LOC devices for biological sample analysis aim to shrink bench-top instrumentation into a single portable platform. The ultimate goal is the integration of one or more functionalities—e.g. sample preparation, reaction/excitation, detection—onto a complete and stand-alone analysis system. The intrinsic advantages of this method have proved to be attractive and particularly suited for the biological and chemical research field. These include: the low sample volume consumption, the faster analysis response time, high sensitivity and portability, due to the reduced device dimensions1. Among the broad range of LOC methods, the branch that exploits optical detection techniques is known as optofluidics2. This method well fits to biological/chemical sample analysis because of its good sensitivity, due to the improved limit of detection, greater robustness and high throughput. In the last two decades, many studies have presented integrated optical elements to achieve more compact and portable devices3. However, the usual working operation of almost all of them is still closely related to the presence of an external power supply. This dependence prevents a more advanced miniaturization and compromises the real portability of the chip. As a result, one of the biggest field challenges—i.e. to reach the independence from external excitation source—is not completely satisfied yet. Furthermore, moving the light source in the same substrate where the microfluidic structures are fabricated leads to an increase of the light probe efficiency by reducing leakages and background noises. For this reason, several interesting solutions have been proposed. Currently, the two most used on-a-chip light sources are dye lasers4,5,6,7,8 and organic light-emitting devices (OLEDs)9, 10, whose outputs are respectively coherent and incoherent light signals. Other attempts are represented by fluorescent liquid–liquid (({mathrm{L}}^{2}))11 or liquid–air (LA) waveguides12, in which an external laser source promotes fluorescence in a specific region of the microfluidic channel. Then, the incoherent light produced can be guided towards the sample interrogation area, thanks to the refractive index difference of the flowing fluids. Another curious approach is shown by Pagliara et al.13 in which the light is emitted by polymeric nanofibers, deposited near the microfluidic channels.

These implementations, while showing the above listed advantages, have all the same critical point: the dependence from an external energy source to feed the emitted light. In fact, for dye lasers, ({mathrm{L}}^{2}), LA waveguides and polymeric nanofibers light emission needs to be pumped by another collimated external light source. OLEDs, in the same fashion, can work as emitters only if a voltage is applied. Whereby, to fully satisfy even the portability requirement, innovative light sources are still crucial. In this context, exploiting chemiluminescence properties of some organic compounds could give a significant contribution.

CL is a well-known chemical phenomenon in which, as schematically depicted in Fig. 1, two reagents (A and B) produce an electronically excited compound (P*) that will emit—directly or indirectly—light, due to relaxation processes14, 15. In our case, the light emission is achieved when the intermediate product P* reacts with a fluorophore (F) present in the solution as cofactor. Then, the fluorophore, excited by the reaction, emits photons through relaxation. This process is known as Indirect Chemiluminescence. Generally, the CL emission reaches an intensity peak after few seconds and the overall emission duration lasts tens of seconds16. Thus, this chemical reaction represents a simple, fast and cheap way to produce incoherent light. Furthermore, what makes this mechanism fascinating for LOC application is the typical liquid physical state of the reagents and the absolute independence from external feeding energy sources.

Figure 1

General scheme of a CL reaction mechanism. Two reagents (A and B), with same cofactors, react to produce an electronically excited compound (P*) that will emitdirectly or indirectly—light. If P* emits itself the CL light the reaction it is known as direct chemiluminescence. Instead when the CL light is emitted by a fluorophore (F) present in the solution as cofactor, thanks to an exchange of energy with P*, the reaction is called indirect chemiluminescence. (Inset) General model of the light emission phenomenon (flash-type) linked with the CL reaction. Emission peak (({t}_{max})) is reached after few seconds the reaction starting (({t}_{0})). In most of CL reactions the emission duration (({t}_{end})({t}_{0})) lasts tens of seconds16.

This peculiar “light system” finds its widespread use in detection assays, especially in the biological samples analysis. Specific chemiluminescent compounds are used as a probe, as they emit light once they meet the analytes17,18,19,20,21,22,23,24,25,26. Nonetheless, in literature there can be found some studies in which CL is used as an excitation source in macro environments. Zhang et al. report a new photoelectrochemical DNA biosensor using chemiluminescence reaction as a light source to produce photocurrents27.

Moving to the micro scale, the current attempts to exploit CL potential as excitation optic source are very few and all concern the lab-on-a-paper field. Lan et al. cunningly embed the chemiluminescent reaction of Luminol in the chain of reactions useful for their purposes. Thus, the light deriving from the CL process plays an important role in the photoelectric chemical system presented in their work28. These types of application are strictly related to photoelectrochemical reactions and require that the luminescent reagents well match the other components of the chemical system. This may be far from the effective use of CL as stand-alone light source because it restricts its potential to a very narrow spectrum of applications.

One of the most relevant limits of this type of chemical reaction is its duration. Indeed, most known CL reactions exhibit a flash-type light emission. Of course, this intrinsic property affects all the possible applications of this principle as a stable light source. On a macroscopic scale, an interesting solution to obviate the lasting issue was to fabricate hydrogels containing chemiluminescent compounds. Thus, exploiting a slow-diffusion controlled catalytic mechanism, it has been possible to reach durable light emission29. Though this appreciable result, a long-lasting CL system (several hours) on a microfluidic platform (microscopic scale) is yet to be demonstrated.

Hence, we present here the fabrication and the characterization of a fully integrated and long-lasting optofluidic incoherent light source for LOC applications, by merging the microvolumes control capabilities given by microfluidics with the 3D geometry shaping freedom of the femtosecond micromachining technique.

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