The structure and unzipping behavior of dumbbell and hairpin DNA revealed by real-time nanopore sensing.

Hairpin structures play an essential role in DNA replication, transcription, and recombination. Single-molecule studies enable the real-time measurement and observation of the energetics and dynamics of hairpin structures, including folding and DNA-protein interactions. Nanopore sensing is emerging as a powerful tool for DNA sensing and sequencing, and previous research into hairpins using an α-hemolysin (α-HL) nanopore suggested that hairpin DNA enters from its stem side. In this work, the translocation and interaction of hairpin and dumbbell DNA samples with varying stems, loops, and toeholds were investigated systematically using a Mycobacterium smegmatis porin A (MspA) nanopore. It was found that these DNA constructs could translocate through the pore under a bias voltage above +80 mV, and blockage events with two conductance states could be observed. The events of the lower blockage were correlated with the loop size of the hairpin or dumbbell DNA (7 nt to 25 nt), which could be attributed to non-specific collisions with the pore, whereas the dwell time of events with the higher blockage were correlated with the stem length, thus indicating effective translocation. Furthermore, dumbbell DNA with and without a stem opening generated different dwell times when driven through the MspA nanopore. Finally, a new strategy based on the dwell time difference was developed to detect single nucleotide polymorphisms (SNPs). These results demonstrated that the unzipping behaviors and DNA-protein interactions of hairpin and dumbbell DNA could be revealed using nanopore technology, and this could be further developed to create sensors for the secondary structures of nucleic acids.

Ultrasensitive Nanopore Sensing of Mucin 1 and Circulating Tumor Cells in Whole Blood of Breast Cancer Patients by Analyte-Triggered Triplex-DNA Release.

The characterization of circulating tumor cells (CTCs) by liquid biopsy has a great potential for precision medicine in oncology. Here, a universal and tandem logic-based strategy is developed by combining multiple nanomaterials and nanopore sensing for the determination of mucin 1 protein (MUC1) and breast cancer CTCs in real samples. The strategy consists of analyte-triggered signal conversion, cascaded amplification via nanomaterials including copper sulfide nanoparticles (CuS NPs), silver nanoparticles (Ag NPs), and biomaterials including DNA hydrogel and DNAzyme, and single-molecule-level detection by nanopore sensing. The amplification of the non-DNA nanomaterial gives this method considerable stability, significantly lowers the limit of detection (LOD), and enhances the anti-interference performance for complicated samples. As a result, the ultrasensitive detection of MUC1 could be achieved in the range of 0.0005-0.5 pg/mL, with an LOD of 0.1 fg/mL. Moreover, we further tested MUC1 as a biomarker for the clinical diagnosis of breast cancer CTCs under double-blind conditions on the basis of this strategy, and MCF-7 cells could be accurately detected in the range from 5 to 2000 cells/mL, with an LOD of 2 cells/mL within 6 h. The detection results of the 19 clinical samples were highly consistent with those of the clinical pathological sections, nuclear magnetic resonance imaging, and color ultrasound. These results demonstrate the validity and reliability of our method and further proved the feasibility of MUC1 as a clinical diagnostic biomarker for CTCs.

Real-time sensing of neurotransmitters by functionalized nanopores embedded in a single live cell.

Interface between neuron cells and biomaterials is the key to real-time sensing, transmitting and manipulating of neuron activities, which are the long-term pursue of scientists and gain intense research focus recently. It is of great interest to develop a sensor with exquisite sensitivity and excellent selectivity for real-time monitoring neurotransmitters transport through single live cell. Sensing techniques including electrode-based methods, optogenetics, and nanowire cell penetration systems have been developed to monitor the neuron activities. However, their biocompatibilities remain a challenge. Protein nanopores with membrane compatibility and lumen tunability provide real-time, single-molecule sensitivities for biosensing of DNA, RNA, peptides and small molecules. In this study, an engineered protein nanopore MspA (Mycobacterium smegmatis porin A) through site-directed mutation with histidine selectively bind with Cu2+ in its internal lumen. Chelation of neurotransmitters such as L-glutamate (L-Glu), dopamine (DA) and norepinephrine (NE) with the Cu2+ creates specific current signals, showing different transient current blockade and dwell time in single channel electrophysiological recording. Furthermore, the functionalized M2MspA-N91H nanopores have been embedded in live HEK293T cell membrane for real-time, in situ monitoring of extracellular L-glutamate translocating through the nanopore. This biomimetic neurotransmitter nanopore has provided a new platform for future development of neuron sensors, drug carrier and artificial synapse.

Development of a fluorescent DNA nanomachine for ultrasensitive detection of Salmonella enteritidis without labeling and enzymes.

A capture probe complex containing a specific Salmonella enteritidis (S. enteritidis) aptamer and partly hybridized signal trigger sequence was designed with the ability to directly detect viable S. enteritidis. In the presence of the target S. enteritidis, single-stranded trigger sequences were liberated and in turn reacted with hairpins I, II, and III to initiate the triple strand migration reaction; this in turn produced numerous hairpin I·II·III complexes with scaffolds of copper nanoparticles (CuNPs) and replaced the trigger sequence which initiated the next cycle of triple migration reaction. Cyclically, the reuse of the trigger sequences and the successive, cascading production of scaffolds of CuNPs achieved the synthesis of highly fluorescent CuNPs, thus providing significantly enhanced fluorescent signals to achieve ultrasensitive detection of live S. enteritidis as low as 25 CFU/mL with a linear range of detection from 50 to 104 CFU/mL with an emission wavelength at 590 nm. By integrating the triple cascade strand migration amplification with recyclable trigger sequences, aptamer-based target recognition, and self-protection mediated by CuNPs hairpin scaffolds, this is the first report on a non-labeled, non-enzymatic, modification-free, and DNA extraction-free ultrasensitive fluorescent biosensor for the direct detection of live Salmonella, which is distinguished from dead Salmonella. It also provides a new strategy to detect viable bacteria by applying the CuNPs, thus extending the application of metal nanoparticles. Graphical abstract.

Detection of Circulating Tumor Cells in Breast Cancer Patients by Nanopore Sensing with Aptamer-Mediated Amplification.

Circulating tumor cells (CTCs) have been utilized in the diagnosis and prognosis of tumor. However, the CTC concentration is extremely low to be detected in peripheral blood. Many existing methods suffer from either expensive labeling or complex operation. In this study, we constructed a label- and enzyme-free and sensitive method to detect the breast cancer CTCs. First of all, a probe containing a breast cancer cell-specific aptamer and a complementary single-stranded DNA (trigger DNA P1) were designed. When the target cells are present, the aptamer binds to the CTCs and releases P1 which triggers the strand displacement amplification. This process generates three-way junction structure DNA, the specific translocation signals of which are identified by nanopore assay. The detection limit of tumor cells is 5 in the current experimental setup and can be further reduced. Furthermore, the method is demonstrated in a clinical sample test with high recovery rate and accuracy. Our results suggest that this method could be applied to early diagnosis of metastatic recurrence and prognosis determination.

Active DNA unwinding and transport by a membrane-adapted helicase nanopore.

Nanoscale transport through nanopores and live-cell membranes plays a vital role in both key biological processes as well as biosensing and DNA sequencing. Active translocation of DNA through these nanopores usually needs enzyme assistance. Here we present a nanopore derived from truncated helicase E1 of bovine papillomavirus (BPV) with a lumen diameter of c.a. 1.3 nm. Cryogenic electron microscopy (cryo-EM) imaging and single channel recording confirm its insertion into planar lipid bilayer (BLM). The helicase nanopore in BLM allows the passive single-stranded DNA (ssDNA) transport and retains the helicase activity in vitro. Furthermore, we incorporate this helicase nanopore into the live cell membrane of HEK293T cells, and monitor the ssDNA delivery into the cell real-time at single molecule level. This type of nanopore is expected to provide an interesting tool to study the biophysics of biomotors in vitro, with potential applications in biosensing, drug delivery and real-time single cell analysis.