A single biomolecule interface for advancing the sensitivity, selectivity and accuracy of sensors

The performance of a sensor is mainly determined by its interface with sensing molecules, which comprises reactive molecules for the recognition, active molecules for the signal responses and/or functional materials for signal enhancement (Fig. 1a(i)). For example, a double layer is set up at the electrode/electrolyte interface, across which charge-transfer reactions occur. In a previous study [1], a membrane interface was constructed onto the electrode, in which ubiquinone was embedded in a hybrid bilayer membrane, facilitating the charge/electron transfer from the NADH (nicotinamide-adenine dinucleotide) to the electrode. This interface mimics the initial stages of respiration to achieve the reversible interconversion between NADH and NAD+ at a low overpotential. Therefore, a local chemical environment is established at the sensor interface, where the electrode potential is measured, giving the information about the analyte. Using the macroscale-sensing interface, traditional sensors have been applied in the wide range of detection. (a) The transformation of a sensing interface. (i) The macroscale interface with a sensing molecule for detection of the targeting analyte. (ii) The macroscale interface shrinks and turns over, (iii) then rolls into a channel as the micro-/nanoscale interface. (iv) A single-biomolecule interface is derived from a single recognition molecule at the nano interface. A self-assembled aerolysin membrane protein is regarded as an example of a single-biomolecule interface. The oligonucleotide is taken as an example for illustrating the analyte. (b) Example of analysing an oligonucleotide through a single aerolysin interface with site-direct mutagenesis. A wild-type aerolysin with two positively charged amino acids produces distinguishable blockage current (red box). The speed of a single negatively charged oligonucleotide translocation aerolysin pore is as slow as ∼ 2 ms/base due to the strong electrostatic interactions caused by the positively charged residues at the entrances and inside the lumen. (c) The mutant I with negatively charged glutamic acid located at the entrance of the pore, which generates a high entropic energy barrier for the sensing of negatively charged oligonucleotide molecules. Consequentially, barely no current blockages occur with the continuous time-current recording. (d) The mutant II with negatively charged glutamic acid at the lumen of the single-molecule interface leads to a prolonged duration (red box). The dynamic conformational changes of the oligonucleotide may be enhanced inside the pore due to the electrostatic repulsion, inducing further current oscillations (blue box). However, there is an urgent demand to miniaturize sensor interfaces for permitting detection at different scales. If a micro/nano area is taken from the traditional macroscale sensor, it could then turn over and roll into a small structure for reducing the size of the sensing interface (Fig. 1a). To achieve this, continuous efforts have produced many examples of high-performance sensing systems at nano- and microscale interfaces. For example, solid-state nanopores employed interface effects based on charge, wettability, receptors for the selective sensing of ions, single peptides/proteins/DNAs, IgGs, etc. [2]. Recently, modulating the charge gradient along the wall of silver-coated quartz nanopores allows the distinction of as low as 14 H2 molecules and 28 Ag+ [3,4]. Moreover, the nanopipette demonstrates valuable advantages in probing individual cells [5]. To achieve a high selectivity, the modification of a specific group presenting in the whole region of the micro/nano interface should be designed. However, all these micro-/nanoscale sensors barely achieve site-directed modification at submolecule resolution. Therefore, it is a challenge to manipulate every single modification group or to control the certain properties of the sensing interface at the sub-nanometer scale. This feature is vital for designing the sophisticated interface for advanced sensing. For example, we can command several molecules at a nano interface for enhancing the possibility of analytes reaching to the sensing interface, thereby increasing the sensitivity. Meanwhile, controlling other multiple kinds of molecules requires the selective recognition of different targets. As a result, the interface could process both high sensitivity for single-molecule sensing and selectivity for multiple analytes. More importantly, this strategy may improve spatial resolution, since it could well control the nanometer- or sub-nanometer-sized sensing site at submolecule resolution. To achieve this goal, in this Perspective, we propose the concept of a single-biomolecule interface. Here, the whole interface originates from a single molecule. As a reasonable model, a single-membrane protein that is assembled from several monomers is regarded as a single-biomolecule interface (Fig. 1b). Therefore, the side chain of every single amino acid group could be designed respectively by single-site mutation. The single-biomolecule interface has been achieved in biological nanopore sensors. A single-membrane protein molecule could be regarded as a single-molecule interface. At applied voltage, a single analyte is electrochemically driven through the single-protein pore in the electrolyte solution [2]. The transit stay of a molecule at the nanopore interface modulates the distribution of the ionic concentration, which results in a current modulation. Differently from the dilution strategy to prepare an ultralow-concentration solution for single-molecule analysis, the single-molecule interface as a protein nanopore provides a confined space to capture a single molecule from the bulk solution [6]. As shown in Fig. 1b, the cap domain of an aerolysin is responsible for the capturing of the single oligonucleotide while the β-barrel region dominates the translocation process. Since the detection and capturing of a single molecule are predominantly diffusion-limited, it is expected that the capture ability of the pore towards the single analyte could be increased if the key amino acid specifically substitutes for the desirable group. Also, the change of side chain at the inner wall of the pore could optimize the geometry of the pore as well as realize an optimal interaction between the single-biomolecule interface and the analyte [7]. This improvement allows the high spatial and current resolution of nanopore sensors, which would ensure the possibility of the dynamic study of a single biomolecule including their metastable conformation, charge distribution and interactions. Recent examples of a single-biomolecule interface show its advantages to address the goal towards sensitivity, specificity and rapidness [6]. Especially for single-molecule detection, the single-biomolecule interface exhibits strong sensing ability for revealing the hidden heterogeneous properties of the analyte at a high throughput. Despite that, single-biomolecule interface based single-molecule detection is challenging, for fundamental, practical and applied reasons. The fundamental challenges come from revealing the intrinsic property of a single molecule with its dynamic structure. For example, the function–structure relationship of a redox enzyme should uncover the electron-transfer property of a dynamic redox enzyme. To achieve this goal, a novel sensing mechanism should be developed by incorporating electrochemistry, fluorescence spectroscopy, plasmonic scattering and mass spectroscopy, etc. Recent attempts at wireless nanopore electrodes that confine bipolar electrochemistry into the nanopore show promise in revealing the redox property of a single molecule [3,4]. The practical perspective comes from dealing with a big data set that involves the transient behaviors of a single molecule. Our continuous development of data-processing methods uncover these characteristics [8] and events, which could further predict the dynamic behavior of a single molecule if machine learning is incorporated. A more applied perspective allows the development of smart, auto-magical and small devices for a single-molecule interface to achieve real sample detections. A recent study shows a simple electric circuit that ensures the integration of the ultra-sensitive electrical detection device with a fabrication process in a portable device [9]. In conclusion, a single-molecule interface facilitates the design of a single sensing zone that ensures high spatial and temporal resolution towards single-molecule analysis. The single-site modification along the single-molecule interface manipulates each site of the interface for the precise responses that meet the multiple-sensing requirement. More importantly, a single-biomolecule interface provides confinement for a single-molecule reaction, which offers the possibility of studying reaction kinetics in high temporal resolution. These advances provide a new concept in designing advanced sensors. Moreover, by combining with other single-molecule methods such as fluorescence or plasmonic technology, it is possible to construct an integrated single-biomolecule interface for multi-element readouts, which is expected to further provide rich structural information on single molecules [10]. We illustrate that a single-molecule interface likes a tuba (Fig. 2). As the single target molecule flows into the ‘tuba’, its dynamic structural information could be performed by modulating the interactions (‘button’) between ‘molecule flow’ and the resonance space at the single-molecule interface (‘tuba’). Therefore, the beautiful music of a single molecule will be played with a typical rhythm and melody. Single-molecule music. A single-molecule interface likes a tuba. As each molecule flows through the single-molecule interface, man one can control the ‘button’ along the ‘tuba’ to organize a beautiful rhythm for single-molecule music. This work was supported by the National Natural Science Foundation of China (21421004 and 21327807), the Innovation Program of Shanghai Municipal Education Commission (2017-01-07-00-02-E00023) and the Fundamental Research Funds for the Central Universities (222201718001 and 222201717003). Conflict of interest statement. None declared.