Ultrasensitive Detection of Cancer Cells Combining Enzymatic Signal Amplification with an Aerolysin Nanopore

Abstract

Sensitive detection of cancer cells at extremely low concentrations would greatly facilitate the screening and early diagnosis of cancer. Herein, we present a novel nanopore-based strategy for ultrasensitive detection of Ramos cells (human Burkitt's lymphoma cells), by combining the enzymatic signal amplification with an aerolysin nanopore sensor. In this assay, an aptamer for Ramos cells was prehybridized with a short complementary DNA. The presence of target cells causes the target-aptamer complex to unwind to free the complementary DNA, which would subsequently trigger the enzymatic cycling amplification. This process eventually generated a large number of output DNA, which could quantitatively produce characteristic current events when translocated through aerolysin. The proposed method exhibits excellent sensitivity, and as few as 5 Ramos cells could be detected. With good selectivity, the approach can allow for the determination of cancer cells in human serum, offering a powerful tool for biomedical research and clinical diagnosis.

A General Strategy of Aerolysin Nanopore Detection for Oligonucleotides with the Secondary Structure

“…When a biomolecule is electrophoretically driven through a nanoscale pore, a transient blockade of ionic current is produced to yield a unique current signature for each analyzed target molecule. Due to the high reproducibility and excellent spatial resolution, the biological nanopore has been widely utilized to study DNAs microRNAs, poly(ethylene glycol) (PEGs), peptides, etc. Particularly, our group discovered that the aerolysin nanopore exhibits outstanding capability of discriminating short oligonucleotides (≤10 nt) at single nucleotide resolution, In spite of this effort, however, a direct analysis of secondary structured oliogonucleotide with a longer length (>10 nt) became one of major challenges which hinders aerolysin to achieve the DNA‐based sensing and diagnosing.…”

The Effects of Tetramethylammonium Cation on Oligonucleotide Analysis with Aerolysin Nanopore

“…[8] In addition to the widely used αhemolysin, varieties of membrane proteins have been used as biological nanopore for single-molecule analysis, such as mycobacterium smegmatis porin A (MspA), [9][10] Outer membrane protein G (OmpG), [11][12] cytolysin A (ClyA) [13][14] and Aerolysin. [15][16] Aerolysin is a β-pore-forming toxin from Aeromonas hydrophila, [17] which could spontaneously oligomerize and insert into the lipid bilayer forming a nanopore, which narrowest diameter is only 1.0 nm and possess a long and uniform βbarrel. [18] In 2006, Aerolysin was firstly applied to analyze the structure of α-helix peptides as a nanopore sensor.…”

Enzyme and AIEgens Modulated Solid‐State Nanochannels: In Situ and Noninvasive Monitoring of H2O2 Released from Living Cells

“…The recent decades have seen the great development of sensing field using functionalized solid‐state nanochannels . So far, the functionalized solid‐state nanochannels have revealed tremendous abilities in the sensing of ions, small biomolecules, biological macromolecules, and even cellular targets with the unique merits of rapid response, high sensitivity, miniaturization and low‐cost .…”