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Beating the Reaction Limits of Biosensor Sensitivity with Dynamic Tracking of Single Binding Events

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Beating the Reaction Limits of Biosensor Sensitivity with Dynamic Tracking of Single Binding Events Beating the reaction limits of biosensor sensitivity with dynamic tracking of single binding events Derin Sevenlera,b,1, Jacob Truebc, and M. Selim Ünlüa,1 aDepartment of Electrical and Computer Engineering, Boston University, Boston, MA 02215; bCenter for Engineering in Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02129; and cDepartment of Mechanical Engineering, Boston University, Boston, MA 02215 Edited by David R. Walt, Brigham and Women’s Hospital–Harvard Medical School, Boston, MA, and accepted by Editorial Board Member John A. Rogers January 19, 2019 (received for review September 13, 2018) The clinical need for ultrasensitive molecular analysis has moti- species (e.g., genomic DNA) with single-copy sensitivity and vated the development of several endpoint-assay technologies precision (10), while the detection limits of other analytes (e.g., capable of single-molecule readout. These endpoint assays are microRNA) are many orders of magnitude worse (11–13). Probe now primarily limited by the affinity and specificity of the affinity can also vary between samples. Variations in extensive molecular-recognition agents for the analyte of interest. In properties of the sample such as pH and ion content change the contrast, a kinetic assay with single-molecule readout could free energy of binding, and variable amounts of nonspecific distinguish between low-abundance, high-affinity (specific ana- background binding further complicates quantitation. lyte) and high-abundance, low-affinity (nonspecific background) Current leading single-molecule detection technologies rely on binding by measuring the duration of individual binding events at signal-amplification reactions. These are endpoint assays: the equilibrium. Here, we describe such a kinetic assay, in which probe molecules are incubated with the sample for a set amount individual binding events are detected and monitored during of time, after which the reaction is halted so that amplification sample incubation. This method uses plasmonic gold nanorods and detection can be performed. What is measured is the and interferometric reflectance imaging to detect thousands of amount of bound analyte at the instant the incubation is halted. individual binding events across a multiplex solid-phase sensor In contrast to endpoint assays, kinetic assays directly measure with a large area approaching that of leading bead-based – endpoint-assay technologies. A dynamic tracking procedure is probe analyte interactions during course of the incubation. Ki- ENGINEERING used to measure the duration of each event. From this, the total netic assays collect more information than endpoint assays: they rates of binding and debinding as well as the distribution of can measure not just concentration but also molecular affinity binding-event durations are determined. We observe a limit of via rates of association and dissociation. This could allow for detection of 19 fM for a proof-of-concept synthetic DNA analyte in intersample variations in probe affinity or nonspecific binding to a 12-plex assay format. be identified and mitigated without additional tests. For low concentrations of analyte, kinetic assays could also be capable of single-molecule detection | biosensor | nanotechnology | nanophotonics | distinguishing low-abundance specific binding from a larger molecular recognition background of nonspecific binding, or even measuring analytes below the so-called “critical concentration” at which there is everal of the promises of precision medicine rely on ultra- fewer than one analyte molecule bound at equilibrium—a feat Ssensitive molecular diagnostic technologies. Liquid biopsies impossible for endpoint assays (14). of circulating genomic, transcriptomic, or proteomic biomarkers of cancer promise earlier detection and treatment, as well as Significance improved guidance of targeted therapies in treating minimum residual disease (1, 2). Similarly, sensitive and specific molecular Low-abundance molecular biomarkers are a promising reser- diagnostic tests for infectious pathogens are vital for the identi- voir of diagnostic information for the early detection and fication and management of pre- or asymptomatic individuals (3, precise treatment of a range of diseases. Yet, most leading 4). Likewise, panel assays of circulating biomarkers could soon assay technologies are currently limited by the affinity and improve the accuracy of diagnosis of injuries such as acute liver specificity of the molecular-recognition agents they use. This failure or traumatic brain injury (5, 6). binding “reaction limit” could be beaten by measuring the These pressing clinical needs have motivated the development duration of individual molecular-binding events as they hap- of a variety of ultrasensitive assay technologies, culminating in pen in real time. We developed a technology to measure the technologies capable of single-molecule detection. A unifying duration of thousands of individual binding events across a characteristic of essentially all of these assay technologies is that large sensor area. These duration measurements allowed spe- they employ molecular-recognition agents (or “capture probes”) cific analyte binding to be distinguished from nonspecific such as antibodies, nanobodies, peptides, oligonucleotides, background binding. The sensor is large enough to accommo- aptamers, or other agents that bind specifically to the molecule date 12 ultrasensitive tests at once. of interest. Single-molecule detection technologies commonly then use droplet emulsions (7) or microwell arrays (8) to isolate Author contributions: D.S. and M.S.Ü. designed research; D.S. performed research; D.S. and J.T. contributed new reagents/analytic tools; D.S. and M.S.Ü. analyzed data; and D.S., and then enumerate the precise number of analyte molecules J.T., and M.S.Ü. wrote the paper. bound to the capture probes. The authors declare no conflict of interest. In terms of signal transduction, it is clear that single-molecule This article is a PNAS Direct Submission. D.R.W. is a guest editor invited by the detection is “as good as it gets” (9). However, transducing the Editorial Board. — amount of captured analyte is only one half of the picture the Published under the PNAS license. analyte must be captured in the first place. Even with single- 1To whom correspondence may be addressed. Email: [email protected] or molecule detection, assay performance is still limited by the af- [email protected]. finity of the capture probes. This causes sensitivity and specificity This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. to vary widely between different probe–analyte pairs. For ex- 1073/pnas.1815329116/-/DCSupplemental. ample, it is now relatively routine to quantify some molecular www.pnas.org/cgi/doi/10.1073/pnas.1815329116 PNAS Latest Articles | 1of6 Downloaded by guest on September 23, 2021 However, single-molecule kinetic measurements are techni- Results cally demanding: without amplification reactions, specific bind- Detection of Individual Binding Events Across a Large Field of View. ing events are more difficult to discern against a background of We recently described the development of a “digital microarray” nonspecific interactions. Indeed, an exquisitely sensitive trans- assay technology, which rapidly enumerates individual captured duction mechanism is required to directly detect single binding molecules across hundreds of microarray spots (21). This tech- events at all. A range of scientific apparatuses have been de- nology uses probe-conjugated gold nanorods as molecular labels veloped to investigate single-molecule binding kinetics. How- and an interferometric reflectance imaging sensor (IRIS) to ever, none of these techniques are useful for ultrasensitive rapidly detect individual nanorods with a low-magnification clinical assays because the sensors are too small (14). To in- (10–20×) objective. The large field of view enabled a similar vestigate nanoscale phenomena, these devices are themselves throughput to commercial fluorescence readers while en- nano- or microscale: their active sensors are the size of single hancing the limit of detection (LOD) and dynamic range by a nanoparticles or nanowires (15–17), or else they require high- factor of ∼10,000. The assay reaction is based on the bio- magnification and high-numerical aperture optics with a small barcode assay developed by others (22) and is compatible with field of view (0.001–0.01 mm2) (18–20). This is problematic be- a range of different analytes. cause small sensors only have space for a small number of cap- We adapted the IRIS digital microarray platform for dynamic ture probes. Maximizing the number of probes is vital for measurements by designing a perfusion chamber that consists of ultrasensitivity: at low concentrations, the amount of captured an IRIS chip, a patterned silicone gasket, and an antireflection- coated coverglass window (Fig. 1A). Two holes for the chamber analyte at equilibrium is proportional to the number of probe inlet and outlet are drilled in the IRIS chips by wafer-scale laser molecules. Single-molecule assay technologies therefore use micromachining. The assembly is held by a custom clamp fixture large sensor areas packed with capture probes. For example, the ∼ that makes fluidic connections to the inlet and outlet on the Quanterix Simoa technology interrogates 25,000 beads, each bottom of the chip (SI Appendix, Fig. S1). 2.7 μm in diameter, corresponding to a total
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