Nipple fluid for breast cancer diagnosis using the nanopore of Phi29 DNA-packaging motor

Abstract

Detection of cancer in its early stage is a challenging task for oncologists. Inflammatory breast cancer has symptoms that are similar to mastitis and can be mistaken for microbial infection. Currently, the differential diagnosis between mastitis and Inflammatory breast cancer via nipple aspirate fluid (NAF) is difficult. Here, we report a label-free and amplification-free detection platform using an engineered nanopore of the phi29 DNA-packaging motor with biomarker Galectin3 (GAL3), Thomsen-Friedenreich (TF) binding peptide as the probe fused at its C-terminus. The binding of the biomarker in NAF samples from breast cancer patients to the probe results in the connector's conformational change with a current blockage of 32 %. Utilization of dwell time, blockage ratio, and peak signature enable us to detect basal levels of biomarkers from patient NAF samples at the single-molecule level. This platform will allow for breast cancers to be resolved at an early stage with accuracy and thoroughness.

Keywords: Biomarker; DNA-packaging motor; Nanopore sensing; Noninvasive; Tumor diagnosis.

Fig. 1.

Illustration and characterization of the channel of engineered phi29 bacterial virus DNA packaging motor. (A) Schematic diagram of real-time detection breast cancer biomarkers from clinic NAF samples. (B) Cross-section structure of the GP 10 connector showing the location of the probe (blue); (C) Construction of the modified gp10 gene by insertion of His tag at the N-terminus, 2-Gly linker and GAL3 or TF probe at the C-terminus; (D) Molecular weight of purified N-His-GP10-GAL3 protein (lane 1,38 kDa), unpurified N-His-GP10-GAL3 protein (lane 2), purified N-His-GP10-TF protein (lane 3,38 kDa), unpurified N-His-GP10-TF protein (lane 4), and control GP10 protein(lane 5) on 10 % SDS-PAGE.

Fig. 2.

Evaluation of biomarkers binding to the motor by ELISA. (A) Asialoglycophorin, glycophorin, asialofetuin or no protein were coated on a plate o/n at 4 °C. After washing, TF channel or biotinylated PNA (BPNA) were added and incubated in an appropriate secondary antibody. Plates were read at 405 nm. (B) GAL3 protein was coated on a plate and various concentrations of biotinylated peptide, channel, or anti-GAL3 antibody were added to well to test the binding efficiency. SA-HRP, anti-HisAb-HRP or antiRat-HRP were used as secondary antibodies. Plates were read at 405 nm.

Fig. 3.

Real-time sensing of cancer biomarkers interaction with engineered phi29 connector channels: (A, D) Current trace results after the addition of the GAL3 protein (A) or TF-HSA (D) antigen. (B, E) Histograms of current blockage after the addition of antigen to corresponding channels; (C, F) Scatter of dwell time versus current blockage result after the addition of GAL3 protein (C) or TF-HSA (F) complex to corresponding channels.

Fig. 4.

Free TF antigen bind to engineered phi29 connector channels: (A) Continuous current trace showing two insertions of TF reengineered connector channels into planar lipid bilayer and the appearance of the signal after the second chamber insertion. (B) Representative current trace result from (A). (C) Histograms of current blockage after the addition of free TF antigen to channel. (D) Scatter of dwell time versus current blockage result after the addition of free TF antigen.

Fig. 5.

GAL3 protein detection in the presence of nonspecific control CA protein with same MW: (A) After addition of CA protein; (B) Histograms of current blockage after the addition of CA protein; (C) Scatter of dwell time versus current blockage after addition of CA protein; (D) Specific GAL3 protein binding events in the presence of CA protein; (E) Histograms of current blockage after the addition of GAL3 protein in the presence of CA protein; (F) Scatter of dwell time versus current blockage after addition of GAL3 protein in the presence of CA protein.

Fig. 6.

Breast cancer biomarker detection from the health and breast cancer patient NAF samples using engineered phi29 connector. (A, B) Current trace results showing representative specific binding events before (A) and after (B) the addition of NAF samples from breast cancer patients to the TF channel. (D, E) Current trace results showing representative specific binding events before (D) and after (E) the addition of NAF samples from breast cancer patients to the GAL3 channel. (C, D) Statistical results of specific binding events from health NAF samples or breast cancer NAF samples in 10 min on N-his-GP10-TF channel (C) and N-his-GP10-GAL3 channel (D). *p < 0.05.