**4. Discussion**

Salivary diagnostics is being increasingly touted as a suitable alternative to whole blood assays. However, the study of salivary functions could also be challenging because of the high physiological variability of saliva in comparison with other bodily fluids [38]. However, given that the saliva samples can be obtained non-invasively, patients are much more likely to cooperate for frequent extraction of saliva, for performing assays regularly. We also observed this trend among the volunteers, who were unanimously relieved with the painless approach of collecting saliva samples for our experiments. Nevertheless, to exploit this convenience of salivary analysis, it is also equally important to develop a

suitable automated point-of-care testing methodology that would enable rapid diagnostics to reduce the time and e ffort normally put into laboratory-based assays. The cumulative results of our present work demonstrates that the hybrid integrated optical biosensing principle proposed herein could be readily applied to potassium measurement from whole saliva samples for real-time point-of-care testing applications based on the principle of colorimetry.

The colorimetric optical absorption-based detection principle presented here is simple and can be easily extended to any other fluid sample. While the results of traditional point-of-care colorimetric assays reported to the naked eye (such as pregnancy tests or blood glucose tests), integrating smart-phones [39] for automated testings have lately been gaining huge popularity. But, the implementation of the colorimetric technique would not be e ffective unless uniform, controlled mixing of samples with the appropriate chromogenic agents is achieved. The acoustofluidic actuator thus lends a major advantage to this device for achieving the required homogenous sample mixing. The bulk acoustic wave micromixer configuration described in this work is simple and cost-e ffective to fabricate. For the device setup presented here, the ease of operation and feasibility of seamless hybrid integration with other complementary modules of the biosensor platform lend advantages to the bulk acoustic wave micromixer over the popularly used surface acoustic wave micromixing formats. However, mixing e fficiency could be further improved by adopting simple modifications to the existing setup, such as incorporation of passive microfluidic elements, surface treatment resulting in increased hydrophobicity [40], and using acoustic index matching glues to improve piezoacoustic energy transfer. Our initial experiments showed that the cavitation-based acoustofluidic micromixing can be used with a wide variety of fluids with di fferent physical properties such as miscibility, viscosities, vapor pressures etc. (Supplementary Figure S1B). While the present work focused only on cavitation-enabled fluid mixing, it was also observed during the initial characterization experiments that the cavitation phenomenon also induced other physical e ffects in the fluids. For example, it was possible to take advantage of the energy released from collapse of the cavitation bubbles for cleaning the surface of the microfluidic channels to ge<sup>t</sup> rid of non-specifically bound adherent particles [26]. This functionality could be extremely useful for conducting biological assays repeatedly without cross-contamination inside the microfluidic channels.

However, there are a few shortcomings to the present approach. Firstly, the device format in its present state does not enable the realization of cheap, disposable biosensors. But, since the device is sterilizable for repeated use, integrating light sources and sensor units on-chip along with suitable optoelectronic packaging could enable further help with the miniaturization of this device for developing a more cost-e ffective version. Secondly, optical absorption-based detection may hit the limits when conducting assays for measuring extremely low analyte concentrations in the samples. Despite the relatively poor sensitivity of microfluidic absorbance detection compared to fluorescence, the principle of working of optical absorption and its instrumentation simplicity gives it an advantage in applications requiring point-of-use analysis. Also, wherever applicable, it is possible to carry out a comprehensive study of time-dependent biochemical interactions using the optical absorption method. One can also take confidence from the fact that a number of absorbance-based microfluidic point-of-care products are commercially available [41]. Our device design also allows for implementing more than one optical sensing principle on the same platform. In an alternate waveguide arrangemen<sup>t</sup> to that used in the present work, the output waveguide was designed at an angle with respect to the inlet waveguide so that light is not directly coupled onto the output waveguide. This configuration, although not used in this work, could be useful for sensitive low absorption measurements or fluorescence-based optical detections where specific emission wavelengths are sensed spectroscopically without being saturated by the excitation wavelengths. Thus, overall our present work clearly demonstrates the feasibility of developing integrated microfluidic optical detection systems for rapid biosensing and real-time diagnostics, which could be incorporated into the next generation point-of-care devices.
