstration, the mixture of DNA targets associated with three different foodborne pathogens was included in a single PCR solution. Then, the solution flowed through microchannels incorporated onto three temperature zones in an oscillatory manner. The effect factors of this oscillatory-flow multiplex PCR thermocycling have been demonstrated, including effects of polymerase concentration, cycling times, number of cycles, and DNA template concentration. The experimental results have shown that the oscillatory-flow multiplex PCR, with a volume of only 5 μl, could be completed in about 13 min after 35 cycles (25 cycles) at 100 μl/min (70 μl/min), which is about one-sixth of the time required on the conventional machine (70 min). By using the presently designed DNA sample model, the minimum target concentration that could be detected at 30 μl/min was 9.8 × 10⁻² ng/μl (278-bp, S. enterica), 11.2 × 10⁻² ng/μl (168-bp, E. coli O157: H7), and 2.88 × 10⁻² ng/μl (106-bp, L. monocytogenes), which corresponds to approximately 3.72 × 10⁴ copies/μl, 3.58 × 10⁴ copies/μl, and 1.79 × 10⁴ copies/μl, respectively. This level of speed and sensitivity is comparable to that achievable in most other continuous-flow PCR systems. In addition, the four individual channels were used to achieve multi-target PCR analysis of three different DNA samples from different food sources in parallel, thereby achieving another level of multiplexing.     

The effects of interleukin (IL)-12 and IL-4 deficiency on worm development and granuloma formation in Schistosoma japonicum-infected mice

CD4⁺ T-helper (Th) cell is widely recognized to be capable of influencing worm development and egg granuloma formation after schistosome infection. Interleukin (IL)-12 and IL-4 play key roles in regulation of Th cell differentiation. In the present study, we subcutaneously inoculated mice with hybridoma cells secreting monoclonal antibodies to neutralize IL-12 and IL-4 and explored the effects of IL-12 and IL-4 deficiency on the worm development and granuloma formation in mice infected with cercariae of Schistosoma japonicum. It was found that deficiency of host IL-12 and IL-4 supported normal parasite survival and fecundity. However, worm development (length and female fecundity) was significantly enhanced in anti-IL-12-treated mice. Mean length of worms in anti-IL-12-treated group was significantly greater than that of intact controls on day 28 after infection (females, 11.84 ± 1.20 mm vs. 9.45 ± 1.34; males, 9.35 ± 1.21 mm vs. 8.10 ± 0.85 mm, p < 0.05). Liver egg load per pair of worms (1,770.12 ± 470.67 vs. 806.08 ± 232.37, p < 0.05) and uterine egg load of ovigerous females (93.08 ± 27.85 vs. 46.05 ± 34.24, p < 0.05) in anti-IL-12-treated mice were significantly higher than those in intact control 28 days postinfection. But these effects diminished 42 days postinfection (p > 0.05). Granuloma size in anti-IL-12-treated mice was significantly larger than that in intact mice 42 days postinfection (398.3 ± 80.7 μm vs. 294.4 ± 72.2 μm, p < 0.05). Granuloma fibrosis dramatically intensified in anti-IL-12-treated mice but diminished in anti-IL-4-treated mice. The results suggest that IL-12 may play an impeditive role in the development of S. japonicum and in granuloma formation as well as fibrosis. IL-4 may promote granuloma formation but have no effect on worm development.     

Rapid detection of pathogens using antibody-coated microbeads with bioluminescence in microfluidic chips

Detection of pathogens was demonstrated in a polydimethylsiloxane (PDMS)/glass microfluidic chip with which microbead-based immunoseparation platform and the bioluminescence technology were integrated. Escherichia coli (E. coli) O157:H7 was used as the model bacteria. The microchamber in microfluidic chip was filled with glass beads coated with antibodies which could capture specific organism, and the capture efficiency of the chip for the bacteria was about 91.75%∼95.62%. Then the concentration of bacteria was determined by detecting adenosine triphosphate (ATP) employing bioluminescence reaction of firefly luciferin-lucifera-ATP on chip. The method allowed reliable detection of E. coli O157:H7 concentrations from 3.2 × 10¹ cfu/μL to 3.2 × 10⁵ cfu/μL within 20 min. This research demonstrated excellent reproducibility, stability, and specificity, and could accurately detect the pathogenic bacteria in food samples. The microfluidic chip and the equipments used in this method are easy to miniaturize, thus the method has great potential to be developed to a portable device for rapid detection of pathogens.