Determining antibody levels in humans is crucial for monitoring immunity against Covid-19 and tackling the national crisis. Antibody levels report about a previous infection and help decide whether people can return to work or live with others without spreading the disease. To be effective for national screening, antibody testing should deliver accurate results to individuals ideally within minutes, and be portable as well as high-throughput. Existing techniques based on immunosorbent assays do not deliver these benefits due to the need for multiple liquid handling steps, signal amplification, insufficient accuracy, or read-out with bulky optical equipment. This project will deliver fast, portable, high-throughput and accurate antibody sensing by pioneering step-changing sensor nanopores from the lead PI at University College London Chemistry (UCLC), and by integrating them into memory-stick-sized on-the-market kits from industrial partner and biotech unicorn Oxford Nanopore Technologies (ONT). These MinION analysis kits have ushered in a revolution in portable DNA sequencing and are currently used for unravelling the Covid-19 sequence. The PI has a strong working relationship with the company and has licensed sequencing pore technology which has been one key component to make the MinION a success. In this project, the technology will be adapted with wider nanopores tailored for Covid- 19 antibodies. The new sensor pores can be plugged into the existing MinION kits without the need for redesigning the device, thereby ensuring production to scale. The devices will be clinically tested and benchmarked by Co-PI and intensive care and monitoring specialist Prof. Mervyn Singer at UCL Medicine (UCLM). All project partners have previously successfully worked together in joint grants, for publications, or via technology licensing contracts.

Proteins are of paramount importance in our lives. They carry out the main functions in our bodies and control our movement, energy conversion, immune defence, and thinking. In medicine, functionally aberrant proteins cause disease. The proteins can, however, be targeted by drugs to cure cells. Enzymes are also of biotechnological importance in the cost-efficient synthesis of drug molecules or for the energy-saving cleaning of fabrics. Detecting and analysing proteins is the first step towards predicting diseases, developing cures, and engineering proteins for industry. The analysis of proteins improves our understanding their structure, dynamics, and function. Ideally, sensing of proteins should be simple and fast and be conducted using inexpensive and portable equipment. This increase research efficiency and opens up point-of-care sensing in diagnosis and homeland security. This project will provide a new way to sense proteins in a portable yet scientifically accurate fashion thereby overcoming problems of existing approaches. Classical approaches have issues such as the requirement to label the proteins with a fluorescent tag which can interfere with the structure and function of the proteins. Optical detection can also increase the weight and cost of the analytical device. Another limitation of conventional sensing approaches is that they average over millions of molecules and have difficulties to detect biologically important sub-groups. We will develop a new approach to sense proteins in a label and optics-free electrical fashion using portable equipment capable of uncovering proteins down to the level of individual molecules. The proposed strategy is currently being used for DNA strand sequencing. Our industry partner Oxford Nanopore Technologies has developed a hand-held device for genome sequencing. The analytes are detected when individual strands pass through nanoscale pores in a thin membrane. The temporary blockade of the pores alters the electronic read-out signal similar to the reduction of water flow when a stone is inside a tube. We will be able to sense proteins which are wide enough to accommodate proteins. The new pores will be composed of DNA stands, thereby exploiting the exquisite ability of DNA to function as a nanoscale construction material. Chemical modification will be key to achieve the functional performance of the pores. To maximise the benefit for academia, industry and society, we will strongly collaborate with our commercial partner to test the new pores in the portable electrical sensing devices.

Doctoral Training Partnerships: a range of postgraduate training is funded by the Research Councils. For information on current funding routes, see the common terminology at https://www.ukri.org/apply-for-funding/how-we-fund-studentships/. Training grants may be to one organisation or to a consortia of research organisations. This portal will show the lead organisation only.

Stochastic sensing with nanopores is a versatile technology that can be used for the recognition and quantification of a wide range of substances (known as analytes) through the detection of individual molecules. Our partner company, Oxford Nanopore Technologies, has been incorporating stochastic sensing into next-generation hand-held devices. The most highly developed application at Oxford Nanopore is cheap, extremely rapid DNA sequencing, which promises to revolutionise numerous areas of biology including aspects of medicine, ancestry and forensics. Currently, a portable sequencer is being tested at hundreds of sites worldwide. In stochastic sensing, analytes are detected as they enter and leave a single narrow pore perturbing a current that flows through it. The diameters of the pores, known as nanopores, are similar to those of a small molecule, about one-fifty thousandth of the diameter of a human hair, providing the basis for detection by current perturbation. Typically current changes of the order of one trillionth of an ampere are measured. Analytes have included drug molecules and small molecules found in the body that act as markers for disease. In the case of DNA sequencing, individual bases are detected as an extended DNA strand is threaded through a nanopore. Protein pores are advantageous for stochastic sensing, because they can be modified for particular applications with atomic precision and prepared in near homogeneous form. Until now, very narrow protein pores have been used and therefore stochastic sensing has been limited to analytes of small size or, in the case of DNA, to extended polymer chains. In the proposed work, we will endeavour to make a new class of functional nanopores, DNA-Protein hybrid nanopores. These pores will be constructed from folded DNA, known as DNA origami, and protein components. The DNA will act as a scaffold for the protein, ensuring that the new pores are up to fifteen times larger in internal diameter than the pores used before. Further, each pore will be of identical size and no incompletes pores will be present, a goal that has not be achieved previously. Finally, it will be possible to modify the new pores at precisely determined sites, which cannot be done with competing technologies, such as solid-state pores. The DNA-protein hybrid nanopores will enable a critical step forward for stochastic sensing by allowing the detection of a wide range of large biological molecules that can enter the pores, including proteins, DNAs and polymeric sugars. Conversely, it will also be possible to lodge these large molecules within the hybrid pores, where they will act as binding sites for a variety of additional analytes. In a futuristic application, it may prove possible to sequence double-stranded DNAs with hybrid pores, which will provide a significant advantage over the manipulations currently required for nanopore sequencing. Our industrial partner, Oxford Nanopore, will evaluate and test our most promising DNA-protein hybrid nanopores in their hand-held sensing devices, which are capable of monitoring the outputs of hundreds of pores in parallel, offering the prospect of step changes in sensing technology in areas including biological warfare defense, food authentication, plant and animal breeding and medical diagnostics.

Genome sequencing methods have led to a rapid expansion in our understanding of the subtleties and variations in how genetic profiles affect disease. This has been spurred in part by new technological advances. Nanopore sequencing has been highlighted as one upcoming technology with the potential to go further, and improve the speed and cost effectiveness of seqencing. In nanopore sensing, the flow of ions through a nanoscopic protein or solid-state pore is disrupted by the presence of an analyte. If this analyte is DNA, blockades in the current corresponding to the sequence are detected. Long reads of many thousands of bases can be made without amplification or labelling. Our research track record marks a long-standing interest in developing methods capable of detecting individual molecules using fluorescence microscopy. We have used this to improve our understanding of membrane protein function, and in particular, how specialized protein pores work. Most recently we used these methods to demonstrate DNA base detection in a nanopore using an optical, rather than an electrical readout. These methods have the potential to improve the parallelization of nanopore sensing. In collaboration with Oxford Nanopore Technologies we have identified two essential challenges for current nanopore sensing where an optical readout provides significant impact: 1. We will combine optical single channel recording and single-molecule fluorescence imaging to understand and then optimise the mechanistic steps of nanopore sensing; 2. We will go beyond the current speed and sensitivity limitations of optical single channel recording and develop new single-molecule microscopy methods for nanopore sensing.