Light-Addressable Actuator-Sensor Platform for Monitoring and Manipulation of pH Gradients in Microfluidics: A Case Study with the Enzyme Penicillinase

Abstract

The feasibility of light-addressed detection and manipulation of pH gradients inside an electrochemical microfluidic cell was studied. Local pH changes, induced by a light-addressable electrode (LAE), were detected using a light-addressable potentiometric sensor (LAPS) with different measurement modes representing an actuator-sensor system. Biosensor functionality was examined depending on locally induced pH gradients with the help of the model enzyme penicillinase, which had been immobilized in the microfluidic channel. The surface morphology of the LAE and enzyme-functionalized LAPS was studied by scanning electron microscopy. Furthermore, the penicillin sensitivity of the LAPS inside the microfluidic channel was determined with regard to the analyte's pH influence on the enzymatic reaction rate. In a final experiment, the LAE-controlled pH inhibition of the enzyme activity was monitored by the LAPS.

Keywords: actuator-sensor system; enzyme kinetics; light-addressable electrode; light-addressable potentiometric sensor; microfluidics.

Figure 1

(a) Schematic of the microfluidic setup with a light-addressable potentiometric sensor (LAPS)/microfluidic foil/ light-addressable electrode (LAE)-sandwich structure. Tobacco mosaic virus (TMV) particles functionalized with the enzyme penicillinase are immobilized inside the microchannel. (b) Typical shape of a photocurrent-voltage curve for a n-type LAPS with characteristic regions of inversion, depletion and accumulation.

Figure 2

(a) Scanning electron microscope (SEM) image (magnification of 20,000× of the LAE showing the TiO₂ film surface on the SnO₂:F glass substrate. (b) SEM image depicting the part of the microfluidic channel where the enzyme-modified Si₃N₄ surface of the LAPS is located with a magnification of 60× (left) and with a zoom-in, showing the adsorbed TMV particles carrying the immobilized penicillinase with a magnification of 35,000× (right).

Figure 3

(a) Chemical images and (b) photocurrent-voltage curves for penicillin concentrations ranging from 0.1 to 5.0 mM after 5 min of enzymatic reaction in phosphate buffered saline (PBS) buffer, pH 7.0. (c) Mean calibration curve evaluated from the photocurrent-voltage curves (n = 4) with an average penicillin sensitivity of 42.3 mV/dec. The inlet represents the photocurrent change in dependence of the penicillin concentration, evaluated from Figure 3a.

Figure 4

(a) Chemical images for 1.0 mM penicillin in pH 4 to pH 8 PBS buffer after 5 min of enzymatic reaction. Photocurrent-voltage curve for (b) pH 4 and (c) pH 8 PBS buffer with and without 1.0 mM penicillin.

Figure 5

(a) Transient photocurrent signal for 60 s of illumination. (b) Chemical images of static (top) and dynamic (bottom) pH changes inside the microfluidic channel induced by the LAE.

Figure 6

Constant potential LAPS measurements. (a) Photocurrent response for 1.0 mM penicillin in PBS buffer, pH 7.1. In (b–d) the blue curves depict the transient photocurrent decrease due to pH changes induced by the LAE with an illumination width of 250 µm, 500 µm and 1500 µm, respectively. The orange curves show the concatenated, additional change in photocurrent when 1.0 mM penicillin is added to the PBS buffer.