**4. Conclusions**

In this work, a LAE/microfluidic foil/LAPS sandwich structure was utilized for the detection and manipulation of pH gradients inside a microfluidic system. The LAPS offers the ability to detect spatially resolved pH changes inside the microfluidic channel. In contrast, locally induced pH changes can be triggered using the LAE, whereby the location may be controlled by the illuminated area. To study this sensing-actuating interplay, as a model bioreceptor, the enzyme penicillinase was immobilized inside the microchannel using plant viral (TMV) particles as enzyme nanocarriers. The enzymatic cleavage of penicillin to penicilloic acid, yielding H+ ions, leads to local pH changes, which can be detected by the LAPS. By inducing a further pH shift via the LAE, the enzymatic activity can be inhibited.

The novel actuator-sensor platform was characterized performing photocurrentvoltage-, photocurrent-time measurements and chemical imaging with the LAPS and by transient current measurements with the LAE. The surface morphology of the LAE and TMV-modified LAPS was analyzed by means of SEM.

In the concentration range from 0.1 to 5.0 mM penicillin, the TMV-penicillinasemodified LAPS sensor achieved a penicillin sensitivity of 42.3 mV/dec, proofing the

functionality as penicillin sensor inside the microfluidic setup. Additionally, the chemical images visualize, that the TMV-immobilized enzymes were confined to the area, predefined through drop-coating during assembly of the system. This extends the use of beneficial plant viral enzyme nanocarriers to a further type of microsystem. For solutions of varying pH, the inhibition of the enzymatic reaction was demonstrated at pH 4.0, whereas enzyme activity increased in LAPS measurements when pH is shifted towards the enzyme's pH optimum. Furthermore, a strategy for a spatially resolved photoelectrocatalytical pH manipulation induced by the LAE was developed. Such a pH gradient inside the microchannel was utilized to control the enzymatic reaction.

By this exemplary application, the feasibility and potential of combining two lightaddressable technologies, LAPS and LAE, was highlighted to be of grea<sup>t</sup> benefit for further integration in lab-on-a-chip systems. The advantage of this system lies in the adaptability of both technologies, as the region of interest inside, e.g., the microfluidic channel, can be regulated in time and geometrical locus by changing the illuminated area.

In future studies, such as e.g., enzyme arrays inside a microfluidic channel, each particular enzyme might be controlled individually. Dependent on the adjusted pH value by the LAE, the enzyme activity can be either shifted to the enzyme's pH optimum (leading to increased reaction rates) or to pH values, where inhibition of enzyme takes place.

A further interesting approach for such actuator-sensing platform lies in the field of enantioselective enzymes which catalyse multiple reactions (e.g., acetoin reductase for acetoin and diacetyl determination [34,35]). Here, local pH variations triggered by the LAE could shift the pH optima corresponding to the respective substrate molecule of interest. A separation by different microchannels will address individual enzymes having a two-dimensional monitoring of each single reaction.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10 .3390/bios11060171/s1, Figure S1: Reference chemical image of the microfluidic structure recorded at an applied potential of −1.65 V in 0.33 mM PBS buffer, pH 7.0. Figure S2: Photocurrent-time curve for 1.0 mM penicillin in PBS buffer, pH 7.1, for different flow rates.

**Author Contributions:** Conceptualization, R.W., M.J., T.W. and M.J.S.; methodology, R.W., M.J., C.W.; T.W. and M.J.S.; validation, R.W., M.J., T.W. and M.J.S.; formal analysis, R.W., M.J., T.W. and M.J.S.; investigation, R.W., M.J.; writing, R.W., M.J., C.W., M.K., P.H.W., T.W. and M.J.S.; supervision, M.K., P.H.W., T.W. and M.J.S.; All authors have read and agreed to the published version of the manuscript.

**Funding:** Part of this research project was funded by the German Federal Ministry of Education and Research (BMBF)–13N12585. Part of this work was funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation)–446507449.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Acknowledgments:** The authors would like to thank Arshak Poghossian for valuable discussions, and Heiko Iken, David Rolka, Benno Schneider, Rebecca Hummel and Jürgen Schubert for technical support. Part of this work was funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation)–446507449. Part of this research project was funded by the German Federal Ministry of Education and Research (BMBF) within the research frame of "Nano-MatFutur": 13N12585. R. Welden thanks Aachen University of Applied Sciences for financial support.

**Conflicts of Interest:** The authors declare no conflict of interest.
