Abstract Cytokine monitoring has attracted great attention due to its significance in the diagnosis and treatment of many diseases, such as tumors, microbial infections, and immunological diseases. Enzyme‐linked immunosorbent assay (ELISA) is one of the most popular methods in cytokine detection, ascribing to the lavish signal amplification methods in the ELISA platform. In addition to classical enzymes, other signal amplifiers such as fluorescent probes, artificial nano‐enzymes, and photothermal reagents have been applied to reduce the detection limit and produce more sensitive ELISA kits. Due to the accumulative effect of heat, photothermal reagents are promising materials in the signal amplification of ELISA. However, the lack of efficient photothermal generation material at an aggregate scale may delay the further development of this area. In this contribution, based on an efficient organic photothermal aggregate material, an enzyme‐free photothermally amplified fluorescent immunosorbent assay system consisting of an assay microfluidic chip and detecting platform was developed. The photothermal nanoparticles with highly efficient photothermal conversion by harvesting energy via excited‐state intramolecular motions and enlarging molar absorptivity were successfully prepared. The detection concentration at 50 pg/mL of interleukin‐2 was achieved, realizing a signal improvement of detection limits by 20‐fold compared to that of previously reported photothermal ELISA. The microscopic imaging integrated with plane sweeping technology provided high spatial resolution and precision, indicating the potential of achieving high throughput profiling at the microscale. Moreover, as an alternative excitation source, light‐emitting diode not only provided a more affordable and miniaturized detection system but also revealed the great feasibility of intramolecular motion‐induced photothermy nanoparticles for biological analyses.
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