Nanopore-based detection of periodontitis biomarker miR31 in saliva samples.

MicroRNAs (miRNAs) play important roles in posttranscriptional gene regulation. Aberrations in the miRNA levels have been the cause behind various diseases, including periodontitis. Therefore, sensitive, specific, and accurate detection of disease-associated miRNAs is vital to early diagnosis and can facilitate inhibitor screening and drug design. In this study, we developed a label-free, real-time sensing method for the detection of miR31, which has been frequently linked to periodontitis, using an engineered protein nanopore and in the presence of a complementary ssDNA as a molecular probe. Our method is rapid and highly sensitive with nanomolar concentration of miR31 that could be determined in minutes. Furthermore, our sensor showed high selectivity toward the target miR31 sequence even in the presence of interfering nucleic acids. In addition, artificial saliva and human saliva samples were successfully analyzed. Our developed nanopore sensing platform could be used to detect other miRNAs and offers a potential application for the clinical diagnosis of disease biomarkers.

A highly sensitive nanopore platform for measuring RNase A activity.

Ribonuclease A (RNase A) plays significant roles in several physiological and pathological conditions and can be used as a valuable diagnostic biomarker for human diseases such as myocardial infarction and cancer. Hence, it is of great importance to develop a rapid and cost-effective method for the highly sensitive detection of RNase A. The significance of RNase A assay is further enhanced by the growing attention from the biotechnology and pharmaceutical industries to develop RNA-based vaccines and drugs in large part as a result of the successful development of mRNA vaccines in the COVID-19 pandemic. Herein, we report a label-free method for the detection of RNase A by monitoring its proteolytic cleavage of an RNA substrate in a nanopore. The method is ultra-sensitive with the limit of detection reaching as low as 30 fg per milliliter. Furthermore, sensor selectivity and the effects of temperature, incubation time, metal ion, salt concentration on sensor sensitivity were also investigated.

Probe-assisted detection of Fe3+ ions in a multi-functionalized nanopore.

Iron is an essential element that plays critical roles in many biological/metabolic processes, ranging from oxygen transport, mitochondrial respiration, to host defense and cell signaling. Maintaining an appropriate iron level in the body is vital to the human health. Iron deficiency or overload can cause life-threatening conditions. Thus, developing a new, rapid, cost-effective, and easy to use method for iron detection is significant not only for environmental monitoring but also for disease prevention. In this study, we report an innovative Fe3+ detection strategy by using both a ligand probe and an engineered nanopore with two binding sites. In our design, one binding site of the nanopore has a strong interaction with the ligand probe, while the other is more selective toward interfering species. Based on the difference in the number of ligand DTPMPA events in the absence and presence of ferric ions, micromolar concentrations of Fe3+ could be detected within minutes. Our method is selective: micromolar concentrations of Mg2+, Ca2+, Cd2+, Zn2+, Ni2+, Co2+, Mn2+, and Cu2+ would not interfere with the detection of ferric ions. Furthermore, Cu2+, Ni2+, Co2+, Zn2+, and Mn2+ produced current blockage events with quite different signatures from each other, enabling their simultaneous detection. In addition, simulated water and serum samples were successfully analyzed. The nanopore sensing strategy developed in this work should find useful application in the development of stochastic sensors for other substances, especially in situations where multi-analyte concurrent detection is desired.

Nanopore Stochastic Sensing Based on Non-covalent Interactions.

A variety of species could be detected by using nanopores engineered with various recognition sites based upon non-covalent interactions, including electrostatic, aromatic, and hydrophobic interactions. The existence of these engineered non-covalent bonding sites was supported by the single-channel recording technique. The advantage of the non-covalent interaction-based sensing strategy was that the recognition site of the engineered nanopore was not specific for a particular molecule but instead selective for a class of species (e.g., cationic, anionic, aromatic, and hydrophobic). Since different species produce current modulations with quite different signatures represented by amplitude, residence time, and even characteristic voltage-dependence curve, the non-covalent interaction-based nanopore sensor could not only differentiate individual molecules in the same category but also enable differentiation between species with similar structures or molecular weights. Hence, our developed non-covalent interaction-based nanopore sensing strategy may find useful application in the detection of molecules of medical and/or environmental importance.