Optical visualization and quantification of enzyme activity using dynamic droplet lenses

Determination of α-Amylase Activity via Disruption of Host–Guest Interactions.

To determine whether the reconfiguration of the droplet lenses can be used to measure enzyme activity, we began with an emulsion system sensitive to the activity of α-amylase. α-Amylase, an enzyme that catalyzes the hydrolytic cleavage of internal α-1,4-glycosidic linkages in polysaccharides, is an important diagnostic enzyme and relevant biomarker for many gastrointestinal tract conditions. The ability of amylase to affect surface tensions by disrupting host–guest complexes between cyclodextrins and surfactants has previously been explored (15), making it an excellent model system to test the proposed sensing scheme. We used α-amylase from Aspergillus oryzae as the enzyme, and we chose γ-cyclodextrin (γ-CD) as the enzyme substrate because it forms a 1:1 inclusion complex with Triton X-100 surfactant (16) and the larger γ-CD is enzymatically degraded more rapidly (with a higher V_max) than the smaller α- and β-CD (17).

To sensitize the droplets to the amylase enzyme, we prepared droplets in an aqueous sodium acetate (NaOAc) buffered solution containing a bound (caged) hydrocarbon surfactant (Triton X-100/ γ-CD) and an inert fluorosurfactant, Zonyl FS-300 (Zonyl) (Fig. 2A). Droplets were composed of heptane and FC770 (a mixed fluorocarbon solvent) in a 1:1 volume ratio. Droplets experiencing this Triton X-100/γ-CD complex in conjunction with Zonyl surfactant exhibited an H/F/W morphology, revealing that the surfactant effectiveness of the Triton X-100 is compromised when complexed with γ-CD (16). Upon introduction of α-amylase, hydrolysis of the γ-CD “uncages” the Triton X-100 molecules and triggers a change in the balance of interfacial tensions. Over time, droplets transitioned through a Janus morphology and finally assumed an F/H/W droplet morphology that is favored by a stabilization of the hydrocarbon water interface (γ_H << γ_F) by liberated Triton X-100.

• Download figure
• Open in new tab
• Download powerpoint

Fig. 2.

Measurement of α-amylase activity using variations in transmission of responsive droplet lenses. (A) When Triton X-100 surfactant is complexed with γ-CD it is no longer an effective surfactant, and droplets in the presence of this Triton X-100/ γ-CD complex along with Zonyl fluorosurfactant exhibit H/F/W morphology. When the γ-CD is hydrolyzed by α-amylase, the Triton X-100 becomes freely available, thereby reducing γ_H relative to y_F and pushing the droplets closer toward the F/H/W state. (B) Schematic of the experimental setup; a stabilized light source was used for illumination and a USB spectrometer was used to measure the intensity of transmitted 650-nm light. (C) Exemplary transmission versus time data collected for a sample with 20 FAU/L α-amylase activity. Droplets began in the opaque H/F/W state, transitioned through the transmissive Janus state, and ended in an F/H/W configuration. We define t* to equal the time difference between when maximum and half-maximum transmission is reached. (D) Exemplary transmission versus time plots for varying concentrations of amylase all using the same reaction scheme shown in B. Data were collected at 37 °C. (E) The time parameter t* was determined for 22 independent samples across a range of relevant amylase activities (Left). The rate of the reaction, characterized by 1/t*, is plotted versus amylase activity and yields a linear correlation with R² = 0.989 (Right).

To demonstrate that the droplet shape could be a useful indicator in a quantitative assay, we investigated the rate of change in the droplet morphology with respect to enzyme activity. Given the previously established relationship between a droplet’s shape and its optical properties (14), we chose to use macroscale optical transmission measurements as an indicator of the droplet morphology rather than direct imaging of the droplets themselves in a microscope. We measured the optical transmission at 650 nm through a glass-bottom dish (SI Appendix, Fig. S1) containing several layers of microscale polydisperse droplets using a spectrometer and a stabilized tungsten light source in an incubator at 37 °C (Fig. 2B). The transmission intensity was tracked over time for a sample containing a known α-amylase activity, defined in fungal α-amylase units (FAU) per liter (Fig. 2C). Droplets began in an opaque H/F/W double-emulsion morphology, transitioned through the translucent Janus morphology, and finally ended in an opaque F/H/W morphology, as expected.

To quantitatively describe the transmission curve and provide a basis for comparison between samples, we defined a time parameter t* to be equal to the time difference between maximum and half-maximum transmission. Effectively, t* is a measure of how long it takes for the droplets to change between two specific morphologies (Fig. 2C). The amount of degraded γ-CD (which is directly related to the amount of liberated Triton X-100) correlates to the droplet shape and as a result, measuring t* is equivalent to measuring the amount of time required for the enzyme to process a defined amount of substrate. Following this paradigm, we subjected droplets to a range of varying α-amylase activities (Fig. 2D), observing that with higher α-amylase activity, the change in droplet morphology occurs more quickly, resulting in a smaller t* (Fig. 2E, Left). A plot of 1/t* (representing the rate at which the γ-CD is degraded) vs. known enzyme activity yielded a linear calibration curve (Fig. 2E, Right).

Determination of Lipase and Sulfatase Activity via Direct Surfactant Degradation.

Having demonstrated a proof-of-concept assay for α-amylase via uncaging of a surfactant, we next explored assays for lipase activity based on direct enzymatic cleavage of a surfactant. Lipase is an important diagnostic enzyme and biomarker for conditions involving the pancreas, such as pancreatitis. We used lipase from Candida sp. as the enzyme, tetra(ethylene glycol)mono-n-octanoate (1) as the lipase-degradable hydrocarbon surfactant (18), and Zonyl as the inert fluorinated surfactant within PBS solution (Fig. 3A). Droplets were composed of a 1:1 volume ratio of (2:1 hexane:heptane) and perfluorohexane. Experiments were conducted at room temperature (20–21 °C) with the same spectrometer configuration as used for the α-amylase measurements. In this case, droplets exhibited the reverse morphology trajectory compared with the α-amylase scheme. Specifically, when degraded by lipase, the surfactant (1) is transformed into a molecule that is an ineffective surfactant and droplets transitioned to the H/F/W state over time (Fig. 3B). Nevertheless, the same definition of t*, as was used in the amylase assay, remains applicable. Exemplary linear transmission versus time curves are shown in Fig. 3C and the correlation between t* and 1/t* with lipase activity as expressed in lipase units (LU) is shown in Fig. 3D. Control experiments that hinder catalytic activity of lipase with a covalent inhibitor, methyl 4-nitrophenyl hexylphosphonate, resulted in neither change in droplet shape nor change in transmission, confirming that variations in droplet morphology are a direct result of the lipase activity and not from hydrolysis of the surfactant over time in aqueous solution (SI Appendix, Fig. S2).

• Download figure
• Open in new tab
• Download powerpoint

Fig. 3.

Measurement of lipase and sulfatase activity via direct enzymatic degradation of surfactants. (A) Droplets in the presence of an ester-linked surfactant (1) and Zonyl initially exhibit a Janus-type morphology. When the ester-linked surfactant is hydrolyzed by lipase, γ_H increases relative to y_F and the droplets transition toward the H/F/W state. (B) Exemplary transmission versus time plot for emulsions sensitized by the lipase-responsive surfactants shown in A at 70 LU/L. Although the droplets follow the reverse trajectory as demonstrated in Fig. 2, we can still apply the same definition of t*. (C) Transmission versus time plots for samples containing varying concentrations of lipase and using the reaction scheme shown in A. Data were collected at 21 °C. (D) The time parameter t* was determined for 22 independent samples across a range of relevant lipase activities (Left). The rate of the reaction, characterized by 1/t*, is plotted versus lipase activity and yields a linear correlation with R² = 0.99 (Right). (E and F) Measurement of sulfatase activity. (E) Droplets in the presence of sulfate surfactant (2) and Zonyl initially exhibit a Janus-type morphology. When the sulfate surfactant is hydrolyzed by sulfatase, γ_H increases relative to y_F and the droplets transition toward the H/F/W state. (F) The time parameter t* was determined for 18 independent samples across a range of sulfatase activities (Left). The rate of the reaction, characterized by 1/t*, is plotted versus sulfatase activity and yields a linear correlation with R² = 0.977 (Right). Data were collected at 37 °C.

Using a similar surfactant-degradation approach, we next designed a system responsive to sulfatase. Sulfatases hydrolyze sulfate esters found on steroids, carbohydrates, and proteins. Extracellular sulfatases are up-regulated in some tumors, whereas sulfatase deficiency is associated with lysosomal storage diseases (19⇓–21). Novel fluorogenic probes for bacterial sulfatase activity, which occurs at a distinct pH range compared with mammalian sulfatases, have enabled the rapid discrimination of tuberculosis strains (22, 23). The mechanism for droplet reconfiguration in the sulfatase assay is analogous to the lipase scheme. In particular, sulfatase-cleavable surfactant 4-decyl-2,6-difluorophenyl sulfate (2) was used along with inert Zonyl fluorosurfactant and sulfatase from Helix pomatia in sodium acetate buffer (Fig. 3E). The droplets were composed of a 1:1 volume ratio of heptane and FC770, and measurements were again collected in an incubator at 37 °C. The calculated t* values for various sulfatase activities and the corresponding rates (1/t*) are shown in Fig. 3F, where 1 unit is defined as the amount of enzyme that can hydrolyze 1.0 µmol of p-nitrocatechol sulfate per hour at pH 5 and 37 °C. We again observed a linear correlation between 1/t* and enzyme activity.

Enzyme Sensing with a Personal Electronic Device.

To explore the possibility that such dynamic droplet lenses could potentially be useful in field experiments and for point-of-care diagnostics, we aimed to demonstrate that such droplet transmission data could be easily collected with commonplace equipment. Our initial experiments with lipase, amylase, and sulfatase were all conducted with a spectrometer, computer software data collection, and an intensity-stabilized light source. This equipment provided the necessary precision for the initial elucidation of the droplet response and activity correlation, but are expensive and impractical for low-cost point-of-care diagnostics. Because all that is required for the proposed assay scheme is the tracking of light intensity over time, we investigated if the light sensors that are now standard equipment on personal electronic devices such as camera phones or tablets could be used in place of the spectrometer and computer. Using the free Physics Toolbox Light Sensor Android application on a Samsung Note tablet 2014 Edition (model SM-P600) and the ambient fluorescent room lighting for illumination, we collected light-transmission versus time data for an exemplary sample, 200 LU/L lipase at room temperature (20 °C) (Fig. 4). Although the light sensor lacks the precision of the spectrometer and so the intensity measurements are more discretized, the overall shape of the curve is nearly equivalent with the data shown in Fig. 3B and still allows determination of t*. A fieldable tablet or phone-based assay will still require some development and standardization, but nevertheless the fact that simple measurement of light intensity appears sufficient for application of this assay is promising for the development of low-cost point-of-care diagnostics.

• Download figure
• Open in new tab
• Download powerpoint

Fig. 4.

Portable electronic devices such as smartphones and tablets have light sensors that can be used along with ambient lighting conditions to collect enzyme activity data similar to that achieved using a spectrometer and a computer. The data shown at right were collected for 200 U/L lipase at 21 °C using a Samsung tablet.