Peptide sequencing in an electrolytic cell with two nanopores in tandem and exopeptidase.

Open defecation is practised by over 600 million people in India and there is a strong political drive to eliminate this through the provision of on-site sanitation in rural areas. However, there are concerns that the subsequent leaching of excreta from subsurface storage could be adversely impacting underlying groundwater resources upon which rural populations are almost completely dependent for domestic water supply. We investigated this link in four villages undergoing sanitary interventions in Bihar State, India. A total of 150 supplies were sampled for thermotolerant (faecal) coliforms (TTC) and tryptophan-like fluorescence (TLF): an emerging real-time indicator of faecal contamination. Sanitary risk inspections were also performed at all sites, including whether a supply was located within 10 m of a toilet, the recommended minimum separation. Overall, 18% of water supplies contained TTCs, 91% of which were located within 10 m of a toilet, 58% had TLF above detection limit, and sanitary risk scores were high. Statistical analysis demonstrated TLF was an effective indicator of TTC presence-absence, with a possibility of TTCs only where TLF exceeded 0.4 μg/L dissolved tryptophan. Analysis also indicated proximity to a toilet was the only significant sanitary risk factor predicting TTC presence-absence and the most significant predictor of TLF. Faecal contamination was considered a result of individual water supply vulnerability rather than indicative of widespread leaching into the aquifer. Therefore, increasing faecal contamination of groundwater-derived potable supplies is inevitable across the country as uptake of on-site sanitation intensifies. Communities need to be aware of this link and implement suitable decentralised low-cost treatment of water prior to consumption and improve the construction and protection of new supplies.

A tandem electrolytic cell with the structure [cis1, upstream nanopore (UNP), trans1 ¼ cis2, downstream nanopore (DNP), trans2], an exonuclease enzyme attached to the downstream side of UNP, and a chemical adapter in or a profiled voltage over DNP can be used with bandwidths of a few kHz to sequence bases in ssDNA in natural order with high accuracy and without loss in trans1/cis2 or regression into DNP from trans2.

A major obstacle faced by nanopore-based polymer sequencing and analysis is the high speed of translocation of an analyte (nucleotide, DNA, amino acid (AA), peptide) through the pore; the rate currently exceeds available detector bandwidth. Except for one method that uses an enzyme ratchet to sequence DNA, attempts to resolve the problem satisfactorily have been largely unsuccessful. Here a counterintuitive method based on reversing the pore voltage, and, with some analytes, increasing their mobility, is described. A simplified Fokker-Planck model shows a significant increase in translocation times for single nucleotides and AAs (up from ∼10 ns to ∼1 ms). Simulations show that with a bi-level positive-negative pore voltage profile all four nucleotides and the 20 proteinogenic amino acids can be trapped inside the pore long enough for detection with bandwidths of ∼1-10 Khz. The method presented here provides, at least in theory, a potentially viable solution to a problem that has prevented nanopore-based polymer sequencing methods from realizing their full potential.

Experiments have shown that DNA can be sequenced using an electrolytic cell with a nanopore and an exonuclease enzyme in the cis chamber that cleaves the leading mononucleotide in a strand of DNA. The base therein can be identified with an accuracy of 80-90% by the level of the current blockade caused in the pore; a biological adapter inside slows down the cleaved mononucleotide and lowers the detection bandwidth required. In this approach, which has been mathematically modeled, analyzed, and simulated, mononucleotides are likely to be lost to diffusion or enter the pore out of order. To remedy this, a modified cell with three stacked nanopores (named UNP, MNP, and DNP) and the enzyme attached to the trans side of UNP is proposed and modeled. Mononucleotide translocation is simulated with the random walk of a dimensionless particle; the results show that the cleaved mononucleotides translocate through MNP and DNP in sequence order without loss. If this holds in practice then with a suitably designed adapter and compatible enzyme turnover rates sequencing accuracy would be limited only by the accuracy of mononucleotide discrimination. Potential implementation issues are discussed.