# Nanotechnology Solutions for Controlled Cytokine Delivery: An Applied Perspective
## 4 More Than Meets the Eye: Cytokines and Nanocarriers as Novel Biosensors  
The cytokines present in body fluids and tissues play an important role as potentially useful biomarkers for several diseases. The monitoring of their levels is of great importance for early clinical diagnosis and for assessing therapeutic efficacy. However, quantification of cytokine secretion in the respective microenvironment still remains a challenge due to the use of laborious and time-consuming detection platforms, such as enzyme-linked immunosorbent assay (ELISA). Although well-established and effective, ELISA includes a long assay time (3-8 h), a large amount of sample volume and low sensitivity [68; 69; 70] when compared to some recently developed biosensors based on nanotechnology. Overall, a biosensor is composed of a biorecognition element, which binds to or interacts with the analyte, a signal transducer, which transduces the biorecognition event into a signal, and a detector, which detects the signal via several modalities [71]. A biosensor must follow some general criteria, which include sensitivity in detecting the concentration of analyte molecules and selectivity for the discrimination between different analytes. Other important features are the dynamic range (concentration range within the linear range of response), response time (time required to indicate a defined percentage of final response), accuracy (agreement between the measured result and the true value), precision (reproducibility of measurements), detection limit (the lowest concentration that produces a measurable signal response), and lifetime (the time length over which the sensor can be operated without significant deterioration in performance) [69]. Nanomaterial-based sensors have attracted increasing attention in molecular diagnosis, since they offer excellent sensitivity, stability and their reduced size is useful to improve their limit detection and analyte-sensor interactions [68]. [PERSON] and co-workers developed a graphene-based field effect transistor nanosensor capable of sampling and quantifying cytokine biomarkers in human body fluids, such as sweat [70]. The nanosensor was functionalized with a short DNA aptamer consisting of 25 nucleotides that acted as a cytokine-specific receptor in order to achieve affinity recognition of the biomarker and enhanced sensitivity of cytokine detection. The researchers proved that the nanosensor was capable of responding to changes in TNF-\(\upalpha\) concentration within 5 min with a limit of detection as low as 26 pM [70]. In turn, [PERSON] and co-workers reported the use of graphene quantum dot nanosensors for in vitro and ex vivo detection of very small amounts of INF-\(\upgamma\)[72]. By conjugating graphene quantum dots (GQD) with aptamers of INF-\(\upgamma\) (Ap-GQD conjugate) and with epitopes of INF \(\upgamma\) (Ep-GQD conjugate), the authors developed a \"switch-on\" cytokine nanosensor based on the aggregation and disaggregation of GQDs: in the absence of INF-\(\upgamma\), Ap-GQD and Ep-GQD specifically interact, forming aggregates with no resulting fluorescence. In the presence of the cytokine, the competitive affinity binding between INF-\(\upgamma\) and Ap-GQD results in the disaggregation of the Ep-GQD and Ap-GQD complex, turning on fluorescence, owing to the strong fluorescent signal of each individual conjugate [72]. The nanosensor was efficiently delivered into living cells, presenting great solubility, stability, biocompatibility and high sensitivity, capable of detecting concentrations of intracellular INF-\(\upgamma\) as low as 2 pg/mL [72]. With the increasing knowledge on biosensors, a translation from in vitro to in vivo is required, as the first is not able to comply with the complexity of the latter. Consequently, in vivo biosensing has been the focus of research aiming at the continuous long-term monitoring of biological processes.