Hierarchical Amplification-Interlinked CRISPR-Cas14a Luminescent Biosensor Coupled with Portable Photonic Crystal Biochip-Boosted Time-Delayed Signaling for the Diagnosis of Pediatric Mycoplasma pneumoniae Pneumonia.

Mycoplasma pneumoniae pneumonia (MPP) is a respiratory infection that readily propagates within pediatric populations, and thus, the development of a robust assay approach for its timely diagnosis is of clinical importance. In this contribution, we build a hierarchical amplification-interlinked CRISPR-Cas14a luminescent biosensor coupled with portable photonic crystal biochip-boosted time-delayed signaling to fulfill this need. To first perform highly sensitive detection, an initial DNA walker module and a later rolling circle amplification module are integrated to construct a hierarchical amplification, which is then utilized to interlink CRISPR-Cas14a systems. To further improve applicability in complicated biosamples, a time-delayed signaling actualized by afterglow luminescence is introduced to circumvent background autoluminescence from biological media. After employing a photonic crystal self-assembled from polystyrene nanospheres to manufacture a portable biochip, the naturally attenuated afterglow luminescence is dramatically boosted. Leveraging these strategies, our biosensing platform achieves a limit of detection as low as 2.27 fM for the M. pneumoniae sequence while maintaining good specificity. Moreover, the luminescent biosensor permits precise analysis of targets in throat swab samples from a pediatric cohort (n = 250) comprising severe and mild MPP patients as well as healthy controls, based on which a convolutional neural network-implemented deep learning model is finally established for accurate automated disease stratification, thereby holding great potential as an efficient and promising diagnostic tool.