Time-resolved digital immunoassay for rapid and sensitive quantitation of procalcitonin with plasmonic imaging

Jing, Wenwen; Wang, Yan; Yang, Yuanyuan; Wang, Yi; Ma, Guangzhong; Wang, Shaopeng; Tao, Nongjian

doi:10.1101/483347

Cited by 3 publications

(4 citation statements)

References 28 publications

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“…The discrimination of specific binding by the lifetime of molecular complex leads to improved limit of detection (LoD). The LoD by direct counting of all binding events within the 0-300 s lifetime window was 14.88 pM, which is similar to the previously reported digital assay 24,36 .…”

Section: The Effectiveness Of Micromanipulationsupporting

confidence: 86%

“…The single-molecule detection was performed in a typical sandwich scheme composed of a high-affinity primary probe immobilized on a planar sensor chip, the analyte and a secondary probe tethered to nanoparticles (Figure 1a, and supplementary information). A microscope with single nanoparticle imaging capability, such as the surface plasmon resonance microscope (SPRM) used herein, dynamically records the image sequences of nanoparticles to report single molecular interactions 24,25,26,27 . Considering the first-order binding kinetics between the analyte and the secondary probe, SSM 3 reports the sum of total association and dissociation events during the observation by digital counting at the single-molecule level (Eq.1),…”

Section: The Principlementioning

confidence: 99%

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Dynamic single-molecule sensing via nanoparticle micromanipulation for rapid and ultrasensitive biomarker detection

Zeng

Zhou

Yang

et al. 2021

Preprint

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The ability to measure many single molecules simultaneously in larger 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, the transportation rate of analyte molecules and the kinetics of molecular interaction are fine-tuned by the nanoparticle micromanipulation. The heterogeneous lifetime of molecular complexes is quantified to discriminate specific binding from nonspecific background noise. By the highly-specific digital counting of single molecules, we demonstrate 15-minute assays for direct detection of microRNAs and amyloid-β proteins via electrical or magnetic micromanipulation, with the limit of detection at the subfemtomolar level. The presented approach could inspire more practical applications of single molecule sensors.

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Section: The Effectiveness Of Micromanipulationsupporting

confidence: 86%

Section: The Principlementioning

confidence: 99%

Dynamic single-molecule sensing via nanoparticle micromanipulation for rapid and ultrasensitive biomarker detection

Zeng

Zhou

Yang

et al. 2021

Preprint

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“…antibodies. 27-29 We studied the binding of troponin T (TnT), a biomarker for heart diseases, 30 to its antibody using a sandwich immunoassay provided by a commercial ELISA kit. First, the capture antibody was immobilized on the gold surface via NHS/EDC chemistry.…”

Section: Resultsmentioning

confidence: 99%

Three-dimensional tracking of tethered particles for probing nanometer-scale single-molecule dynamics using plasmonic microscope

Wan

Yang

et al. 2021

Preprint

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Three-dimensional (3D) tracking of surface-tethered single-particle reveals the dynamics of the molecular tether. However, most 3D tracking techniques lack precision, especially in axial direction, for measuring the dynamics of biomolecules with spatial scale of several nanometers. Here we present a plasmonic imaging technique that can track the motion of ~100 tethered particles in 3D simultaneously with sub-nanometer axial precision at millisecond time resolution. By tracking the 3D coordinates of tethered particle with high spatial resolution, we are able to determine the dynamics of single short DNA and study its interaction with enzyme. We further show that the particle motion pattern can be used to identify specific and non-specific interactions in immunoassays. We anticipate that our 3D tracking technique can contribute to the understanding of molecular dynamics and interactions at the single-molecule level.

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“…Researchers have developed rapid (14)(15)(16)(17), point-of-care (18)(19)(20), multiplex (21,22) immunoassays powered by microfluidics. Nonetheless, it is still challenging for these assays to simultaneously achieve a combination of high multiplexity and sensitivity with a rapid assay turnaround time in a clinical setting.…”

Section: Introductionmentioning

confidence: 99%

Machine Learning-Based Protein Microarray Digital Assay Analysis

Song

Zhao

Cai

et al. 2020

Preprint

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Serial measurement of a large panel of protein biomarkers near the bedside could provide a promising pathway to transform the critical care of acutely ill patients. However, attaining the combination of high sensitivity and multiplexity with a short assay turnaround poses a formidable technological challenge. Here, we developed a rapid, accurate, and highly multiplexed microfluidic digital immunoassay by incorporating machine learning-based autonomous image analysis. The assay achieved 14-plexed biomarker detection at concentrations < 10pg/mL with a sample volume < 10 μL, including all processes from sampling to analyzed data delivery within 30 min, while only requiring a 5-min assay incubation. The assay procedure applied both a spatial-spectral microfluidic encoding scheme and an image data analysis algorithm based on machine learning with a convolutional neural network (CNN) for pre-equilibrated single-molecule protein digital counting. This unique approach remarkably reduced errors facing the high-capacity multiplexing of digital immunoassay at low protein concentrations. Longitudinal data obtained for a panel of 14 serum cytokines in human patients receiving chimeric antigen receptor-T (CAR-T) cell therapy manifested the powerful biomarker profiling capability and great potential of the assay for its translation to near-real-time bedside immune status monitoring.

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Time-resolved digital immunoassay for rapid and sensitive quantitation of procalcitonin with plasmonic imaging

Cited by 3 publications

References 28 publications

Dynamic single-molecule sensing via nanoparticle micromanipulation for rapid and ultrasensitive biomarker detection

Dynamic single-molecule sensing via nanoparticle micromanipulation for rapid and ultrasensitive biomarker detection

Three-dimensional tracking of tethered particles for probing nanometer-scale single-molecule dynamics using plasmonic microscope

Machine Learning-Based Protein Microarray Digital Assay Analysis

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