Integrated microdevice with a windmill-like hole array for the clog-free, efficient, and self-mixing enrichment of circulating tumor cells

Abstract

Circulating tumor cells (CTCs) have tremendous potential to indicate disease progression and monitor therapeutic response using minimally invasive approaches. Considering the limitations of affinity strategies based on their cost, effectiveness, and simplicity, size-based enrichment methods that involve low-cost, label-free, and relatively simple protocols have been further promoted. Nevertheless, the key challenges of these methods are clogging issues and cell aggregation, which reduce the recovery rates and purity. Inspired by the natural phenomenon that the airflow around a windmill is disturbed, in this study, a windmill-like hole array on the SU-8 membrane was designed to perturb the fluid such that cells in a fluid would be able to self-mix and that the pressure acting on cells or the membrane would be dispersed to allow a greater velocity. In addition, based on the advantages of fluid coatings, a lipid coating was used to modify the membrane surface to prevent cell aggregation and clogging of the holes. Under the optimal conditions, recovery rates of 93% and 90% were found for A549 and HeLa cells in a clinical simulation test of our platform with a CTC concentration of 20–100 cells per milliliter of blood. The white blood cell (WBC) depletion rate was 98.7% (n = 15), and the CTC detection limit was less than 10 cells per milliliter of blood (n = 6). Moreover, compared with conventional membrane filtration, the advantages of the proposed device for the rapid (2 mL/min) and efficient enrichment of CTCs without clogging were shown both experimentally and theoretically. Due to its advantages in the efficient, rapid, uniform, and clog-free enrichment of CTCs, our platform offers great potential for metastatic detection and therapy analyses.

The integrated on-chip isolation and detection of circulating tumour cells

“…56,119,122 Physical methods of isolation completed on a single chip are reportedly capable of obtaining CTCs with capture efficiencies of 25-99% (ref. 41,54,55,120,[137][138][139]146) and associated purities spanning 20-92%. 54,120,138 While the purity metric is typically lower with physical means of isolation, their utility should not be underestimated as they allow for the recovery of label-free CTCs, delivering an ideal starting point for interference-free downstream analysis.…”

“…In these reports, samples are flowed through microfluidic chips and CTCs are captured in vortices generated by chamber geometries, 138 or by settling into hole arrays. 55,139 These integrated methods have achieved capture efficiencies of 25-99% from a few mL of whole blood and obtained purities of 35-92%, with a WBC depletion of up to 98.7%. 55,[138][139][140] A recent study utilized a nanostructured array to isolate individual cells within nanodroplets, functioning as individual reactors for evaluating the secretion of matrix metalloproteinase 9 (MMP6), an enzyme crucial in the EMT process of cancer cells.…”

“…55,139 These integrated methods have achieved capture efficiencies of 25-99% from a few mL of whole blood and obtained purities of 35-92%, with a WBC depletion of up to 98.7%. 55,[138][139][140] A recent study utilized a nanostructured array to isolate individual cells within nanodroplets, functioning as individual reactors for evaluating the secretion of matrix metalloproteinase 9 (MMP6), an enzyme crucial in the EMT process of cancer cells. The methodology involved the use of a specially designed FRET probe to assess enzyme activity without affecting cell integrity or contents, thus enabling the recovery of individual cells for subsequent analyses, as the method maintains the integrity of cells without requiring any surface modifications.…”

“…However, sample input across these works has not been consistent, with some groups completing their enrichment on unmodified patient samples, 137,139 whilst other studies utilised pre-processing, including RBC lysis, various washing steps, 41 and PBS dilution of blood. 55,138 As with integrated platforms applying antigen-dependent isolation, the vast majority of physical methods utilised within integrated formats have been paired with optical detection methods, 41,55,[137][138][139][140]142,149 with only two reports of electrochemical means of detection. 54,146 3 Detection of CTCs…”

“…Optical methods are most commonly fulfilled through well-established immunofluorescence (IF) imaging, using multichannel fluorescence staining of cellular markers. 55,56,115,116,119,121,122,137,139,140,149 CTCs are typically detected based on CK (cytokeratin) positive , EpCAM positive , nuclear DAPI positive , and CD45 negative signatures. 69 However, IF requires cell fixation, numerous manual transfer steps and ultimately, interpretation by a clinical cytologist or pathologist.…”