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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. The NAF sample was collected by a nipple aspiration device and then subjected to nanopore sensing using the re-engineered durable channel of the Phi29 DNA packaging motor. Breast cancer was diagnosed with the utilization of signature signals after biomarkers binding to the nanopore. [Display omitted] [ABSTRACT FROM AUTHOR]

In optical devices such as camera or microscope, an aperture is used to regulate light intensity for imaging. Here we report the discovery and construction of a durable bio-aperture at nanometerscale that can regulate current at the pico-ampere scale. The nano-aperture is made of 12 identical protein subunits that form a 3.6-nm channel with a shutter and "one-way traffic" property. This shutter responds to electrical potential differences across the aperture and can be turned off for double stranded DNA translocation. This voltage enables directional control, and three-step regulation for opening and closing. The nano-aperture was constructed in vitro and purified into homogeneity. The aperture was stable at pH2-12, and a temperature of −85C–60C. When an electrical potential was held, three reproducible discrete steps of current flowing through the channel were recorded. Each step reduced 32% of the channel dimension evident by the reduction of the measured current flowing through the aperture. The current change is due to the change of the resistance of aperture size. The transition between these three distinct steps and the direction of the current was controlled via the polarity of the voltage applied across the aperture. When the C-terminal of the aperture was fused to an antigen, the antibody and antigen interaction resulted in a 32% reduction of the channel size. This phenomenon was used for disease diagnosis since the incubation of the antigen-nano-aperture with a specific cancer antibody resulted in a change of 32% of current. The purified truncated cone-shape aperture automatically self-assembled efficiently into a sheet of the tetragonal array via head-to-tail self-interaction. The nano-aperture discovery with a controllable shutter, discrete-step current regulation, formation of tetragonal sheet, and one-way current traffic provides a nanoscale electrical circuit rectifier for nanodevices and disease diagnosis. [ABSTRACT FROM AUTHOR]

Protein post-translational modification (PTM) is crucial to modulate protein interactions and activity in various biological processes. Emerging evidence has revealed PTM patterns participate in the pathology onset and progression of various diseases. Current PTM identification relies mainly on mass spectrometry-based approaches that limit the assessment to the entire protein population in question. Here we report a label-free method for the detection of the single peptide with only one amino acid modification via electronic fingerprinting using reengineered durable channel of phi29 DNA packaging motor, which bears the deletion of 25-amino acids (AA) at the C-terminus or 17-AA at the internal loop of the channel. The mutant channels were used to detect propionylation modification via single-molecule fingerprinting in either the traditional patch-clamp or the portable MinION™ platform of Oxford Nanopore Technologies. Up to 2000 channels are available in the MinION™ Flow Cells. The current signatures and dwell time of individual channels were identified. Peptides with only one propionylation were differentiated. Excitingly, identification of single or multiple modifications on the MinION™ system was achieved. The successful application of PTM differentiation on the MinION™ system represents a significant advance towards developing a label-free and high-throughput detection platform utilizing nanopores for clinical diagnosis based on PTM. [ABSTRACT FROM AUTHOR]