The detection of organic molecules is important in many areas, including medicine, environmental monitoring and defence. Stochastic sensing is an approach that relies on the observation of individual binding events between analyte molecules and a single receptor. Engineered transmembrane protein pores are promising sensor elements for stochastic detection, and in their simplest manifestation they produce a fluctuating binary ('on/off') response in the transmembrane electrical current. The frequency of occurrence of the fluctuations reveals the concentration of the analyte, and its identity can be deduced from the characteristic magnitude and/or duration of the fluctuations. Genetically engineered versions of the bacterial pore-forming protein alpha-haemolysin have been used to identify and quantify divalent metal ions in solution. But it is not immediately obvious how versatile binding sites for organic ligands might be obtained by engineering of the pore structure. Here we show that stochastic sensing of organic molecules can be procured from alpha-haemolysin by equipping the channel with an internal, non-covalently bound molecular 'adapter' which mediates channel blocking by the analyte. We use cyclodextrins as the adapters because these fit comfortably inside the pore and present a hydrophobic cavity suitable for binding a variety of organic analytes. Moreover, a single sensing element of this sort can be used to analyse a mixture of organic molecules with different binding characteristics. We envisage the use of other adapters, so that the pore could be 'programmed' for a range of sensing functions.
MicroRNAs are short RNA molecules that regulate gene expression. They have been investigated as potential biomarkers because their expression levels are correlated with various diseases. However, the detection of microRNAs in the bloodstream remains difficult because current methods are not sufficiently selective or sensitive. Here, we show that a nanopore sensor based on the alpha-hemolysin protein selectively detected microRNAs at the single molecular level in plasma samples from lung cancer patients without the need for labelling or amplification. The sensor, which used a programmable oligonucleotide probe to generate a target-specific signature signal, was able to quantify sub-picomolar levels of cancer-associated microRNAs and to discriminate single nucleotide differences between microRNA family members. This approach could prove useful for quantitative microRNA detection, biomarker discovery, and the non-invasive early diagnosis of cancer.
Stochastic sensing is an emerging analytical technique that relies upon single-molecule detection. Transmembrane pores, into which binding sites for analytes have been placed by genetic engineering, have been developed as stochastic sensing elements. Reversible occupation of an engineered binding site modulates the ionic current passing through a pore in a transmembrane potential and thereby provides both the concentration of an analyte and, through a characteristic signature, its identity. Here, we show that the concentrations of two or more divalent metal ions in solution can be determined simultaneously with a single sensor element. Further, the sensor element can be permanently calibrated without a detailed understanding of the kinetics of interaction of the metal ions with the engineered pore.
In this study, the charge selectivity of staphylococcal ␣-hemolysin (␣HL), a bacterial pore-forming toxin, is manipulated by using cyclodextrins as noncovalent molecular adapters. Anionselective versions of ␣HL, including the wild-type pore and various mutants, become more anion selective when -cyclodextrin (CD) is lodged within the channel lumen. By contrast, the negatively charged adapter, hepta-6-sulfato--cyclodextrin (s7CD), produces cation selectivity. The cyclodextrin adapters have similar effects when placed in cation-selective mutant ␣HL pores. Most probably, hydrated Cl ؊ ions partition into the central cavity of CD more readily than K ؉ ions, whereas s7CD introduces a charged ring near the midpoint of the channel lumen and confers cation selectivity through electrostatic interactions. The molecular adapters generate permeability ratios (P K؉/PCl؊) over a 200-fold range and should be useful in the de novo design of membrane channels both for basic studies of ion permeation and for applications in biotechnology.
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