Micro- and nanochamber array system for single enzyme assays

Abstract

Arrays of small reaction containers, ranging from 624 femtoliters (10^-15 L) to 270 attoliters (10^-18 L), for capturing a single enzyme molecule and measuring the activity were developed along with a new reversible sealing system based on a pneumatic valve actuator made of polydimethylsiloxane (PDMS). The valve was actuated by PBS solution, effectively preventing evaporation of the solution from the micro- and nanochambers and allowing the assay to be performed over a long period of time. The hydrolysis rates of β-D-galactosidase (β-gal), k_cat, were decreased according to the decrease of the chamber size, and the overall tendency seems to be symmetrically related to the specific surface area of the chambers even under the prevented condition of non-specific adsorption. The spatial localization of the protons in the chambers, which might could affect the dissociation state of the proteins, was also investigated to explain the decrease in the hydrolysis rate. The developed chamber system developed here may be useful for artificially reproducing the confined intracellular environment and molecular crowding conditions.

Figure 1

(A) Hydrolysis reaction scheme by β-gal used in this study. (B) Fabrication process of the micro and nanochambers equipped with a pneumatic valve. (C) A scanning microscope (SEM) image of dry-etched Si mold for the microchambers. (D) A SEM image of PDMS microchambers and a cross section image (insert). (E) Working principle of the microchambers with pneumatic valves. (F) Overview of the device.

Figure 2

Chemical structures of (A) Poly(2-Methacryloyloxyethyl phosphorylcholine-random-n-butyl methacrylate) and (B) Poly(N-hydroxyethyl acrylamide), which are used for the dynamic coating of the microchambers. Fluorescent microscope images of the PDMS chambers with (C) non-coating, (D) MPC polymer, and (E) PHEA after 10-min incubation of 1 µg/mL FITC-BSA and washing by phosphate buffer.

Figure 3

Single-enzyme assay in the 624-fL chambers. The fluorescent images of the microchamber array enclosing β-gal after times (A) 0 and (B) 2 min. (C) Histogram of the fluorescent intensity changes for 2 min. The concentrations of β-Gal and FDG were 2.0 ng/mL and 200 μM, respectively. The dotted lines indicate the thresholds of fluorescence intensity increase with different numbers of β-gal molecules in the microchambers, assuming a Poisson distributed encapsulation of β-gal molecules in the microchamber. (D) Occupancy distribution of the microchambers under the condition of 2.0 ng/mL β-gal. The bars show the ratio of the microchambers with an occupancy of N enzymes (N = 0, 1, 2, 3). The circles indicate the probability of the ratio of the microchambers that were captured N enzymes at λ = 0.737, assuming it was a Poisson distribution. The detail explanation is described in the main text. All the fluorescent images were captured under the illumination of a 72.7 μW excitation laser.

Figure 4

Single-enzyme assay in the 61-fL chambers. The fluorescent images of the microchamber array enclosing β-gal after times (A) 0 and (B) 2 min. (C) Histogram of the fluorescent intensity changes for 2 min. The concentrations of β-Gal and FDG were 6.0 ng/mL and 200 μM, respectively. The dotted lines indicate the thresholds of fluorescence intensity increase with different numbers of β-gal molecules in the microchambers, assuming a Poisson distributed encapsulation of β-gal molecules in the microchamber. (D) Occupancy distribution of the microchambers under the condition of 6.0 ng/mL β-gal. The bars show the ratio of the microchambers with an occupancy of N enzymes (N = 0, 1, 2, 3). The circles indicate the probability of the ratio of the microchambers that were captured N enzymes at λ = 0.966, assuming it was a Poisson distribution. The detail explanation is described in the main text. All the fluorescent images were captured under the illumination of a 17.8 μW excitation laser.

Figure 5

Single-enzyme assay in the 270-aL chambers. The fluorescent images of the microchamber array enclosing β-gal after times (A) 0 and (B) 2 min. (C) Histogram of the fluorescent intensity changes for 2 min. The concentrations of β-Gal and FDG were 400 ng/mL and 200 μM, respectively. The dotted lines indicate the thresholds of fluorescence intensity increase with different numbers of β-gal molecules in the microchambers, assuming a Poisson distributed encapsulation of β-gal molecules in the microchamber. (D) Occupancy distribution of the microchambers under the condition of 400 ng/mL β-gal. The bars show the ratio of the microchambers with an occupancy of N enzymes (N = 0, 1, 2, 3). The circles indicate the probability of the ratio of the microchambers that were captured N enzymes at λ = 0.270, assuming it was a Poisson distribution. The detail explanation is described in the main text. All the fluorescent images were captured under the illumination of a 1.8 μW excitation laser.

Figure 6

Hydrolysis rate and specific surface area as a function of volume of chamber without or with PHEA coating. The error bars show the standard deviation of the triplicate measurements. Some dots overlapped with the error bars.

Figure 7

Fluorescence spectra of 5-(and-6)-Carboxy SNARF-1 at 10 µM dissolved in phosphate buffer (pH = 7.4) and excited at 488 nm (A) in 1-cm cuvettes and (B) microchambers. (C) Calculated pH values by the following equation: $\mathrm{pH}={\mathrm{p}K}_a-\log \left[\frac{R-R_B}{R_A-R},\times ,\frac{F_{B\left(\lambda ,2\right)}}{F_{A\left(\lambda ,2\right)}}\right]$, where R is the ratio $\frac{F_{\lambda 1}}{F_{\lambda 2}}$ of fluorescence intensities measured at two wavelengths. λ₁ (568 nm) and λ₂ (616 nm), and the subscripts A and B represent the limiting values at the acidic and basic end points of the titration, respectively. The calibration was performed using a dual-emission ratio with λ₁ = 568 nm and λ₂ = 616 nm excited at 488 nm. The error bars show the standard deviation of the triplicate measurements.