Single-Cell Liquid Biopsy of Lung Cancer: Ultra-Simplified Efficient Enrichment of Circulating Tumor Cells and Hand-Held Fluorometer Portable Testing

Abstract

Herein, we propose a paper-based laboratory via enzyme-free nucleic acid amplification and nanomaterial-assisted cation exchange reactions (CERs) assisted single-cell-level analysis (PLACS). This method allowed for the rapid detection of mucin 1 and trace circulating tumor cells (CTCs) in the peripheral blood of lung cancer patients. Initially, an independently developed method requiring one centrifuge, two reagents (lymphocyte separation solution and erythrocyte lysate), and a three-step, 45 min sample pretreatment was employed. The core of the detection approach consisted of two competitive selective identifications: copper sulfide nanoparticles (CuS NPs) to C-Ag⁺-C and Ag⁺, and dual quantum dots (QDs) to Cu²⁺ and CuS NPs. To facilitate multimodal point-of-care testing (POCT), we integrated solution visualization, test strip length reading, and a self-developed hand-held fluorometer readout. These methods were detectable down to ag/mL of mucin 1 concentration and the single-cell level. Forty-seven clinical samples were assayed by fluorometer, yielding 94% (30/32) sensitivity and 100% (15/15) specificity with an area under the curve (AUC) of 0.945. Nine and 15 samples were retested by a test strip and hand-held fluorometer, respectively, with an AUC of 0.95. All test results were consistent with the clinical imaging and the folate receptor (FR)-PCR kit findings, supporting its potential in early diagnosis and postoperative monitoring.

Keywords: CTC enrichment; POCT; cascade amplification; clinical sample; hand-held instrument.