Faculty Bibliography

A microfluidic device capable of storing picoliter droplets containing single bacteria at constant volumes has been fabricated in PDMS. Once captured in droplets that remain static in the device, bacteria express both a red fluorescent protein (mRFP1) and the enzyme, alkaline phosphatase (AP), from a biscistronic construct. By measuring the fluorescence intensity of both the mRFP1 inside the cells and a fluorescent product formed as a result of the enzymatic activity outside the cells, gene expression and enzymatic activity can be simultaneously and continuously monitored. By collecting data from many individual cells, the distribution of activities in a cell is quantified and the difference in activity between two AP mutants is measured.

Fully integrated: Mass spectrometry has been integrated into a detection scheme for microdroplets that are created within microfluidic channels (see picture, scale bar 200 microm). This technique allows droplets to be identified based on the compounds they contain, and combines fluorescence screening with MS analysis. These experiments indicate how similar approaches can be applied to the ambitious goals of on-chip protein evolution and chemical synthesis.

Water-in-oil microdroplets in microfluidics are well-defined individual picoliter reaction compartments and, as such, have great potential for quantitative high-throughput biological screening. This, however, depends upon contents of the droplets not leaking out into the oil phase. To assess the mechanism of possible leaking, the retention of various fluorescein derivatives from droplets formed in mineral oil and stored for hours in a reservoir on chip was studied. Leaking into the oil phase was observed and was shown to be dependent on the nature of the compounds and on the concentration of the silicone-based polymeric surfactant Abil EM 90 used. In experiments in which droplets filled with fluorescein were mixed with droplets filled with only buffer, the rate of efflux from filled droplets to empty droplets was dependent on the number of neighboring droplets of different composition. Buffer droplets with five fluorescein-containing neighbors took up the fluorophore 4.5 times faster than buffer droplets without fluorescein neighbors. The addition of bovine serum albumin (BSA) substantially reduced leaking. A formulation with 5% BSA reduces leaking of the fluorophore from 45% to 3%. Inclusion of BSA enabled experiments to be carried out over periods up to 18 h, without substantial leaking (<5%). We demonstrate the utility of this additive by following the enzymatic activity of alkaline phosphatase expressed by Escherichia coli cells. The ability to reliably compartmentalize genotype (cell) and phenotype (reaction product) is the basis for using microdroplets in directed evolution studies, and the approaches described herein provide a test system for assessing emulsion formulations for such purposes.

We describe the design, fabrication and use of a single-layered poly(dimethylsiloxane) microfluidic structure for the entrapment and release of microdroplets in an array format controlled entirely by liquid flow. Aqueous picoliter droplets are trapped en masse and optically monitored for extended periods of time. Such an array-based approach is used to characterize droplet shrinkage, aggregation of encapsulated E. coli cells and enzymatic reactions. We also demonstrate that trapped droplets may be recovered from the microfluidic array for further processing.

PURPOSE/OBJECTIVE:Increased blood lactate concentration has been suggested as a primary stimulus for the exercise-induced growth hormone response (EIGR). Patients with McArdle disease are unable to produce lactate in response to exercise and thus offer a unique model to assess the role of lactate in the EIGR. Accordingly, McArdle's patients were exercised to test the hypothesis that lactate is a major stimulus of the EIGR. METHODS:11 patients with McArdle disease (3 male, 8 female; age: 35.5 (SD 13.9) years, height: 166 (8) cm, body mass: 75.2 (13.1) kg) were recruited for the study. The patients walked initially at 0.42 m/s, increasing by 0.14 m/s per 3 min stage. Exercise was terminated when participants completed 3 minutes at 1.80 m/s or when a Borg CR10 pain scale rating of "4" was reached. Stages were separated by 60 s for capillary blood sampling for analysis of hGH and blood lactate concentration. RESULTS:McArdle's patients' blood lactate levels remained at resting levels (0.3-1.2 mmol/l) as exercise intensity increased. Nine out of 11 participants failed to demonstrate an EIGR obtaining hGH values below the clinical definition of a response (>3 microg/l). CONCLUSION/CONCLUSIONS:The absence of an EIGR in nine out of 11 participants suggests that lactate could play a major role in the EIGR in humans.

A growing body of evidence reporting altered cardiac function and myocardial damage after arduous exercise, together with the increased prevalence of arrhythmias observed in highly trained athletes, suggests that repetitive bouts of prolonged, arduous exercise may be deleterious to long-term cardiac health. We report the case of an experienced, highly trained marathon runner who died suddenly while running. On post-mortem examination, left ventricle hypertrophy and idiopathic interstitial myocardial fibrosis was found. We believe that life-long, repetitive bouts of arduous physical activity resulted in fibrous replacement of the myocardium, causing a pathological substrate for the propagation of fatal arrhythmias.

We describe the development of an enzyme assay inside picoliter microdroplets. The enzyme alkaline phosphatase is expressed in Escherichia coli cells and presented in the periplasm. Droplets act as discrete reactors which retain and localize any reaction product. The catalytic turnover of the substrate is measured in individual droplets by monitoring the fluorescence at several time points within the device and exhibits kinetic behavior similar to that observed in bulk solution. Studies on wild type and a mutant enzyme successfully demonstrated the feasibility of using microfluidic droplets to provide time-resolved kinetic measurements.

Microdroplets have great potential for high-throughput biochemical screening. We report the design of an integrated microfluidic device for droplet formation, incubation and screening. Picolitre water-in-oil droplets can be stored in a reservoir that contains approximately 10(6) droplets. In this reservoir droplets are stable for at least 6 h, which gives an extended timescale for biochemical experiments. We demonstrate the utility of the system by following the in vitro expression of green fluorescent protein. The high efficiency allows protein expression from a single molecule of DNA template, creating "monoclonal droplets" in which genotype and phenotype are combined in one emulsion compartment.

The present study aimed to examine maximum heart rate (HRmax) in elite athletes. 130 (68 male, 23.2 +/- 4.8 years, 62 female, 21.0 +/- 5.1 years) endurance trained athletes, 40 (24 male, 24.0 +/- 5.6 years, 16 female, 22.8 +/- 4.6 years) anaerobically trained athletes, and 95 (39 male, 24.8 +/- 4.8 years, 56 female, 23.0 +/- 4.8 years) sedentary participants entered the study. All participants undertook a standard ramp protocol to volitional exhaustion to establish HRmax. Significant differences in HRmax were identified due to mode of exercise (p < 0.001) and gender (p = 0.001). The mean HRmax for the three modes of exercise were; aerobic 190.3 (SEE = 0.66), anaerobic 190.1 (SEE = 1.12) and sedentary 194.8 (SEE = 0.73) beats . min (-1) estimated at the average age of 23.1 years. The slope parameter for age varied between genders, the beta slope for females being significantly more negative than male subjects (- 1.1 beats . min (-1) . year (-1) vs. - 0.55 beats . min (-1) . year (-1), respectively). The predictive HRmax equation for male athletes was HRmax = 202 - 0.55 x age, and for female athletes it was HRmax = 216 - 1.09 x age. HRmax is similar between aerobically and anaerobically trained athletes. HRmax is significantly lower in athletes compared with age matched sedentary counterparts. The mechanisms underlying the lower HRmax remain to be elucidated.