Circulating rare cells in the blood are of great significance for both materials research and clinical applications. For example, circulating tumor cells (CTCs) have been demonstrated as useful biomarkers for "liquid biopsy" of the tumor. Circulating fetal nucleated cells (CFNCs) have shown potential in noninvasive prenatal diagnostics. However, it is technically challenging to detect and isolate circulating rare cells due to their extremely low abundance compared to hematologic cells. Nanostructured substrates offer a unique solution to address these challenges by providing local topographic interactions to strengthen cell adhesion and large surface areas for grafting capture agents, resulting in improved cell capture efficiency, purity, sensitivity, and reproducibility. In addition, rare-cell retrieval strategies, including stimulus-responsiveness and additive reagent-triggered release on different nanostructured substrates, allow for on-demand retrieval of the captured CTCs/CFNCs with high cell viability and molecular integrity. Several nanostructured substrate-enabled CTC/CFNC assays are observed maturing from enumeration and subclassification to molecular analyses. These can one day become powerful tools in disease diagnosis, prognostic prediction, and dynamic monitoring of therapeutic response-paving the way for personalized medical care.

Circulating tumor cells (CTCs) are cancer cells shredded from either a primary tumor or a metastatic site and circulate in the blood as the potential cellular origin of metastasis. By detecting and analyzing CTCs, we will be able to noninvasively monitor disease progression in individual cancer patients and obtain insightful information for assessing disease status, thus realizing the concept of "tumor liquid biopsy". However, it is technically challenging to identify CTCs in patient blood samples because of the extremely low abundance of CTCs among a large number of hematologic cells. In order to address this challenge, our research team at UCLA pioneered a unique concept of "NanoVelcro" cell-affinity substrates, in which CTC capture agent-coated nanostructured substrates were utilized to immobilize CTCs with remarkable efficiency. Four generations of NanoVelcro CTC assays have been developed over the past decade for a variety of clinical utilities. The 1st-gen NanoVelcro Chips, composed of a silicon nanowire substrate (SiNS) and an overlaid microfluidic chaotic mixer, were created for CTC enumeration. The 2nd-gen NanoVelcro Chips (i.e., NanoVelcro-LMD), based on polymer nanosubstrates, were developed for single-CTC isolation in conjunction with the use of the laser microdissection (LMD) technique. By grafting thermoresponsive polymer brushes onto SiNS, the 3rd-gen Thermoresponsive NanoVelcro Chips have demonstrated the capture and release of CTCs at 37 and 4 °C respectively, thereby allowing for rapid CTC purification while maintaining cell viability and molecular integrity. Fabricated with boronic acid-grafted conducting polymer-based nanomaterial on chip surface, the 4th-gen NanoVelcro Chips (Sweet chip) were able to purify CTCs with well-preserved RNA transcripts, which could be used for downstream analysis of several cancer specific RNA biomarkers. In this review article, we will summarize the development of the four generations of NanoVelcro CTC assays, and the clinical applications of each generation of devices.

Tumor-derived extracellular vesicles (EVs) play essential roles in intercellular communication during tumor growth and metastatic evolution. Currently, little is known about the possible roles of tumor-derived EVs in sarcoma because the lack of specific surface markers makes it technically challenging to purify sarcoma-derived EVs. In this study, a specific purification system is developed for Ewing sarcoma (ES)-derived EVs by coupling covalent chemistry-mediated EV capture/ release within a nanostructure-embedded microchip. The purification platform-ES-EV Click Chip-takes advantage of specific anti-LINGO-1 recognition and sensitive click chemistry-mediated EV capture, followed by disulfide cleavage-driven EV release. Since the device is capable of specific and efficient purification of intact ES EVs with high purity, ES-EV Click Chip is ideal for conducting downstream functional studies of ES EVs. Absolute quantification of the molecular hallmark of ES (i.e., EWS rearrangements) using reverse transcription Droplet Digital PCR enables specific quantification of ES EVs. The purified ES EVs can be internalized by recipient cells and transfer their mRNA cargoes, exhibiting their biological intactness and potential role as biological shuttles in intercellular communication.

Well-preserved mRNA in circulating tumor cells (CTCs) offers an ideal material for conducting molecular profiling of tumors, thereby providing a noninvasive diagnostic solution for guiding treatment intervention and monitoring disease progression. However, it is technically challenging to purify CTCs while retaining high-quality mRNA.Here, we demonstrate a covalent chemistry-based nanostructured silicon substrate ("Click Chip") for CTC purification that leverages bioorthogonal ligation-mediated CTC capture and disulfide cleavage-driven CTC release. This platform is ideal for CTC mRNA assays because of its efficient, specific, and rapid purification of pooled CTCs, enabling downstream molecular quantification using reverse transcription Droplet Digital polymerase chain reaction. Rearrangements of ALK/ROS1 were quantified using CTC mRNA and matched with those identified in biopsy specimens from 12 patients with late-stage non-small cell lung cancer. Moreover, CTC counts and copy numbers of ALK/ROS1 rearrangements could be used together for evaluating treatment responses and disease progression.

Tumor-derived extracellular vesicles (EVs) present in bodily fluids are emerging liquid biopsy markers for non-invasive cancer diagnosis and treatment monitoring. Because the majority of EVs in circulation are not of tumor origin, it is critical to develop new platforms capable of enriching tumor-derived EVs from the blood. Herein, we introduce a biostructure-inspired NanoVilli Chip, capable of highly efficient and reproducible immunoaffinity capture of tumor-derived EVs from blood plasma samples. Anti-EpCAM-grafted silicon nanowire arrays were engineered to mimic the distinctive structures of intestinal microvilli, dramatically increasing surface area and enhancing tumor-derived EV capture. RNA in the captured EVs can be recovered for downstream molecular analyses by reverse transcription Droplet Digital PCR. We demonstrate that this assay can be applied to monitor the dynamic changes of ROS1 rearrangements and epidermal growth factor receptor T790M mutations that predict treatment responses and disease progression in non-small cell lung cancer patients.

Placenta accreta spectrum (PAS) is a high-risk obstetrical condition associated with significant morbidity and mortality. Current clinical screening modalities for PAS are not always conclusive. Here, we report a nanostructure-embedded microchip that efficiently enriches both single and clustered circulating trophoblasts (cTBs) from maternal blood for detecting PAS. We discover a uniquely high prevalence of cTB-clusters in PAS and subsequently optimize the device to preserve the intactness of these clusters. Our feasibility study on the enumeration of cTBs and cTB-clusters from 168 pregnant women demonstrates excellent diagnostic performance for distinguishing PAS from non-PAS. A logistic regression model is constructed using a training cohort and then cross-validated and tested using an independent cohort. The combined cTB assay achieves an Area Under ROC Curve of 0.942 (throughout gestation) and 0.924 (early gestation) for distinguishing PAS from non-PAS. Our assay holds the potential to improve current diagnostic modalities for the early detection of PAS.