Li-Qun Gu

Stochastic sensing of organic analytes by a pore-forming protein containing a molecular adapter.

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 α-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 α-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.

Nanopore-based detection of circulating microRNAs in lung cancer patients

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.

Simultaneous stochastic sensing of divalent metal ions.

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.

Capturing Single Molecules of Immunoglobulin and Ricin with an Aptamer-Encoded Glass Nanopore

Nanopore-based single-molecule biosensors have been extensively studied. Protein pores that have receptors attached to them are target-selective, but their real-time applications are limited by the fragility of the lipid membrane into which the protein pores are embedded. Synthetic nanopores are more stable and provide flexible pore sizes, but the selectivity is low when detecting in the translocation mode. In spite of modifications with probing molecules, such as antibodies, to potentiate specific targeting, these nanopores fail to bind individual target molecules. Distinguishing between binding and translocation blocks remains unsolved. Here, we propose an aptamer-encoded nanopore that overcomes these challenges. Aptamers are well-known probing oligonucleotides that have high sensitivity and selectivity. In contrast to antibodies, aptamers are much smaller than their targets, rendering target blockades in the nanopore much more distinguishable. We used aptamer-encoded nanopores to detect single molecules of immunoglobulin E and the bioterrorist agent ricin, sequentially captured by the immobilized aptamer in the sensing zone of the pore. The functional nanopore also probed sequence-dependent aptamer-protein interactions. These findings will facilitate the development of a universal nanopore for multitarget detection.

Stochastic sensing of TNT with a genetically engineered pore

Engineered versions of the transmembrane protein pore α‐hemolysin (αHL) can be used as stochastic sensing elements for the identification and quantification of a wide variety of analytes at the single‐molecule level. Until now, nitroaromatic analytes have eluded detection by this approach. We now report that binding sites for nitroaromatics can be built within the lumen of the αHL pore from simple rings of seven aromatic amino acid side chains (Phe, Tyr or Trp). By monitoring the ionic current that passes through a single pore at a fixed applied potential, various nitroaromatics can be distinguished from TNT on the basis of the amplitude and duration of individual current‐blocking events. Rings of less than seven aromatics bind the analytes more weakly; this suggests that direct aromatic–aromatic interactions are involved. The engineered pores should be useful for the detection of explosives and, in combination with computational approaches and structural analysis, they could further our understanding of noncovalent interactions between aromatic molecules.

Single-molecule detection of folding and unfolding of the G-quadruplex aptamer in a nanopore nanocavity

Guanine-rich nucleic acids can form G-quadruplexes that are important in gene regulation, biosensor design and nano-structure construction. In this article, we report on the development of a nanopore encapsulating single-molecule method for exploring how cations regulate the folding and unfolding of the G-quadruplex formed by the thrombin-binding aptamer (TBA, GGTTGGTGTGGTTGG). The signature blocks in the nanopore revealed that the G-quadruplex formation is cation-selective. The selectivity sequence is K+ > NH4+ ∼ Ba2+ > Cs+ ∼ Na+ > Li+, and G-quadruplex was not detected in Mg2+ and Ca2+. Ba2+ can form a long-lived G-quadruplex with TBA. However, the capability is affected by the cation–DNA interaction. The cation-selective formation of the G-quadruplex is correlated with the G-quadruplex volume, which varies with cation species. The high formation capability of the K+-induced G-quadruplex is contributed largely by the slow unfolding reaction. Although the Na+- and Li+-quadruplexes feature similar equilibrium properties, they undergo radically different pathways. The Na+-quadruplex folds and unfolds most rapidly, while the Li+-quadruplex performs both reactions at the slowest rates. Understanding these ion-regulated properties of oligonucleotides is beneficial for constructing fine-tuned biosensors and nano-structures. The methodology in this work can be used for studying other quadruplexes and protein–aptamer interactions.

Electroosmotic enhancement of the binding of a neutral molecule to a transmembrane pore

The flux of solvent water coupled to the transit of ions through protein pores is considerable. The effect of this electroosmotic solvent flow on the binding of a neutral molecule [β-cyclodextrin (βCD)] to sites within the staphylococcal α-hemolysin pore was investigated. Mutant α-hemolysin pores were used to which βCD can bind from either entrance and through which the direction of water flow can be controlled by choosing the charge selectivity of the pore and the polarity of the applied potential. The Kd values for βCD for individual mutant pores varied by >100-fold with the applied potential over a range of –120 to +120 mV. In all cases, the signs of the changes in binding free energy and the influence of potential on the association and dissociation rate constants for βCD were consistent with an electroosmotic effect.

Method of Creating a Nanopore-Terminated Probe for Single-Molecule Enantiomer Discrimination

Nanopores are increasingly utilized as tools for single-molecule detection in biotechnology. Many nanopores are fabricated through procedures that require special materials, expensive facilities and experienced operators, which limit their usefulness on a wider scale. We have developed a simple method of fabricating a robust, low-noise nanopore by externally penetrating a nanocavity enclosed in the terminal of a capillary pipet. The nanocavity was shown to have a pore size on the scale of a single molecule, verified by translocation of molecules of known sizes, including double-stranded DNA (2 nm), gold nanoparticles (10 nm), and ring-shaped cyclodextrin (1.5 nm). The small pore size allows entrapment of a single cyclodextrin molecule. The glass nanopore with a trapped cyclodextrin proves useful in single-molecule discrimination of chiral enantiomers.

Molecular bases of cyclodextrin adapter interactions with engineered protein nanopores

Engineered protein pores have several potential applications in biotechnology: as sensor elements in stochastic detection and ultrarapid DNA sequencing, as nanoreactors to observe single-molecule chemistry, and in the construction of nano- and micro-devices. One important class of pores contains molecular adapters, which provide internal binding sites for small molecules. Mutants of the α-hemolysin (αHL) pore that bind the adapter β-cyclodextrin (βCD) ∼104 times more tightly than the wild type have been obtained. We now use single-channel electrical recording, protein engineering including unnatural amino acid mutagenesis, and high-resolution x-ray crystallography to provide definitive structural information on these engineered protein nanopores in unparalleled detail.

Programming nanopore ion flow for encoded multiplex microRNA detection.

Many efforts are being made in translating the nanopore into an ultrasensitive single-molecule platform for various genetic and epigenetic detections. However, compared with current approaches including PCR, the low throughput limits the nanopore applications in biological research and clinical settings, which usually requires simultaneous detection of multiple biomarkers for accurate disease diagnostics. Herein we report a barcode probe approach for multiple nucleic acid detection in one nanopore. Instead of directly identifying different targets in a nanopore, we designed a series of barcode probes to encode different targets. When the probe is bound with the target, the barcode group polyethylene glycol attached on the probe through click chemistry can specifically modulate nanopore ion flow. The resulting signature serves as a marker for the encoded target. Therefore counting different signatures in a current recording allows simultaneous analysis of multiple targets in one nanopore. The principle of this approach was verified by using a panel of cancer-derived microRNAs as the target, a type of biomarker for cancer detection.