Dynamic single-molecule sensing by actively tuning binding kinetics for ultrasensitive biomarker detection

The ability to measure many single molecules simultaneously in large and complex samples is critical to the translation of single-molecule sensors for practical applications in biomarker detection. The challenges lie in the limits imposed by mass transportation and thermodynamics, resulting in long assay time and/or insufficient sensitivity. Here, we report an approach called Sensing Single Molecule under MicroManipulation (SSM³) to circumvent the above limits. In SSM³, single-molecule binding processes were dynamically recorded by surface plasmon resonance microscopy in a nanoparticle-mediated sandwich scheme. The binding kinetics between analyte and probes are fine-tuned by nanoparticle micromanipulations to promote the repetitive binding and dissociation. Quantifying the heterogeneous lifetime of each molecular complex allows the discrimination of specific binding from nonspecific background noise. By digitally counting the number of repetitive specific binding events, we demonstrate the direct detection of microRNAs and amyloid-β proteins with the limit of detection at the subfemtomolar level in buffer and spiked human serum. Together with the nanoparticle micromanipulation to promote the transportation rate of analyte molecules, the assay could be performed within as short as 15 min without the need for preincubation. The advantages over other single-molecule sensors include short assay time, compatible with common probes and ultrasensitive detection. With further improvement on the throughput and automation, we anticipate the proposed approach could find wide applications in fundamental biological research and clinical testing of disease-related biomarkers.

The analytical methods have converged from ensemble measurements of numerous entities to quantized measurements at the single-molecule level. Single-molecule measurements could reveal heterogeneities and stochastic processes within biological systems (1, 2) and set the ultimate detection limit of chemical and biological sensors. By reducing the measurement volume to a few femtoliters, the detection of a single molecule has been realized in various forms [i.e., single-molecule fluorescence (3, 4), nanopores (5, 6), localized surface plasmon resonance (7, 8), and surface-enhanced Raman scattering (9, 10)]. These measurements typically require quantifying many single-molecule events to gain new molecular and mechanistic insights or to achieve better analytical performance. However, it has been difficult to perform quantitative analysis with sufficient efficiency and statistical accuracy because of the concentration limit from mass transportation (11, 12) and the thermodynamic limit from probe affinity (13). For quantification of biomarkers in biological media, in which the required concentrations are usually at the femtomolar level or even lower (14), the single-molecule measurements could take inordinately long, and the nonspecific binding of unwanted species degrades the accuracy.In the past two decades, several single-molecule approaches for biomarker detection have been developed to surpass the above limits by biasing the equilibrium and driving binding reactions (15, 16). A typical scheme involves the usage of nanoparticles to collect the analyte followed by a digital measurement of single molecules at a confined space (17), such as the commercialized, single-molecule enzyme-linked immunosorbent analysis (digital ELISA) (18). The digital ELISA uses the antibody-modified magnetic beads to capture the analyte in solution and loads them into femtoliter-sized reaction chambers termed single-molecule arrays. It effectively improves the sensitivity of conventional ELISA by three orders with a limit of detection (LoD) at the subfemtomolar level but requires sophisticated devices and excessive operation to remove free analyte molecules. Besides, the performance is still limited by the probe affinity and false positive arising from detection antibodies that bind nonspecifically to assay surface.A distinct yet effective strategy is to explore the in-depth heterogeneous information of single-molecule interaction (19). Walter et al. first demonstrated a kinetic fingerprinting approach to perform highly specific and sensitive detection of biomarkers via single-molecule fluorescence microscopy (20–22). This single-molecule recognition through equilibrium Poisson sampling technique surpasses the thermodynamic limit by exploiting the repetitive binding of fluorescently labeled, low-affinity probes to the analyte (23) and discriminating specific binding from background noise by a kinetic signature. The detection limits of microRNAs (miRNAs) and proteins also reach the subfemtomolar level, but screening probes with unique kinetic property is not compatible with current pipelines, and the concentration limit implies long incubation time before detection.Herein, we present the integration of single-molecule manipulation and dynamic sensing to allow rapid and ultrasensitive detection of biomarkers beyond the concentration and thermodynamic limits. In this Sensing Single Molecule under MicroManipulation (SSM³) approach, an external force is applied on the molecular bound between analyte and probes through tethered nanoparticles to actively tune the binding kinetics. This strategy, together with a dynamic sensing approach to exploit the heterogeneity at the single-molecule level, is able to beat the limits in both assay time and sensitivity. We show the principle and realization of the SSM³ technique and demonstrate 15-min assays to directly measure miRNAs and proteins at the subfemtomolar concentration.