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.     

Development of a monoclonal antibody-based ELISA to detect Escherichia coli O157:H7

A pair of monoclonal antibodies (mAb) from 10 murine hybridomas secreting Escherichia coli O157:H7 (E. coli O157:H7)-specific mAbs were selected for the development of the sandwich ELISA to detect E. coli O157:H7. On the basis of pairwise interaction analysis, mAb-1 was selected as a capture antibody while mAb-6 was used as a detection antibody. The buffer system which provided the greatest difference between the specific E. coli O157:H7-positive antigen and the negative control was chosen. This sandwich ELISA showed good linearity when the concentration of E. coli O157:H7 was in the range of 10⁵–10⁸ cfu/mL, and the sensitivity was 1×10⁴ cfu/mL. With 8-h enrichment of bacteria, this ELISA was found to detect 0.4 cfu/g E. coli O157:H7 in artificially contaminated green tea samples.     

Design and validation of an endothelial progenitor cell capture chip and its application in patients with pulmonary arterial hypertension

The number of circulating endothelial progenitor cells (EPCs) inversely correlates with cardiovascular risk and clinical outcome, and thus has been proposed as a valuable biomarker for risk assessment, disease progression, and response to therapy. However, current strategies for isolation of these rare cells are limited to complex, laborious approaches. The goal of this study was the design and validation of a disposable microfluidic platform capable of selectively capturing and enumerating EPCs directly from human whole blood in healthy and diseased subjects, eliminating sample preprocessing. We then applied the “EPC capture chip” clinically and determined EPC numbers in blood from patients with pulmonary arterial hypertension (PAH). Blood was collected in tubes and injected into polymeric microfluidic chips containing microcolumns pre-coated with anti-CD34 antibody. Captured cells were immunofluorescently stained for the expression of stem and endothelial antigens, identified and counted. The EPC capture chip was validated with conventional flow cytometry counts (r = 0.83). The inter- and intra-day reliability of the microfluidic devices was confirmed at different time points in triplicates over 1–5 months. In a cohort of 43 patients with three forms of PAH (idiopathic/heritable, drug-induced, and connective tissue disease), EPC numbers are ≈50% lower in PAH subjects vs. matched controls and inversely related to two potential disease modifiers: body mass index and postmenopausal status. The EPC capture chip (5 × 30 × 0.05 mm³) requires only 200 μL of human blood and has the strong potential to serve as a rapid bedside test for the screening and monitoring of patients with PAH and other proliferative cardiovascular, pulmonary, malignant, and neurodegenerative diseases.     

A dietary probiotic (Bifidobacterium lactis HN019) reduces the severity of Escherichia coli O157:H7 infection in mice

The protective effects of the probiotic Bifidobacterium lactis HN019 against Escherichia coli O157:H7 were investigated in murine challenge infection models. BALB/c or C57BL/6 mice were fed milk-based diets supplemented with B. lactis HN019 (3 × 10⁸ cfu/g) for 7 days prior to and following oral challenge with E. coli O157:H7. Behavioral parameters (morbidity, feed intake) were measured for 7 days following challenge; immunological responses (phagocytosis, antibody) and pathogen translocation were measured in a sub-sample of ostensibly healthy animals 1 week post-challenge. Results showed that HN019-fed mice maintained significantly higher post-challenge feed intake and exhibited a lower cumulative morbidity rate, compared to control mice which did not receive the probiotic. Significantly higher proportions of phagocytically active cells in the blood and peritoneum, and higher intestinal tract IgA anti-E. coli antibody responses, were recorded among HN019-fed mice compared to controls. Among HN019-fed mice, pathogen translocation was identified in one of five BALB/c and one of five C57 mice; the comparative figures in control mice were two of five and three of five, respectively, and the mean bacterial burdens in these mice were over 100-fold higher than in HN019-fed mice. These results demonstrate that HN019 can reduce the severity of infection due to the enterohemolytic pathogen E. coli O157:H7, and suggest that this reduction may be associated with enhanced immune protection conferred by the probiotic. Received: 24 October 2000     

An integrated microsystem with dielectrophoresis enrichment and impedance detection for detection of <Emphasis Type="Italic">Escherichia coli</Emphasis>

An integrated microsystem device with matched interdigitated microelectrode chip was fabricated for enrichment and detection of Escherichia coli O157:H7. The microsystem has integrated with positive dielectrophoresis (pDEP) enrichment and in situ impedance detection, whose total volume is only 3.0 × 10^?3 m³, and could provide impedance testing voltages of 0 ~ 10 V, detection frequencies of 1 KHz ~ 1 MHz, DEP excitation signals with amplitude of 0 ~ 10 Vpp and frequencies of 1KHz ~ 1 MHz, which fully meets the demands of pDEP enrichment and impedance detection for bacteria. The microfluidic chip with interdigitated microelectrodes was manufactured by microfabrication methods. The interdigital microelectrode array has sufficient contact area with a bacterial suspension to improve enrichment efficiency and detection sensitivity. Bacteria in the interdigital microelectrode area of the microfluidic chip were firstly captured and enriched by pDEP. Then, in situ impedance detection of the enriched bacteria was realized by switching test conditions. Using the self-assembly microsystem, a novel quantitative detection method was established and demonstrated to detect Escherichia coli O157:H7. Experimental results showed that the detection limits of Escherichia coli O157:H7 was 5 × 10⁴ cfu mL^?1, and testing time was only 6 min under the optimized detection voltage of 100 mV and frequency of 500 KHz. The method was successfully used to detect Escherichia coli O157:H7 in synthetic chicken synthetic samples.     

Interdigitated microelectrode array-coupled bipolar semiconductor photodiode array (IMEA-PDA) microchip for on-chip electrochemiluminescence detection

This paper reports the design, fabrication and testing of a microchip wherein interdigitated microelectrode arrays (IMEA) were integrated with bipolar semiconductor photodiode array (PDA) chip to fabricate a highly compact embodiment for on-chip handling of solutions and electrochemiluminescence (ECL) detection. A 12 × 12 micro array of photodiodes, each coupled with an interdigitated microelectrode array (IMEA), an array of current amplifiers, and a photodiode element-addressing circuit were integrated into a single 2 × 2 cm² IC chip. Each photodiode had dimensions of 300 × 300 μm² and the photodiode-to-photodiode distance was 100 μm. The chip was successfully applied to the on-chip quantification of electro-chemiluminescing probe-labeled single stranded oligonucleotides. The minimum detectable limit at signal/noise ≥ 3 was found to be 5 × 10⁻¹⁴ moles of oligonucleotides with a sample volume as low as 5 μl (i.e., 10 fmole/μl). The attractive features of the developed IMEA-PDA microchip are that a plurality of samples can be analyzed simultaneously using a chip and that for a given sample the data can be averaged from values obtained from multiple, individually addressed pixels. These in turn bring in speed and statistical confidence in analysis. The IMEA-PDA microchip system has the potential to be used as a versatile and highly compact chemical analysis tool for chemical sensing and metrology applications.     

Antibody immobilization on to polystyrene substrate—on-chip immunoassay for horse IgG based on fluorescence

A simple microfluidic immunoassay card was developed based on polystyrene (PS) substrate for the detection of horse IgG, an inexpensive model analyte using fluorescence microscope. The primary antibody was captured onto the PS based on covalent bonding via a self-assembled monolayer (SAM) of thiol to pattern the surface chemistry on a gold-coated PS. The immunosensor chip layers were fabricated from sheets by CO₂ laser ablation. The functionalized PS surfaces after each step were characterized by contact angle measurement, X-ray photoelectron spectroscopy (XPS), and atomic force microscopy (AFM). After the antibody–antigen interaction as a sandwich immunoassay with a fluorescein isothiocyanate (FITC)-conjugated secondary antibody, the intensity of fluorescence was measured on-chip to determine the concentration of the target analyte. The present immunosensor chip showed a linear response range for horse IgG between 1 μg/ml and 80 μg/ml (r = 0.971, n = 3). The detection limit was found to be 0.71 μg/ml. The developed microfluidic system can be extended for various applications including medical diagnostics, microarray detection and observing protein–protein interactions.     

An Electrokinetically-Controlled Immunoassay for Simultaneous Detection of Multiple Microbial Antigens

An electrokinetically-controlled heterogeneous immunoassay microchip for multiple analyte detection was developed in this study. Numerical simulation was employed to study the transport process in a microfluidic network (μFN). The operation parameters obtained from numerical simulation was then applied to immunoassay experiment. The effectiveness of the automatic electrokinetic control was demonstrated in a separate experiment using fluorescein dye. The immunoassay microchip was made of poly(dimethylsiloxane)(PDMS)/PDMS-coated glass using soft lithography and replica molding. Multi-antigen immobilization was accomplished by adsorbing the antigen molecules onto a PDMS-coated glass slide and by using a μFN. Immobilized lysate antigen of Escherichia coli O157: H7 at different concentrations was assayed and the lower detect limit was 3 μg/mL. The assay also displayed very good specificity, when different microbial lysate antigens were immobilized, including Escherichia coli and Helicobacter pylori, and the primary and secondary antibodies were mixtures of different species. The time required for the immunoassay, from antigen coating to signal detection, was only one hour. While still an un-optimized prototype, this automatic-operating, high-throughput immunoassay microchip shows a great potential in detecting multiple pathogenic infections efficiently for clinical applications.     

Screening cell surface receptors using micromosaic immunoassays

This report presents a general method for screening cell surface receptors using so-called micromosaic immunoassays. This method employs a microfluidic chip having n (n = 11) independent flow paths to move cells over m (m = 11) lines of surface-patterned antibodies for screening individual cells in a parallel, combinatorial, fast and flexible manner. The antibodies are patterned as 30-μm-wide lines on a poly(dimethylsiloxane) layer used to seal the area of the chip in which screening is being monitored. Mouse hybridoma cells having CD44 cell surface receptors and anti-CD44 antibodies were used to establish a proof-of-concept for this method. Both the capture antibodies and the cells were fluorescently labelled to allow the position of the cells to be accurately tracked over the binding sites using an inverted fluorescence microscope. The chips and cells were maintained at a constant temperature between 20 to 37°C, and flow velocities of the cells over the capture areas were 100–280 μm~s⁻¹, resulting in a ∼0.1–0.3 s residency time of the cells on each of the eleven 30 × 30 μm s2 capture areas. Binding of the cells appeared to be specific to the capture areas, with a yield of 30% when the assay was performed at a temperature of 37°C and with a slow flow velocity. We suggest that this proof-of-concept is broadly applicable to the screening of cells for medical/diagnostic purposes as well as for basic research on the interaction of cells with surfaces. Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users.     

Comparative Cell Shape and Diffusional Water Permeability of Red Blood Cells from Indian Elephant ( Elephas maximus ) and Man ( Homo sapiens )

Red blood cells (RBC) from an Indian elephant (Elephas maximus) were studied by light microscopy (LM), scanning electron microscopy (SEM) and a new nuclear magnetic resonance (NMR) ‘imaging’ method based on the translational diffusion of water, NMR q-space analysis. Also, the transmembrane diffusional permeability, P _d of water in RBC was measured by using a Mn²⁺-doping NMR technique, taking human RBC as a reference. The main diameter of the elephant RBC was measured as 9.3 ± 0.7 μm by LM, 9.3 ± 0.7 μm by ‘shrinkage-corrected’ SEM, and 9.3 ± 0.4 μm by q-space anlaysis. The value is ∼1.4 μm larger than that for the human RBC. The values of P _d were, in the case of elephant RBC, 3.2 × 10⁻³ cm/s at 25 °C, 3.9 × 10⁻³ cm/s at 30 °C, 5.2 × 10⁻³ cm/s at 37 °C and 6.5 × 10⁻³ cm/s at 42 °C; all values were significantly lower than the corresponding values of P _d for human RBC, namely 4.3 × 10⁻³ cm/s at 25 °C, 5.2 × 10⁻³ cm/s at 30 °C, 6.1 × 10⁻³ cm/s at 37 °C, 7.8 × 10⁻³ cm/s at 42°C. The maximal inhibition of P _d (56%) was reached in 30 min at 37 °C with 2 mm p-chloromercuribenzene sulphonate (PCMBS) for both species of RBC. The basal permeability to water at 37 °C was estimated to be 2.3 × 10⁻³ cm/s for elephant and 2.6 × 10⁻³ cm/s for human RBC. The values of the activation energy for water permeability (E _a,d ) was significantly higher for elephant RBC (31.9 kJ/mol) than for human RBC (25.9 kJ/mol). This indicated that features other than the number of transporters per cell are likely to be important in defining the differences in water permeability in the RBC from the two species.     

Comparative Cell Shape and Diffusional Water Permeability of Red Blood Cells from Indian Elephant (Elephas maximus) and Man (Homo sapiens)

Red blood cells (RBC) from an Indian elephant (Elephas maximus) were studied by light microscopy (LM), scanning electron microscopy (SEM) and a new nuclear magnetic resonance (NMR) ‘imaging’ method based on the translational diffusion of water, NMR q-space analysis. Also, the transmembrane diffusional permeability, P _d of water in RBC was measured by using a Mn²⁺-doping NMR technique, taking human RBC as a reference. The main diameter of the elephant RBC was measured as 9.3 ± 0.7 μm by LM, 9.3 ± 0.7 μm by ‘shrinkage-corrected’ SEM, and 9.3 ± 0.4 μm by q-space anlaysis. The value is ∼1.4 μm larger than that for the human RBC. The values of P _d were, in the case of elephant RBC, 3.2 × 10⁻³ cm/s at 25 °C, 3.9 × 10⁻³ cm/s at 30 °C, 5.2 × 10⁻³ cm/s at 37 °C and 6.5 × 10⁻³ cm/s at 42 °C; all values were significantly lower than the corresponding values of P _d for human RBC, namely 4.3 × 10⁻³ cm/s at 25 °C, 5.2 × 10⁻³ cm/s at 30 °C, 6.1 × 10⁻³ cm/s at 37 °C, 7.8 × 10⁻³ cm/s at 42°C. The maximal inhibition of P _d (56%) was reached in 30 min at 37 °C with 2 mm p-chloromercuribenzene sulphonate (PCMBS) for both species of RBC. The basal permeability to water at 37 °C was estimated to be 2.3 × 10⁻³ cm/s for elephant and 2.6 × 10⁻³ cm/s for human RBC. The values of the activation energy for water permeability (E _a,d ) was significantly higher for elephant RBC (31.9 kJ/mol) than for human RBC (25.9 kJ/mol). This indicated that features other than the number of transporters per cell are likely to be important in defining the differences in water permeability in the RBC from the two species.     

In Vitro Adherence of Enterococcus faecalis and Enterococcus faecium to Urinary Catheters

 The in vitro adherence of ten strains of Enterococcus faecalis and ten strains of Enterococcus faecium to siliconized latex urinary catheters and to silicone elastomer was evaluated. Bacterial suspensions (2.5×10⁵ cfu/ml) in tryptic soy broth containing 0.5 cm segments from each type of catheter were incubated at 37 °C. At specified intervals, the segments were washed to remove nonadherent bacteria and sonicated for 1 min, and colony-forming units were quantified. Bacterial adherence occurred rapidly, reaching maximal peaks after 24 h of incubation. Enterococcus faecium adherence to both biomaterials was significantly lower than that of Enterococcus faecalis. No differences were observed between the two elastomers. Bacterial adherence was not related to bacterial surface hydrophobicity, hemolysin or gelatinase production.     

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.     

Beneficial Effects of TLR-2/6 Ligation in Pulmonary Bacterial Infection and Immunization with <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis>

Pseudomonas aeruginosa (P. aeruginosa) is the major pathogen in nosocomial and life-threatening infections of immunocompromised or critically ill patients. The macrophage-activating lipopeptide-2 (MALP-2) activates the immune system via Toll-like receptors (TLR) 2 and 6 and leads to an accumulation of immune cells in lungs of young adult (8–10 week old) rats after intratracheal application. This is characterized by a high increase of granulocyte numbers in the BAL 24 h after MALP-2 treatment. It was hypothesized that MALP-2 may have a positive effect on the clinical course of an experimental infection. Therefore, rats were treated with MALP-2 at different time points following an infection with P. aeruginosa. The effect of MALP-2 in combination with immunization with inactivated P. aeruginosa was also investigated. Rats (n = 10) were infected intratracheally (i.t.) with 1 × 10⁸ CFU P. aeruginosa on day 0. They were treated on day −3, −1, 0 and +1 with 2.5 μg MALP-2 or the vehicle i.t. In additional experiments, rats were immunized on day −21 and −14 with 1 × 10⁸ CFU of inactivated P. aeruginosa bacteria and 2.5 μg MALP-2 or vehicle with 1 × 10⁸ CFU of inactivated bacteria and isopropanol. The clinical score, rectal temperature and weight of the rats were checked in both treatment and immunization experiments twice a day. On day 2 they were sacrificed, CFU were determined in the left lung, the right lung being used for histology. In the group treated with MALP-2 1 day prior to infection significant effects were seen: The rectal temperature was about 2°C higher in comparison to the controls at 6 h and also 1 day after infection. Both the symptoms of the infection and the weight loss were significantly reduced. In addition, the CFU and the inflammation in the lung tissue were significantly lower. These effects were not observed after treatment on day −3, 0 or +1. The MALP-2 enhanced immunization only resulted in a tendency to clinical improvement. In conclusion, local immunostimulation at the appropriate time can enhance the host defense against bacteria in the lung.     

Cryptosporidium parvum oocysts in zebra mussels (Dreissena polymorpha): evidence from the St. Lawrence River

Molluscan shellfish can recover and concentrate environmentally derived waterborne pathogens and can be used for the sanitary assessment of water quality. Oocysts of Cryptosporidium parvum (genotype 1) were identified in zebra mussels (Dreissena polymorpha) from the St. Lawrence River, Quebec. Approximately 67 oocysts/ml of hemolymph and 129 oocysts/g of soft tissue were recovered. The adjusted concentration of oocysts per gram of tissue was 2.2 × 10², and approximately 4.4 × 10² oocysts were recovered from a single mussel. Zebra mussels can serve as biological indicators of waterborne contamination with Cryptosporidium. Received: 22 May 2000 / Accepted: 17 July 2000     

Urinary Excretion and Bactericidal Activity of Intravenous Ciprofloxacin Compared with Oral Ciprofloxacin

 Twelve healthy volunteers participated in a randomized crossover study to compare urinary concentrations, serum parameters, and urinary bactericidal activity of ciprofloxacin after single intravenous (i.v.) doses of 200 mg and 400 mg and an oral (p.o.) dose of 500 mg. The median serum concentrations at 1 h after administration were 1 μg/ml, 4.3 μg/ml, and 2.2 μg/ml, respectively. Between the first collection period (0–2 h) and the last collection period (38–48 h), the median urinary concentrations decreased from 394 μg/ml, 675 μg/ml, and 585 μg/ml, respectively, to 0.3 μg/ml, 0.6 μg/ml, and 1 μg/ml, respectively. The urinary concentrations after the 400 mg i.v. and the 500 mg p.o. doses were not statistically different but were significantly higher than those after the 200 mg i.v. dose. The urinary bactericidal titers (UBTs), defined as the highest urinary dilution bactericidal for the organism tested, were determined against Escherichia coli (ATCC 25922) and eight uropathogens up to 48 h after administration of ciprofloxacin. The UBTs after the 400 mg i.v. and the 500 mg p.o. doses were similar and were significantly higher (P<0.05) than those following the 200 mg i.v. dose. After 400 mg i.v. and 500 mg p.o., median UBTs of ≥1 : 4 were present up to 48 h for all strains for which the MIC was ≤0.5 μg/ml, except for one nalidixic-acid resistant Escherichia coli strain for which the MIC was 0.25 μg/ml. Species for which the MIC is ≥1 μg/ml showed median UBTs of ≥1 : 4 for 8–16 h. Median UBTs of ≥1 : 4 were present up to 8 and 12 h for both Pseudomonas strains tested. A once-daily dosage of 400 mg i.v. or 500 mg p.o. might be sufficient for treatment of urinary tract infections caused by highly susceptible pathogens. A twice-daily dosing scheme seems to be preferable for complicated infections caused by pathogens with intermediate susceptibilty (MIC≥1 μg/ml) or for empiric therapy.     

Cell loss in integrated microfluidic device

Cell loss during sample transporting from macro-components to micro-components in integrated microfluidic devices can considerably deteriorate cell detection sensitivity. This intrinsic cell loss was studied and effectively minimized through (a) increasing the tubing diameter connecting the sample storage and the micro-device, (b) applying a hydrodynamic focusing approach for sample delivering to reduce cells contacting and adhesion on the walls of micro-channel and chip inlet; (c) optimizing the filter design with a zigzag arrangement of pillars (13 μm in chamber depth and 0.8 μm in gap) to prolong the effective filter length, and iv) the use of diamond shaped pillar instead of normally used rectangular shape to reduce the gap length between any two given pillar (i.e. pressure drop) at the filter region. Cell trapping and immunofluorescent detection of 12 Giardia lamblia and 12 Cryptosporidium parvum cells in 150 μl solution and 50 MCF-7 breast cancer cells in 150 μl solution was completed within 15 min with trapping efficiencies improved from 79 ± 11%, 50.8 ± 5.5% and 41.3 ± 3.6% without hydrodynamic focusing, respectively, to 90.8 ± 5.8%, 89.8 ± 16.6% and 77.0 ± 9.2% with hydrodynamic focusing.     

On-chip acidification rate measurements from single cardiac cells confined in sub-nanoliter volumes

The metabolic activity of cells can be monitored by measuring the pH in the extracellular environment. Microfabrication and microfluidic technologies allow the sensor size and the extracellular volumes to be comparable to single cells. A glass substrate with thin film pH sensitive IrO _x electrodes was sealed to a replica-molded polydimethylsiloxane (PDMS) microfluidic network with integrated valves. The device, termed NanoPhysiometer, allows the trapping of single cardiac myocytes and the measurement of the pH in a detection volume of 0.36 nL. For wild-type (WT) single cardiac myocytes an acidification rate of 6.45 ± 0.38 mpH/min was measured in comparison to 19.5 ± 0.38 mpH/min for very long chain Acyl-CoA dehydrogenase (VLCAD) deficient mice in 0.8 mM of Ca²⁺. VLCAD deficiency is a fatty acid oxidation disease leading to cardiomyopathy and arrhythmias. The acidification rate increased to 11.96 ± 1.33 mpH/min for WT and to 32.0 ± 4.64 mpH/min for VLCAD −/− in 1.8 mM of Ca²⁺. The NanoPhysiometer concept can be extended to study ischemia/reperfusion injury or disorders of other biological systems to identify strategies for treatment and possible pharmacological targets.     

Determination of the effect of probiotic (Saccharomyces cerevisiae) on growth performance and hematological parameters of rabbits

Insufficient supply of animal protein is a major problem in developing countries including Nigeria. Rabbits are adjudged to be a convenient source of palatable and nutritious meat, high in protein, and contain low fat and cholesterol. A doe can produce more than 15 times her own weight in offspring in a year. However, its productivity may be limited by inadequate nutrition. The objective of this study was to determine the effect of probiotic (Saccharomyces cerevisiae) supplementation on growth performance and some hematological parameters of rabbit. The appropriate level of the probiotic inclusion for excellent health status and optimum productivity was also determined. A total of 40 male rabbits were randomly divided into four groups (A–D) of ten rabbits each. Each group was subdivided into two replicates of five rabbits each. They were fed pelleted grower mash ad libitum. The feed for groups A to C were supplemented with bioactive yeast (probiotic) at inclusion levels of 0.08, 0.12, and 0.16 g yeast/kg diet, respectively. Group D had no yeast (control). Daily feed intake was determined. The rabbits were weighed weekly. The packed cell volume (PCV), hemoglobin concentration, white blood cell total, and differential counts were determined at the 8th week, 16th week, and 22nd week following standard procedures. The three results which did not have any significant difference were pooled together. Group A which had 0.08 g yeast/kg of diet had a significantly lower (P ≤ 0.05) PCV than groups B (which had 0.12 g yeast/kg of diet) and C (which had 0.16 g yeast/kg of diet) as well as D (the control). Total WBC count for groups B and C (14.35 ± 0.100 × 10³/μl and 14.65 ± 0.786 × 10³/μl, respectively) were significantly higher (P ≤ 0.05) than groups A and D (6.33 ± 0.335 × 10³/μl and 10.40 ± 0.296 × 10³/μl, respectively). Also the absolute neutrophils and lymphocytes counts were significantly higher (P ≤ 0.05) in groups B and C than in groups A and D. Group B had significantly higher (P ≤ 0.05) weight gain (1.025 ± 0.006 kg/rabbit) followed by group A (0.950 ± 0.092 kg/rabbit). The control (group D) had the least weight gain of 0.623 ± 0.0.099 kg/rabbit. These results showed that like most probiotics, bioactive yeast at an appropriate level of inclusion had a significant beneficial effect on health status and growth rate of rabbit. Probiotic supplementation level of 0.12 g yeast/kg of diet was recommended for optimum rabbit production.